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QUALITY ASSURANCE METHODS MANUAL
FOR
LABORATORY ANALYTICAL TECHNIQUES
Wayne P. Robarge
Department of Soil Science
North Carolina State University
Raleigh, NC 27695
and
Ivan Fernandez
Department of Plant & Soil Science
University of Maine-Orono
Orono, HE 04469
July 1986
This Laboratory Analytical Techniques Methods Manual is one of four
Quality Assurance.Methods Manuals prepared for the U.S. EPA and USDA
Forest Service Sorest Response Program. Quality Assurance for the
Forest Response Program is being administered through Corvallis
Environmental Research Laboratory, Corvallis, OR. Coordination of the
vorkshops, technical editing of the manuals, and administrative support
were provided by the North Carolina State University Acid Deposition
Program, Raleigh, NC.
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TABLE OF CONTENTS
Section Pages Revision Date
PREFACE 6 . . . 0 . July 1986 . iv
1. GOOD LABORATORY PRACTICES 13 . . . 0 . July 1986 . 1
1.1 Documenting Analysis Procedures 1
1.2 Maintaining Distilled Water Quality. . . 2
1.3 Cleaning and Using Glassvare and Plasticvare 3
1.4 Using Laboratory Reagents, Solvents, and Acids 5
1.5 Maintaining Analytical Standards . 6
1.6 Weighing . ..... ....... 6
1.7 Maintaining Laboratory Cleanliness 7
1.8 Controlling Laboratory Sample Handling/Custody 6
1.9 Recording and Reporting Data 10
1.10 References 13
2. FOLIAR INORGANIC ANALYSIS 47 . . . 0 . July 1986 . 14
2.1 Standard Operating Procedure for Multi-Element Analysis:
N (Kjeldahl-N), P Using an AutoAnalyzer 14
2.2 Standard Operating Procedure for Multi-Element Analysis:
C, N (Total-N), S Using an Elemental Analyzer 22
2.3 Standard Operating Procedure for Multi-Element Analysis:
N (Kjeldahl-N), P, Ca, Mg, K Using VFT Digestion 25
2.4 Standard Operating Procedure for Multi-Element Analysis:
K, P, Ca, Mg, B, Na, Cu, Zn Using Dry Combustion 31
2.5 Standard Operating Procedure for Multi-Element Analysis:
K, Ca, Mg, Cu, Fe, Mn, Zn, Al, Cd, Na, Ni, Pb, V, Ba, Co, Rb
Using Dry Combustion 36
2.6 Standard Operating Procedure for Measurement of Boron 42
2.7 Standard Operating Procedure for Measurement of Chloride ..... 48
2.8 Standard Operating Procedure for Measurement of Sulphur. ..... 54
3. FOLIAR ORGANIC ANALYSIS .35 . . . 0 . July 1986 . 61
3.1 Standard Operating Procedure for Determination of Total
Chlorophyll . . . 61
3.2 Standard Operating Procedure for Quantitative Analysis of
n-Alkane Concentration and Total Epicuticular Wax of Red
Spruce Foliage ..... 69
3.3 Standard Operating Procedure for Measurement of Starch and Total
Sugars 84
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TABLE OF CONTENTS (cont'd)
Section Pages Revision Date Page
4. SOIL PHYSICAL ANALYSIS 34 . . . 0 . July 1986 . 96
4.1 Standard Operating Procedure for Sample Preparation 96
4.2 Standard Operating Procedure for Measurement of Soil Moisture
Content 103
4.3 Standard Operating Procedure for Measurement of Organic Matter by
Loss-On-Ignition 106
4.4 Standard Operating Procedure for Measurement of Soil Bulk
Density (Core Method) 109
4.5 Standard Operating Procedure for Measurement of Field Water
Content (Gravimetric Approach) 117
4.6 Standard Operating Procedure for Particle Size Analysis
(Hydrometer Method) 122
5. SOIL CHEMICAL ANALYSIS. 58 . . . 0. . July 1986 . 130
5.1 Standard Operating Procedure for Measurement of pH (DI water,
0.01M CaCl:, IN KCl-air dry soils) 130
5.2 Standard Operating Procedure for Measurement of Exchangeable
Bases (Ca, Mg, Na, K) by IN NH^Cl 136
5.3 Standard Operating Procedure for Measurement; of Exchangeable
Acidity 142
5.4 Standard Operating Procedure for Measurement of Extractable
Metals (Fe, Zn, Cu, Pb, Cd, Ni, Mn) 150
5.5 Standard Operating Procedure for Measurement of Extractable
Phosphorus ('Bray 1'). . . 156
5.6 Standard Operating Procedure for Measurement of Extractable
Sulfate-S. 161
5.7 Standard Operating Procedure for Measurement of Total (Kjeldahl)
Nitrogen 168
5.8 Standard Operating Procedure for Measurement of Total Carbon/
Nitrogen/Sulfur Content Using an Elemental Analyzer 176
5.9 Standard Operating Procedure for Measurement of Elemental Content
by Total Dissolution (Ca, K, Mg, Al, Fe, S, P, Cu, Mn, Zn, Na,
Cd, Ni, Pb, V) 179
REFERENCES 7 . . . 0. . July 1986 . 188
APPENDICES 9 ... .0. . July 1986 . 195
A - List of Attendees • 196
B - Data Quality Objectives 198
C - Variable Codes (Computer Database Codes) 202
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PREFACE
The following Forest Response Program Quality Assurance Methods
Manual for Laboratory Analytical Techniques was developed in response to
the Environmental Protection Agency's (EPA) requirements for Quality
Assurance (QA) in environmental research. The Laboratory Analytical
workshop which provided the basis for this document was held on March 13-
14, 1986 in Raleigh, NC. The workshop was attended by 22 individuals
consisting of principal investigators and program administrators (see
Appendix A). The Laboratory Analytical workshop was one of four workshops
conducted by the North Carolina State University Acid Deposition Program
for the EPA to develop Quality Assurance Methods Manuals for the Forest
Response Program. The workshops and ensuing reviews of the methods
manuals were designed to allow scientists input in the selection of
standard research methods and the development of QA procedures for these
methods. Drs. Vayne Robarge and Ivan Fernandez provided workshop
leadership and compiled this methods manual from the comments and concerns
voiced at the workshop, and other sources.
Technical and support services to conduct the workshops and produce
these methods manuals were provided by the North Carolina State University
Acid Deposition Program under the direction of Ann M. Bartuska, Program
Coordinator. Janet M. McFayden coordinated the meeting. Kimberly Joyner
edited the draft and final documents. Additional editorial assistance was
provided by Gary B. Blank, School of Forest Resources, North Carolina
State University.
The protocols and Standard Operating Procedures '(SOPs) presented in
the methods manuals represent a consensus on the "best" technique for
operating a specified system of equipment or measuring a specified
variable, given the various objectives of the Forest Response Program.
"Best" is defined for these manuals according to the following criteria:
1) the quality or soundness of the protocol or SOP;
2) the availability of staff and facilities to adhere to the
protocol or SOP; and
3) the feasibility of assuring data quality through tests for
accuracy, precision, and consistency among sites.
The protocols and SOPs written in this first edition are general in
nature and encompass most existing research plans. As the program
progresses, the protocols and procedures will be revised and the manuals
will be expanded to include additional protocols and SOPs.
The purpose of the quality assurance methods manuals is
1) to provide standardization of research methods to allow for the
synthesis and integration of results for assessment purposes;
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2) to provide standardized quality assurance/quality control
techniques within standardized protocols and SOPs to allow for
the assessment and documentation of data quality; and
3) to prevent duplication of documentation efforts among
investigators using common techniques.
Appropriate standardization of research methods among projects which
contribute data to a centralized database is critical to the
synthesis/integration/assessment effort for the Forest Response Program.
Vithout comparability among sites, an overall assessment of research
results would be impossible. The National Forest Response Program
Research Plan provides details on the integration and assessment effort.
Standard quality assurance activities, providing guidelines for the
minimum amount of activity required to participate in the integrated
research program, ensures the ability of investigators and program
administrators to assess data quality as data are produced. This
knowledge directly influences the level of confidence for assessment
decisions, which is determined from the quality of the individual parts.
Other aspects of the QA program (research plan preparation, auditing, and
sample exchanges between sites) contribute to the process of ensuring that
data are of known and sufficient quality to meet the program's objectives.
Finally, these methods manuals will serve the investigators in simply
reducing the amount of documentation required for the assessment of data
quality. Many of the techniques employed by investigators examining
similar hypotheses are nearly identical. The manual^ identify and
document these similarities and can be referenced by the investigators in
their research plans. Much of the information required by the QA program
is included in these manuals; however, there are instances where
generalities should be clarified in individual plans. For example,
"sufficient training to operate the required equipment" as might be found
in a manual, must be specified for projects and facilities in the
individual research plan since large differences may exist between sites
and projects.
All investigators vith research projects funded through the Forest
Response Program Research Cooperatives will be required to adhere to the
protocols and the SOPs as described in these manuals. Vhere necessary,
investigators can deviate from the manual, by providing
1) a justification for the deviation;
2) a full explanation of the alternative protocol or procedure with
QA activities clearly described; and
3) an assessment of the impact of the deviation on data quality, by
comparing the alternative protocol or procedure with the
original.
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This will create an additional documentation burden for the investigator
and an assessment burden for administrators and, therefore, should be used
only when absolutely necessary.
The Forest Response Program Quality Assurance staff would like to
thank the workshop leaders, Drs. Robarge and Fernandez, and the
participants for their technical efforts in developing and refining the
material in this manual, and the Acid Deposition Program staff for
organizing and supporting the effort. Special thanks are extended to Dr.
Lee Allen (Section 2.3), Dr. Elaine fiirk (Section 3.3), Dr. Stephen A.
Modena (Section 3.1), Dr. M. Rutzke (Section 2.4) and Dr. Ronald Vilkinson
(Section 3.2) for their substantial contributions to this Methods Manual.
Special thanks are also extended to Dr. Ruth Alscher for her help during
the workshop, and to Dr. Michele Schoeneberger and to Mr. John Bower.
Ve appreciate the cooperation and patience of all the investigators
during the slow process of developing the QA program, including this
manual. Ve look forward to the ensuing years of quality research and an
Integration and assessment effort worthy of that quality.
NOTE:
The use of brand names in this manual is not an endorsement of a
particular product but a guide to the type of equipment required to
perform a particular measurement.
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1. GOOD LABORATORY PRACTICES
Good laboratory practices can be defined as a set of rules, operating
procedures, and practices that are adequate to ensure the quality and
integrity of data generated by a laboratory (Garfield, 1984). Good
laboratory practices are designed to complement the specific quality
control measures included within each standard operating procedure (SOP),
and should be considered as part of the overall quality assurance program
for the analytical laboratory.
1.1. DOCUMENTING ANALYSIS PROCEDURES
All standard operating procedures (SOPs) used in analysis of
samples shall be prepared using the following 8 point protocol:
1. Scope and Purpose
2. Materials and Supplies
2.1. Equipment
2.2. Chemi cals/Reagent s
2.3. Other
3. Procedure
3.1. Sample Preparation
3.2. Equipment Operation (include training and QC checks)
4. Preventive Maintenance
5. Calibration Procedures
6. Calculations/Units
7. Error Allovance and Data Quality
8. References
Revisions to a SOP shall be incorporated in the above 8 .point format
with specific reference to the subsection(s) being altered, added, or
deleted and forwarded immediately to the Quality Assurance Specialist,
along with justification for changing the SOP. Included within the
justification for changing the SOP should be sufficient information to
document that the proposed changes do not compromise the original Data
Quality Objectives defined for the SOP by the Quality Assurance Officer.
Additions and revisions to a SOP are considered to include the following:
a) information on how to train personnel in the use of special
instrumentation or sample preparation.
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b) revisions to portions of a SOP and how personnel were instructed
in revisions to SOP.
c) changes in the periodic performance evaluation of personnel in the
use of special instrumentation or sample preparation.
d) changes in the use of particular reagents, acids, glassware,
plasticvare, etc. found either unsuitable or to give optimum
results for a SOP.
1.2. MAINTAINING DISTILLED WATER QUALITY
Reference is made in the attached SOPs to distilled-deionized (DI)
water. Most analytical chemical laboratories have available tap distilled
water. The source of this distilled water is usually condensed steam from
the central heating plant which is condensed and passed through a
deionizing resin column, or steam which is condensed, redistilled and
passed through a deionizing column. Tap distilled water is often further
purified by laboratory distillation units or passage through a series of
resin and charcoal columns (e.g., Millipore Super-Q System, Hillipore
Corp., Bedford, Mass.).
In general, preparation of distilled-deionized water in this fashion
is acceptable for most inorganic analyses of samples, although the
composition of the distribution network and distilled water faucets should
be noted. Part of the quality assurance plan for a laboratory should
include maintenance guidelines and schedules for production of tap DI
water. Purified water or ACS reagent grade water can be defined as water
having a specific resistance of more than 500,000 ohms or a conductivity
of 2.0 microhms (Garfield, 1984). It is recommended that these values be
set as the minimum acceptable for DI water. Quality of the DI water
should be checked on a regular schedule using a suitable conductivity
probe (cell constant of 0.1).
Guidelines should also be developed for use of tap DI water,
especially if demand for DI water by other laboratories exceeds in-house
capacity to generate DI water. It is not uncommon for the temperature of
DI water produced in this fashion to change dramatically during the day.
Such changes in temperature may translate into changes in DI water
quality, and personnel should be instructed as to conditions under which
tap DI water is not to be used. It is also recommended that standard
laboratory practice include discarding the first three (3) liters of water
drawn from an in-house DI tap at the start of each working day.
Distilled-deionized water prepared by the above procedures is
generally not suitable for analyses requiring ammonia-free or low organic
background DI water. Use of an alkaline permanganate still is strongly
recommended for preparation of organic-free DI water. Simple
redistillation of DI water is ineffectual. Passage through charcoal beds
results in short term reduction of the organic content of DI water, but
charcoal beds will also release soluble organic matter with continued use.
Failure to remove organic background from DI water will result in high
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blanks for soluble organic matter determination in samples, and fouling of
separator columns in liquid chromatographs.
Sources of ammonia in DI water include preservatives added to steam
by heating plants, amines released from deionizing resin beds, and
absorption of ammonia from within the laboratory itself. Use of an
alkaline still, with discarding of the first 500 ml of distillate, is
sufficient for generating ammonia-free water. Use of fresh deionizing
resin is also acceptable. Use of concentrated ammonia solutions or
ammonia based floor waxes should be avoided when preparing ammonia-free DI
water.
1.3. CLEANING AND USING GLASSVARE AND PLASTICWARE
1.3.1. CLEANING PROCEDURES
New glassware and plasticware should be washed with mild detergent
before use. It is recommended that a low residue detergent be used for
cleaning new glassware and plasticware. New plasticware should also be
soaked in IN HN03 for several days to remove possible metal residues left
from molds during manufacturing.
It is recommended that all glassware and plasticware be permanently
labelled with a coding scheme that will permit identification of a
particular piece of labware at any time during an analysis. Such coding
schemes are useful in keeping track of samples during an analysis and in
randomizing reaction vessels to prevent the same vessels from being used
for the same purpose in each analysis. Random assignment of reaction
vessels to samples is recommended to prevent bias from use of particular
pieces of labware from going undetected.
Contaminated glassware and plasticware should be cleaned by a
suitable detergent, organic solvent, or acid. Use of cleaning agents is
especially recommended after use with sample aliquots containing soluble
organic matter or dispersed soil. It is recommended that glassware used
for soil analyses be kept separate from glassware for water and foliage
analyses.
Glassware and plasticware used in trace element analysis may become
contaminated by using certain cleaning procedures (e.g., chromic acid
cleaning solution). It is recommended that labware used in trace element
analysis be cleaned by carrying out one analysis using only blanks for
samples. This approach uses the actual chemical procedure to remove
contaminants from the labware. This approach also reduces the chance of
having a bias from contaminated labware in the analyses of the first set
of samples.
Care should be taken when cleaning volumetric glassware with strong
alkaline solutions to remove grease. Strong alkaline solutions attack
glassware, and extensive use of or prolonged contact with alkaline
solutions can alter calibration of volumetric glassware. Contact time
should be less than 15 minutes.
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Dry clean glassware or plasticware in a convection oven, or allow to
drain dry on an absorbent surface.
Cleaned labware should be stored in dust free areas with controlled
access, especially if labware is used in trace element analysis. Labware
removed from storage should be rinsed with DI water before use to remove
any particulate contamination that may have accumulated during storage.
1.3.2. USAGE - VOLUMETRIC GLASSWARE AND PLASTICWARE
Personnel should be instructed on the following points when using
volumetric ware (pipettes, flasks, burets, etc.).
° Never heat glassware unless stipulated in SOP.
0 Almost all volumetric ware is calibrated at 20eC. Guidelines
should be developed for what limits in temperature are acceptable
for use of volumetric ware. Note that temperature limits to be
set refer to temperature of solutions, not to the air temperature
of the laboratory. It is important to alert technicians to the
fact that tap DI water may change temperature if the source of the
DI water is a considerable distance from the laboratory.
0 Volumetric ware is either calibrated to contain (TC) or to deliver
(TD) set volumes of solution. Personnel should be instructed in
the difference, and when to use the appropriate type of volumetric
ware.
° Volumetric pipettes are to be drained and NOT blown out, unless
special blow out pipettes are called for in SOP.
0 Burets require specific time periods for accurate delivery of
contents. Personnel must allow sufficient time for burets to
drain for accurate results.
0 Most plastic volumetric ware does not have a curved meniscus.
Accurate use of plastic volumetric ware should be checked with a
balance as part of training of personnel in the use of same.
1.3.3. USAGE - MECHANICAL PIPETTES WITH DISPOSABLE TIPS
Mechanical pipettes with disposable tips are acceptable in place of
standard volumetric pipettes, provided the mechanical pipettes are
properly calibrated. Mechanical pipettes require regular maintenance and
suitable maintenance schedules should be established to check calibration
and overall condition. This latter step is especially important for
microliter mechanical pipettes. Follow manufacturer's recommended
guidelines for calibration and maintenance. Check calibration of pipettes
daily using a balance (+ 0.001 g).
Selection of disposable tips for use with mechanical pipettes is a
function of the type of analysis to be performed. Selection of colored
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tips may result in contamination of samples as the color is due to the
presence of certain metals (e.g., Cd or Co) in the plastic matrix. Clear
disposable tips that have been soaked in IN HN03 are recommended for trace
metal analysis. Tips should be used no more than 3 times before
discarding. Tips that will not vet properly with the sample solution
should be discarded.
1.4. USING LABORATORY REAGENTS, SOLVENTS, AND ACIDS
Unless otherwise stated, it is assumed that all reagents listed in
the SOP's are analytical reagent grade (AR). If special purity reagents,
solvents, or acids are required for an analysis, the level of purity and
possible source for chemical are to be listed in the SOP. Special
preparation of organic solvents (redistillation, filtration or degassing)
required for a particular analysis is to be fully documented in the SOP.
All purchased analytical reagent containers should be dated upon
arrival in the laboratory. A second date should be noted when the
container is opened. Estimated shelf life for each analytical reagent
should be recorded to prevent use of reagent stocks that may no longer
meet AR specifications.
Individual AR grade acid bottles should be checked for metal content.
It cannot be assumed that acid bottles with the same lot number have the
same metal content.
Use of high purity acids in digestion procedures is only necessary if
AR grade acids that are free from contamination cannot be found. Use of
high purity acids is warranted only if the rest of a'particular analysis
is carried out under conditions that do not compromise the purity of the
original acid. The use of a portable class +100 clean hood is recommended
during sample preparation, transfer, and other steps in a SOP where
contamination from the laboratory environment may compromise the purity of
the original acid. All high purity acids should be transferred to cleaned
Teflon ware for prolonged storage.
Preparation of high purity solutions of hydrochloric acid and ammonia
hydroxide is possible using isopiestic distillation (Zief and Mitchell,
1976). Redistilling of concentrated nitric acid is also relatively easy
and does not require the use'of expensive acid stills. These alternatives
to purchasing high purity acids should be considered when developing
SOP's.
All prepared reagents should be permanently labelled with the
following information:
0 chemical composition and solvent matrix
° date of preparation and date to be used by
0 initials of person who formulated reagent
° special storage conditions if required.
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1.5. MAINTAINING ANALYTICAL STANDARDS
It is recommended that all inorganic standards used for atomic
absorption and emission spectroscopy be of spectroquality. It is assumed
that preparation of organic standards vill be from sources of equal
quality.
Schemes for preparing working analytical standard solutions from
stock standard solutions should be included in the SOP. Possible
influence of volumetric vare and storage containers on stability of
vorking analytical standards should also be included in SOP. Dilutions of
concentrated stock standards to form vorking standards using small volumes
(<2 ml) should be avoided. Instead, working analytical standard solutions
should be prepared via serial dilutions of stock standard solutions.
Vorking analytical standards should be prepared daily, or with each
analysis if the actual standard concentrations are relatively low
(<1 mg/1), or in a sample matrix where prolonged storage (several days)
may result in changes in the solutions.
Personnel should be trained in the proper use of stock standard
solutions and working analytical standard solutions. Direct contact with
the bulk stock standard solution is to be avoided. Aliquots of stock
standard are to be used when preparing serial dilutions for working
analytical standard solutions. Viping of storage container openings and
caps is strongly recommended as part of the protocol for handling
standards. This step prevents droplets of standard solution from drying
and forming salt deposits near the storage container opening and inside
the cap. Such residues can have a marked influence on the concentration
of standard solutions that is not immediately apparent.
1.6. WEIGHING
Because almost every measuring operation in the analytical laboratory
is ultimately related to a weighing operation, the proper use of the
analytical balance should be strongly emphasized. Maintenance of the
balance, including periodic standardization, should be repeatedly
emphasized to all personnel. Purchase of a set of weights (at least Class
S) which are calibrated against NBS standards is highly recommended.
Calibration of a balance should be checked and duly noted each time the
balance is used.
Adsorption of moisture by samples and sample containers during
weighing, especially by ground plant material, can lead to bias in the
final results. Special precautions should be taken when weighing out
small (<200 mg) sample sizes for an analysis (e.g., placing desiccant
inside the sample compartment of the balance, using a special low humidity
weighing room, or placing balance and samples inside a glove box). It is
also recommended that personnel actually weighing samples wear nylon
gloves to prevent direct contact with the sample weighing container. Use
of an antistatic device may also be required to prevent static charge on
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the dried sample from influencing the final observed weight (Zief and
Mitchell, 1976).
1.7. MAINTAINING LABORATORY CLEANLINESS
Most modern analytical laboratories are equipped for temperature and
humidity control, but fev have controls for regulating airborne
contamination. Airborne contaminants in the form of dusts, mists, and
fumes can freely enter most laboratories, and are often dravn into
laboratories as a result of the draft from chemical exhaust hoods. Thus
the composition of the air in the laboratory often approximates that of
the surrounding atmosphere. The situation is even worse if fine powdery
material, like soil, is handled routinely in the laboratory (Zief and
Mitchell, 1976).
Most modern analytical laboratories also have metal fixtures for
bench top access to water, bottled gas and compressed air. Such fixtures
often corrode due to the presence of acid fumes in the laboratory, and are
ready sources of highly concentrated contaminants. Other metal surfaces
in the laboratory which often corrode and are potential sources of
contaminants include metal handles on laboratory drawers and closets,
safety showers, buret clamps, ring stands, instrument casings, shelving
supports and metal power stripping. Peeling paint from walls, ceilings,
heating pipes and air conditioning vents is also a potential contaminant.
Before starting an analysis, the laboratory should be inspected for
potential sources of contamination that may influence the results of
planned experiments or compromise the integrity of the samples themselves.
Careful attention should be given to the presence of 'corroded surfaces,
such as those mentioned, near or above the sample work area. Such
surfaces should be removed, covered or wiped clean before starting work.
It is also recommended that counter top working surfaces be wiped daily to
prevent possible dust or chemical contamination of samples. The latter is
especially a problem in laboratories using concentrated salts (IN) as
extractants. Dried salt crystals represent very concentrated sources of
contamination that may exist on the outside of reagent bottles or on the
open bench top. Hand contact with dried salt crystals is a common
transmission mode for contamination of samples.
Vhen scheduling sample analyses, steps should be taken to avoid
creating a situation where reagents for one procedure represent a major
potential source of contaminants for another procedure. Analyses of
foliar material and soil samples should not be done in the same laboratory
at the same time.
Cleaning of the laboratory should be done on a regular basis, and
when there is to be a major shift in emphasis within the laboratory (e.g.,
when shifting from soil analyses to foliar analyses). Laboratory
cleanliness should not be left to the janitorial staff. Mops, plastic
scrub pails and a vacuum cleaner should be considered essential equipment
in the laboratory (Van Loon, 1985).
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1.8. CONTROLLING LABORATORY SAMPLE HANDLING/CUSTODY
1.8.1. SAMPLE LOGGING
Below are general guidelines to be used in training laboratory
personnel in the proper procedures for receiving samples at the
laboratory.
0 Each sample should be assigned a unique laboratory number to be
used as the primary means of identification of that sample.
0 A protocol should be established to follow in assigning laboratory
identification numbers if the sample is to be further subdivided
into distinct sub-units during preliminary sample preparation
(e.g., dividing a tree branch into separate foliage and stem
samples).
0 The field identification number and other information from field
notes about a particular sample should be recorded. This
information must be traceable to the laboratory identification
number assigned to a sample.
° A physical examination of the sample(s) as received should be
conducted and the results compared with the description of the
sample(s) as provided in field notes. Samples missing, damaged,
altered, contaminated, or destroyed should be duly noted.
° Cleaning procedures should be established for preparing laboratory
containers for transfer of samples if original field containers
are not to be used for storage.
0 Proper storage procedures should be established for preservation
of samples.
• All new sample laboratory identification numbers should be entered
into the database management system and scheduling of samples for
analysis. Ranking of analyses should be developed if amount of
sample collected is insufficient to complete all analyses
scheduled.
0 All new sample laboratory identification numbers should be entered
into the sample custody log book.
1.8.2. SAMPLE CUSTODY
Protocols are to be developed that will make it possible to determine
the disposition of a sample within the laboratory at any given point in
time. It is recommended that a sample custody log book be maintained
within the laboratory.
New samples received by the laboratory are to be recorded by their
respective laboratory identification numbers in the sample custody log
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book and placed in storage. Vhen a sample or group of samples is removed
from storage the folloving information is to be recorded in the sample
custody log book.:
° laboratory identification numbers of samples removed
° signature of individual removing samples
0 time of removal
0 purpose for removing samples
0 estimated time of return
° actual time of return
0 signature of individual returning sample
0 laboratory identification numbers of samples returned.
Vhen a sample or group of samples leaves the laboratory the following
information is to be recorded in the sample custody log book:
0 laboratory identification numbers of samples removed
0 reason for withdrawal and disposal of samples
° signature of person removing samples
° signature of supervisor authorizing disposal of samples.
It may be necessary to maintain both storage sample custody log books
and laboratory sample custody log books if the sample storage area is not
in the same building as the laboratory. Such would be the case if samples
are sent to other laboratories at different locations for analysis. It is
the responsibility of the principal investigator to review and approve the
sample custody procedures to be used by other laboratories in analyzing
samples.
The above information will also be recorded if a sample is consumed
during chemical analysis or archived at a different location.
A separate log should be kept for periodic checks on conditions under
which samples are stored. Periodic maintenance schedules should also be
included in the log if samples require special equipment for long-term
storage. One individual should be assigned primary responsibility for
monitoring the sample storage area.
1.8.3. LABORATORY SAMPLE HANDLING
Protocols are to be developed for handling samples in order to avoid
contamination from the laboratory environment. Special attention should
be given to weighing of samples, as the same balances are often used for
weighing out laboratory reagents. Special attention should also be given
to potential destruction of large sets of samples in the laboratory due to
chemical spills or physical accidents. In general, samples should be
handled in small groups and kept on stable surfaces to avoid possible loss
through handling. If possible, only aliquots of bulk samples should be
taken into the main chemical analysis laboratory.
Once an aliquot of a sample is removed it must NEVER be returned to
the original sample container.
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1.8.4. ARCHIVING OF SAMPLES
Protocols should be developed for archiving of samples after
completion of scheduled analyses. The following general guidelines are
provided as aids in developing a suitable protocol for long-term storage
of samples.
0 Areas selected for long term storage should be relatively clean
and have controlled access.
0 Individual sample storage containers should be placed inside of
large storage containers to permit ease of handling, prevent loss,
and protect against contamination. It is recommended that
individual sample containers be sealed within large plastic bags
before being placed in large storage containers.
0 Full documentation listing the sample identification numbers,
sample descriptions, project title, name of principal
investigators, funding agency, and contract number should be
included with each set of samples inside each separate large
storage container. Contained in this documentation should be
explicit instructions NOT to discard samples.
° The outside of each large storage container should be clearly
labelled with the project title, name of the principal
investigators, funding agency, and contract number, along with
warnings NOT to discard samples. This information should be
written on the walls of the large storage container as well as on
attached labels or tags.
° Areas selected; for long-term storage of samples should be visited
at least once a year to check storage conditions and to determine
if the samples: have been damaged or inadvertently discarded.
1.9. RECORDING AND REPORTING DATA
1.9.1. OBSERVATIONS T,0 BE RECORDED
Observations to be recorded from an analysis include raw data and
quality control data. There are several important types of quality
control data to analyze and record:
0 BLANK SAMPLES^-l) calibration—a "0" mg/1 standard containing
only the matrix of the calibration standards, and 2) reagent—a
sample composed of all the reagents (in the same quantities) used
in preparing a "real" sample for analysis. These data establish a
zero baseline or a background value, respectively, that is used to
adjust or correct routine analytical results.
0 BLIND SAMPLES—a standard submitted for analysis as a "real"
sample without the analyst's knowledge. Blinds will be submitted
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to cooperating labs (interlaboratory exchanges) to assess accuracy
of analytical methods.
° CALIBRATION STANDARDS—standards used to quantify the relationship
between the output of a sensor and a property to be measured.
These standards should be traceable to a standard reference
material (SRM) and analyzed at concentrations that bracket the
concentrations expected from the samples.
° CALIBRATION QUALITY CONTROL SAMPLES (WORKING STANDARDS)—known
sample standards containing the analyte of interest, but prepared
or obtained from a source independent of, but traceable to, the
calibration standards. These standards are used primarily as
routine intralaboratory checks of accuracy. They are commonly
analyzed at two concentrations—near mid-calibration range and the
detection limit.
° SPIKED SAMPLES—a "real" sample to which the analyte of interest
is added to check the performance of a routine analysis or the
recovery efficiency of a method.
0 REPLICATE SAMPLES—repeated, but independent, determinations of
the same sample by the same analyst at essentially the same time
and same conditions. Replicates may be performed in duplicate,
triplicate, or more. These data are used to assess the precision
of the analytical system.
° DUPLICATE SAMPLES — repeated, but independent, analysis of
different samples, from which sample variation is calculated.
1.9.2. RESEARCH NOTEBOOK POLICY
The following guidelines are recommended for maintaining laboratory
and field notebooks.
0 All observations are to be recorded in serially numbered, bound
laboratory and field notebooks. As an alternative, one may use
data sheets provided they are consecutively numbered and bound
before storage. Electronic recording of data is preferred when
possible, provided backup and hardcopy of the data are available.
NOTE: The phrase, "all observations," refers to all data
generated during an analysis, including raw data,
calibration standards, calibration checks, blanks, and
quality control checks.
0 Entries into notebooks are to be made in waterproof ink. Mistakes
should be crossed out with a single line and initialed.
Exceptions to this rule will be determined by the principal
investigator and listed in the SOP.
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° Spaces and pages left blank, are to be crossed out to prevent
entries from being made at a later time. Date of entries shall be
provided on each page.
0 Supporting records can be included in the laboratory notebook.
These records are to be attached with glue or tape, signed and
dated.
° Supporting results and conclusions (e.g., computer printouts, data
sheets, calibration records) are to be referenced in sufficient
detail to allow retrieval of the record.
° If more them one individual uses a notebook, the individual
responsible for the entry must sign the page or individual entry.
° Notebooks and quality control charts should be periodically
reviewed and signed by the principal investigator. The review
process should look for both short-term and long-term trends in
the data. Trends in the data related to such things as changes in
personnel, receipt of a new lot of laboratory chemicals, times of
the day, or days of the week may indicate need for corrective
action that is not readily apparent by restricting review
procedures to a daily basis.
0 Pages are not to be removed from any notebook.
This laboratory notebook policy is also required of laboratories
contracted by the principal investigator for analysis of samples. It is
the responsibility of the principal investigator to a'scertain that all
information concerning the analyses of samples be readily available for
quality assurance audits.
1.9.2. ACTUAL OBSERVATIONS TO BE RECORDED
Each SOP lists what observations are to be recorded during execution
of an analytical procedure. Guidelines are to be established for when to
record a reading and to how many significant figures.
Additional information should be recorded in laboratory notebooks if
° Samples were not analyzed within the recommended time period for a
given procedure,
° Sample readings were not within the range limits of the
calibration for a particular procedure,
° Handling errors or the composition of the sample matrix may have
created errors,
° Unusual circumstances present during the course of the analysis
may have created errors. For example, failure of central air
conditioning or heating, activities in adjacent laboratories which
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created excessive fumes or dust in the air, building construction
or remodeling within the same building or immediately adjacent to
it, or cleaning of laboratory at night by janitors are all
possible activities that are not under the immediate control of
the principal investigator but could have a direct impact of the
laboratory environment where analyses are performed. This
information would be useful if data are later flagged as
questionable.
1.9.3. DATA REPORTING
Guidelines for data validation, verification, and data preparation
for final reports are discussed in a separate manual (Baes et al., 1986).
1.10. REFERENCES
Baes, C.F., R. Olson, and K. Riitters. 1986. Quality Assurance Methods
Manual for Experimental Design and Data Management. NCSU Acid
Deposition Program, Raleigh, NC.
Dux, J.P. 1986. Handbook of Quality Assurance for the Analytical
Chemistry Laboratory. Van Nostrand Reinhold Co., New York.
Garfield, F.C. 1984. Quality Assurance Principles for Analytical
Laboratories. AOAC. 1111 North Nineteenth Street, Suite 210.
Arlington, VA. 22209.
Kolthoff, I.M., E.B. Sandell, E.J. Meehan, and S. Bruckenstein. 1969.
Quantitative Chemical Analysis. The Macmillan Company, Collier-
Macmillan Limited, London.
U.S. EPA. 1979. Handbook for Analytical Quality Control in Water and
Wastewater Laboratories. EPA-600/4-79-019.
Van Loon, J.C. 1985. Selected Methods of Trace Metal Analysis.
Biological and Environmental Samples. John Wiley & Sons, New York.
Zief, M. and J.W. Mitchell. 1976. Contamination Control in Trace Element
Analysis. John Wiley & Sons, New York.
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2. FOLIAR INORGANIC ANALYSIS
2.1. STANDARD OPERATING PROCEDURE FOR MULTI-ELEMENT ANALYSIS: N
(KJELDAHL-N), P USING AN AUTOANALYZER
2.1.1. SCOPE AND PURPOSE
This standard operating procedure is designed to determine the
nitrogen and phosphorus content of foliar tissue. Inclusion of salicylic
acid in this procedure results in 95% or greater of nitrate-N being
recovered from a sample (Eastin, 197B). The procedure does not yield a
true 'total' analysis for nitrogen because certain refractory N compounds
are not broken down by the digestion step (McKenzie and Wallace, 1954;
Minagava et al., 1984). However, the procedure as described is used
routinely in the literature to determine the N content of foliar tissue.
Published reports on efficiency of recovery by this procedure have
been concerned only with nitrogen. It is assumed that recovery of P is
100% (Gales and Booth, 1978).
The procedure described in this section is one of many variations on
the original Kjeldahl method (Nelson and Sommers, 1980). Investigators
wishing to use another variation of this method (change in catalyst,
digestion time, method of determination of NH4-N and ortho-P in
digestates) should provide the Quality Assurance Specialist with the
necessary data to verify the accuracy and precision of their technique and
the comparability to this described procedure.
2.1.2. MATERIALS AND SUPPLIES
2.1.2.1. EQUIPMENT
0 digestion block with controller (400°C)
0 vortex mixer
° Technicon AutoAnalyzer (or equivalent)
° analytical balance (0.001 g)
0 calibrated digestion tubes (usually 75 ml)
0 metal funnel with long stem (25 cm)
° Parafilm
° thermometer (100-500°C)
2.1.2.2. CHEMICALS/REAGENTS
° 18M Sulfuric acid (H„S0^), low N content
° Salicylic acid (C7H,6„ - 2-hydroxybenzoic acid)
° Kel-Pak No. 1 digestion catalyst (9.9g I^SO^O^lg Hg0:0.08g
CuSO^) (available from Curtin Matheson Scientific, Inc.)
° Distilled-deionized (DI) water
° Reagents for Technicon AutoAnalyzer Method 321-74A and 328-74A.
(Consult Technicon procedures sheet for proper formulation of
reagents.)
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° 18M Sulfuric acid + salicylic acid - dissolve 70 g of salicylic
acid per 9 lb bottle of concentrated sulfuric acid. (Constant
mixing is required to dissolve salicyclic acid in concentrated
sulfuric acid.)
° Ammonium sulfate ((NH^KSO,)
0 Potassium dihydrogen phospnate (KI^PO^)
2.1.3. PROCEDURES
2.1.3.1. SAMPLE PREPARATION
1. Foliar sample should be oven-dried (70°C) and ground to pass a
1 mm screen.
2. Place 3.5 g of Kel-Pak catalyst into each digestion tube.
3. Weigh 0.5 g of sample and transfer into digestion tube using
metal funnel vith long stem. Vhen using 75 ml digestion tubes,
one rack holds 40 tubes. Weigh out samples in prescribed order
according to tube position.
4. Add 10 ml of 18H H-SO^+salicylic acid to each tube. Rotate tube
vhile adding acid to remove sample from sides of tube. CAUTION:
Contact vith concentrated sulfuric acid causes severe burns.
Wear gloves at all times during this phase of the procedure.
5. Allov tubes to stand 20-30 minutes for preliminary charring of
sample.
6. Transfer rack of digestion tubes to preheated (385°C) digestion
block. Remove rack of digestion tubes from block every 20
seconds or less during first several minutes of heating samples.
This is necessary to prevent samples from spilling out of tubes.
Continue monitoring (approximately 10 minutes) the digestion
reaction, and removing tubes if necessary, until charred sample
no longer rises towards the top of the digestion tube. Record
time for start of the digestion sequence.
7. Vhen reaction has stabilized, small amounts of acid can be added
to rinse dovn charred sample that may have accumulated at the
top of the digestion tubes. Refluxing action within the tubes
during digestion will clean lover two thirds of tubes.
8. Continue digestion for 65 minutes. Total digestion time is not
to exceed 95 minutes. Record time at the end of digestion
sequence.
9. Remove rack and allov tubes to cool until warm to touch.
Digestate should be clear. Do NOT allov tubes to reach room
temperature because a precipitate will form that is difficult to
dissolve.
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10. To lukewarm sample, slowly add DI water while mixing contents
with a vortex mixer. Add DI water in increments to allow
thorough mixing with the viscous acid.
11. Bring to volume with DI water, cap top of tubes tightly with
Parafilm or comparable sealer and place in rack. After capping
with Parafilm, invert each tube, or the entire rack using a
template and clamps to hold the tubes in place, 10 times for
final mixing of contents.
12. Transfer a 15 ml aliquot to a polycarbonate vial and cover vial
with a leak proof cap. To avoid possible mislabeling of
samples, arrange vials in groups of 40 in holders similar to
digestion rack. Transfer 15 ml aliquots of sample in same order
as samples were assigned to digestion tubes.
13. Store vials at room temperature until analysis. Discard
remaining digestate. Digestate contains mercury and should be
disposed of according to the guidelines established for the
participating institution.
14. Follow guidelines detailed in 'Good Laboratory Practices'
section for recording of all raw data. Note that raw data
include the following: (a) analytical observations necessary to
calculate the final results, (b) calibration data, (c)
calibration checks, and (d) quality control checks. Deviations
from standard operating procedures during sample preparation,
calibration or actual analyses are to be fqlly documented and
initialed by laboratory personnel. Samples suspected of being
in error or outside of the calibration range are to be
'flagged', and this notation carried through all records to the
final report. Retain all written materials (graphs, tables,
etc.) generated as part of an analysis. Do not discard portions
of laboratory notebooks or any other information directly
related to calculation of the final result for a set of samples.
2.1.3.2. EQUIPMENT OPERATION
2.1.3.2.1. Digestion Block
Follow manufacturer's manual for start-up, shut-down, maintenance,
and calibration procedures.
2.1.3.2. Technicon AutoAnalyzer (or equivalent)
Follow manufacturer's manual for start-up, shut-down, maintenance and
calibration procedures.
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2.1.4. PREVENTIVE MAINTENANCE
2.1.4.1. DIGESTION BLOCK
Monitor temperature of block daily with thermometer. Place
thermometer in digestion tube approximately one-third full of fine sand.
Position digestion tube with thermometer in center of block and at several
other positions to check for uneven heating. Record all observations in
maintenance log.
2.1.4.2. TECHNICON AUTOANALYZER
Follov all maintenance guidelines as stipulated in operating manuals.
Keep tracings of output on file for comparison of patterns over time.
Replace pump tubing on regular basis. Record date of preparation of all
reagents and remake when necessary.
2.1.5. CALIBRATION PROCEDURES
2.1.5.1. DIGESTION BLOCK
Set temperature of block at 385°C using controller and thermometer.
Block takes approximately 0.5 hour to reach full temperature. Use of a
timer is recommended to turn on block an hour before start of digestions.
2.1.5.2. TECHNICON AUTOANALYZER
Use the following solutions to calibrate AutoAnalyzer.
1. Combined N and P stock standard (7500 mg/1 N, 750 mg/1 P) -
Dissolve 35.394 g of (NH,)»S0^ and 3.295 g of KH-PO^ in 1 liter
of DI water.
2. Store stock standard in the refrigerator.
3. Standards are to be carried through digestion procedure to
matrix match samples. The following chart gives the ml of the
stock N and P standard to be added to separate digestion tubes
to give the indicated standards.
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cone (mg/1) of
ml stock N & P equivalent cone
std in std soln (+) in plant tissue (*)
6 600 N - 60 P 9.0% N - .90% P
5 500 N - 50 P 7.5% N - .75% P
4 400 N - 40 P 6.0% N - .60% P
3 300 N - 30 P 4.5% N - .45% P
2 200 N - 20 P 3.0% N - .30% P
1 100 N - 10 P 1.5% N - .15% P
0.5 50 N - 5 P .75% N - .075% P
0.25 25 N - 2.5 P .375% N - .0375% P
0 ON OP 0% N - 0% P
(+) - assumes final volume of 75 ml in digestion tube.
(*) - concentration in plant tissue assuming sample weight of 0.500 g
4. Typically 4 tubes each of the above standards are prepared and
combined at the end of the following digestion procedure to
yield standard solutions.
5. Carefully add 10 ml of 18M sulfuric acid + salicylic acid to
each tube. Do not exceed 10 ml of acid because solutions may.
not clear with excess of acid.
6. Place digestion tubes on preheated block set at 180°C. NOTE
that the initial temperature for standard preparation is
different than for digestion of samples.
7. Allow water to boil off for at least 1 hour. Solutions no
longer contain water if they are not boiling.
8. Raise temperature of block to 385°C. Digest for 1.5 hours.
(Assuming 0.5 hour to reach 385°C, allows 1 hour for digestion).
9. Check color during digestion. Continue heating for 0.5 hour
after clearing of solution. (Solution will proceed from brown
to green to clear). Recommended digestion time is usually
sufficient to yield clear digestate.
10. After digestion, allow tubes to gooI and add DI water as
described in steps 9-11 of Section 2.1.3.1.
11. Typically the digestion tubes are combined from 4 digestion
tubes to make enough standard to last for several analyses of
samples. It is recommended that each 75 ml of a particular
standard be checked with the others in a set before combining
together.
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12. Filtering of standards through No. 42 filter paper is
recommended before use.
13. Actual calibration of AutoAnalyzer involves use of each standard
and blank in sample tray at the beginning and end of analyses,
and the use of individual standards and blanks repeated among
samples to maintain calibration. Actual use of standards
depends on readout device employed for recording of data. Use
of computer programs to record output may limit the number of
standard calibrations that can be made for a set of samples.
14. Calibration procedure to be used by individual investigators
should be provided in detail to the Quality Assurance
Specialist.
2.1.6. CALCULATIONS/UNITS
Calibration of -AutoAnalyzer output is usually set equal to the final
concentration of N and P (expressed as % oven-dried sample) in sample
using the values given in step 3 of Section 2.1.5. Final calculations are
not necessary when this is done.
NOTE: The calibration standards in step 3 of Section 2.1.5. are
designed for a particular configuration of the Technicon AutoAnalyzer II.
Sample injection using this segmented continuous flow procedure is done
through a series of dilutions vhile adding reagents to develop final
color. The concentration of standards chosen yields the optimum linear
response for the configuration used by AutoAnalyzer II. Standards may be
different for other AutoAnalyzers, such as flow injection analyzers.
2.1.7. ERROR ALLOWANCE AND DATA QUALITY
2.1.7.1. CONSIDERATIONS
Blanks, replicates, and in-house secondary standards should be
included in analyses of all samples. Vithin one group of approximately 40
samples (20 samples if 250 ml digestion tubes are used) there should be
one blank, tvo in-house secondary standards, and 3 replicates. Certified
NBS reference materials should be included on a monthly basis. At least
tvo NBS SRM's vith different matrices are recommended for use, with this
procedure.
Samples selected as replicates should also be run on different days
to estimate day-to-day precision.
Replicate samples will be used to monitor precision. Percent
coefficient of variation should be calculated using the industrial
statistic I, where %CV = 200I//2 and I=|A-B|/A+B, A and B being the
results for the replicate samples (U.S. EPA, 1979).
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Precision will also be monitored using Shewhart control values for R
(range) values, if the total number of sample groups to be analyzed will
generate enough points to make use of such charts worthwhile.
Critical ranges for repeated measurement error and accuracy will be
set by Quality Assurance Officer and listed in the Data Quality Objectives
(DQO) table.
Accuracy will be evaluated through the use of interlaboratory sample
exchanges and use of NBS SRM's. Sample exchanges will be coordinated by
the Quality Assurance Specialist assigned to the project.
The length of digestion time is critical. The conditions in this
procedure have been optimized to yield maximum recovery of N and P in the
shortest period of time possible. Exceeding the total digestion time (1.5
hours) by 0.5 hour will probably result in loss of N. Loss of N due to
prolonged heating is usually complete and is evident in N content of
in-house standards. Recording time at the start and at the end of a
digestion sequence step is necessary to insure accuracy and precision in
the analysis.
Storage of sample aliqvots at room temperature in capped
polycarbonate vials is acceptable for periods of up to 3 weeks. Prolonged
storage beyond this time usually results in partial evaporation of
contents and concentrating of sample. Cooling of aliquot (4°C) is not
generally recommended due to the possible formation of precipitates in the
sample solutions.
2.1.7.2. DATA QUALITY OBJECTIVES
Variable
Reporting
Units
Repeated Measurement
Error at
Lower Limit Upper Limit
Measurement
Accuracy
Tolerance
N
P
X (v/v)
X (w/w)
10% (cv)
102 (cv)
10Z (cv)
10Z (cv)
10%
10X
2.1.7.3.
COMPUTER DATABASE
CODES
Variable
Code
Kjeldahl-N
Total Elemental P
FTKN
FTTP
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2.1.8. REFERENCES
Eastln, E.F. 1978. Total nitrogen determination for plant material
containing nitrate. Anal. Biochem. 85:591-594.
Gales, M.E., Jr. and R.L. Booth. 1978. Evaluation of the Technicon block
digestor system for total Kjeldahl nitrogen and total phosphorus.
U.S. EPA. Cincinnati, OH 45268. EPA-600/4-78-015.
McKenzie, H.A. and H.S. Wallace. 1954. The Kjeldahl determination of
nitrogen: a critical study of digestion conditions — temperature,
catalyst, and oxidizing agent. Aust. J. Chem. 7:55-70.
Minagava, M., D.A. Vinter and I.R. Kaplan. 1984. Comparison of Kjeldahl
and combustion methods for measurement of nitrogen isotope ratios in
organic matter. Anal. Chem. 56:1859-1861.
Nelson, D.V. and L.E. Sommers. 1980. Total nitrogen analysis of-soil and
plant tissues. J. Assoc. Off. Anal. Chem. 63:770-778.
Technicon AutoAnalyzer 11 Industrial Method No. 321-74A. 1974.
Ammoniacal Nitrogen/BD Acid Digests. BD-20/BD-40 (Dialyzer).
Technicon Industrial Systems. Tarrytovn, NY 10591.
Technicon AutoAnalyzer II Industrial Method No. 328-74A. 1974.
Phosphorus/BD Acid Digests. BD-20/BD-40 (Dialyzer). Technicon
Industrial Systems. Tarrytovn, NY 10591.
U.S. Environmental Protection Agency. 1979. Handbodk for Analytical
Quality Control in Vater and Vastevater Laboratories. Environ. Mon.
and Sup. Lab. U.S. EPA. Cincinnati, OH 45268. EPA-600/4-79-019.
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2.2. STANDARD OPERATING PROCEDURE FOR MULTI-ELEMENT ANALYSIS: C, N
(TOTAL-N), S USING AN ELEMENTAL ANALYZER
2.2.1. SCOPE AND PURPOSE
A number of manufacturers currently produce instruments capable of
analyzing finely ground plant materials for total content of carbon and
nitrogen as well as for sulfur, which often requires the purchase of
appropriate accessories. Because of the uniformity in measurement which
can be obtained using automated elemental analyzers, it is recommended
that this equipment be used for the analysis of carbon, nitrogen, and
sulfur in vegetation samples where instrumentation is available. Where
elemental analyzers are not available for one or more of these elements,
alternative procedures for element content measurements as outlined in
this document should be employed.
2.2.2. MATERIALS AND SUPPLIES
2.2.2.1. EQUIPMENT
0 elemental analyzer for C-N-S
0 analytical balance (0.0001 g)
• Wiley Mill
2.2.2.2 CHEMICALS/REAGENTS
As per manufacturer's instructions
2.2.3. PROCEDURES
2.2.3.1. SAMPLE PREPARATION
Most elemental analyzers use relatively small sample sizes compared
to the other SOPs in this section (50-100 mg vs. 0.5 - 1.0 g).
Preparation of sample, therefore, is critical to ensure acceptable
accuracy and precision.
Pollov manufacturer's guidelines for sample drying, grinding, mixing
and final preparation.
2.2.3.2. EQUIPMENT OPERATION
Several commercial elemental analyzers are currently available for
analysis of foliar tissue. Follow manufacturer's instructions for proper
operation of particular analytical instrument selected.
2.2.4. PREVENTIVE MAINTENANCE
Carry out all maintenance procedures as recommended by the
manufacturer. Record maintenance operations in maintenance log.
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2.2.5.. CALIBRATION PROCEDURES
Follow manufacturer's instructions for proper calibration of the
instrument.
2.2.6. CALCULATIONS/UNITS
Need for calculations will depend on instructions for calibration of
instrument. Follow manufacturer's recommended guidelines.
2.2.7. ERROR ALLOWANCE AND DATA QUALITY
2.2.7.1. CONSIDERATIONS
Blanks, replicates, and in-house secondary standards should be
included in analyses of all samples. The number of samples run per day
will vary depending on the elements measured and equipment used. At a
minimum, one blank, one in-house secondary standard, and one replicate
should be included for every 25 samples. Certified NBS reference
materials should be included on a monthly basis, as appropriate.
Replicate samples will be used to monitor precision. Percent
coefficient of variation will be calculated using the industrial statistic
I, where % CV = 200 I//2 and I=|A-B|/A+B, A and B being the results for
the replicate samples.
Precision will also be monitored using Shewhart control values for R
(range) values, if the total number of sample groups to be analyzed will
generate enough points to make use of such charts worthwhile.
Critical ranges for repeated measurement error and precision will be
set by each Quality Assurance Officer in a Data Quality Objectives (DQO)
table.
2.2.7.2. DATA QUALITY OBJECTIVES
Repeated Measurement
Measurement
Reporting
Error
at
Accuracy
Variable
Units
Lower Limit
Upper Limit
Tolerance
N
% (w/w)
10* (cv)
10% (cv)
10%
S
mg/kg
15% (cv)
10% (cv)
15%
C
mg/kg
5% (cv)
5% (cv)
5%
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2.2.7.3. COMPUTER DATABASE CODES
Variable
Code
Total Elemental
N
FTTN
Total Elemental
S
FTTS
Total Elemental
C
FTTC
2.2.8. REFERENCES
U.S. Environmental Protection Agency. 1979. Handbook for Analytical
Quality Control in Water and Wastewater Laboratories. U.S.
Environmental Protection Agency, Cincinnati, OH. EPA-600/4-79-019.
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2.3. STANDARD OPERATING PROCEDURE FOR MULTI-ELEMENT ANALYSIS: N (KJELDAHL-
N), P, Ca, Mg, K USING VET DIGESTION
2.3.1. SCOPE AND PURPOSE
This standard operating procedure vill be used by the School of
Forest Resources, NCSU, Raleigh, NC, for the multi-element analysis of
foliar tissue. The advantages of the procedure over dry-ashing include
(a) only one solution is used for all elements measured, (b) there is no
formation of complex insoluble silicates since the oxidation reaction is
carried out in solution, (c) no volatilization losses occur, and (d) the
procedure is rapid and easy, making it suitable for routine analysis
(Parkinson and Allen, 1975). The procedure as tested yields acceptable
results for N (Kjeldahl N), P, Ca, Mg, and K. Use of this procedure for
elements other than those listed is not recommended.
2.3.2. MATERIALS AND METHODS
2.3.2.1. EQUIPMENT
° digestion block with digestion tubes calibrated at 50 ml
0 dispenser (50 ml)
° repipette (5 ml)
0 vortex mixer
0 analytical balance (+ 0.001 g)
° 1 liter flat-bottom Foiling flask
° 1 liter glass stoppered bottle
° 500 ml volumetric flask
° 200 ml volumetric flasks
2.3.2.2. CHEMICALS/REAGENTS
° Lithium sulfate (Li-SO^-^O)
° Selenium powder (Se;
0 30% Hydrogen Peroxide (H„0»)
° 18M Sulfuric acid (H,S0,7
° 12M Hydrochloric acid (nCl)
0 Lanthanum oxide (La-O,)
° Sodium phosphate dibasic (Na„HP0,)
° Ammonium sulfate [(NH^KSO^]
° Certified atomic absorption standard solutions, 1000 mg/1 (Ca, Mg,
K)
° Digestion Reagent: Add 14 g of Li„S0, and 0.42 g of selenium
powder to a flat-bottom boiling flask. Add 350 ml of 30% H-O-
and mix by swirling flask. Slowly add 210 ml of 18M HjSO^ by
pouring down side of flask so as not to mix the acid and
peroxide. Place flask under running cold water and then mix by
swirling. Vhen mixture cools, add another 210 ml of 18M ^SO^
in a similar fashion. Vhen cool, store in labelled glass
bottle in refrigerator at 1°C.
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° Lanthanum releasing agent: Place 11.728 g of La„0, into a 1 liter
volumetric flask. Add 100 ml of DI water. Slowly add 50 ml of
12M HCl to dissolve LajO^. Swirl flask and let stand. Add
another 100 ml of DI water and swirl flask. Let suspension
stand overnight. Bring to volume with DI water.
° Stock standard solution: (200 mg/1 Ca, 400 mg/1 K, 80 mg/1 Mg, 80
mg/1 Pf 500 mg/1 N): Transfer 0.1833 g of Na-HPO, and 1.1793 g
of (NH^)„S0^ to a 500 ml volumetric flask. Add 30 ml of DI
water ana swirl flask to dissolve crystals. -Add 200 ml of 1000
mg/1 commercially prepared K standard. Swirl flask to mix
contents. Add 100 ml of 1000 mg/1 Ca and 40 ml of 1000 mg/1 Kg
commercially prepared standards. Swirl flask to mix contents
and bring to volume with DI water. Mix well and store at 4°C
until use.
2.3.3. PROCEDURES
2.3.3.1 SAMPLE PREPARATION
1. Foliar sample should be oven-dried (70°C) and ground to pass a 1
nun screen.
2. Weigh out 0.200 (+ 0.005) g of sample and transfer to a digestion
tube.
3. Add 4.4 ml of digestion reagent and let stand until suspension
begins to boil.
4. Place rack of digestion tubes on preheated 200°C digestion block.
5. Increase temperature to 340°C and heat until solution clears.
Continue heating for 1 hour after clearing. Total time on
digestion block is approximately 2 hours.
6. Remove tubes from block and let cool. Bring each tube to volume
using DI water. Do this in stages mixing well between each
addition of water. Let tubes cool after first addition of water.
7. Mix well on vortex mixer and transfer to a polypropylene bottle
for storage. It is recommended that the final solution be
transferred back to the digestion tube from the polypropylene
bottle at least once to ensure complete mixing of sample.
8. Solutions can be stored up to one month at room temperature.
9. Follow guidelines detailed in Good Laboratory Practices section
for recording of all raw data. Note that raw data include the
following: (a) analytical observations necessary to calculate the
final results, (b) calibration data, (c) calibration checks, and
(d) quality control checks. Deviations from standard operating
procedures during sample preparation, calibration, or actual
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analyses are to be fully documented and initialed by laboratory
personnel. Samples suspected of being in error or outside of the
calibration range are to be 'flagged', and this notation carried
through all records to the final report. Retain all vritten
materials (graphs, tables, etc.) generated as part of an
analysis. Do not discard portions of laboratory notebooks or any
other information directly related to calculation of the final
result for a set of samples.
2.3.3.2. EQUIPMENT OPERATION
Consult operating manual for detailed instructions on operation and
calibration of digestion block.
The concentration of N and P in the digestate is determined using a
Technicon AutoAnalyzer. Reagent formulations and flow diagrams are
described in Nicholson (1978). The only modification suggested is the
substitution of Brij-35 for use as the surfactant.
It is assumed that either emission or atomic absorption spectroscopy
vi.ll be used to determine the concentration of Ca, Mg and K in the
digestates. Consult operating manuals for detailed instructions on
operation of instruments.
If alternative methods of analysis are selected for Ca, Mg and K,
precision of the chosen methods must be equal to that available using
emission or atomic absorption spectroscopy.
Use of lanthanum as a releasing agent is recommended to remove
chemical interferences from phosphate when determining the concentration
of Ca or Mg vith an atomic absorption spectrophotometer (Beaty, 1978).
Rather than adding lanthanum to each sample, a different approach is
taken. The capillary tubing feeding the nebulizer on the atomic
absorption unit is fitted with a small glass 'T' fitting commonly used on
an AutoAnalyzer. One end of the 'T' fitting is connected to a supply of
lanthanum releasing agent. The remaining end is attached to the tubing
used as the sample probe. The result is that the effective volume of
sample is halved during aspiration. Samples and standards can be treated
identically such that no adjustments to final calculations are required.
2.3.4. PREVENTIVE MAINTENANCE
Carry out all maintenance procedures as recommended by the
manufacturer. Record maintenance operations in maintenance log.
2.3.5. CALIBRATION PROCEDURES
1. Preparation of standards requires matrix matching to the sample
digestates.
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2. Prepare 'Blank! digest' by heating 4.4 ml of the digestion reagent
in a digestion tube until the hydrogen peroxide boils off. It is
recommended that an entire rack of 'Blank digest' be prepared at
one time.
3. Remove tubes from block and let cool. Combine tubes into one
solution, mix veil, and store at room temperature. Approximately
9.6 ml of 'Blank digest' will be required for each 200 ml of
standard.
4. Calibrate AutoAnalyzer and atomic absorption or emission
spectrometers ,using the following standards:
Element
ml of
stock standard N P K Ca Mg
final concentration in 200 ml volumetric flask
0
0
0
0
0
0
1
2.5
0.4
2
1
0.4
2
5
0.8
4
2
0.8
5
12.5
2
10
5
2
10
25
4
20
10
4
15
37.5
6
30
15
6
20
50
8
40
20
8
25
62.5
10
50
25
10
30
75
12
60
30
12
35
87.5
14
70
35
14
40
100
16
80
40
16
5. Add 40 ml of DI water to each 200 ml volumetric flask after
adding aliquot of stock standard.
6. Add 9.6 ml of 'Blank digest' to each flask. Mix veil and let
cool to room temperature.
7. Bring to volume with DI water.
8. Four standards plus the zero should be used to calibrate
instrumentation for the analysis of each element. Standards
should be run at the beginning and end of each sample group and
after a set number of samples within each group.
2.3.6. CALCULATIONS/UNITS
The following is an example of how to calculate the final
concentration of each element in the sample:
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A x 3 0
-= jr- = % of element in sample on weight basis
D X t
vhere A = ug of a base cation per ml of digestate as determined from the
standard curves, 8 = sample weight in mg (200 mg), and C = dilution factor
(o 1 if no dilution made).
Report all results as X oven dry weight.
2.3.7. ERROR ALLOWANCE AND DATA QUALITY
2.3.7.1. CONSIDERATIONS
Blanks, replicates, and in-house secondary standards should be
Included in analyses of all samples. Within one group of approximately 40
samples there should be one blank, two in-house secondary standards, and
three replicates. Certified NBS reference materials should be included on
a monthly basis. At least two NBS SRM's with different matrices are
recommended for use with this procedure.
Samples selected as replicates should also be run on different days
to estimate day-to-day precision.
Replicate samples will be used to monitor precision. Percent
coefficient of variation will be calculated using the industrial statistic
I, where %CV = 200I//2 and 1=|A-B|/A+B, A and B being the results for the
replicate samples (U.S. EPA, 1979).
Precision will also be monitored using Shewhart control values for R
(range) values, if the total number of sample groups to be analyzed will
generate enough points to make use of such charts worthwhile.
Critical ranges for repeated measurement error and accuracy will be
set by Quality Assurance Officer and listed in the Data Quality Objectives
(DQO) table.
Accuracy will be evaluated through the use of interlaboratory sample
exchanges and use of NBS SRM's. Sample exchanges will be coordinated by
the Quality Assurance Specialist assigned to the project.
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2.3.7.2. DATA QUALITY OBJECTIVES
Repeated Measurement
Measurement
Reporting
Error at
Accuracy
Variable Units
Lover Limit
Upper Limit
Tolerance
N
0.1% (w/w)
10% (cv)
10% (cv)
10%
P
0.01% (w/w)
20% (cv)
10% (cv)
10%
K
0.01% (w/w)
10% (cv)
10% (cv)
15%
Ca
1 nig/kg
10% (cv)
10% (cv)
15%
Mg
1 mg/kg
10% (cv)
10% (cv)
15%
2.3.7.
3. COMPUTER DATABASE CODES
Variable
Code
Kjeldahl-N
FTKN
Total Elemental P
FTTP
Total Elemental K
FTTK
Total Elemental Ca
FTCA
Total Elemental Mg
FTMG
2.3.8.
REFERENCES
Beaty,
R.D. 1978. Concepts,
Instrumentation and Techniques
in Atomic
Absorption Spectrophotometry. Perkin-Elmer Corp., Norvalk, CT.
Nicholson, G. 1978. Methods of Soil, Plant and Vater Analyses. New
Zealand Forest Service. Forestry Research Institute. Soils and Site
Productivity Report # 100. Rotorua, NZ.
Parkinson, J.A. and S.E. Allen. 1975. A vet oxidation procedure suitable
for the determination of nitrogen and mineral nutrients in biological
material. Commun. Soil Sci. Plant Analysis. 6(1): 1—11.
U.S. Environmental Protection Agency. 1979. Handbook for Analytical
Quality Control in Water and Wastewater Laboratories. Environ. Mon.
and Sup. Lab.. U.S. EPA. Cincinnati, OH 45268. EPA-600/4-79-019.
30
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2.4. STANDARD OPERATING PROCEDURE FOR MULTI-ELEMENT ANALYSIS: K, P, Ca,
Mg, B, Na, Cu, Zn USING DRY COMBUSTION
2.4.1. SCOPE AND PURPOSE
This standard operating procedure will be used by the Department of
Pomology, Cornell University, Ithaca, NY, for the multi-element analysis
of foliar tissue. The procedure as tested using NBS SRM's 1571 (Orchard
leaves), 1573 (Tomato), 1568 (Rice), and 1575 (Pine needles), yields
acceptable results for K, P, Ca, Mg, B, Na, Cu, and Zn when using ion-
coupled plasma emission spectroscopy. Recovery for other elements is
either less than or in excess of 1002. Use of this procedure for elements
other than those listed is not recommended.
2.4.2. MATERIAL AND METHODS
2.4.2.1. EQUIPMENT
° quartz test tubes or crucibles
° muffle furnace (100-1000°C)
° analytical balance (+ 0.001 g)
° micro-pipette (0.25 ml)
° repipette (10 ml)
0 repipette (1 ml)
2.4.2.2. CHEMICALS/REAGENTS
° 30% Hydrogen Peroxide (H202)
° 37% (v/v) Hydrochloric acid (HC1)
° Distilled-deionized (DI) water
° First combined calibration standard: 500 mg/1 K and Ca, 200 mg/1
Mg, 100 mg/1 P, 50 mg/1 Fe and Al, and 10 mg/1 Na and Mn.
0 Second combined calibration standard: 1 mg/1 Cu and 2.0 mg/1 B and
Zn in 5% BCL.
° Internal standard solution: 4 Mg of yttrium (Y) per 9.5 ml of DI
water.
2.4.3. PROCEDURES
2.4.3.1. SAMPLE PREPARATION
1. Foliar sample should be oven-dried (70°C) and ground to pass a 1
mm screen.
2. Veigh 0.4 g of sample into a quartz test tube or crucible.
3. Place in cool muffle furnace and ash sample at 450°C.
4. Remove and let cool. Add 0.25 ml of 30% H202 and reash sample at
450°C for 2 hours.
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5. Remove and let cool- Add 0.500 ml of 372 HC1. Let stand for 1
hour.
6. Add 9.5 ml of DI water containing ytrriura internal standard..
7. Nix sample and let stand. Settling time is required so that
suspended material will not clog nebulizer on emission
spectrometer if samples are not centrifuged.
8. Samples are to be analyzed immediately after suspended material
has settled.
9. Follow guidelines detailed in Good Laboratory Practices section
for recording of all raw data. Note that raw data include the
following: (a) analytical observations necessary to calculate the
final results, (b) calibration data, (c) calibration checks, and
(d) quality control checks. Deviations from standard operating
procedures during sample preparation, calibration, or actual
analyses are to be fully documented and initialed by laboratory
personnel. Samples suspected of being in error or outside of the
calibration range ace to be 'flagged', and this notation carried
through all records to the final report. Retain all written
materials (graphs, tables, etc.) generated as part of an
analysis. Do not discard portions of laboratory notebooks or any
other information directly related to calculation of the final
result for a set of samples.
2.4.3.2. EQUIPMENT OPERATION
Consult operating manual for detailed instructions on operation and
calibration of muffle furnace.
The following instrument settings are for a Jarrell-Ash 975 Atom Comp
simultaneous inductively coupled argon plasma emission spectrometer:
0 Forward Power - 1.2 kw
0 Coolant gas - 17 1pm
0 Plasma gas - 0.8 1pm
° Sample gas - 0.25 lprs
° Uptake rate - 2.2 ml/min using peristaltic pump and a fixed cross
flow nebulizer.
2.4.4. PREVENTIVE MAINTENANCE
Carry out all maintenance procedures as recommended by the
manufacturer. Record maintenance operations in maintenance log.
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2.4.5. CALIBRATION PROCEDURES
1. Let instrument warm up for 2 hours before use.
2. Using a 2.0 mg/1 Cd solution, align entrance slit so that all of
spectral lines are centered on their respective photomultiplier
tubes. This procedure is necessary to ensure maximum
sensitivity.
3. Calibrate instrument with 2 multielement standard solutions and
one reagent blank.
4. Program instrument for the following spectral interferences:
Effect of On Factor
Fe B .0017
5. Monitor instrument drift by checking with standards on a regular
basis. Drift of more than 3.55% for P, Ca, Mg, K, or Mn, or more
than 5% for B, Cu, Na, or Zn indicates need to recalibrate
instrument.
2.4.6. CALCULATIONS/UNITS
Final concentration of an element in a sample can be calculated using
the following equation:
= mg of element per g of sample
where A = yg of element per ml of digestate as determined from the
calibration curve, C = total volume of digestate (10 ml), and B = weight
of foliar sample.
Report results for P, Ca, Mg, and K on a X weight basis ( IX by
weight = 10000 mg/kg).
Report results for Mn, B, Cu, Na, and Zn as mg/kg of dried sample.
2.4.7. ERROR ALLOWANCE AND DATA QUALITY
2.4.7.1. CONSIDERATIONS
Blanks, replicates, and in-house secondary standards should be
included in analyses of all samples. Vithin one group of approximately 40
samples there should be one blank, two in-house secondary standards, and 3
replicates. Certified NBS reference materials should be included on a
monthly basis. At least two NBS SRM's with different matrices are
recommended for use with this procedure.
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Samples selected as replicates should also be run on different days
to estimate day-to-day precision.
Replicate samples will be used to monitor precision. Percent
coefficient of variation will be calculated using the industrial statistic
I, where %CV = 200I//2 and I=|A-B|/A+B, A and B being the results for the
replicate samples (U.S. EPA, 1979).
Precision will also be monitored using Shewhart control values for R
(range) values, if the total number of sample groups to be analyzed will
generate enough points to make use of such charts worthwhile.
Critical ranges for repeated mesurement error and accuracy will be
set by Quality Assurance Officer and listed in the Data Quality Objectives
(DQO) table.
Accuracy will be evaluated through the use of interlaboratory sample
exchanges and use of NBS SRM's. Sample exchanges will be coordinated by
the Quality Assurance Specialist assigned to the project.
2.4.7.2. DATA QUALITY OBJECTIVES
Repeated Measurement
Measurement
Reporting
Error
at
Accuracy
Variable
Units
Lower Limit
Upper Limit
Tolerance
P
0.01% (v/w)
20% (cv)
10% (cv)
15%
K
0.01% (w/w)
10* (cv)
10% (cv)
15%
Ca
1 mg/kg :
10% (cv)
10% (cv)
15%
Mg
1 mg/kg
10% (cv)
10% (cv)
15%
B
1 mg/kg
10% (cv)
10% (cv)
15%
Na
1 mg/kg
20% (cv)
10% (cv)
15%
Cu
0.1 mg/kg
20% (cv)
10% (cv)
15%
Zn
1 mg/kg
10% (cv)
10% (cv)
15%
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2.4.7.3. COMPUTER DATABASE CODES
Variable Code
Total Elemental
P
FTTP
Total Elemental
K
FTTK
Total Elemental
Ca
FTCA
Total Elemental Mg
FTMG
Total Elemental
B
FTTB
Total Elemental
Na
FTNA
Total Elemental
Cu
FTCU
Total Elemental
Zn
FTZN
2.4.8. REFERENCES
Commun. Soil Sci. Plant Anal. 1983. 14(7):629-644.
Dhalquist, R.L. and J.V. Knoll. 1978. Inductively coupled plasma-atomic
emission spectrometry: analysis of biological materials and soils for
major, trace, and ultra-trace elements. Applied Spectroscopy 32(1).
Isaac, R.A. and V.C. Johnson. 1985. Elemental analysis of plant tissue
by plasma emission spectroscopy: collaborative study. J. Assoc. Off.
Anal. Chem. 68(3).
Taketoshi, N. 1983. The determination of trace amounts of tin by
inductively coupled argon plasma atomic emission spectroscopy with
volatile hydride method. Applied Spectroscopy 37(6): 539-545.
U.S. Environmental Protection Agency. 1979. Handbook for Analytical
Quality Control in Water and Wastewater Laboratories. Environ. Mon.
and Sup. Lab. U.S. EPA. Cincinnati, OH 45268. EPA-600/4-79-019.
35
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2.5. STANDARD OPERATING PROCEDURE FOR MULTI-ELEMENT ANALYSIS: K, Ca, Mg,
Cu, Pe, Hn, Zn, Al, Cd, Na, Ni, Pb, V, Ba, Cs, Rb USING DRY
COMBUSTION
2.5.1. SCOPE AND PURPOSE
This method is designed to yield the total content of the following
elements in foliar material: K, Ca, Mg, Cu, Fe, Mn, Zn, Al, Cd, Na, Ni,
Pb, V, Ba, Cs, and Rb.
The procedure as described is based on a combination of dry
combustion and dissolution of the residue in strong acid. Alternative
dissolution procedures are acceptable.
2.5.2. MATERIALS AND SUPPLIES
2.5.2.1. EQUIPMENT
0 muffle Furnace
0 hot Plate
° open pan balance (+ 0.01 g)
® automatic Diluter/Dispenser
0 250 ml Teflon Beakers
° porcelain crucibles (70 ml capacity)
° plastic stirring rods
0 plastic 100 ml centrifuge tubes
0 polyethylene screv cap bottles (60 ml capacity)
0 polyethylene funnels
0 No. 42 filter paper
° parafilm
2.5.2.2. CHEMICALS/REAGENTS
0 12 M HCl, Analytical Reagent Grade
0 49£ HF, Analytical Reagent Grade
0 15 M HN03, Analytical Reagent Grade
° Lanthanum Oxide (99.99% pure)
0 Distilled-deionized (DI) vater
2.5.3. PROCEDURES
2.5.3.1. SAMPLE PREPARATION
1. Foliar sample should be dried (70°C) and ground in a stainless
steel Viley Mill with a 1 mm stainless steel sieve. Other metal
cutting surfaces should be avoided to minimize contamination of
sample.
Sample must be mixed well after grinding. Passage of sample
through grinder can segregate material in layers in receiving
container. Mix veil before placing in storage vial. Extent of
mixing should be checked on random samples to verify that
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heterogeneity of sample will not contribute to total error
variance.
2. Weigh 3 g of sample into a tared porcelain crucible. Transfer
crucible to muffle furnace and ash at 475°C for 8 to 12 hours.
3. After cooling, dissolve the ashed samples in 4 ml of 6N HCl on a
hot plate (90°C), then quantitatively transfer to a 250 ml Teflon
beaker using a minimal amount of 4N HCL to rinse porcelain
crucible.
4. Transfer Teflon beakers to a hot plate (120°C) and take to
dryness.
5. Allow beakers to cool and add 5 ml of 12 ml HCl and 5 ml of 49%
HF to each beaker. Place on hot plate and allow to go to
dryness.
6. Repeat the preceding step as needed to dissolve any residue
remaining after addition of another 5 ml of 12M HCl. If no
residue remains, allow beaker to go to dryness.
7. Allow beakers to cool, then add 2 ml of 12 M HCl and 10 ml of
distilled-deioni2ed water. Heat until residue dissolves and
solution clears. Transfer solution to a tared 100 ml
polyethylene centrifuge tube and add DI water to bring net weight
to 50 g.
8. Cap tubes with Parafilm, mix end-over-end lCf times, then filter
contents through No. 42 filter paper into acid washed (IN HN03)
60 ml polyethylene bottles for storage until analysis.
9. Follow guidelines detailed in Good Laboratory Practices section
for recording of all raw data. Note that raw data include the
following: (a) analytical observations necessary to calculate the
final results, (b) calibration data, (c) calibration checks, and
(d) quality control checks. Deviations from standard operating
procedures during sample preparation, calibration, or actual
analyses are to be fully documented and initialed by laboratory
personnel. Samples suspected of being in error or outside of the
calibration range are to be 'flagged', and this notation carried
through all records to the final report. Retain all written
materials (graphs, tables, etc.) generated as part of an
analysis. Do not discard portions of laboratory notebooks or any
other information directly related to calculation of the final
result Cor a set of samples.
2.5.3.2. EQUIPMENT OPERATION
It is assumed that either emission or atomic absorption spectroscopy
will be used to analyze contents of filtrates. Consult operating manuals
for detailed instructions on operation of instruments.
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If alternative methods of elemental analysis are selected, precision
of the chosen methods must be equal to that available using emission or
atomic absorption spectroscopy.
2.5.4. PREVENTIVE MAINTENANCE
Carry out all maintenance procedures as recommended by the
manufacturer. Record maintenance operations in maintenance log.
Calibrate muffle furnaces on a monthly basis using a pyrometer and
record changes with time.
Calibrate hot plate temperatures settings with appropriate
thermometer inserted into sand bath placed onto hot plate. Adjust
conditions of measurement to those of actual analysis to detect effect of
air flow across hot plate when making measurements. Excess temperatures
may cause Teflon beakers to melt or cause loss of sample due to excessive
boiling, especially as salt residues begin to dry. Note that aluminum
topped hotplates will corrode with prolonged exposure to the digestion
mixture described in this procedure. Covering the top of the hotplate
with aluminum foil daily is recommended to prevent contamination of
samples from material that might accumulate on the bottom of the Teflon
beakers.
2.5.5. CALIBRATION PROCEDURES
1. Four standards plus a reagent zero should be used to calibrate
instrumentation for the analysis of each element. Standards
should define linear operating range of the instrument.
Standards should be run at the beginning and end of each sample
group and after a set number of samples within each group.
2. Prepare standards from certified commercial stock sources or from
primary reagents.
3. Standard solutions should be prepared using class A volumetric
glassware and transferred to cleaned (IN HN03) polyethylene
bottles for storage between analyses. Special care should be
taken that temperature of solutions during preparation of
standards meets that stated for the calibration of the glassware.
2.5.6. CALCULATIONS/UNITS
Standard curves should be fitted by the method of least squares after
hand graphing to check for continuity.
Final concentration of an element in a sample can be calculated as
follows:
= mg of element per g of solid
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vhere A = yg of element per ml of digestate as determined from the
calibration curve, C = total volume of digestate (assume density of
1 g/cc), and B = total weight of sample weighed into porcelain crucible.
Final results are expressed as mg/kg, except for K which is usually
expressed on a % dry weight basis.
2.5.7. ERROR ALLOWANCE AND DATA QUALITY
2.5.7.1. CONSIDERATIONS
Blanks, replicates, and in-house secondary standards should be
included in analyses of all samples. Vithin one group of approximately 40
samples there should be one blank, two in-house secondary standards, and
three replicates. Certified NBS reference materials should be included on
a monthly basis. At least two NBS SRM's with different matrices are
recommended for use with this procedure.
Samples selected as replicates should also be run on different days
to estimate day-to-day precision.
Replicate samples will be used to monitor precision. Percent
coefficient of variation should be calculated using the industrial
statistic I, where XCV = 200I//2 and I=|A-B|/A+B, A and B being the
results for the replicate samples (U.S. EPA, 1979).
Precision will also be monitored using Shewhart control values for R
(range) values, if the total number of sample groups to be analyzed will
generate enough points to make use of such charts worthwhile.
Critical ranges for repeated error measurement and accuracy will be
set by Quality Assurance Officer and listed in the Data Quality Objectives
(DQO) table.
Accuracy will be evaluated through the use of interlaboratory sample
exchanges and use of NBS SRM's. Sample exchanges will be coordinated by
the Quality Assurance Specialist assigned to the project.
The advantage of this approach is that fairly large samples can be
handled without need of special digestion flasks. Using a larger sample
size also increases the amount of element present and decreases the need
for more time consuming and costly means of analysis.
One disadvantage of this procedure is that prolonged dissolution in
the open Teflon beakers increases the chance of trace metal contamination
from the immediate laboratory environment. This is especially so since
the digestion must be carried out in a chemical hood. Care must be taken
to ensure that potential contamination from the laboratory be kept to a
minimum. This is especially true for trace metals such as Pb and Cu.
Consideration should be.given to thoroughly cleaning the laboratory
(wiping down all surfaces, vacuuming, then mopping the floor) before
actually doing the analyses. The laboratory should be isolated from
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direct contact with outside air, especially when the laboratory is located
near areas of high vehicular traffic. Consideration should also be given
to performing the analysis when actual movement of other technicians in
the laboratory is minimal.
Another disadvantage of this technique is the potential loss of
certain trace metals—e.g., Cd—during dry ashing in the muffle furnace.
There are conflicting reports in the literature as to whether Cd and Fb
are lost during dry ashing of biological material without the specific use
of ashing aids. Vet digestion procedures avoid this problem but often
require the use of perchloric acid to achieve total dissolution. Most
laboratories are not equipped to safely handle digestions based on
perchloric acid mixtures. Reference standards and blanks should be used
in preliminary trials to determine whether contamination or loss of
elements during digestion procedures is occurring.
Ultrapure Grade acids are not recommended because of cost and the
fact that most analyses will be performed under conditions that will
quickly compromise the quality of such reagents. Individual bottles of
acids should be checked as to their purity by concentrating test aliquots.
Of particular concern with 12M HC1 is the presence of Zn in individual
bottles. Presence of contaminates in individual bottles is often not
related to lot numbers (Dr. T. Rains, NBS, Gaithersburg, MD, personal
communication). Redistilling of acids is possible provided storage
containers are of equal quality (i.e., Teflon). Use of blanks in quality
control procedures will help serve to determine whether contamination from
reagents used in digestion will be detected using instrumentation
selected.
2.5.7.2. DATA QUALITY OBJECTIVES
Repeated Measurement
Measurement
Reporting
Error
at
Accuracy
Variable
Units
Lower Limit
Upper Limit
Tolerance
K
0.01% (w/w)
10* (cv)
10* (cv)
15*
Ca
1 mg/kg
10* (cv)
10* (cv)
15*
Mg
1 mg/kg
10* (cv)
10* (cv)
15*
Cu
0.1 mg/kg
20* (cv)
10* (cv)
15*
Fe
1 mg/kg
10* (cv)
10* (cv)
15*
Hn
1 mg/kg
10* (cv)
10* (cv)
15*
Zn
1 mg/kg
10* (cv)
10* (cv)
15*
A1
1 mg/kg
20* (cv)
10* (cv)
15*
Cd
0.01 mg/kg
20* (cv)
10* (cv)
15*
Na
1 mg/kg
20* (cv)
10* (cv)
15*
Ni
0.01 mg/kg
20* (cv)
20* (cv)
15*
Pb
0.01 mg/kg
25* (cv)
25* (cv)
15*
V
0.01 mg/kg
20* (cv)
20* (cv)
15*
Ba
0.01 mg/kg
15* (cv)
10* (cv)
15*
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DATA QUALITY OBJECTIVES (cont'd)
Repeated Measurement Measurement
Reporting Error at Accuracy
Variable Units Lover Limit Upper Limit Tolerance
Cs 0.01 mg/kg 10* (cv) 10* (cv) 15%
Rb 1 mg/kg 10% (cv) 10% (cv) 15%
2.5.7.3. COMPUTER DATABASE CODES
Variable Code
Total
Elemental
K
FTTK
Total
Elemental
Ca
FTCA
Total
Elemental
Mg
FTMG
Total
Elemental
Cu
FTCU
Total
Elemental
Fe
FTFE
Total
Elemental
Mn
FTMN
Total
Elemental
Zn
FTZN
Total
Elemental
A1
FTA1
Total
Elemental
Cd
FTCD
Total
Elemental
Na
FTNA
Total
Elemental
PB
FTPB
Total
Elemental
V
FTTV
Total
Elemental
Ba
FTBA
Total
Elemental
Cs
FTCS
Total
Elemental
Rb
FTRB
2.5.8. REFERENCES
Greveling, T. 1976. Chemical Analysis of Plant Tissue. Search. 6:1-35.
Cornell University, Ithaca, NY.
Hatcher, J.T. and L.V. Wilcox. 1950. Colorimetric determination of boron
using carmine. Anal. Chem. 22:567-569.
U.S. Environmental Protection Agency. 1979. Handbook for Analytical
Quality Control in Water and Wastewater Laboratories. Environ. Hon.
and Sup. Lab. U.S. EPA. Cincinnati, OH 45268. EPA-600/4-79-019.
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2.6. STANDARD OPERATING PROCEDURE FOR MEASUREMENT OF BORON
2.6.1. SCOPE AND PURPOSE
The procedure discussed in this section will determine the
concentration of boron in plant tissue samples. This method uses a
digestion step similar to that for analysis of foliar material in Section
2.5. However, treatment with HF is excluded because use of this acid will
result in the loss of B when the acid solutions are taken to dryness.
Final determination of B in the filtrate uses the chromophore formed
between B and carmine in concentrated acid (Hatcher and Wilcox, 1950).
2.6.2. MATERIALS AND SUPPLIES
2.1 EQUIPMENT
° Muffle Furnace
° Open pan balance (+ 0.01 g)
° UV/Vis spectrophotometer
° volumetric glassware
° volumetric pipettes
° 50 ml Erlenmeyer flasks
° Oxford Repipetter (10 ml)
° filter funnels (9 cm)
° funnel rack
° Porcelain crucibles (70 ml capacity)
0 Plastic stirring rods
0 Plastic 100 ml centrifuge tubes
° Polyethylene screw cap bottles (60 ml capacity)
0 No. 42 filter paper
° Parafilm
° 1 cm open-top spectrometer cell
2.6.2.2. CHEMICALS/REAGENTS
° 12M Hydrochloric acid (HCl)
° 18M Sulfuric acid (H-SO,)
° Distilled deionized (DIJ water
° Calcium hydroxide (Ca(0H)2)
° Carmine
0 Carmine working solution - dissolve 0.920 g of carmine in 1 liter
of 18M sulfuric acid. This solution is unstable in light and
air. Store in dark and make fresh every 2-3 days.
0 Stock B standard (100 mg/1) - dry reagent grade boric acid
crystals over dessiccant for 24 hours. Transfer 0.5716 g into
a 1 liter volumetric flask. Make to volume with DI water.
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2.6.3. PROCEDURE
2.6.3.1. SAMPLE PREPARATION
1. Veigh 2.5 g of ground (<1 mm) oven-dried (70°C)) sample into a
tared porcelain crucible. Transfer crucible to muffle furnace
and ash at 475°C for 8 to 12 hours.
2. After cooling, dissolve the ashed samples in 4 ml of 6N HC1 on a
hot plate (90°C), then quantitatively transfer solution to a
tared 100 ml polyethylene centrifuge tube and add DI vater to
bring net weight to 50 g.
3. Cap tubes vith Parafilm, mix end-over-end 10 times, then filter
contents through No. 42 filter paper into acid washed (IN HN03)
60 ml polyethylene bottles for storage until analysis.
4. Transfer 2 ml of sample filtrate or standard to a 50 ml
Erlenmeyer flask.
5. Add 10 ml of 18M sulfuric acid using an Oxford Repipetter.
Rotate Erlenmeyer flask while adding acid so that acid runs down
sides of flask. This ensures that no sample is left remaining
on the sides of the flask and prevents splattering if acid were
added directly to sample.
6. CAUTION! Vorking vith concentrated sulfuric acid requires
extreme care. Vear gloves at all times! Several layers of thin
latex gloves provide adequate protection arfd manual dexterity
when adding acid. Running tap water should be close at hand to
minimize burns in case of contact vith skin.
7. Allow acid solution to cool.
8. Mix acid solution by slowly swirling flask to remove any bubbles
formed during addition of concentrated acid.
9. Add 10 ml of carmine vorking solution using Oxford repipetter.
As before, rotate Erlenmeyer flask while adding acid so that
solution runs down sides of flask.
10. CAUTION! Vorking vith concentrated sulfuric acid requires
extreme care. Wear gloves at all times!
11. Mix acid solution in flask. Allow minimum of 1 hour for color
development. Color is stable for several hours.
12. Follow guidelines detailed in Good Laboratory Practices section
for recording of all raw data. Note that raw data, include the
following: (a) analytical observations necessary to calculate
the final results, (b) calibration data, (c) calibration checks,
and (d) quality control checks. Deviations from standard
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operating procedures during sample preparation, calibration, or
actual analyses are to be fully documented and initialed by
laboratory personnel. Samples suspected of being in error or
outside of the calibration range are to be 'flagged', and this
notation carried through all records to the final report.
Retain all written materials (graphs, tables, etc.) generated as
part of an analysis. Do not discard portions of laboratory
notebooks or any other information directly related to
calculation of the final result for a set of samples.
2.6.3.2. EQUIPMENT OPERATION
1. Consult operating manual of spectrophotometer for proper
adjustment of 0%T and 1002T before taking readings.
2. Flow through spectrophotometer cells will not work with the acid
solution in Section 2.6.3.1., step 11. Use a 1 cm open-top
spectrophotometer cell.
3. Zero instrument using 18M sulfuric acid in cell. Rinse between
samples and standards with 18M sulfuric acid.
4. Do not put water into cell at any time during reading of
standards and samples.
5. CAUTION! Working with concentrated sulfuric acid requires extreme
care. Wear gloves at all times!
6. Read samples and standards at 585 nm.
7. Rinse 1 cm cell with sample or standard. Fill with another
aliquot of sample or standard and insert cell in
spectrophotometer. Record reading when output is stable.
2.6.4. PREVENTIVE MAINTENANCE
Perform all maintenance procedures specified in the spectrophotometer
operating manual. Keep a log book of all adjustments made. Check 0%T,
100XT and 1 Abs settings weekly during useage. Check these settings daily
if the spectrophotometer is a common use instrument.
2.6.5. CALIBRATION
1. Prepare working standards using the following chart. Stock
source is 100 mg/1 B. Procedure is to add ml of stock to desired
volumetric flask. Add recommended ml of 12M HCl and add half of
the DI water necessary to bring to volume. Let flasks cool, then
make to volume with DI water.
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Std cone ml stock std ml 12M HC1 Vol. flask size
0 0 8 200
0.2 2 AO 1000
0.4 2 20 500
0.6 3 20 500
1 2 8 200
2 4 8 200
3 6 8 200
5 10 8 200
7 7 4 100
2. Prepare standards the same as samples in Section 2.6.3.1.,
step 4.
3. Read set of standards at the beginning and end of analyses. Read
individual standards with samples during analysis.
4. Standard blanks and samples blanks should agree within 0.003
absorbance units. Failure to have similar readings indicates
contamination with B somewhere in the procedure. Stop analysis
and find source of contamination.
2.6.6. CALCULATIONS/UNITS
Standard curves should be fitted by the method Qf least squares after
hand graphing to check for continuity.
Blank values are not subtracted from readings because sample blanks
and standard blanks should be nearly identical.
The concentration of B in a sample (mg B/kg oven-dried (70°C) plant
tissue) can be calculated using the following equation:
A x V
—j,— = mg B/kg oven-dried sample
where A => concentration of B in original filtrate as determined from
calibration curve, V = volume of original filtrate (50 ml, assuming
density = 1 g/cc), and C = weight of original sample (2.5 g).
2.6.7. ERROR ALLOWANCE AND DATA QUALITY
2.6.7.1. CONSIDERATIONS
Blanks, replicates, and in-house secondary standards should be
included in analyses of all samples. Within one group of approximately 40
samples (20 samples if 250 ml digestion tubes are used) there should be
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one blank, two in-house secondary standards, and three replicates.
Certified NBS reference materials should be included on a monthly basis.
At least two NBS SRM's with different matrices are recommended for use
with this procedure.
Samples selected as replicates should also be run on different days
to estimate day-to-day precision.
Replicate samples will be used to monitor precision. Percent
coefficient of variation should be calculated using the industrial
statistic I, where %CV = 200I//2 and 1=|A-B|/A+B, A and B being the
results for the replicate samples (U.S. EPA, 1979).
Precision will also be monitored using Shewhart control values for R
(range) values, if the total number of sample groups to be analyzed will
generate enough points to make useage of such charts worthwhile.
Critical ranges for repeated measurement error and accuracy will be
set by Quality Assurance Officer and listed in the Data Quality Objectives
(DQO) table.
Accuracy will be evaluated through the use of interlaboratory sample
exchanges and use of NBS SRM's. Sample exchanges will be coordinated by
the Quality Assurance Specialist assigned to the project.
Actual density of 18M sulfuric acid has an influence on sensitivity
and precision of this procedure. Concentrated acid should be between 96-
982 acid.
Procedure as described uses standard Pyrex glassware. This glassware
is a potential source of boron contamination. Corning alkali resistant
glass no. 7280 or plastic labware is recommended for long term storage of
solutions or samples. However, daily use of Pyrex glassware does not
appear to contaminate samples with B, at least within the sensitivity
limits of this procedure.
i
Another possible source of B contamination is porcelain crucibles if
the glaze has been worn avay.
Boron may be lost upon ashing plant material low in base cations. If
loss on ignition is suspected, use an ashing aid such as 5 ml of a
saturated solution of calcium hydroxide. Mix with weighed sample,
evaporate to dryness, then include with remainder of samples. Prepare
blanks with 5 ml of calcium hydroxide solution to check its B content.
The carmine procedure as described forms a stable complex for several
hours and is relatively unaffected by the presence of a wide variety of
electrolytes. The principal objection to the procedure is the requirement
of developing the complex in the presence of concentrated sulfuric acid.
Use of this acid requires that the procedure be carried out in a
deliberate and careful manner.
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2.6.7.2.
DATA QUALITY
OBJECTIVES
Revision: 0
Date: July 1986
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Repeated Measurement
Measurement
Reporting
Error at
Accuracy
Variable
Units
Lover Limit Upper Limit
Tolerance
Boron
mg/kg
10% (cv) 10% (cv)
15%
2.6.7.3. COMPUTER DATABASE CODES
Variable
Code
Total Elemental B
FTTB
2.6.8. REFERENCES
Greveling, T. 1976. Chemical Analysis of Plant Tissue. Search. 6:1-35.
Cornell University, Ithaca, NY.
Hatcher, J.T. and L.V. Wilcox. 1950. Colorimetric determination of boron
using carmine. Anal. Chem. 22:567-569.
U.S. Environmental Protection Agency. 1979. Handbook for Analytical
Quality Control in Vater and Wastewater Laboratories. Environ. Hon.
and Sup. Lab. U.S. EPA. Cincinnati, OH 45268. EPA-600/4-79-019.
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2.7. STANDARD OPERATING PROCEDURE FOR MEASUREMENT OF CHLORIDE
2.7.1. SCOPE AND PURPOSE
This procedure has been designed to determine the concentration of
chloride anion (CL~) in foliar tissue. This method is based on an
extraction of ground foliar tissue because chloride is lost during dry
ashing of samples. Determination of chloride in the extract is by use of
a chloridometer.
2.7.2. MATERIAL AND SUPPLIES
2.7.2.1. EQUIPMENT
0 chloridometer
° titrating vials for chloridometer
0 volumetric flasks (1 liter, 200 ml)
° volumetric pipettes (100 ml, 1-40 ml)
° 250 ml Erlenmeyer flask
° analytical balance (+ 0.001 g)
° filter funnels (9 cm diameter)
° funnel rack
0 50 ml Erlenmeyer flasks
° Oxford pipette with disposable tips (5 ml)
0 convection oven (110°C)
° Buchner funnel
0 side-arm suction flask
° aspirator
° No. 40 Vhatman filter paper (9 cm diameter)
2.7.2.2. CHEMICALS/REAGENTS
0 15M Nitric acid (HN03)
° Glacial acetic acid
° Distilled deionized (DI) water
° Decolorizing Charcoal Neutral (Fisher SCI. #C-170) - wash charcoal
as described in Section 2.7.4., step 1.
° Gelatin (ground gelatin, thymol blue, thymol mixture; ratio
60:1:1, respectively). Can be purchased premixed from Fisher
Sci. Co. (# 09-311-126).
0 Gelatin working solution - Add 100 ml of boiling DI water to 0.62
g of gelatin. Mix well and store in covered glass vial in a
4°C refrigerator. Solution is stable for 6 months when
refrigerated but deteriorates in 1 day at room temperature.
° Extracting solution (0.1N HN03, 10% acetic acid) - Produce as much
extracting solution as needed using the following ratios: 6.4
ml 15M HN03:100 ml glacial acetic acid:894 ml DI water.
° Stock chloride standard (1000 mg/1) - dry reagent grade NaCl
overnight at 105°C. Transfer 1.6484 g dried NaCl in a 1 liter
volumetric flask and bring to volume with extracting solution.
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2.7.3. PROCEDURES
2.7.3.1. SAMPLE PREPARATION
1. Weigh 0.500 g of ground (<1 mm) oven-dried (70°C) sample into a
250 ml Erlenmeyer flask.
2. Add approximately 1 ml of dried charcoal (pea sized scoop).
3. Add 100 ml of extracting solution using a 100 ml volumetric
pipette. Carefully rinse down sides of flask vhile adding
extracting solution to remove any sample material. Do NOT svirl
when finished.
4. Cover flasks with large sheet of plastic and let stand overnight.
5. Filter suspension thru No. 40 filter paper into 50 ml Erlenmeyer
flasks (or any suitable glass container).
6. Follov guidelines detailed in Good Laboratory Practices section
for recording of all raw data. Note that rav data include the
following: (a) analytical observations necessary to calculate the
final results, (b) calibration data, (c) calibration checks, and
(d) quality control checks. Deviations from standard operating
procedures during sample preparation, calibration, or actual
analyses are to be fully documented and initialed by laboratory
personnel. Samples suspected of being in error or outside of the
calibration range are to be 'flagged', and this notation carried
through all records to the final report. Re'tain all vritten
materials (graphs, tables, etc.) generated as part of an
analysis. Do not discard portions of laboratory notebooks or any
other information directly related to calculation of the final
result for a set of samples.
2.7.3.2. EQUIPMENT OPERATION
2.7.3.2.1. Haake Buchler Digital Chloridometer
1. Ready instrument as per manufacturer's instructions.
2. Clean electrode with silver polish.
3. Pipette 4 ml (Oxford pipette) of sample or standard extracts (see
Section 2.7.5.) into titration vial.
4. Add 4 drops of gelatin reagent.
5. Place vial in holder and set in position for titration
(electrodes submersed).
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7
8
9
t .
1
2
3
4
5
6
1
2
3
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Set 'Titration' switch to Start. Digits will blank and
instrument will begin titration. Instrument stops, automatically
when endpoint is reached.
Readout of digital counter is 0-999.9. Record readout when
instrument is finished.
Remove vial and wipe electrodes. Clean with silver polish as
needed (see Section 2.7.4.).
Vhen finished, set switches to standby and turn off power. Be
sure electrodes are immersed in water.
PREVENTIVE MAINTENANCE
Charcoal Washing Procedure - Add sufficient amount of extracting
solution to original container holder charcoal to make a slurry.
Mix slurry in container using glass rod.
Filter suspension through No. 40 filter paper in a Buchner
funnel. Be sure filter paper is wet before applying suction.
Add suspension to Buchner funnel as soon as wet filter paper is
drawn against, bottom of Buchner funnel.
Add suspension to Buchner funnel until filter cake fills inside
of funnel. Remove drained filter cake. Repeat procedure until
entire suspension is filtered.
Allow filter cake of charcoal to dry. Drying may take several
days. Use of a convection oven is permissible.
Electrodes of chloridometer become fouled with AgCl, especially
when extracts contain > 200 mg/1 Cl (800 yg total). Output
becomes erratic and unreproducible when electrodes are fouled.
Cleaning with silver polish will remove problem.
Carry out all maintenance procedures as recommended by the
manufacturer. Record maintenance operations in maintenance log.
CALIBRATION PROCEDURES
The chloridometer described in this procedure has two range
settings: L0V and HIGH.
Selection of range is a function of Cl content of extract. The
following guidelines are based on use of 4 ml volume of extract
for titration.
LOW range: 1 yg/ml Cl (4 yg total) - 50 yg/ml Cl (200 yg total).
Blank sample is extracting solution and typically gives a reading
of 50 units for reagent grade acids and DI water. The 50 yg/ml
Cl standard in the LOW range typically has a readout of 650 units
for linear response.
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4. HIGH range: 10 pg/ml CI (40 pg total) - 200 ug/ml Cl (800 pg
total). Blank sample is extracting solution and typically gives
a reading of 8 units. The 200 pg/ml Cl standard in the HIGH
range typically has a readout of 250 units for a linear response.
5. Higher readouts are possible vith HIGH range setting but attempts
thus far to exceed 200 pg/ml have met vith poor reproducibility
and excessive fouling of electrode vith AgCl.
6. Vorking Standards - LOW range (0,5,10,15,30,50 wg/ml Cl) Aliquots
of stock standard (1000 mg/1 Cl) are placed in a 200 ml
volumetric and brought to volume vith extracting solution.
Std Cone ml of stock
0 0
5 1
10 2
15 3
30 6
50 10
7. Vorking Standards - HIGH range (0,25,50,100,200 pg/ml Cl)
Aliquots of stock standard (1000 mg/1 Cl) are placed in a 200 ml
volumetric and brought to volume vith extracting solution.
Std Cone
0
30
50
100
200
ml of stock
0
6
10
20
40
8. Filtration of standard solutions through No. 40 filter paper is
not necessary.
9. Run set of standards at the beginning and end of each set of
samples. Run individual standards vith samples during course of
analysis. One standard contains enough volume for approximately
50 determinations, vhich should be sufficient for most analyses
without making nev standards.
2.7.6. CALCULATIONS/UNITS
1. Standard curves should be fitted by the method of least squares
after hand graphing to check for continuity.
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2. Blank values are not subtracted from readings because sample
blanks and standard blanks are identical.
3. The concentration of Cl in a sample (mg Cl/kg oven-dried (70°C)
plant tissue) will be calculated using the following equation:
A x V
—r— = mg Cl/kg oven-dried sample
vhere A = concentration of Cl determined in extract, V = total volume of
extract (100 ml), and B = weight of sample (0.500 g).
2.7.7. ERROR ALLOWANCE AND DATA QUALITY
2.7.7.1. CONSIDERATIONS
It is recommended that replicates be run for all samples. If
replicates fail to agree within 5%, the samples should be rerun and
operation of the instrument (especially fouling of electrode) checked.
Blank values should be consistent between sets of analyses and not
change by more than 5X on a daily basis. Significant changes in daily
values of blank values indicate contamination from Cl anion somewhere in
the procedure. Analysis should be stopped until constant blank values are
obtained. Daily blank values should be recorded and placed on charts for
visual assessment of quality control of analysis.
In-house secondary standards should be included in analyses of all
samples. Approximately 35 samples will compose each group of analyses.
Within one group there should be one blank, and 2 in-house secondary
standards. Certified NBS reference materials should be included on a
monthly basis, when appropriate.
Acceptable range for accuracy measurements will be listed in the Data
Quality Objectives (DQ0) table.
This procedure is capable of detecting small differences in the
concentration of Cl in samples. Possible contamination of glassware
should be avoided during all phases of this analysis. Two possible
sources of Cl contamination are tap water residues on glassware and direct
contact of glassware or reagents with human skin. All glassware is to be
rinsed well with DI water as final cleaning step. Handling of glassware
with hands is to be done so as to avoid any possibility of contamination
of interior glass surfaces.
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2.7.7.2.
DATA QUALITY
OBJECTIVES
Revision: 0
Date: July 1986
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Repeated Measurement
Measurement
Reporting
Error at
Accuracy
Variable
Units
Lower Limit Upper Limit
Tolerance
Chloride
mg/kg
5£ (cv) 5% (cv)
15%
2.7.7.3. COMPUTER DATABASE CODES
Variable
Code
Extractable Cl
FECL
2.7.8. REFERENCES
Greveling, T. 1976. Chemical Analysis of Plant Tissue. Search. 6:1-35.
Cornell University, Ithaca, NY.
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2.8. STANDARD OPERATING PROCEDURE FOR MEASUREMENT OF SULPHUR
2.8.1. SCOPE AND PURPOSE
This method will provide a value for the total elemental content of S
in foliar tissue. The method uses a combination of vet and dry ashing,
with sulfate-S being determined in the digestates by turbidimetric
measurement of a barium sulfate precipitate. The procedure as described
can be performed in most laboratories without the need for special
equipment.
Direct determination of S in the digestates is possible using an
emission spectrometer. However, the ashing aid used in this procedure
will clog most cross-flow nebulizers used with ion-coupled plasma emission
spectrometers.
2.8.2. MATERIALS AND SUPPLIES
2.8.2.1. EQUIPMENT
0 Muffle furnace.
0 Hot plate or steam bath
° Magnetic stirrers
° UV/Vis Spectrophotometer with flow through cell chronometer
° analytical balance (+ 0.001 g)
° 1 liter volumetric flasks
0 100 ml volumetric flasks
° repipettes (5 ml)
° 10 ml volumetric pipettes
° 150 ml Pyrex beakers
° 100 ml Pyrex beakers
° watch glasses (for covering 150 ml Pyrex beakers)
0 No. 41 Vhatman filter paper
2.8.2.2. CHEMICALS/REAGENTS
0 15M Nitric acid (HN03)
° 12M Hydrochloric acid (HC1)
0 302 Hydrogen peroxide (H202)
° Distilled-deionized water (DI)
° 20% Magnesium nitrate solution - dissolve 200 g of Mg(N03)-6H20 in
1000 ml of DI water.
0 Polyvinyl Pyrrolidone (l-ethyl-2 pyrrolidone polymer) (PVP)
° Barium Chloride (BaClj*2H20)
° Polyvinyl Pyrrolidone/Barium Chloride solution. Dissolve 3.0 g
PVP in 100 ml of hot water (not boiling). Add 20 g of
BaClj*2H20 and mix while adding 800 ml of DI water. Solution
is to be prepared fresh daily for maximum sensitivity in
analytical working curve.
° Diethylene triamine penatacetic acid, Na salt, 40% solution (DTPA)
(dilute 50 ml DTPA to 1 liter of DI water. Solution is
stable.)
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0 Stock. S standard (1000 mg/l)~ weigh 5.4354 g of dried and
transfer to a 1 liter volumetric flask. Heat gently with
stirring to dissolve.
0 Spike S solution - transfer 10 ml of stock S standard (1000 mg/1)
to a 1 liter volumetric flask. Add 13.3 ml of 12H HCl and 500
ml of DI water. Allow solution to cool and bring to volume
vith DI water.
2.8.3. PROCEDURES
2.8.3.1. SAMPLE PREPARATION
1. Weigh 0.25 g of ground (<1 mm) oven-dried (70°C)) sample into a
150 ml Pyrex beaker.
2. Place samples and standards (prepared as described in Section
2.5) in a hood and treat each beaker vith 4 ml of 15N EN03-
Cover with watch glass and let stand overnight.
3. Add 1 ml of 30% H202 to each beaker in hood. Do not svirl
beakers but move each beaker in such a fashion as to ensure even
distribution of H202 in beaker.
4. Dry contents of beaker (100°C).
5. Add 5 ml of 20% Mg(N03)2 solution and dry contents of beaker.
6. Ash in muffle furnace at 500°C for 6-12 hours. Do not let
beakers sit on floor of muffle furnace. Us'e suitable metal rack
(nichrome wire) supported by empty crucibles to keep beakers off
of furnace floor.
7. Cool and add 4 ml of 4N HCl and dry on steam bath.
8. Add 4 ml of 4N HCl, heat until hot to touch and quantatively
transfer to a 100 ml volumetric flask.
9. Allow volumetric flasks to cool, then bring to volume vith DI
water. Cover volumetric flasks and invert at least 10 times to
mix contents. Solutions are stable.
10. Filter contents of 100 ml volumetric flasks through No. 41
Vhatman filter paper until 20-30 ml of filtrate is obtained.
11. Pipette 10 ml of sample or standard filtrate into a 100 ml Pyrex
beaker. Add 3 ml of spike S solution.
12. Begin readings by placing a magnetic stirring bar in a 100 ml
Pyrex beaker vith 10 ml of sample or standard, and place beaker
on a magnetic stirrer.
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13. Immediately after placing beaker on magnetic stirrer add 5 ml
PVP-BaCl2 solution with Oxford Repipettor. Add PVP-BaCl2
aliquot in one continuous motion at the center of beaker.
14. Continue to mix for a total of one minute.
15. Remove beaker and let stand for one minute. Place second beaker
on magnetic stirrer and treat as described in step 13.
16. Place first 100 ml beaker on second magnetic stirrer and let mix
for one minute. Remove beaker from first magnetic stirrer and
let stand. Start third beaker on first magnetic stirrer as
described in step 13.
17. Remove beaker and let stand for one minute. Advance other
beakers in series to next treatment point.
18. Read absorbance of first beaker at 440 nm and record reading
when display output is stable. Use of a 1 cm flow through cell
is highly recommended for this step.
19. Rinse cell with DTPA solution between samples, letting DTPA sit
in cell 15 seconds before rinsing with DI water and reading the
next sample.
20. Follow guidelines detailed in Good Laboratory Practices section
for recording of all raw data. Note that raw data include the
following: (a) analytical observations necessary to calculate
the final results, (b) calibration data, (c) calibration checks,
and (d) quality control checks. Deviations from standard
operating procedures during sample preparation, calibration, or
actual analyses are to be fully documented and initialed by
laboratory personnel. Samples suspected of being in error or
outside of the calibration range are to be 'flagged', and this
notation carried through all records to the final report.
Retain all written materials (graphs, tables, etc.) generated as
part of an analysis. Do not discard portions of laboratory
notebooks or any other information directly related to
calculation of the final result for a set of samples.
2.8.3.2. EQUIPMENT OPERATION
Consult operating manual of spectrophotometer for proper adjustment
of 0%T and 100%T before taking readings.
Magnetic stirrers' are set at slowest speed for analysis. Violent
mixing is to be avoided, as well as rapid stirring. Uniformity in
stirring rates between samples is critical to preserve precision and
accuracy.
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2.8.4. PREVENTIVE MAINTENANCE
Perform all maintenance procedures specified in the spectrophotometer
operating manual. Keep a log book of all adjustments made. Check 0%T,
100%T and 1 Abs settings weekly during use. Check these settings daily if
the spectrophotometer is in a common use area.
The ISO ml Pyrex beakers used for ashing sample need to be scrubbed
with a brush, soaked in a IN HCl bath for 30 minutes, and rinsed 4 times
with DI vater.
The 100 ml beakers used to hold the 10 ml sample and standard
aliquots are to be rinsed with tap vater, soaked in IN HCl bath for 30
minutes, and rinsed 4 times with DI vater.
2.8.5. CALIBRATION PROCEDURES
1. Prepare vorking standard (200 mg/1 S) by placing 20 ml of stock S
solution (1000 mg/1) in a 100 ml volumetric flask and bringing to
volume vith DI vater.
2. Prepare calibration curve by pipetting following aliquots into
150 ml Pyrex beakers:
mg/1 S ml of 200 mg/1 Std
0
0
2
1
6
3
10
5
16
8
20
10
30
15
3. Prepare several sets of standards and include in analysis
procedure starting at step 2 in Section 2.8.3. Place at least
one set of standards in each muffle furnace used for ashing step
(step 6 in Section 2.8.3.).
4. Include individual standard beakers throughout set of sample
beakers.
2.8.6. CALCULATIONS/UNITS
1. Standard curve has 'S' shape for this type of analyses. It is
recommended that standard curve be plotted by hand. Standards
listed in Section 2.8.5., item 2. are designed to fall within
linear portion of 'S' curve for concentration of S typically
found in plant tissue samples.
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2. Standards should be run at the beginning and end of each set of
samples. Individual standards should be run amongst samples.
Plot average values for each standard point.
3. If more than one muffle furnace is used for ashing, and the
muffle furnaces are not set at equal temperatures, the standard
curves from each muffle furnace may not agree. If this happens,
plot separate standard curves for each muffle furnace and for the
samples ashed in that furnace.
4. Final concentration (mg/kg oven-dried sample) of S in plant
tissue can be calculated as follows:
a v .i on
= = raS S/kg oven-dried (70°C) sample
where A = concentration of S in the 100 ml volumetric flask as determined
from the standard curve, and B = weight of oven-dried sample (0.25 g).
Note that the preparation of the standards and samples is the same; thus
the mg/1 S indicated in Section 2.8.5. is the concentration of S in the
100 ml volumetric flask.
2.8.7. ERROR ALLOWANCE AND DATA QUALITY
2.8.7.1. CONSIDERATIONS
Blanks, replicates, and in-house secondary standards should be
included in analyses of all samples. Within one group of approximately 40
samples (20 samples if 250 ml digestion tubes are used) there should be
one blank, two in-house secondary standards, and three replicates.
Certified NBS reference materials should be included on a monthly basis.
At least two NBS SRM's with different matrices are recommended for use
with this procedure.
Samples selected as replicates should also be run on different days
to estimate day-to-day precision.
Replicate samples will be used to monitor precision. Percent
coefficient of variation should be calculated using the industrial
statistic I, where %CV = 200I//2 and I=|A-B|/A+B, A and B being the
results for the replicate samples (U.S. EPA, 1979).
Precision will also be monitored using Shewhart control values for R
(range) values, if the total number of sample groups to be analyzed will
generate enough points to make use of such charts worthwhile.
Critical ranges for repeated measurement error and accuracy will be
set by Quality Assurance Officer and listed in the Data Quality Objectives
(DQ0) table.
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Accuracy vill be evaluated through the use of interlaboratory sample
exchanges and use of NBS SRM's. Sample exchanges vill be coordinated by
the Quality Assurance Specialist assigned to the project.
The procedure as presented is rather long and tedious but effective
in achieving accurate analyses of the S content of plant material. The
predigestion step vith HN03 is necessary and should not be left out.
Magnesium nitrate is added to retain S during the ashing step. It can be
assumed that the nitrate anion also serves as an ashing aid, but it is not
equivalent to the HN03 treatment.
The HN03 treatment is included to ensure recovery of apparently
volatile S fractions which are lost during the ashing step. These
fractions do not occur in significant amounts in all plant material. The
effect can best be demonstrated using NBS SRM Citrus Leaves. Omission of
the HN03 treatment vill not recover all of the S reported for this SRM.
Results are very precise but inaccurate. NBS SRM Pine Needles yield
similar results vith or vithout the HN03 treatment, but it is recommended
that the step be retained vhen doing unknovn samples.
Alternative digestion procedures are becoming available for
destroying the sample matrix in preparation for S analysis. It is
recommended that if an alternate digestion procedure is selected, a series
of NBS SRM plant material be run to ensure that S recovery is satisfactory
for a variety of matrices.
Adaptation of alternative digestion procedures to the above method
are to be approached vith caution, as the final absorbance values are a
function of pH of the final test aliquot. The procedure developed in this
section has been optimized for pB in the final test solution.
Use of ICP emission spectroscopy to determine S content is acceptable
provided the sample solutions are optimal for this approach. The
solutions prepared as outlined in this procedure are not acceptable for
ICP emission spectroscopy because of the range of S standards and salt
content of the final sample solutions.
2.8.7.2. DATA QUALITY OBJECTIVES
Repeated Measurement
Measurement
Reporting
Error at
Accuracy
Variable
Units
Lover Limit Upper Limit
Tolerance
Sulphur
mg/kg
15% (cv) 10* (cv)
152
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COMPUTER DATABASE CODES
Variable
Code
Total Elemental S
FTTS
2.8.8. REFERENCES
Gaines, T.P. and G.A. Mitchell. 1979. Ghemical Methods for Soil and
Plant Analysis. Coastal Plain Experiment Station. University of
Georgia, Tifton, GA. Agronomy Handbook No. 1.
Greveling, T. 1976. Chemical Analysis of Plant Tissue. Search. 6:1-35.
Cornell Universityi Ithaca, NY.
Lambert, M.J. 1978. Methods for Chemical Analysis. Technical Paper No.
25. Forestry Commission of New South Vales. NSW, Australia.
U.S. Environmental Protection Agency. 1979. Handbook for Analytical
Quality Control in Water and Wastewater Laboratories. Environ. Hon.
and Sup. Lab. U.S. EPA. Cincinnati, OH 45268. EPA-600/4-79-019.
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3. FOLIAR ORGANIC ANALYSIS
3.1. STANDARD OPERATING PROCEDURE FOR DETERMINATION OF TOTAL CHLOROPHYLL
3.1.1. SCOPE AND PURPOSE
Determining total chlorophyll is important both for the normalization
of physiological and biochemical parameters and as an index of plant
health. This procedure for determining the total chlorophyll content of
conifer needles is an indirect method; i.e., chlorophyll-a and
chlorophyll-b are not quantitatively purified. Instead,
spectrophotometric measurements are made on a complex leaf extract
containing these chlorophylls and other chromophores. The absorptivity
coefficients used here to calculate total chlorophyll content are those of
Vellburn and Lichtenthaler (1984), who have pointed out and substantiated
that Arnon's (1949) widely-used method is technically flawed.
3.1.2. MATERIALS AND SUPPLIES
3.1.2.1. EQUIPMENT
0 centrifuge, capable of at least 5000 x G, refrigerated
0 centrifuge tubes, 50 mL, vide-mouthed, acetone-resistant, with
closures
0 funnel, plastic, vide mouth, vide bore
0 spectrophotometer, capable of reliable measurements at 662 nm
when the beam slit width is set for a half-intensity spectral
bandwidth of 2-nm or less
0 spectrophotometer cuvettes, 10 mm light path, acetone resistant
0 parafilm
0 pasteur pipets, with rubber bulb
0 polytron tissue grinder (Brinkmann Instruments)
° repipet dispenser, acetone resistant
3.1.2.2. CHEMICALS/REAGENTS
0 acetone
° chlorophyll-a, substantially free of chlorophyll-b (Sigma C5753;
Aldrich 25,825-3)
0 chlorophyll-b, substantially free of chlorophyll-a (Sigma C5878;
Aldrich 25,826-1)
3.1.3. PROCEDURES
3.1.3.1. SAMPLE PREPARATION
Plant pigments are readily oxidized by light, heat, oxygen, or
combinations of the three. For this reason samples should always be
treated in such a manner that exposure to these factors is minimal and
consistent from sample to sample.
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In the procedure that follows, one should minimize the length of time
between the isolation of chlorophyll and the measurement of absorption
values. Absorption values are available for several solvents (Wellburn
and Lichtenthaler, 1984). Chlorophyll extracts in methanol and in ethanol
were visibly degraded after standing for 16 hours at room temperature.
The spectrum of chlorophyll extract in 100% acetone was minimally degraded
after 16 hours at room temperature. Therefore, sample degradation can be
monitored. This is a specific recommendation against long delays between
extraction and measurement.
Bring a voucher sample of conifer needles that has been held in
suitable storage to ambient temperature. Divide the sample into two
subsamples. The mass of needles needed is discussed in Section 3.1.5.
Weigh both subsamples, one after the other. One subsample will be dried
to constant mass to estimate the dry matter mass of the second subsample.
The second subsample is subjected to the following procedure.
1. Hold the subsample of conifer needles as a bundle and cut the
needles with clean scissors into very short pieces of
approximately 3 mm length. Do this over a very wide mouth, wide
bore plastic funnel sitting in the throat of a 50 mL plastic
centrifuge tube. There should be no loss of material.
2. Remove the funnel and add 15.0 mL (see step 6) of acetone to the
centrifuge tube using a Repipet dispenser. Reclose the centrifuge
tube and stick it in a bed of crushed ice for cooling, perhaps in
a light-tight container.
3. IN THIS STEP THE OPERATOR OF A PIECE OF POWER EQUIPMENT SHOULD
WEAR EYE GOGGLES OR A PROTECTIVE FACE SHIELD AND ALSO A CHAIN
MAIL GLOVE ON THE HAND THAT HOLDS THE CENTRIFUGE TUBE DURING
TISSUE REDUCTION. Remove the cap and insert the shaft of the
Polytron grinder into the mouth of the centrifuge tube. Start
the Polytron at low speed and raise the centrifuge tube to
immerse the Polytron shaft. In a smooth motion increase the
Polytron speed for a 5 second burst, but take care not to lose
any acetone or sample by splash or swell. Shut the Polytron off
and lower the centrifuge tube for a visual inspection. No green,
partial needles should be trapped in the Polytron tip, but
clinging colorless fibers are permitted to remain. In the
sediment in the bottom of the centrifuge tube, there should be no
green pieces or particles of conifer needle visible. However,
the supernatant should be green. If there are green pieces or
particles, repeat the above. Don't heat the acetone with
excessive grinding. Close the centrifuge tube as soon as
possible and return it to the ice bed. It is recommended that
one practice ;this step thoroughly with throw-away samples before
working up the actual samples.
4. Centrifuge the sample tubes at 5000 x G for 4 minutes with
refrigeration (5°C). The object of this step is to obtain a non-
turbid supernatant. If the pellet at the bottom of the centrifuge
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tube tends to break up vhile handling the tube, decant the
supernatant into a second centrifuge tube and centrifuge again.
Increase speed or duration (doubled rpms are exponentially more
effective than doubled duration) of centrifugation as necessary
to obtain a non-turbid supernatant.
5. Using a Pastuer pipette vith a rubber bulb, remove a sample
aliquot and fill a spectrophotometer cuvette. Cap the cuvette
tightly. Depending on ambient conditions of temperature and
humidity, the cuvette may fog up when it is filled vith cold
supernatant. If moisture condenses on the outside of the
cuvette, the absorbance reading will be incorrect. By
experimentation, determine hov long it takes the cuvette and its
contents to come to room temperature.
6. The volume of actetone recommended in step 2 may be adjusted
according to the procedure outlined in Section 3.1.5., steps 3 to
11. The standard volume used is determined by the average
chlorophyll content of the pine needles, the number of needles
per sample required by the experimental design, and achieving a
final absorption value of 0.5 to 0.8 at 662 nm.
7. Follow guidelines detailed in Good Laboratory Practices section
for recording of all rav data. Note that rav data include the
following: (a) analytical observations necessary to calculate the
final results, (b) calibration" data, (c) calibration checks, and
(d) quality control checks. Deviations from standard operating
procedures during sample preparation, calibration, or actual
analyses are to be fully documented and initialed by laboratory
personnel. Samples suspected of being in error or outside of the
calibration range are to be 'flagged', and this notation carried
through all records to the final report. Retain all written
materials (graphs, tables, etc.) generated as part of an
analysis. Do not discard portions of laboratory notebooks or any
other information directly related to calculation of the final
result for a set of samples.
3.1.3.2. EQUIPMENT OPERATION
1. A specific spectrophotometer model or manufacturer has not been
recommended. However, the spectrophotometer must be qualified on
the following basis. Usable absorbance readings at 662 nm must
be obtainable vith the slit width set no wider than would allow a
half-intensity spectral bandwidth of 2 nm maximum. The user
manual vill contain a graph or a data table from which this
maximum slit width can be determined. If the user manual does
not provide this information, automatically assume that the
spectrophotometer in question is not suitable for this procedure.
On newer models, set the slit width and use only the gain control
to adjust the full scale setting on the blank sample cuvette. On
older models, some of which may not have electronic amplifiers,
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the slit width may be adjusted to less that the maximum permitted
slit width, but it should never exceed the maximum slit width.
2. Set the spectrophotometer to 662 nm (Section 3.1.5., steps 3 to
11). Assuming that a matched set of cuvettes is being used,
adjust the transmission to zero with the light path shutter
closed. Then open the shutter and adjust the transmission to 1.0
with the "blank" (acetone only) cuvette in the measurement path.
Make an absorbance measurement with the sample cuvette in the
light path.
3. Set the spectrophotometer to 645 nm and otherwise repeat the
previous step.
4. A matched set of cuvettes is defined as all members of the group
fall within + 0.003 transmission units of the mean transmission
value of the group as measured at 662 nm with empty cuvettes
(Anon, 1964). A calibrated set of cuvettes may be used, in which
case the relative absorbance of each empty cuvette is known in
relation to the cuvette that is used for the "blank." In a
calibrated set, the most absorbing cuvette is used for the
"blank." This permits the arithmetic correction of the
absorbance value determined in a specific cuvette (Anon, 1964).
3.1.4. PREVENTIVE MAINTENANCE
Carry out all maintenance procedures as recommended by the
manufacturer for the spectrophotometer, Polytron tissue grinder, and the
centrifuge. Record maintenance operations in maintenance log.
3.1.5. CALIBRATION PROCEDURES
1. Follow manufacturer's instructions for operation of
spectrophotometer. If any aspect of the optical or detection
systems is hot within specified tolerances, make the adjustments
suggested by the manual. If the machine can not be brought into
specification, contract for factory adjustment or repair.
2. Specific calibration of wavelength settings for chlorophyll
measurement uses highly purified samples of chlorophyll-a and
chlorophyll-b, each substantially free of the other. One may
obtain these from a commercial source or one may follow the
procedure of Lichtenthaler and Pfister (1978) for
chromatographic purification.
3. Make a solution of chlorophyll-a in 1002 acetone so that 662 nm
absorbance value will be in the range of 0.5 to 0.8. Adjust the
slit width of the spectrophotometer to achieve a 2-nm or
narrower half intensity spectral bandwidth for 667 nm, as
described in Section 3.1.3.
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4. Beginning at 667 nm and proceeding to 657 nm in 1 nm increments,
collect absorbance data without readjusting the slit width.
5. Then beginning at 657 nm and proceeding to 667 nm in 1 nm
increments, collect absorbance data without readjusting the slit
width.
6. Repeat this procedure using chlorophyll-b, spanning 650 nm to
640 nm with the slit width maximum determined for 650 nm.
7. Graph these partial spectra and determine the dial settings for
maximum absorbance that correspond to 662 nm for chlorophyll-a
and to 645 for chlorophyll-b. Use these dial settings during the
analytical procedure to determine total chlorophyll content.
Retain these notebook pages as documentation of wavelength
calibration.
8. To calibrate the response of the detector system, follow the
user's guide recommendation for neutral density filters. Be
sure to calibrate at 662 nm and 645 nm. Retain these notebook
pages as documentation of detector response calibration.
9. Pool single needles from vouchered samples that are
representative of the next group of samples to be analyzed.
Record the sample identities. Determine the mean needle mass
(divide total mass of needles by number of needles). Cut up the
batch of needles as in step 1, Section 3.1.3. and mix veil.
Determine the equivalent mean dry mass of a needle (Section
3.1.3.).
10. Use enough needle pieces for chlorophyll extraction (Section
3.1.3.1.) to exceed the final chlorophyll concentration needed
for routine analyses by at least a factor of two. By serial
dilution, create a descending concentration series with many
intervals. Make absorbance measurements at 662 nm and 645 nm
(3.1.3.) and calculate the apparant concentrations of
chlorophyll-a, chlorophyll-b and total chlorophyll (3.1.6.).
11. Graph these data against relative dilution with lx representing
the most concentrated solution. (These graphs are not expected
to be straight lines (Anon, 1964).) From the data collected,
determine the whole number of needles (on a constant dry mass
basis) needed for an average sample such that during the
determination of total chlorophyll the 662 nm absorbance value
will fall within the range 0.5 to 0.8. Retain the notebook
pages of these analyses and calculations as part of the
documentation needed for interpretation of the analytical data.
3.1.6. CALCULATIONS/UNITS
Total chlorophyll content should be expressed as mg chlorophyll per
kg dry mass.
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The calculations used for determining chlorophyll-a, chlorophyll-b,
and total chlorophyll in 100£ acetone are arrived at by solving
simultaneous equations using selected absorptivity coefficients. The
equations of Vellburn and Lichtenthaler (1984) are as follows:
Chl-a = (11.75 x A662) - (2.35 x A645) (1)
Chl-b = (18.61 x A645) - (3.96 x A662). (2)
Therefore,
Chl-t = (7.79 x A662) + (16.26 x A645). (3)
Chl-a, Chl-b and Chl-t are chlorophyll-a, chlorophyll-b and total
chlorophyll, respectively. A662 and A645 are the absorbance values
measured at 662 nm and! 645 nra (see Section 3.1.5., step 3), repectively,
for a sample light path of 10 mm. For spectrophotometers calibrated in
transmittance (T), rather than absorbance (A), equations 4 and 5 describe
these terms (Anon, 1964).
T = P / Po
A = logl0( 1 / T).
Po is the incident radiant energy entering the first surface of the
sample, and P is the radiant energy leaving the second surface of the
sample (Anon, 1964).
Equations 1-3 give results expressed as yg chlorophyll per mL. The
final report for total chlorophyll is expressed as mg chlorophyll per kg
dry mass of sample. This is obtained using equation 6:
F = (C x V x 1000) / V. (6)
(4)
(5)
C is total concentration of chlorophyll in the subsample extract, as yg
per mL; V is the total volume of subsample extract, as mL; W is the dry
mass of subsample, as kg; and F is the final datum, reported as mg
chlorophyll per kg dry mass.
3.1.7. ERROR ALLOWANCE AND DATA QUALITY
Blanks and replicates should be included in analyses of all samples.
Samples should be choosen at random for replicates. At least 10 percent of
all tissue samples should be replicated.
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Samples selected as replicates should also be run on different days
to estimate day-to-day precision.
Replicate samples will be used to monitor precision. Percent
coefficient of variation vill be calculated using the industrial statistic
I, vhere £CV= 200I//2 and I=|A-B|/A+B, A and B being the results for the
replicate samples (U.S. EPA, 1979).
Precision vill also be monitored using Shevhart control values for R
(range) values, if the total number of sample groups to be analyzed vill
generate enough points to make useage of such charts vorthvhile.
Critical ranges for repeated measurement error and accuracy vill be
set by Quality Assurance Officer and listed in the Data Quality Objectives
(DQO) table.
Accuracy vill be evaluated through the use of interlaboratory sample
exchanges. These exchanges vill be coordinated by the Quality Assurance
Specialist assigned to the project.
3.1.7.2. DATA QUALITY OBJECTIVES
Repeated Measurement
Measurement
Reporting
Error
at
Accuracy
Variable
Units
Lover Limit
Upper Limit
Tolerance
Chlorophyll
mg/kg
10X (cv)
10* (cv)
N/A
3.1.7.3. COMPUTER DATABASE CODES
Variable
Code
Total Chlorophyll
FCHL
3.1.8 REFERENCES
Anonymous. 1964. Model DU-2 ultraviolet spectrophotometer. Beckman
preliminary Instructions 1291. Beckman Instruments, Inc., Scientific
and Process Instruments Division, Fullerton, CA.
Arnon, D.I. 1949. Copper enzymes in isolated chloroplasts.
Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24:1-15.
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Cochran, V.G. and G.M. Cox. 1957. Experimental Designs. 2nd ed. John
Wiley & Sons, New York.
Lichtenthaler, H.K. and K. Pfister. 1978. Practium der Photosynthese.
Quelle Meyer Verlag, Heidelberg.
U.S. Environmental Protection Agency. 1979. Handbook for Analytical
Quality Control in Water and Wastewater Laboratories. Environmental
Monitoring and Support Laboratory. U.S. EPA. Cincinnati, OH 45268.
EPA-600/4-79-019.
Wellburn, A.R. and H. Lichtenthaler. 1984. Formulae and program to
determine total carotenoids and chlorophylls a and b of leaf extracts
in different solvents. In: Advances in Photosynthesis Research. Vol.
2, C. Sybesma (ed). Martinus Nijhoff/Dr W. Junk Publishers, The
Hague, pp. II.1.9-12.
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3.2. STANDARD OPERATING PROCEDURE FOR QUANTITATIVE ANALYSES OF n-ALKANE
CONCENTRATION AND TOTAL EPICUTICULAR VAX OF RED SPRUCE FOLIAGE
3.2.1. SCOPE AND PURPOSE
This standard operating procedure for isolation and quantitative
analysis of n-alkanes and total epicuticular waxes of conifer foliage is
designed for evaluation of a large number of individual tree samples
consisting of a smaller number of needles. The method consists of (1)
solvent extraction of waxes from needle surfaces, (2) a gravimetric
measure of total epicuticular waxes, (3) purification and selective
elution of n-alkanes from chromatographic columns, and (4) quantitative
analysis of n-alkane composition of waxes by hydrogen flame ionization
gas-liquid chromatography.
3.2.2. MATERIALS AND SUPPLIES
3.2.2.1 EQUIPMENT
° electronic balance (0.00001 g)
° gas chromatograph - hydrogen flame detector
° temperature programmable oven
0 lov dead volume injection system - Injection system should include
teflon-faced, silicone rubber gas-chromatography septa.for high
temperature applications.
0 capillary column - high resolution capillary column with bonded
silicone phase (e.g., DB05). A wide bore column (0.5-0.75 mm)
is suggested for splitless injection. A wide bore column can
also be adapted to packed column chromatographs. Column should
be at least 20 m in length.
0 stripchart recorder with integrator with following specifications:
Accuracy: + 0.1% of full scale or + 1 count
Reproducibility: 0.01% of full scale
Linearity: + 0.1% of full scale
Response: Zero time lag between motion of recorder
potentiometer and integrator pen.
° carrier gases - Nitrogen or helium should be used as the carrier
gas. Helium is recommended if samples are to be collected for
GC-MS analysis. Use of a C.G.A. 540 or 580 regulator (0-
100 psig outlet) with moisture and hydrocarbon traps installed
between the carrier gas inlet to the chromatograph and the
regulator is highly recommended. Breathing air tank and C.G.A.
540, 580 or 590 regulator (capable of regulating the input
pressure from 0 to 90 psig). Hydrogen tank and C.G.A. 350
regulator (capable of regulating the input pressure from 0 to
90 psig).
0 syringes - Hamilton model 701 N 10 yl or Model 95 N 5 yl syringes
or equivalent
0 solid phase extraction system - Baker - 10 Solid Phase Extraction
system with 6 ml alumina (A1203), silica, or florisil columns,
vacuum manifold, 75 ml reservoirs, and columetric collection
rack (holds 1, 2, 5, and 10 ml volumetric flasks). Side arm
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flasks, 500 ml or larger, designed for vacuum source, water
aspirator or vacuum pump. Columns made for Pasteur pipettes
(7 mm x 10 cm glass tubing plugged with deactivated glass-wool)
2 gm of adsorbents are a satisfactory alternative (Bengtson et
al., 1978). Adsorbents can be either 60-100 mesh or 100-200
mesh neutral chromatographic alumina, silica, or florisil (1,
2, 3).
0 refrigerator or walk-in cold room
° freezer (-10°C)
° convection oven (30-200°C)
0 water bath or rotary-evapo mixer
° volumetric flasks (1 ml and 5 ml)
° breakers (20 ml)
0 filter funnelsi(4.25 cm dia)
0 filter flasks (100 ml)
° No. 1 filter paper (4.25 cm dia)
° borosilicate glass reaction vials (2 ml) with tapered cone design
and teflon faced rubber septa and caps
° snap-cap glass vials - Wheaton 800 glass vials and (48 ml) and
accompanying storage racks
° culture tubes r 13 x 10 mm pyrex tubes with teflon lined screw
caps and storage racks
° clear glass vial - (4 ml) with open-top cap and septum
° glass inserts - (250 yl) borosilicate glass, conical shaped
° vial files
3.2.2.2. CHEMICAL/REAGENTS
° Chloroform - HPLC grade
° Hexane - 99 + mol% pure
° n-Alkane standards (C19-C35) - 99% pure
° n-Alkane hydrocarbon mixtures for calibration of gas chromatograph
(Foxboro/Analabs, North Haven, CT)
3.2.2.3. OTHER
0 pruning pole
° stainless steel surgical shears and forceps
0 insulated cooliers
° diamond-lox plastic labels
° steel wire twist ties
° freezer bags - 2.7 mil, 10 9/16 x 11 inches
° paper bags
3.2.3. PROCEDURES
3.2.3.1 SAMPLE PREPARATION
1. Excise and collect current-year (or other age) branches from
several different aspects at the upper mid-crown level of
saplings or mature trees or near mid-height on seedlings. Avoid
all contact with human skin to prevent contamination.
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Note: The exact number of branches sampled from each tree and
the allocation of funds and time to measurement of among-and
vithin-tree variation should be based on specific experimental
objectives.
2. Seal collected branch samples in plastic bags, making sure that
identification labels (written with waterproof markers) are
securely attached to each branch, and put them into an Insulated
cooler with ice or snow for transport back to the laboratory;
then refrigerate.
3. Extract epicuticular wax from either fresh or air-dryed needle
samples. Minimum disruption to needle surfaces will occur, and
the risk of contamination by cellular extractives will be
reduced, if the branches are allowed to dry and the needles are
collected after natural abscision.
4. Detach fresh needles—the estimated minimum number of needles
required per sample for reliable quantitative analysis is 24—
from each branch collected and store separately, or place the
individual branches in labeled paper bags until the needles dry.
Measure needle length and needle area (if possible). If time
does not allow for separate analysis of each sample, randomly
select an equal number of needles from each branch and pool them
to obtain a sample of needles that is equally represented by each
of the crown collection sites. Replicate as many times as
possible, and obtain dry or fresh weight of each replicate.
5. Place needle samples from each tree in opaque paper bags for at
least two hours to insure that stomates are closed prior to
extraction (Corrigan et al., 1978). Assuming the minimum number
of 24 needles per sample, isolate epicuticular waxes from needle
surfaces by making four successive 5-10 second immersions of each
sample into four 10 ml portions of fresh chloroform at room
temperature in 20 ml beakers (Eglinton and Hamilton, 1967;
Franich et al., 1978).
6. Combine the four chloroform extracts for each sample of needles
and filter through Vhatman No. 1 filter paper to remove
particulate contamination. Place filtered extracts in 48 ml
Wheaton 800 Snap-cap glass vials and store in a freezer (Freeman
et al., 1979).
7. After wax has been removed from all samples of needles, transfer
the extracts to pre-weighed tubes—13 x 10 mm pyrex culture tubes
with teflon lined screw caps. Remove chloroform from extracts
taken from the freezer to an approximate volume of 3 ml—after
warming to room temperature—by evaporation in a hot water bath
or rotary evaporator under vaccuum at 40°C. Dry the extracts
under a stream of nitrogen. Oven dry the samples at 55°C
overnight and weigh the wax in the tared test tubes to obtain net
wax weight in mg/sample and then return to storage.
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Warning: Carry out all work, with proper ventilation (fume hood)
to avoid contact with chloroform fumes.
8. Dilute the wax samples from each tree with 3 ml of 99 Mol% Pure
hexane to dissolve all nonpolar components. Add the samples to
the top of chromatography columns for purification and isolation
of the hydrocarbon from the total lipid.
9. Collect the eluate containing the hydrocarbon fraction of the
total lipid in 5 ml volumetric flasks. Dry the eluate with a
nitrogen stream to a volume of less than 250 yl and
quantitatively transfer it into 250 ul borosilicate glass vials
with open-top caps and septa. Completely dry the eluate with a
nitrogen stream and then redilute with 100 yl of 99 MolX Pure
hexane. The samples are now ready for further separation of the
n-alkane fraction into the homologous series by either isothermal
or temperature-programmed gas-liquid chromatography.
3.2.3.2 EQUIPMENT OPERATION
1. Separation and quantitative analysis of n-alkanes can be
achieved by a variety of gas-chromatographic procedures. The
high resolution afforded by long capillary columns is desirable,
but excellent results have been attained using standard size
glass or stainless steel columns. The large molecular weight of
the alkanes necessitates high temperatures for gas-liquid
chromatographic analysis. This requirement limits the choice of
stationary phases to those with considerable stability, all at
low (1 to 5 percent) loadings. Gas-chromatTographic conditions
and parameters suitable for analyses of the homologous series of
n-alkanes found in epicuticular waxes of red spruce are
described below. Many other examples of gas-chromatographic
operating procedures for separation of n-alkanes found in plant
waxes are in the literature (Bengtson et al., 1978; Chang and
Grunwald, 1976; Corrigan et al., 1978; Gulz, et al., 1979;
Herbin and Robins, 1968; Holloway, 1982; Kohlen and Gulz, 1976;
Leavitt et al., 1978; Macey and Barber, 1970; Purdy and Truter,
1963a, 1963b; Schomburg et al., 1977; Silva Fernandes et
al.,1964; Steinmuller and Tevini, 1985; Trimble et al., 1982;
Tulloch, 1973).
2. Column - use a high resolution capillary column with bonded
silicone phase (e.g., DB-5). Use of a wide bore capillary
column (0.5 - 0.75 mm) allows splitless injection and removes
need for a dual detector system. A vide bore column can also be
adapted to packed column chromatographs. Column should be
between 10-20 m long.
3. Column oven temperature - 150°C at 80°C/minute to 330°C.
Programming the oven temperature is important because it permits
the analysis of samples that contain many components that have
widely spaced boiling points. The technique provides good
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resolution of the lov molecular weight compounds without
crowding and peak overlap, while preventing the dilution of high
molecular weight compounds with carrier gas and consequent peak
spreading and long analysis time.
4. Injection port temperature: 350°C. Detector temperature:
350°C. Carrier gas: nitrogen. Note: Nitrogen is the
preferred carrier gas for the widest linear range.
5. Flame detector response depends on the number of molecules per
unit time entering the detector; consequently, low response and
slight peak broadening during passage through the detector will
result from very low flow rates. Make-up gas should be used
when carrier flow is less than 25 ml per minute and desirable at
flow rates below 50 ml/minute.
6. Hydrogen Flow rate: 10 cc/24 sec. + 0.2 cc/24 sec. at 8 psig.
Air Flow rate: 100 cc/12 sec. + 6 cc/12 sec. at 33 psig.
Detector: hydrogen flame ionization. Vhen the oven temperature
is programmed, drift in the baseline can be a serious detriment
to quantitative analysis. For that reason it is highly
desirable to use an instrument with dual detectors and columns
with the ability to compensate for the effects of substrate
bleeding.
7. Amount injected: 1 yl. This is the largest sample size
recommended by the column manufacturer. Smaller sample sizes
are usable, but trace components are not detectable at the same
detector and electrometer sensitivity settings.
8. Use peak integration to record output. Use of a DISC or digital
electronic integrator is recommended. The DISC integrator is
one of several brands of similar design used with potentiometric
recorders. The integrator automatically computes peak area and
displays this computation as a second trace directly under each
recorder peak.
9. Inject all samples (1 yl) through the injection port septum onto
the analytical column using Hamilton model 701 N 10 yl or model
95 N 5 yl syringes or equivalent with identical performance
specifications. To prevent sample solvent evaporation draw the
samples into the syringe after inserting the syringe needle
directly through the septum of the storage vial (boiling point
of hexane is approximately 69°C).
10. 'If the syringe is thoroughly dried after cleaning with the
solvent used to dissolve or dilute the sample (hexane) proper
quantitative syringe injection is as follows:
a. Draw up a yl of air.
b. Draw in 1 yl of sample.
c. Draw in 1 yl of air.
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d. Insert the needle through the septum supporting the plunger
in the palm of the hand, quickly depress the plunger and
retract the syringe needle. The complete sample will be
forced into the injection port with only air remaining in
the syringe needle.
11. A 10 yl syringe needle contains 0.6 to 0.8 yl of sample or
solvent after the plunger has been depressed. Fart or all of
the 0.6-0.8 yl in the needle can "bleed" into the injection
port. This results in the total sample injection being the
amount read in the syringe barrel plus the fraction of the
needle volume volatilized. The sample should always be pulled
out of the needle into the syringe barrel.
12. If the syringe is not dried after cleaning proper quantitative
syringe injection is as follows:
a. Draw the 0.6-0.8 pi of cleaning solvent left in the needle
of the syringe back into the syringe barrel so that you have
a plug of solvent against the plunger followed by a plug of
air.
b. Draw in .1 yl of sample
c. Draw in 1 yl of air
d. Proceed as in step 10 above. The complete sample will be
forced into the injection port with only solvent remaining
in the syringe needle.
13. Immediately following injection clean the syringe by filling and
emptying 10 successive times each in two different 50 ml beakers
of solvent (hexane) covered with aluminum foil and located in an
exhaust hood with sink drain. Remove excess solvent remaining in
the needle with a small vacuum pump with teflon tubing attached.
14. Excessively dirty syringes should be cleaned at regular
intervals by using commercial syringe cleaning solutions
according to manufacturers' directions or "dry cleaned" by
vaporization of residues under high heat and vacuum.
15. Follow guidelines detailed in Good Laboratory Practices section
for recording of all raw data. Note that raw data include the
following: (a) analytical observations necessary to calculate
the final results, (b) calibration data, (c) calibration checks,
and (d) quality control checks. Deviations from standard
operating procedures during sample preparation, calibration, or
actual analyses are to be fully documented and initialed by
laboratory personnel. Samples suspected of being in error or
outside of the calibration range are to be "flagged", and this
notation carried through all records to the final report.
Retain all written materials (graphs, tables, etc.) generated as
part of an analysis. Do not discard portions of laboratory
notebooks or any other information directly related to
calculation of the final result for a set of samples.
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16. It is recommended that for each wax sample and standard injected
the following information be recorded in a laboratory notebook,
using a new sheet for each day, and on the recorder chart
output:
a. Sample identity number.
b. Date analyzed.
c. Sample volume.
d. Volume injected.
e. Chart speed,
f. Carrier gas rotameter readings (analytical and reference
columns)
g. Sensitivity (range and attenuation).
17. Additional information should be recorded on a daily basis and
is greatly facilitated by purchase of special GLC rubber stamps
shown below:
Operator
Column, #
Length ft.
I.D mm.
Coating
vt. x
Support
Mesh
Carrier Gas
Rate ml/min.
Sensitivity
Sample:
Date
Pressures:
Inlet psig
Outlet psig
Temperatures:
Column °C
Detector °C
Sampler °C
High voltage
Sample Size
Chart Speed.
Temp. Rate. °/min
18. Identify unknown chromatographic peaks of wax samples by
comparing relative retention times with those of standard
n-alkanes C19-C35 run on the same columns and under the same
conditions as the wax samples. In addition, check for the
intensification of appropriate unknown peaks when genuine
n-alkane standards of known carbon number are added individually
to a plant wax extract which is then reinjected and reanalyzed.
Make quantitative determinations for each n-alkane in the wax
sample by comparing recorder peak integrator values with those
of standard curves prepared from known concentrations of
n-alkane standards C19-C35 run on the same columns and under the
same conditions as the wax samples.
3.2.4. PREVENTIVE MAINTENANCE.
The GLC carrier gas flows through a moisture trap before it enters
the oven. The moisture trap usually consists of a length of tubing filled
with molecular sieves (type 5A) to remove moisture and heavy hydrocarbon
impurities that might be in the carrier gas supply. The moisture trap
should be removed periodically and regenerated by heating the molecular
sieve to approximately 300°C. This may be accomplished without removing
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the sieve from the trap if a convenient means of heating the moisture trap
while maintaining a slow flow of an inert gas is available.
Note: If moisture is allowed to enter the column system, certain
column substrates will deteriorate rapidly when exposed to
elevated temperatures.
With the injection port at high temperature, the septum life will be
shortened. It is advisable to replace the septum at least at the
beginning of each day. This will prevent carrier gas leakage from
delaying work and possibly making the validity of previous data
questionable.
Glass wool plugs in column ends become contaminated with non-volatile
residues and should be replaced after every 50-100 samples with new
silanized glass wool. Vhile columns are disconnected, remove injection
port insert liners—except when on-column injection is being used—and
clean them with solvent after they have cooled to room temperature
(injection port temperature is 350°C). The insert liners may also by
partially packed with clean glass wool and used to trap non-volatile
materials that might shorten the columns' useful life.
Columns should be reconditioned periodically (after every 50-100
samples, for example) and/or at the first signs of baseline instability.
The major symptom is drift caused by substrate bleeding that cannot be
satisfactorily reduced by increasing carrier gas flow rate through the
reference column.
Carrier gas, hydrogen, and air flow rates should be checked
periodically for consistency with initial settings with a soap film
flowmeter (100 cc for carrier gas and air and 10 cc for hydrogen) at the
detector for carrier gas or restrictor block for hydrogen and air.
New columns should be held at maximum liquid phase temperature (450°C
for Dexsil 300) for several hours without connecting the columns to the
detectors (conditioned). Overnight conditioning eliminates heavy detector
contamination from volatile column constituents.
Note: The hydrogen flow must be shut off when the chromatograph oven
is heated without columns connected to the detectors, as a
possible hazzard exists if hydrogen flows back into the oven
for a prolonged period.
Check carrier gas rotameter readings every day before proceeding with
sample analyses.
Carry out all maintenance procedures as recommended by the
manufacturer. Record maintenance operations in maintenance log.
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3.2.5. CALIBRATION PROCEDURES
1. Prepare individual n-alkane standards (for peak identification)
by dissolving 0.5 mg of each C19-C36 alkane (can be purchased
from Foxboro/Analabs, North Haven, CT in 99% pure form) in the
amount of 99 Mol% pure hexane required to attain a total volume
of 1 ml in a volumetric flask. This will provide an injection
concentration of 0.5 ug of alkane per 1 yl of solution.
2. Transfer individual n-alkane standard solutions to 2.0 ml glass
vials with tapered conical interior, silicone septum stopper and
screw cap for refrigerated storage.
3. Prepare standard response curves for each n-alkane C19-C36 using
a range of concentrations of analytical GLC calibration mixtures
of hydrocarbons (can be purchased from Foxboro/Analabs, North
Haven, CT).
4. The response of a flame ionization detector depends on the
number of molecules per unit time entering the detector. It is
not a direct function of the concentration of these molecules in
the carrier gas. The degree of ionization (response) is roughly
proportional to the number of carbon atoms per molecule in any
given organic compound. For this reason calibration of response
(by constructing standard curves with external standards) for
each individual component over the entire expected concentration
range of the unknown samples is required for accurate
quantitation.
5. Individual n-alkanes are expected to occur in amounts ranging
from 0.5 ug to 80 pg in red spruce epicuticular wax samples.
Maximum concentration is 80 ug/100 yl. This is equivalent to
0.8 Mg/iil or a 0.08 percent solution based on the injection of 1
ul volumes of sample.
6. The five calibration mixtures to be used are as follows:
#1 10 mg each of
#2 10 mg each of
n-Octadecane (C18)
n-Nonadecane (C19)
n-Eicosane (C20)
n-Heneicosane (C21)
n-Docosane (C22)
n-Oocosane (C22)
n-Tricosane (C23)
n-Tetracosane (C24)
n-Pentacosane (C25)
n-Hexacosane (C26)
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#3 10 mg each of n-Hexacosane (C26)
n-Heptacosane (C27)
n-Octacosane (C28)
n-Nonacosane (C29)
n-Triacontane (C30)
#4 5 mg each of n-Octacosane (C28)
n-Nonacosane (C29)
n-Triacontane (C30)
n-Hentriacontane (C31)
n-Dotriacontane (C32)
#5 6.5 mg each of n-Dotriacontane (C32)
n-Tritriacontane (C33)
n-Tetratriacontane (C34)
n- Hexatriacontane (C36)
Note: Pentatriacontane (C35) is not a component in any of
the calibration mixtures.
7. To prepare standard response curves make solutionis of each
calibration mixture so that individual n-alkanes are diluted to
0.10, 0.05, and 0.025 percent concentrations. This procedure is
as follows:
Weigh 5 mg of mixtures #1-4 and 4 rag of mixture #5.
Dissolve each of these in the amount of 99 Mol% pure hexane
required to attain a total volume of 1 ml in a 1 ml
volumetric flask. Designate and labei this as solution No.
1 for each calibration mixture.
8. The series of five solutions designated No. 1 have
concentrations of 1 mg of each n-alkane in the calibration
mixture per ml of total solution. This is equivalent to 1.0
Ug/yl or a concentration of 0.10%. Injection of 1 yl into the
gas-chromatograph will define the maximum concentration and
response in peak area on the standard curve. It will be higher
(0.10 yg versus .08 yg) than the maximum expected in red spruce
wax samples..
9. To define two additional points on the curve take a 0.5 ml
aliquot of solution No. 1 for each mixture and add 0.5 ml of
hexane. Concentration of this solution (No. 2) will be 0.5
Ug/yl or 0.05%. Then take a 0.5 ml aliquot of solution No. 2
and add 0.5 ml of hexane to obtain a solution (No. 3) with a
concentration of 0.25 ug/yl or 0.025%. Concentrations below
0.025% should yield a linear response for each n-alkane so the
curve can be extended downward through zero response-zero
concentration.
10. Standard response curves for Pentatriacontane (C35) can be
similarly constructed starting with 1.0 mg/ml of hexane, or 6.25
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mg of Pentatriacontane can be added to calibration mixture #5,
which would then be treated in a manner identical to mixtures
#1-4.
11. Transfer calibration standard solutions from volumetric flasks
to 2.0 ml glass vials with silicone septum stopper and screw cap
for refrigerated storage.
12. Calibration mixtures for construction of standard curves and
individual alkane standards should be injected and run on the
same columns and under the exact same conditions as the wax
samples.
13. New standard response curves should be made in an identical
manner after analysis of 25 to 50 wax samples or anytime gas-
chromatographic conditions change or are suspected to have
changed.
14. Standard curves should be checked daily for changes in detector
sensitivity or changes in column variables by injecting 1 ul of
solution No. 2 mixture #1 at the beginning of each day and by
injecting 1 yl of solution No. 2 mixture #5 at the end of each
day.
3.2.6. CALCULATIONS/UNITS.
Express wax yield measured gravimetrically (Section 3.2.3., step 7)
in mg wax/sample; mg wax/mg of fresh or dry weight of needles; mg wax/cm
of needle length; and if possible mg wax/cm2 of needle area.
Standard curves of n-alkanes concentrations prepared from calibration
mixtures will have peak area on the Y axis and concentration yg/yL on the
X axis. The amount of each individual n-alkane/sample can be determined
directly from the standard curve if 1.0 yl samples are injected into the
GLC. Express the amount of each n-alkane as yg/sample; yg/mg of wax;
yg/mg of fresh or dry weight of needles; yg/cm of needle length; and if
possible yg/cm2 of needle area.
3.2.7. ERROR ALLOWANCE AND DATA QUALITY
3.2.7.1. CONSIDERATIONS
It is recommended that replicates be run for all samples. If
replicates fail to agree within 5%, the samples should be rerun and
operation of the instrument checked.
Blank values should be consistent between sets of analyses and not
change by more than 5% on a daily basis. Significant changes in daily
values of blank values indicate contamination from somewhere in the
procedure. Analysis should be stopped until constant blank values are
obtained. Daily blank values should be recorded and placed on charts for
visual assessment of quality control of analysis.
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Samples should also be run on different days to. estimate day-to-day
precision.
Replicate samples will be used to monitor precision. Percent
coefficient of variation should be calculated using the industrial
statistic I, vhere %CV = 200I//2 and I = |A-B|/A+B, A and B being the
results for the replicate samples (U.S. EPA, 1979).
Precision vill also be monitored using Shewhart control values for R
(range) values, if the total number of sample groups to be analyzed will
generate enough points to make useage of such charts worthwhile.
Repeated injection of calibration mixture standards will be used to
determine and monitor precision. Note that each calibration mixture
contains at least one n-alkane in common with a second mixture.
Accuracy will be evaluated through the use of interlaboratory sample
exchanges. Sample exchanges will be coordinated by the Quality Assurance
Specialist assigned to the project.
Critical ranges for repeated measurement error and accuracy will be
set by Quality Assurance Officer and listed in the Data Quality Objectives
(DQO) table.
3.2.7.2. DATA QUALITY OBJECTIVES
Repeated Heasurement
Measurement
Reporting
Error
at
Accuracy
Variable
Units
Lower Limit
Upper Limit
Tolerance
n-alkanes
mg/kg
5% (cv)
52 (cv)
N/A
epicuticular
wax
mg/kg
5% (cv)
52 (cv)
N/A
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3.2.7.3. COMPUTER DATABASE CODES
Variable
Code
Cuticular Vax
FTCV
n-Octadecane
FC18
n-Nonadecane
FC19
n-Eicosane
FC20
n-Heneicosane
FC21
n-Docosane
FC22
n-Tricosane
FC23
n-Tetracosane
FC24
n-Pentacosane
FC25
n-Hexacosane
FC26
n-Heptacosane
FC27
n-Octacosane
FC28
n-Nonacosane
FC29
n-Triacontane
FC30
n-Hentriacontance
FC31
n-Dotriacontane
FC32
n-Tritriacontane
FC33
n-Tetratriacontane
FC34
n-Pentatriacontane
FC35
n-Hexat r i aeon tane
FC36
3.2.8. REFERENCES
Bengtson, C., S. Larsson, and C. Liljenberg. 1978. Effects of water
stress on cuticular transpiration rate and amount and composition of
epicuticular vax in seedlings of six oat varieties. Physiol. Plant.
44:319-324.
Bowman, R.N. 1980. Phylogenetic implications from cuticular vax analyses
in Epilobium Sect. Zauschneria (Onagraceae). Amer. J. Bot. 67(5):671-
685.
Chang, S.Y. and C. Grunvald. 1976. Duvatrienediols in cuticular vax of
Burley tobacco leaves. J. of Lipid Res. 17:7-11.
Corrigan, D., R.F. Timoney, and D.M.X. Donnelly. 1978. N-alkanes and N-
hydroxy alkanoic acids from the needles of twenty-eight Picea species.
Phytochem. 17:907-910.
Eglinton, G. and R.J. Hamilton. 1967. Leaf epicuticular waxes. Science
156:1322-1335.
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Franich, R.A., L.G. Wells, and P.T. Holland. 1978. Epicuticular wax of
Pinus radiata needles. Phytochem. 17:1617-1623.
Freeman, G., L.G. Albrigo, and R.H. Biggs. 1979. Ultrastructure and
chemistry of cuticular waxes of developing Citrus leaves and fruits.
J. Amer. Soc. Hort. Sci. 104(6):801-808.
Gulz, P.G., P. Proksch, and D. Schvarz. 1979. Investigations on
cuticular waxes in the genus Cistus L. (Cistaceae). V. Summary on
the hydrocarbons and wax esters of the epicuticular waxes from leaves
and petals. Z. Pflanzenphysiol. 92:341-347.
Herbin, G.A. and P.A. Robins. 1968. Studies on plant cuticular waxes -
I. The chemotaxonomy of alkanes and alkenes of the genus Aloe
(Liliaceae). Phytochemi. 7:239-255.
Holloway, P.J. 1982. Structure and histochemistry of plant cuticular
membranes: an overview. In: The Plant Cuticle. Eds. D.F. Cutler,
K.L. Alvin, and C.E. Price. Academic Press, N.Y. pp.33-44.
Kohlen, L. and P.G. Gulz. 1976. Investigations about cuticular waxes in
the genus Cistus L. (Cistaceae). I. The composition of the alkanes in
leaf waxes. Z. Pflanzenphysiol. 77:99-106.
Leavitt, J.R.C., D.N. Duncan, D. Penner, and W.F. Meggitt. 1978.
Inhibition of epicuticular wax deposition on cabbage by ethofumesate.
Plant Physiol. 61:1034-1036.
Macey, M.J.K. and H.N. Barber. 1970. Chemical generics of wax formation
on leaves of Brassica oleracea. Phytochem. 9:13-23.
Purdy, S.J. and E.U. Truter. 1963a. Constitution of the surface lipid
from the leaves of Brassica oleracea (var. capitata (Winnigstadt) I.
Isolation and quantitative fractionation. Proc. Roy. Soc. B. 158:536-
543.
Purdy, S.J. and E.V. Truter. 1963b. Constitution of the surface lipid
from the leaves of Brassica oleracea (var. capitata (Winningstadt).
III. Nonacosane and its derivatives. Proc. Roy. Soc. B. 158:553-565.
Schomburg, G., H. Behlau, R. Dielmann, F. Weeke, and H. Husmann. 1977.
Sampling techniques in capillary gas chromatography. J. of
Chromatography 142:87-102.
Silva Fernandes, A.M.S., E.A. Baker, and J.T. Martin. 1964. Studies on
plant cuticle VI. The isolation and fractionation of cuticular waxes.
Ann. of Appl. Biol. 53:43-58.
Steinmuller, D. and M. Tevini. 1985. Action of ultraviolet radiation
(UV-B) upon cuticular waxes in some crop plants. Planta 164:557-564.
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Trimble, J.L., J.M. Skelly, S.A. Tolin, and D.M. Orcutt. 1982. Chemical
and structural characterization of the needle epicuticular vax of two
clones of Pinus strobus differing in sensitivity to ozone. Phytopath.
72:652-656.
Tulloch, A.P. 1973. Comparison of some commercial waxes by gas liquid
chromatography. J. Amer. Oil Chemists Soc. 50:367-371.
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3.3. STANDARD OPERATING PROCEDURE FOR MEASUREMENT OF STARCH AND TOTAL
SUGARS
3.3.1. SCOPE AND PURPOSE
Starch is a major form of storage carbohydate in woody plants. A
tree's ability to withstand particular kinds of stress can be assessed
by evaluating the soluble (non-structural) reserves. This standard
operating procedure is for the measurement of total non-structural
carbohydrates (sugars and starch) in plant tissue samples. Tissues are
extracted in a methanol-chloroform-water (MCW) solution, which removes
the soluble sugars, pigments, phenolics, and other solubles (Dickson,
1979). The residue contains starch.
After removing the pigmented chloroform fraction, a subsample of
the MCW extract is mixed with anthrone reagent to hydrolyze the
sucrose. Total sugar (measured as glucose) is determined
colorimetrically.
Starch in the tissue residue is gelled, then enzymatically hydrolyzed
in an overnight incubation to glucose with a combination of purified
Diazyme L-200 (alpha 1,4 glucan glucohydrolase) and alpha amylase in
concentrations of 5 mg/ml and 2.5 mg/ml, respectively. Starch, (measured
as glucose) is determined colorimetrically using the glucose
oxidase method (Sigma Chemical Co., Box 14508, St. Louis, MO 63178.
Bulletin 510-A).
According to Hassig and Dickson (1979), MCW extracts the tissue
much better than 802 ethanol, which does not completely remove
pigments. A cautionary note: there may be some problems with
interference from phenolics or some other substance in conifer
tissues. It is important to carry out the % starch recovery procedure
to determine the potential for interference.
Another limitation of the procedure as presented is that it does not
allow separate quantification of sucrose and total hexoses — a
potentially important measure of ozone import on carbohydrate physiology.
The procedure is also long, requiring three days to complete once the
samples have been extracted with MCW.
Accuracy of the procedure will depend on minimizing shifts among
and losses of carbohydrate fractions during sample handling. There are
numerous points from harvest to completion of analysis where shifts in the
carbohydrate fractions can occur. Accurate analysis is dependent on
deactivating (throughout this period) the enzymes responsible for pool
shifts. It is important to realize that shifts in carbohydrate fractions
can occur even in freeze-dried material if the material is not stored
properly. Interconversions that result from temperature-induced
carmelization can occur from oven-drying at high temperatures as well as
possibly within the chemical analysis where high temperature reactions
occur.
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During drying of samples, it is critical that all sample material be
dried quickly and evenly. Overpacking of driers can result in uneven
drying which may allow further shifts in carbohydrate fractions. Freezing
then thawing can allow further respiratory shifts to occur as well as
increased enzymatic activity from cell disruption. Microwaving is a
possible method whereby enzymes may be quickly and cheaply deactivated in
plant material. Further work is needed, however, to determine the
minimum/maximum time needed for different species and types of plant
material and also to determine whether microwaving can cause shifts via
carmelization reactions.
3.3.2. MATERIALS AND SUPPLIES
3.3.2.1. EQUIPMENT
° analytical balance (0.001 g)
0 volumetric flasks (2 liter, 200 ml)
° centrifuge tubes to hold 15 ml
° screw top tubes if possible to hold 25 ml (reduce volume of MCV
used if 25 ml tubes not available or not suitable for
centrifuge)
0 2 sets numbered centrifuge tubes (15 ml) required for starch; 1
set screw top (25 ml) and 1 set of 15 ml tubes required for
sugar analysis
° test tube racks (1 rack per 20 samples)
0 centrifuge (1100 g with baskets to hold 30 tubes (desktop model)
0 sonic bath
0 vortex mixer
° automatic pipets (0.05 ml, 0.2-lml, 3-5 ml)
0 repipet
° waterbath incubator (30-37°C)
0 dry incubator (50+l°C) big enough to hold several test tube tacks
c hot plates (2)
0 marbles (diameter not less than internal diameter of glass test
tubes)
° UV/Vis spectrophotometer or AutoAnalyzer
° refrigerator (explosion proof)
° freeze drier
° Parafilm
0 wiley mill (#40 mesh)
3.3.2.2. CHEMICALS/REAGENTS
0 Sucrose
0 Distilled-deionized (DI) water
° 18M Sulfuric acid (H2S04)
0 Absolute methanol
0 Methanol-water - 5 parts methanol to 4 parts DI water. To make
500 ml, use 278 ml methanol and 222 ml deionized water
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Methanol-chloroform-vater (MCW)- To 2 liter flask, add 1.2 liters
of absolute methanol, 0.5 liters of chloroform and 0.3 liters
of DI water. Approximately 130 samples can be extracted with
this volume of reagent. Mix in the hood.
Sodium acetate (NaC2H302:3H20)
Glacial acetic acid
0.1 M sodium acetate-acetic acid buffer - dissolve 13.608 g sodium
acetate in deionized water and make up to 1 liter. Add
approximately 7 ml glacial acetic acid to bring pH to 4.5.
70% ethanol (v/v).
Anthrone reagent - dissolve 1.75 g of anthrone in 1 liter of
cooled sulfuric acid solution (760 ml concentrated H2S04 plus
240 ml DI water, chilled in ice bath after mixing. Add acid
to water, NOT water to acid). Enough for 200 samples. For
100 samples use 120 ml H20, 380 ml acid, 0.88 g anthrone.
Waste should be reserved for disposal as hazardous waste.
Glucose colorimetric kits (available from Sigma Chemical Co.).
Each kit contains color reagent and 10 capsules; each capsule
makes 100 ml of glucose peroxidase. This is enough for 20
samples, to make the combined enzyme-color reagent solution,
add the contents of 1 enzyme capsule from kit per 100 ml DI in
an amber bottle. Invert several times with gentle shaking to
dissolve. Store refrigerated (2°C). Solution is stable up to
1 month unless turbidity develops (date bottle). For color
reagents; the preweighed vial contains 50 mg reagent. Note
this is a possible carcinogen; do not breathe dust.
Reconstitute contents of vial with 20 ml DI. Solution is
stable for 3 months in refrigerator (2°C)>. Date Bottle.
Combine ea.ch 100 ml enzyme solution with *1.6 ml color reagent
solution. Invert several times gently to mix. Solution is
stable up to 1 month refrigerated (2-6°C) unless turbidity
develops. ; Date bottle.
Soluble starch (potato) reagent grade (available from J.T. Baker
Chemical Co., # 4006).
Alpha-amylase (from Aspergillus oryzae, available from Sigma
Chemical Co., # A0273)" Purify before using. Dissolve 1.25 g
in 75 ml of DI water and dialyze against running DI water for 3
days. Test for reducing sugars using the DNS method (Clark,
1964; Eassig and Dickson, 1979).
Diazyme L-200 (alpha-l,4-glucan glucohydolase = amylogucosidase)
(available from Miles Laboratory P.O. Box 2000, Elkhart, IN
46518, # 6083). Purify before using. Precipitate with cold
acetone (1:1 ratio) and let sit in refrigerator overnight.
Pour off acetone, redissolve, and reprecipitate. Lyophilyze
and test for reducing sugars using the DNS method (Clark, 1964;
Hassig and Dickson, 1979). Note: the purified enzyme will be a
hard mass. Purify in small amounts or break apart during
lyophilizing.
Enzyme solution for starch hydrolysis - dissolve 25 g purified
Diazyme-L200 in 0.1 M sodium acetate (pH 4.5); add the purified
a-amylase solution. Make up to 500 ml with 0.1 M sodium
acetate (pH 4.5).
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3.3.3. PROCEDURES
3.3.3.1. SAMPLE PREPARATION
1. NOTE: extraction, starch hydrolysis, and glucose analysis
requires three days to complete for a given set of samples.
Once the extraction is initiated the overall procedure has to be
carried out without interruption. Temporary stopping points are
indicated in the procedure. Sugar analysis can be done after
the starch procedure is completed.
Day 1 - Sugar is extracted from tissue in methanol-chloroform-
vater. Tissue is dried overnight for use in starch
determination. Supernatant is refrigerated for sugar
analysis.
Day 2 (Starch) - Starch in residue is gelled; then incubated
overnight with an enzyme that coverts starch to glucose.
Day 2 (Sugar) - Concentration of sugar is determined
colorimetrically.
Day 3 -Concentration of glucose is determined colorimetrically.
Extraction
2. Make MCV; fill repipetter. Set out numbered tubes in racks of
20. Place .02 + .002 g of freeze-dried ground tissue sample
into numbered centrifuge tubes. Record exact mass to 3 decimal
places. Actual sample mass may vary betveen 5-100 mg depending
on tissue type.
3. Add 5 ml of MCV reagent to first group of 20 samples using
repipet. Immediately place tube in sonic bath for 5 seconds.
(Pick up 2 tubes at a time, dispense MCV, place in sonicator,
dispense MCV into 2 more tubes, remove first 2 tubes from
sonicator, replace with next 2 tubes...continue through 20
tubes).
4. Let stand for 10 minutes.
5. Centrifuge for 10 minutes at 1100 x g. Have ready a set of 20
numbered tubes (volume 25 ml, screw top if possible). Carefully
pour off supernatant into this second set of tubes. Repeat
steps 3 to 5 three times or until supernatant is clear of
pigments. Note the number of MCV washes. (Note: It is
possible to use less than 5 ml MCV in each extraction as long as
the supernatant is clear of pigments. Try 3-4 ml.)
6. The MCV supernatant contains soluble sugars. Add 3 ml of DI
water per 5 ml of supernatant. Mix and centrifuge for 5
minutes. Decant and discard pigmented chloroform (C) phase.
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Save MV phase for soluble sugars. (Total volume should be about
20-25 ml). Note: decrease the volume of DI used proportionally
if less than 5 ml MCV is used in the extractions.
7. Cap the tubes (use parafilm if not screw top; mix by inverting
or use vortex mixer). The MV fraction can be stored in the
refrigerator prior to sugar analysis.
8. The residue remaining in the first set of test tubes (step 5) is
used to determine starch content. Continue with step 9 (drying
sample) after separation of MCV supernatant.
9. Place racks of uncovered tubes containing residue in prewarmed
incubator (50°C) overnight. Be sure to provide adequate
ventilation during drying step. Prepare starch stock standard
(or be sure enough is on hand). Prepare enzyme solution (or be
sure enough is on hand).
Starch Hydrolysis
10. Remove one rack of tubes from incubator. Wet all tubes vith 0.2
ml of 70% ethanol. Add 4 ml of DI water to each tube with
repipettor. Place all tubes in boiling water for 10 minutes to
form gel. (Use hot plates to boil water in 3-4 250 ml beakers.
A strip of paper toweling placed in the bottom of each beaker
will prevent tube bumping and breakage. Check that water will
not overflow when tubes are added).. Cap each test tube with a
marble to prevent evaporation. Prepare 1 set of starch working
standards with each set of 20 samples (see "Section 3.3.5.).
Note: if you are doing more than 1 rack of 20 tubes, gel the
"batches" at 30 minute intervals so that on day 3 there will be
enough time to run each through the colorimeter.
11. Remove from boiling water, and add 1 ml of buffered enzyme
solution to each tube. Mix well. Seal tube with Parafilm (or
equivalent) and incubate in oven at 50°C for 24 hours. Note:
To each rack of tubes, attach a label with the time that
incubation begins. On day 3, the racks must be removed from the
oven at exact 24 hours (+ 10 min).
Glycose Analysis
12. Make enzyme color reagent from Sigma kit; put in repipettor.
Have water bath at 37°C. Have ready racks of clean, numbered
test tubes in sets of 20. Remove first set of tubes from
incubator at 24 hrs + 10 min; mix each tube on vortex mixer
(1100 x g) and centrifuge set for 10 minutes.
13. To the corresponding numbered test tube add 0.1 ml of sample,
starch standard, or enzyme blank plus 0.4 ml DI to make total
volume of 0.5 ml. This ratio of sample to DI can be changed
depending on the concentration, but standards will have to be
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diluted at the same ratio. Note the changes. Prepare one set
of glucose standards (made up in DI) for each set of 20 tubes
and starch standards.
14. To each test tube add 5.0 ml of the combined Enzyme-Color
Reagent and mix veil using vortex mixer. Incubate the rack of
tubes in water bath 37°C for 30 + 5 minutes.
15. Remove set of tubes from water bath and read within 30 minutes
at 450 nm using a spectrophotometer. Note: this step can be
automated using the nitrate manifold on a Technicon
AutoAnalyzer. It is possible to process a group of 20 samples
(including blanks and standards) within 20 minutes. This method
also enables one group of samples to be prepared while another
is analyzed. The number of samples in each group will depend on
how many can be read within the 30 minute time limit.
16. Record absorbance readings for samples, blanks, and standards.
Sugar Analysis
17. Have ready sets of clean, numbered test tubes. Hake anthrone
reagent; put in repipettor. Remove MV fraction from
refrigerator (step 7) and let warm to room temperature.
Transfer 0i5 ml of the top clear layer to a clean test tube
using an automatic pipette. In each set of 20 tubes include a
set of glucose working standards (in MV) and blank.
18. Have ready an ice bath and beakers of boiling water. Note: for
the ice bath, use a plastic dishpan. For the boiling water bath
use 3-4 250 ml beakers on hot plates as in step 10. To each
tube add 5 ml of anthrone reagent which has been cooled in an
ice bath. Mix contents of tube using vortex mixer and place in a
rack in an ice bath (4°C) until anthrone reagent has been added
to all the samples, blanks, and standards.
19. Place test tubes in beakers of boiling water for exactly 12
minutes. Remove to ice bath and mix again.
20. Read within 30 minutes at 625 nm using a spectrophotometer. The
number of samples in each group will depend on how many can be
read within the 30 minute time limit.
21. Record absorbance readings for samples, blanks, and standards.
22. Follow guidelines detailed in Good Laboratory Practices section
for recording of all raw data. Note that raw data include the
following: (a) analytical observations necessary to calculate
the final results, (b) calibration data, (c) calibration checks,
and (d) quality control checks. Deviations from standard
operating procedures during sample preparation, calibration, or
actual analyses are to be fully documented and initialed by
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laboratory personnel. Samples suspected of being in error or
outside of the calibration range are to be 'flagged', and this
notation carried through all records to the final report.
Retain all written materials (graphs, tables, etc.) generated as
part of an analysis. Do not discard portions of laboratory
notebooks or any other information directly related to
calculation of the final result for a set of samples.
3.3.3.2. EQUIPMENT OPERATION
It is assumed that a spectrophotometer will be used to measure
absorbance values for samples, blanks, and standards. Consult operating
manuals for detailed instructions on operation of instrument selected.
If an AutoAnalyzer (or equivalent) is used for glucose and starch
analysis, follow manufacturer's manual for start-up, shut-down,
maintenance, and calibration procedures.
In addition, connect a long glass tube to the bubbler. If bubbler
has an additional inlet, seal with parafilm. Connect the front bubbler
connector to the yellow-yellow sample line (nitrate manifold). Use a red-
red for colorimeter "to waste" line.
3.3.4. PREVENTIVE MAINTENANCE
Carry out all maintenance procedures as recommended by the
manufacturer. Record maintenance operations in maintenance log.
Wash all glassware with hot, soapy water, using "a test tube brush
when necessary to remove residue from the bottom of the tubes. Rinse
glassware 5 times with DI water and dry in an oven.
3.3.5. CALIBRATION PROCEDURES
1. In addition to tissue samples (and replicates) each group of
test tubes analyzed should include two to five tissue blanks
(empty tubes), one enzyme blank, and 5 starch standards.
Starch Standards
2. Stock Standard (0.5% or 5 mg/1 starch): Weigh 1 g of dried MCW -
extracted starch (follow extraction procedure, Section 3.3.3.1.,
steps 2 - 8. Do not save supernatant). Wet with 1 ml ETOH and
add to 100 ml of 0.1M pH A.5 acetate buffer. Heat on hot plate
and bring suspension just to boil to help dissolution. After
cooling in a water bath, transfer to a 200 ml volumetric flask
and bring to volume with the same acetate buffer. Adjust pH to
4.2 with glacial acetic acid. If precipitate forms, discard
solution. Shake well before using. The buffer is stable for
about 2 weeks at room temperature - do not refrigerate.
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3. Prepare set of working standards as follows. Dispense the
required volume of stock standard into a clean, labelled test
tube; add enough DI to bring volume to 4 ml. Use automatic
pipettes.
Starch
DI
Final Cone
-ml-
-5H
0.15
3.9
10 mg/1
0.2
3.8
20 mg/1
0.4
3.6
40 mg/1
0.6
3.4
60 mg/1
0.8
3.2
80 mg/1
1.0
3.0
100 mg/1
Note: 0 mg/1 (enzyme blank) contains 0 ml stock in 4.0 ml DI
water.
4. Insert set of working standards into analyses at Section 3.3.3.,
step 10. When running standards on colorimeter, check to be
sure standard curve is linear.
5. Subtract absorbance reading for enzyme blank from readings for
samples (Section 3.3.3., step 15).
6. To ensure that starch breakdown is complete in Section 3.3.3.,
steps 9 - 12, compare glucose standards with starch standards.
Glucose Standards
7. Use glucose stock standard solution in Sigma 510-A kit, stock
standard concentration is 1000 mg/1. Hake standards in DI for
starch analysis and in methanol-water (MV) for sugar analyses.
8. Prepare set of working standards using following chart:
0 mg/1 ° 0.00 ml of stock in 10 ml
15 mg/1 = 0.15 ml of stock in 10 ml
30 mg/1 = 0.30 ml of stock in 10 ml
45 mg/1 = 0.45 ml of stock in 10 ml
60 mg/1 = 0.60 ml of stock in 10 ml
75 mg/1 = 0.75 ml of stock in 10 ml
(volumetric flask) of DI water or MV
(volumetric flask) of DI water or MW
(volumetric flask) of DI water or MW
(volumetric flask) of DI water or MW
(volumetric flask) of DI water or MW
(volumetric flask) of DI water or MW
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Alternative
0 mg/1 = 0.00 ml
10 mg/1 = 0.10 ml
20 mg/1 = 0.20 ml
AO mg/1 = 0.40 ml
60 mg/1 = 0.60 ml
80 mg/1 = 0.80 ml
100 mg/1 = 1.00 ml
of stock in 10 ml
of stock in 10 ml
of stock in 10 ml
of stock in 10 ml
of stock in 10 ml
of stock in 10 ml
of stock in 10 ml
(volumetric flask)
(volumetric flask)
(volumetric flask)
(volumetric flask)
(volumetric flask)
(volumetric flask)
(volumetric flask)
of DI water or MW
of DI water or MW
of DI water or MW
of DI water or MW
of DI water or MW
of DI water or MW
of DI water or MW
To make up 100 ml, add 1.5, 3.0, 4.5, 6.0, 7.5 ml of stock
respectively to 100 ml volumetric and bring up to volume with MW
or DI.
Sucrose Standards
10. Prepare a 1000 mg/1 stock solution of sucrose using certified
ACS grade sucrose.
11. Prepare set of working standards using following chart:
0 mg/1 = 0.00 ml
15 mg/1 = 0.15 ml
30 mg/1 = 0.30 ml
45 mg/1 = 0.45 ml
60 mg/1 = 0.60 ml
75 mg/1 = 0.75 ml
of stock in 10 ml
of stock in 10 ml
of stock in 10 ml
of stock in 10 ml
of stock in 10 ml
of stock in 10 ml
of methanol-water
of metKanol-vater
of methanol-water
of methanol-water
of methanol-water
of methanol-water
Alternative:
0 mg/1 = 0.00 ml of stock in 10 ml of methanol-water
10 mg/1 a 0.10 ml of stock in 10 ml of methanol-water
20 mg/1 = 0.20 ml of stock in 10 ml of methanol-water
40 mg/1 = 0.40 ml of stock in 10 ml of methanol-water
60 mg/1 = 0.60 ml of stock in 10 ml of methanol-water
80 mg/1 = 0.80 ml of stock in 10 ml of methanol-water
100 mg/1 = 1.00 ml of stock in 10 ml of methanol-water
Note: methanol-water = 5 parts methanol to 4 parts DI water.
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3.3.6. CALCULATIONS/UNITS
The amount of starch in a sample can be calculated using the
following equation:
{A x B x C) = ^ starch / g of freeze-dried plant tissue
where A = amount of starch from the calibration curve in mg/1, B = .005 1
- sample volume after steps 10 and 11, Section 3.3.3., C = factor of 5
dilution at step 13 of Section 3.3.3., and D = freeze-dry weight of sample
(.02 + .002 g)-use actual weight recorded.
The amount of sugar in a sample can be calculated using the following
equation:
= mg sugar / g of oven dried plant tissue
where A = amount of sugar from calibration curve (in mg/1) of glucose
standards, B = total volume of MV solution (20 ml) (.02 1), and C =
freeze-dried weight of sample (g).
Calculate the concentration of total non-structural carbohydrates
(TNC) as follows:
A + B = C
where A = concentration of starch found (mg/g), B = concentration of
sugars found (mg/g), and C = TNC (mg/g).
3.3.7. ERROR ALLOWANCE AND DATA QUALITY
3.3.7.1. CONSIDERATIONS
Blanks and replicates should be included in analyses of each group of
20 samples. Samples should be chosen at random for replicates. At least
10 percent of all tissue samples should be replicated.
Samples selected as replicates should also be run on different days
to estimate day-to-day precision.
Replicate samples will be used to monitor precision. Percent
coefficient of variation will be calculated using the industrial statistic
I, where %CV = 200I//2 and I=|A-B|/A+B, A and B being the results for the
replicate samples (U.S. EPA, 1979).
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Precision will also be monitored using Shevhart control values for R
(range) values, if the total number of sample groups to be analyzed will
generate enough points to make use of such charts worthwhile.
Critical ranges for repeated measurement error and accuracy will be
set by Quality Assurance Officer and listed in the Data Quality Objectives
table.
Accuracy will be evaluated through the use of interlaboratory sample
exchanges. These exchanges will be coordinated by the Quality Assurance
Specialist assigned to the project.
Accuracy of the procedure can also be checked with use of spiked
samples. Spiked samples should be prepared such that the spike will
contain an appropriate amount of starch or sucrose to approximately equal
that normally extracted from tissue samples. The spike should be prepared
as a separate solution and added to the weighed sample in Section 3.3.3.,
step 2 before extraction with MCV. Dry sample after adding spike before
extraction with MCW.
Percent recovery (*P) can be calculated using results from the spiked
samples with the following formula:
XP = 100 x (A~B)
where A = mg of starch or sucrose found for sample plus spike, B = mg of
starch or sucrose found for sample, and S = mg of starch or sucrose added
in spike.
Note: Use of spikes with this procedure is a test of the procedure
itself and does not directly reflect upon the ability of laboratory
personnel to perform the analyses.
3.3.7.2. DATA QUALITY OBJECTIVES
Repeated
Measurement
Measurement
Reporting
Error at
Accuracy
Variable
Units
Lower Limit
Upper Limit
Tolerance
Starch and
mg/g
10* (cv)
10* (cv)
10*
Total Sugars
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3.3.7.3. COMPUTER DATABASE CODES
Variable
Code
Extractable Starch
Total Sugars
FTSR
FSUG
3.3.8. REFERENCES
Birk, E.H. and P.A. Matson. Site fertility affects seasonal carbon
reserves in loblolly pine. Tree Physiology (accepted for
publication).
Clark, J.M. 1964. Experimental Biochemistry. V.B. Freeman and Co., San
Francisco.
Dickson, R.E. 1979. Analytical procedures for the sequential extraction
of 14C labelled constituents from leaves, bark and vood of cottonvood
plants. Physiologia Plantarum. 45:480-488.
Bassig, B.E. and R.E. Dickson. 1979. Starch measurement in plant tissue
using enzymatic hydrolysis. Physiologia Plantarum. 47:151-157.
Hatson, P.A. and R.H. Waring. 1984. Effects of nutrient and light on
mountain hemlock: susceptibility to laminated root rot. Ecology 65:
1517-1524.
U.S. Environmental Protection Agency. 1979. Handbook for Analytical
Quality Control in Water and Wastewater Laboratories. Environ. Mon.
and Sup. Lab. U.S. EPA. Cincinnati, OH. 45268. EPA-600/4-79-019.
Yemm, E.V. and A.J. Willis. 1954. The estimation of carbohydrates in
plant extracts by anthrone. Biochem. Journ. 57:508-514.
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4. SOIL PHYSICAL ANALYSIS
4.1. STANDARD OPERATING PROCEDURE FOR SAMPLE PREPARATION (AIR-DRT SOILS)
4.1.1. SCOPE AND PURPOSE
Soil sampling is the first step in obtaining information about a
given soil. However, it is unrealistic to assume that once a soil sample
has been removed from its location on the landscape that there will not be
some alteration from its field condition (Gillman and Murtha, 1983).
Depending on the types of analyses to be performed on the sample,
alterations that occur after removal from the field may or may not
influence the desired results.
The standard procedure for handling most soil samples is to convert
the samples to an air dry state shortly after collection. Typically a
soil seldom reaches an air-dry state in the field, especially soils in the
spruce-fir ecosystem. Thus, the first step often taken in soil sample
preparation is to convjert the soil to an atypical condition, with the
assumption that this conversion process will not influence the basic
response of the sample to various analytical procedures, and also act to
preserve the sample for long periods of time.
Unfortunately both of these assumptions are incorrect (Bartlett and
James, 1980). Air-drying of soils does alter their response to various
soil extractants as compared to responses of soils retained in the
original field moist condition. Perhaps the best documented differences
between field moist and air-dried samples deal with mineralization rates
and Mn chemistry, but differences have also been noted for P adsorption
capacity, soil acidity, solubility of soil organic matter, and extractable
amounts of Ca, Mg, K, Fe, and A1 (Bartlett and James, 1979, 1980; Gillman
and Murtha, 1983; Nelson, 1977). Field moist soils also differ from air-
dried soils in handling, with the latter often easier to work with,
especially when filtering soil extracts (Bartlett and James, 1980).
Although limited in number, published studies suggest that air-dried
soils are not stable and do change with time, but the results appear to
depend on the parameter measured (Gillman and Murtha, 1983; Bartlett and
James, 1980).
Arguments for using field moist versus air-dried soil samples have
been based on the assumption that field moist samples more closely
approximate conditions found in the field. While this argument can be
justified on the basis of certain physical parameters, it is also fair to
say that few data exist to support the assumption that the results
obtained using field moist soil samples are more representative of field
conditions than those obtained using air-dried samples. Certainly sieving
and mixing of field moist soil samples to obtain a representative
subsample presents a formidable problem which is largely ignored in most
published papers comparing field moist versus air-dried samples. It is
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entirely possible that any gain in accuracy of results using field moist
samples may be lost because of the increase in uncertainty in the final
data.
This standard operating procedure outlines steps to be taken for
preparing air-dried soil samples. The analytical procedures contained in
this methods manual also assume that analyses will be carried out on air-
dried samples. It is recognized that air-drying of soils may impart
changes in the soil matrix that will be reflected in the analytical
results, but methods for handling air-dried soil samples are standardized,
and accuracy and precision limits can be assigned to the various
procedures in this methods manual with a high degree of confidence.
Specific research objectives and associated analytical techniques may
require the use of field moist versus air-dried soil samples. However,
standardized procedures for handling, processing, and storing field moist
soil samples are not available in the peer reviewed literature. Thus,
investigators wishing to use field moist versus air-dried soil samples
must supply the Quality Assurance Specialist for the project with a
detailed plan as to how the integrity of the samples will be preserved
during transit, processing, and storage. Included within the plan should
be a detailed description on how the field moist samples will be mixed to
insure sample homogeneity, and what tests will be used to verify whether
the mixing procedure used is successful. Also, if any of the standard
operating procedures in this methods manual will be used to characterize
field moist samples, the Quality Assurance Specialist should be provided
with written details as to how each standard operating procedure was
modified to account for use of field moist samples.
4.1.2. MATERIALS AND SUPPLIES
4.1.2.1. EQUIPMENT
° 2 mm sieve (brass, stainless steel or plastic)
° wooden rolling pin
0 large mortar with rubber tipped pestle
° large plastic, enamel, or fiberglass trays
° brown wrapping paper (30 inches in width)
° hammer mill
° Viley Mill or equivalent with stainless steel blades and
1 mm stainless steel screen
0 work area to lay out samples to. dry
0 desiccator
4.1.2.1. CHEMICALS/REAGENTS
Desiccant
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4.1.3. PROCEDURES
4.1.3.1. SAMPLE PREPARATION
1. Soil samples are usually made up of a range of different sized
particles. By definition, those particles less than 2 mm in
diameter are considered the fine soil fraction. Discrete
particles larger than 2 mm are usually discarded. Note: clumps
of soil are NOT considered discrete particles and are to be
broken dovn before discarding particles larger than 2 mm in
size.
2. Inspect moist soil sample and remove large coarse fragments such
as stones and pebbles. Discard this fraction unless othervise
noted in standard operating procedures.
3. Discard large roots and stems (> 0.5 cm in diameter) unless a
decision has been oiade to retain these as part of the sample.
Do not remove fine roots unless othervise instructed.
4. Lay out moist soil sample on large trays or sheets of brown
wrapping paper. Place soil in relatively dust free area to dry.
Using brown wrapping paper may contaminate sample with B and S,
but the extent of contamination may not be detectable depending
on the bulk of the original sample.
5. Drying time for samples is a function of sample composition,
weather, and location selected for drying. Soils high in
organic matter will not dry unless heated o'r placed in an area
with low relative humidity. Using a dehumidifier may be
necessary to speed air-drying of samples.
6. Greenhouses are often used to air-dry soil samples. Vhile such
structures offer large areas for spreading out samples, their
use in drying soils should be approached with caution.
Temperature regulation in greenhouses is usually not possible;
thus samples may be exposed to above-ambient temperatures during
the drying phase. Most greenhouses leak when it rains, and
contamination of samples is a distinct possibility. The problem
is not contamination of the sample from rainwater, but from
other surfaces within the greenhouse that come in contact with
rainwater before the latter reaches the soil sample. Zinc
contamination from water dripping off of galvanized metal is
probably the most common source of contamination from rainwater.
7. Greenhouses are often located in large complexes with a
substantial amount of vehicular traffic nearby. This traffic is
a source of dust contamination that could influence the
composition pf the sample. Some dust contamination is to be
expected, but the significance of dust contamination will be a
function of the bulk of the original sample, content of the
contaminating dust, and objectives of the project. Possible
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contamination o£ samples during drying should be noted in the
sample log book, and the samples flagged for evaluation during
generation of the final results.
8. By weighing subsamples daily, the rate of air-drying can be
monitored and a decision made as to when the samples are air-
dry. It should be remembered, however, that the final air-dry
weight will be a function of relative humidity. Subsamples can
be dried in desiccators to estimate percent weight loss to be
expected from bulk samples before they are air-dry.
9. Air-dry forest floor samples cannot be sieved and should be
ground before storage in sample containers. Dried material
should be coarse sieved with a large screen to prevent rocks and
other foreign objects from damaging mill.
10. Samples which are predominately mineral matter should be sieved
through a 2 mm sieve. Stainless steel or plastic sieves are
highly recommended. Brass sieves will contaminate soil samples.
Vhile this may not be important for physical analyses, the
sample will not be suitable for any future trace element
analyses. A note should be made in the sample log if brass
sieves are used to prepare samples.
11. Large clumps of mineral soils can be broken apart with a wooden
rolling pin or mortar and pestle. A rubber tipiped pestle is
highly recommended to prevent larger rock fragments from being
broken down into finer sized particles during processing. Soils
high in clay content will often form clumps* of soil that can
become quite hard. Vorking of samples before they become
completely air-dry will often prevent formation of hard clumps
of soil.
12. Coarse fragments remaining on the 2 mm sieve can be discarded
unless otherwise noted. Fine roots and other organic matter
fragments should be separated from the rock fragments, ground to
pass a 1 mm sieve, and added back to the < 2 mm soil sample. A
note should be recorded in the sample log book if the fine roots
and other organic matter fragments are excluded from the < 2 mm
sample.
13. After sieving, the sample should be well mixed before being
placed into a storage container. Sieving soils actually
segregates the sample and should not be considered as a mixing
step. It is especially important to mix well if only a
subsample of the soil is to be kept, and the remainder of the
sample discarded.
14. A recommended procedure for mixing air-dried soil uses a large
sheet of brown paper with the sample placed in the center of the
paper. The sample is then mixed by lifting the edges of the
brown paper in sequence, rolling the sample across the paper.
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This process is continued for approximately 50 to 100 rotations.
The sample is then centered on the paper and separated with a
spatula into four even piles. Each pile is further separated
into four smaller piles until the desired sample size is
obtained. The sample is then randomly selected from one or more
of the sample piles.
15. The procedure just outlined is tedious, but recommended if the
intention is to keep only subsamples of the original sample.
Recent work by Mullins and Hutchison (1982) suggests that rotary
subsampling and chute splitting may be preferred to the
quartering procedure. However, these same authors also note
that variability in subsampling is not only a function of
subsampler design, but also of the procedure adopted and the
properties of the sample itself.
16. Alternative mixing procedures are acceptable. Suitable
experiments should be run to test the mixing procedure selected.
Failure to maintain quality control for precision may indicate
poor mixing of samples.
17. It is recommended that processed samples be stored in air-tight
containers.
4.1.4. PREVENTIVE MAINTENANCE
All equipment and sampling areas should be cleaned after use. Soil
samples which are naturally acid can corrode metal surfaces, especially
after such surfaces become scratched from repeated contact with soil.
Aluminum trays and pans should be avoided as these corrode and scratch
easily.
Record maintenance operations in maintenance log.
4.1.5. CALIBRATION PROCEDURES
It is recommended that all sieves meet ASTM specifications.
4.1.6. CALCULATIONS/UNITS
Not Applicable
4.1.7. ERROR ALLOWANCES AND DATA QUALITY
4.1.7.1. CONSIDERATIONS
Sample preparation is a crucial step in any analysis, and appropriate
amounts of time and money should be allocated for processing of samples.
Unfortunately, the amount of time and resources needed to handle samples
properly is often underestimated, leading to short cuts in preparation of
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samples. Personnel responsible for processing samples should be
instructed in the proper handling procedures and the need to follow these
procedures for all samples.
Handling of samples in terms of potential contamination should be
evaluated on the basis of what the samples vill be used for. Soil samples
collected for physical analyses do not require special handling to prevent
chemical contamination, unless on a gross scale. Hovever, handling of all
samples should be properly documented so that the potential for use of
samples in future experiments can be evaluated.
Preparation of air-dry soil samples following the steps outlined in
this standard operating procedure vill yield samples which are suitable
for most of the standard operating procedures listed in this methods
manual. However, it should be remembered that soil samples are
essentially collections of discrete particles which ultimately limits
sample homogeneity because the measured property is not associated equally
with each discrete particle (Mullins and Hutchison, 1982). Selection of a
subsample for analysis must be of sufficient size to include a
representative sampling of the various discrete particles that make up the
soil sample. Chemical analyses requiring less than 1 gram quantities of
sample should be prepared from larger subsamples that have been finely
ground (<32 mesh) and mixed. Failure to take a large enough subsample
will introduce a bias into the final results that vill not be detected
unless a separate subsample is taken, finely ground and analyzed. It is
recommended that at least 10 grams be chosen as the minimum subsample size
from which to prepare finely ground samples.
Preparation of finely ground subsamples can be done by hand using an
agate mortar and pestle or with a mechanical grinder, ball mill or mixer
mill (such as a Spex mixer mill or equivalent). However, preparation and
mixing of finely ground samples requires a substantial amount of time, and
should be properly accounted for when designing experiments.
4.1.8. REFERENCES
Bartlett, R. and B. James. 1979. Behavior of chromium in soils: III.
Oxidation. J. Environ. Qual. 8:31-35.
Bartlett, R. and B. James. 1980. Studying dried, stored soil samples -
some pitfalls. Soil Sci. Soc. Am. J. 44:721-724.
Gillman, G.P. and G.G. Murtha. 1983. Effects of sample handling on some
chemical properties of soils from high rainfall coastal North
Queensland. Aust. J. Soil Res. 21:67-72.
Mullins, C.E. and B.J. Hutchison. 1982. The variability introduced by
various subsampling techniques. J. Soil Sci. 33:547-561.
Nelson, L.E. 1977. Changes in water-soluble Mn due to soil sample
preparation and storage. Commun. in Soil Sci. Plant Anal. 8:479-487.
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U.S. Department of Agriculture. 1951. Soil Survey Manual. U.S. Dept.
Agriculture Handbook No. 18., U.S. Dept. of Agriculture. Washington,
DC.
I
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4.2. STANDARD OPERATING PROCEDURE FOR MEASUREMENT OF SOIL MOISTURE
CONTENT
4.2.1. SCOPE AND PURPOSE
This operating procedure is designed to determine the moisture
content of mineral and organic air-dry soil samples for expression of
results on an oven dry basis.
4.2.2. MATERIALS AND SUPPLIES
4.2.2.1. EQUIPMENT
° analytical balance (+ 0.01 g)
0 2 forced draft ovens capable of maintaining 105°C + 2°C
0 2 thermometers (0°C to 200°C)
° glass, metal, or ceramic containers
0 desiccators
4.2.2.2. CHEMICALS/REAGENTS
0 Desiccant
4.2.3. PROCEDURES
4.2.3.1. SAMPLE PREPARATION
1. Record tare veight of each sample container.
2. Weigh approximately 10 g of air-dried soil into tared container.
Record veight to the nearest 0.01 g.
3. Place mineral soil samples (< 40% L0I) into oven set at 10S°C
for 24 hours. Note: use of a convection oven is acceptable, but
drying time may exceed 24 hours.
4. Place organic soil samples (> 40% L0I) into oven set at 70°C for
24 hours. Note: use of a convection oven is acceptable, but
drying time may exceed 24 hours.
5. Remove samples and allow to cool in desiccators.
6. Veigh samples and record weight to nearest 0.01 g.
7. Place samples back in ovens for an additional 2 hour period.
8. Remove and let cool in desiccator. Veigh samples and record
weight to the nearest 0.01 g.
9. Second weighing should be within + 0.1 g of the first weighing.
If not, repeat step 8 until recorded veight does not change by
more than 0.1 g.
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10. Follov guidelines detailed in Good Laboratory Practices section
for recording of all raw data. Note that raw data include the
following: (a) analytical observations necessary to calculate
the final results, (b) calibration data, (c) calibration checks,
and (d) quality control checks. Deviations from standard
operating procedures during sample preparation, calibration, or
actual analyses are to be fully documented and initialed by
laboratory personnel. Samples suspected of being in error or
outside of the calibration range are to be 'flagged', and this
notation carried through all records to the final report.
Retain all written materials (graphs, tables, etc.) generated as
part of an analysis. Do not discard portions of laboratory
notebooks or any other information directly related to
calculation of the final result for a set of samples.
4.2.3.2. EQUIPMENT OPERATION
Follow instruction manual procedures for proper operation of
convection ovens.
4.2.4. PREVENTIVE MAINTENANCE
Check temperature of oven daily and after loading with samples. Note
any prolonged delays in reaching the desired temperature setting after
filling the oven with samples.
Record maintenance operations in maintenance log.
Prevent contamination of samples from oven walls or racks if samples
are to be used for other analyses. Replace all metal parts having
corroded surfaces.
If samples are kept on trays during time in oven, check to see that
the bottom of each tray is clean and does not contain material that could
contaminate samples beneath the tray.
4.2.5. CALIBRATION PROCEDURES
1. Check oven settings with several thermometers of the appropriate
range to ensure selection of correct temperatures.
2. Label ovens to prevent possible mix between mineral and organic
soils.
4.2.6. CALCULATIONS/UNITS
Calculate % moisture content using the following formula:
A'-B
x 100 = X moisture content
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where A = recorded weight of air-dried sample plus sample container, B =
recorded weight of oven-dried sample plus sample container, and TV = tare
weight of sample container.
Calculate equivalent oven-dry weight of an unknown sample using the
following formula:
A
. £MC = equivalent oven-dry weight of an air-dry sample
1 + 100
where A = air-dry weight of soil sample, and %MC = X moisture content as
calculated above.
4.2.7. ERROR ALLOWANCE AND DATA QUALITY
4.2.7.1. CONSIDERATIONS
Replicates should be included with every 10 soil samples.
Samples selected as replicates should also be run on different days
to estimate day-to-day precision.
Replicate samples will be used to monitor precision. Percent
coefficient of variation will be calculated using the industrial statistic
I, where %CV = 200I//2 and I=|A-B|/A+B, A and B being the results for the
replicate samples (U.S. EPA, 1979).
4.2.8. REFERENCES
Blume, L.J. Chemical and Physical Characterization of Soils. Statement of
Work. National Acid Deposition Survey. IFB No. WA 85-566. Environ.
Mon. Sys. Lab. U.S. Environmental Protection Agency. Las Vegas, NV
89114.
U.S. Environmental Protection Agency. 1979. Handbook for Analytical
Quality Control in Water and Wastewater Laboratories. Environ. Mon.
and Sup. Lab. U.S. EPA. Cincinnati, OH. 45268. EPA-600/4-79-019.
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4.3. STANDARD OPERATING PROCEDURE FOR MEASUREMENT OF ORGANIC MATTER BY
LOSS-ON-IGNITION (LOI)
4.3.1. SCOPE AND PURPOSE
This standard operating procedure will be used to estimate the total
organic matter content of soils based on weight loss following ignition in
a muffle furnace. This approach is routinely used in soils research and
provides a relatively easy yet useful index of the proportion of soil mass
attributable to organic materials. Results from this analysis will be
used to group soil samples into mineral soil samples (< 40% LOI) and
organic soil samples (> 40% LOI) (Federer, 1982).
4.3.2. MATERIALS AND SUPPLIES
4.3.2.1. EQUIPMENT
0 quartz test tubes or crucibles
° muffle Furnace (100 to 1000°C)
° analytical Balance (+ 0.001 g)
0 forced draft oven capable of maintaining 105°C + 2°C
4.3.3. PROCEDURES
4.3.3.1. SAMPLE PREPARATION
1. Mineral soil samples (< 40% LOI) should be oven-dried at 105°C,
and organic soil samples (> 40% LOI) should be oven-dried at
70°C. It may be necessary to repeat this procedure for those
soil samples that cannot be grouped into either class by visual
inspection. Such samples should be first dried at 70°C, and then
dried again at 105°C if results from this procedure indicate that
the soil samples are mineral soils.
2. Place numbered crucibles in drying oven overnight.
3. Remove crucibles from drying oven and place in desiccator to
cool.
4. After cooling, weigh crucibles and record the oven-dry weight of
each numbered crucible to the nearest 0.001 g.
5. Veight 3 g + 100 mg of soil sample into a weighed crucible and
place in cool muffle furnace. Record weight of crucible plus
sample to the nearest 0.001 g.
6. Bring muffle furnace gradually to 450°C and ignite samples
overnight (minimum of 12 hours).
7. Remove crucibles from muffle furnace and place in desiccator to
cool. Cover crucibles.
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8. Reweigh cooled crucibles containing ignited soil sample and
record weight to the nearest 0.001 g.
4.3.3.2. EQUIPMENT OPERATION
Follow operating manual instructions for specific muffle furnace
employed as to operations and calibration.
Several test runs of the muffle furnace should be used to determine
the appropriate instrument settings to achieve a constant 450°C
temperature. Rooms housing the muffle furnace should be devoid of
excessive drafts.
4.3.4. PREVENTIVE MAINTENANCE
Carry out all maintenance procedures as recommended by the
manufacturer. Record maintenance operations in maintenance log.
4.3.5. CALIBRATION PROCEDURES
Calibrate muffle furnaces on a monthly basis using a pyrometer and
record changes with time.
4.3.6. CALCULATIONS/UNITS
Calculate Loss-On-Ignition using the following equation:
X LOI = 100 x [1 - ]
where A = wt. of oven-dry crucible, B = wt. of ignited soil sample plus
crucible, and C = wt. of oven-dry soil sample plus crucible.
Estimate X organic matter (X 0M) content by setting X 0M = X LOI.
4.3.7. ERROR ALLOVANCE AND DATA QUALITY
4.3.7.1. CONSIDERATIONS
Blanks, replicates, and in-house secondary standards should be
included in the analyses of all samples. Vithin one group of 40 samples
there should be one blank., two in-house secondary standards, and three
replicates. Note: Use of a blank (empty crucible) in this SOP serves as
a check on sample handling and calibration of balance.
Samples selected as replicates should also be run on different days
to estimate day-to-day precision.
Replicate samples will be used to monitor precision. Percent
coefficient of variation should be calculated using the industrial
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statistic I, where %CV = 2001//2 and I = |A-B|/A+B, A and B being the
results for the replicate samples (U.S. EPA, 1979).
Precision will also be monitored using Shevhart control values for R
(range) values, if the total number of sample groups to be analyzed will
generate enough points to make use of such charts worthwhile.
Critical ranges for repeated measurement error and accuracy will be
set by the Quality Assurance Officer and listed in the Data Quality
Assurance (DQO) table.
4.3.7.2. DATA QUALITY OBJECTIVES
Repeated Measurement
Measurement
Reporting
Error
at
Accuracy
Variable
Units
Lower Limit
Upper Limit
Tolerance
Loss on
X
50% (cv)
5% (cv)
+ 5%
ignition
4.3.7.3. COMPUTER DATABASE CODES
Variable
Code
Loss on Ignition
SL0I
4.3.8. REFERENCES
Federer, C.A. 1982. Subjectivity in the separation of organic horizons
of the forest floor. Soil Sci. Soc. Am. J. 46:1090-1093.
Fernandez, Ivan J. 1983. Field Study Program Elements to Assess the
Sensitivity of Soils to Acidic Deposition Induced Alternatives in the
Forest Productivity. National Council of the Paper Industry for Air
and Stream Improvement Technical Bulletin No. 404, New York. 87 pp.
U.S. Environmental Protection Agency. 1979. Handbook for Analytical
Quality Control in Water and Wastewater Laboratories. Environ. Hon.
and Sup. Lab. U.S. EPA. Cincinnati, OH 45268. EPA-600/4-79-019.
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4.4. STANDARD OPERATING PROCEDURE FOR MEASUREMENT OF SOIL BULK DENSITY
(CORE METHOD)
4.4.1. SCOPE AND PURPOSE
This procedure is designed to determine the bulk density (grams of
oven-dry soil per unit volume) of soil in situ.
Bulk density measurements can be used to assess soil structure, plant
available water, and the amount of a given soil nutrient in a soil
morphological horizon. Bulk density can also be used to express results
on a soil volume or area basis.
Numerous methods are available for measuring bulk density. Selection
of a method in large part depends on the soil itself and the presence or
absence of pebbles, stones, rocks, or boulders. This standard operating
procedure describes measuring bulk density using the minimally disturbed
core method.
The minimally disturbed core method essentially consists of driving a
metal cylinder of known volume into the desired soil horizon to a
specified depth. The cylinder, with soil, is then extracted, and dried at
105°C until a constant veight is obtained. Subtraction of the weight of
the cylinder gives the oven-dry weight of the soil. Dividing the latter
by the volume of the cylinder yields the bulk density.
For the remainder of this standard operating procedure, it is assumed
that the metal sample core will be driven into the ground using a double-
cylinder sampler (U.S. Dept. Agr., 1954). It is also assumed that a
decision has been made as to which soil horizons are t'o be sampled and to
what depths. The latter is important as the double-cylinder sampler is
designed for sampling over fixed depths.
If bulk density measurements are to be made throughout the soil
profile, it is recommended that several soil pits be dug in the sample
area and the soil horizons present identified. It is assumed that the
personnel employed to take the bulk density samples, and/or the principal
investigator have sufficient training to distinguish soil horizons in the
field.
For convenience, bulk density measurements can be taken at the same
time as pit excavation by removing the overburden along one side of the
pit to expose the soil horizon of interest. The only restriction is that
the samples be taken no closer than 10 cm to the edge of trie soil pit.
4.4.2. MATERIALS AND SUPPLIES
4.4.2.1. EQUIPMENT
0 double-cylinder sampler
° aluminum cylinders that fit inside of double-cylinder
0 spacer (5-7.5 cm in length)
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0 oven (50-200°C + 2°C)
° thermometers (0-200°C)
0 analytical balance (+ 0.01 g)
° metal trowel
0 knife or spatula
° desiccators
0 ziplock plastic bags (large enough to hold aluminum cylinders)
0 one-pint, cylindrical, waxed-paper cartons
° weighing pans or glass beakers
0 50 and 100 ml graduated cylinders
0 rose clippers or scissors
4.4.2.2. CHEMICALS/REAGENTS
0 Desiccant
4.4.3. PROCEDURES
4.4.3.1. SAMPLE PREPARATION
1. Number aluminum cylinders with either waterproof ink or metal
dies. Cylinders when purchased usually are prenumbered with
metal dies.
2. Clean cylinders with tap water, rinse with distilled water, and
dry in oven (105^).
3. Remove from oven, let cool, and record weight.
Field Sampling
4. Excavate overburden to the desired depth of soil to be sampled.
5. Place an aluminum cylinder followed by a spacer in the casing of
the double-cylinder sampler. The spacer acts as a buffer while
the cylinder is driven into the ground. Soil accumulated in the
spacer will be discarded. Note the position of the top part of
the spacer (nearest the piston) on the outside of the sampler.
6. Assemble the double-cylinder device and set in vertical position
over the sampling point. Using the piston on the handle shaft,
drive the sampler into the soil using short steady strokes.
Sampler must be held steady while it is being driven into the
soil.
7. Stop driving sampler into the soil when the top part of the
spacer ring (as indicated by the mark on the outside of the
sampler) is flush with soil. Continuing to drive in the sampler
beyond this point will compress the soil in the sample cylinder
and result in a bias in the final calculation.
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8. Using a trowel, carefully excavate around and beneath the casing
of the sampler in the soil. Remove the sampler and unscrew the
outer casing containing the sample cylinder now full of soil.
9. Carefully remove the sample cylinder from the outer casing so as
to preserve the natural structure and packing of the soil as
nearly as possible.
10. Remove the spacer and, with a sharp knife or spatula, trim the
soil extending beyond each end of the sample cylinder such that
the soil is flush vith the edges of the sample cylinder. One
mistake often made by inexperienced field personnel is the
failure to insure that the soil within the sample cylinder is in
fact flush with the edges. The tendency is for a rounded effect
in the sample, with only the edge of the soil core flush with
the edge of the sample cylinder. Field collection personnel
should be instructed in the proper manner of trimming the soil
core.
11. Care must also be taken during this step to avoid disturbing
roots, dislodging gravel, or losing aggregates of soil. Rose
clippers or scissors are sometimes helpful in trimming off
excess material.
12. Loss of. soil during extraction of the sampler from the soil, or
while trimming the soil sample should" be duly noted in the field
log book and the sample discarded. Observation of large rocks
or sections of large roots protruding from or imbedded in the
sample cylinder should be noted and the sample discarded.
13. If the sample passes field inspection, place the sample cylinder
in a ziplock plastic bag and place entire contents into a one-
pint, cylindrical, waxed-paper carton. Use of the carton and
plastic bag minimizes loss of water and disturbance of the
sample during transport to the laboratory.
14. Enter number of sample cylinder in field log book and any other
information necessary for identification of sample.
Laboratory Procedure
15. Drying of sample can be done without extrusion from the aluminum
cylinder, but the amount of drying time required to reach
constant oven-dry weight is increased. It is recommended that
the soil in the aluminum cylinder be extruded and placed in a
tared weighing pan or glass beaker.
16. Extrusion of the sample from the cylinder also allows inspection
of the internal portion of the sample core. The presence of
large rocks or root segments should be noted and the sample
discarded. Dry samples can be checked for shattering. Record
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identification number of discarded sample and reason for
removing from the sample set.
17. Place sample in an oven (105°C) for at least 24 hours. Remove
and let cool in desiccator. Record weight of sample to the
nearest 0.01 g.
18. Select approximately 20% of the samples weighed and place back,
into oven for an additional 24 hour drying period.
19. Remove from oven, allow to cool in desiccator and reweigh to the
nearest 0.01 g. If first and second weighings do not agree
within + 0.5 g, place entire sample set in oven and dry for
another 24 hour period.
20. Continue steps 18-19 until weighings agree within 0.5 g.
21. Completion of step 20 will yield the bulk density of the entire
soil mass in the sample cylinder. In soils with a substantial
proportion of coarse material (stones and pebbles), a corrected
bulk density is calculated by measuring the weight and volume of
the coarse size fraction.
22. Remove oven-dried sample from aluminum cylinder or from weighing
pan and pass through a 2 mm sieve. Break up large clumps of
soil by hand.
23. Record the weight of the coarse fraction remaining on the 2 mm
sieve to the nearest 0.01 g.
24. Place the gravel into a 100 ml graduated cylinder. Fill a 50 ml
graduated cylinder to the 50 ml mark with distilled water.
25. Add enough water from the 50 ml graduated cylinder to the 100 ml
cylinder until the coarse material is covered with distilled
water. Continue adding water to raise volume to the nearest
mark on the 100 ml graduated cylinder.
26. Record volume reading on 100 ml graduated cylinder and on the 50
ml graduated cylinder.
27. Discard coarse1 material and rinse cylinders with distilled water
and allow to dry. Clean aluminum cylinders with tap water and
rinse with distilled water. Dry cores and cans as detailed in
step 2.
28. Follow guidelines detailed in Good Laboratory Practices section
for recording of all raw data. Note that raw data include the
following: (a) analytical observations necessary to calculate
the final results, (b) calibration data, (c) calibration checks,
and (d) quality control checks. Deviations from standard
operating procedures during sample preparation, calibration, or
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actual analyses are to be fully documented and initialed by
laboratory personnel. Samples suspected of being in error or
outside of the calibration range are to be 'flagged', and this
notation carried through all records to the final report.
Retain all written materials (graphs, tables, etc.) generated as
part of an analysis. Do not discard portions of laboratory
notebooks or any other information directly related to
calculation of the final result for a set of samples.
A.4.3.2. EQUIPMENT OPERATION
Follow instruction manual procedures for proper operation of
convection ovens.
4.4.4. PREVENTIVE MAINTENANCE
Check temperature of oven daily and after loading with samples. Note
any prolonged delays in reaching the desired temperature setting after
filling the oven with samples.
Record maintenance operations in maintenance log.
Prevent contamination of samples from oven walls or racks if samples
are to be used for other analyses. Replace all metal parts having
corroded surfaces.
If samples are kept on trays during time in oven, check to see that
the bottom of each tray is clean and does not contain material that could
contaminate samples beneath the tray.
4.4.5. CALIBRATION PROCEDURES
Check oven settings with several thermometers of the appropriate
range to ensure selection of correct temperature (105°C).
4.4.6. CALCULATIONS/UNITS
Bulk density can be calculated using the following formula:
A-T
= bulk density (g/cc) of whole soil
where A = weight of oven-dry soil, T = tare weight of weighing pan, glass
beaker, and/or aluminum cylinder, and V = volume of the aluminum cylinder
in cubic centimeters (cc).
Whole soil is defined as the entire mass retained in the volume of
the aluminum cylinder. If the soil contains a large amount of gravel
sized material, a corrected bulk density can be calculated using the
following formula:
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„ v = bulk density (g/cc^) of soil < 2 mm
V-(Vwg-Vw) J
where A = weight of oven-dry soil, B = weight of coarse fraction
(remaining on 2 mm sieve), V = volume of the aluminum cylinder in cubic
centimeters (cc), Vwg = volume reading on 100 ml graduated cylinder
containing coarse fraction and distilled water, and Vw = volume of water
added to 100 ml graduated cylinder (50 ml minus the volume of distilled
water remaining in the 50 ml graduated cylinder).
Calculation of Vvg and Vw assumes that the density of distilled water
is equal to 1 g/cc.
Report results as megagrams per cubic meter (Mg/m3). Note that one
gram per cubic centimeter is equal one megagram per cubic meter.
4.4.7. ERROR ALLOWANCE AND DATA QUALITY
4.4.7.1. CONSIDERATIONS
Each sample core represents a unique measurement; thus use of
duplicates to monitor quality control is limited. Duplicate cores
obtained from the field will reflect both laboratory and field variation
in the results.
Preliminary sampling studies should be carried out to establish how
many individual cores are required in each sampling set to account for
field variation. Laboratory quality control should focus on carrying out
procedures exactly as written. Field practice using this technique under
supervision of trained personnel must precede use of this procedure by the
field collection crev for collection of samples.
A major source of laboratory variation in this procedure is failure
to obtain a constant oven-dry weight. When using a convection oven, it is
recommended that at least 24 hours should be allowed for drying. Care
should be taken that convection ovens are not opened during the final 12
hours of drying. Also, guidelines should be established as to the load
limit for each oven in use. Overloading an oven will increase the time
necessary to reach constant weight.
Improper adjustment of the balance can also result in a constant bias
in the final calculations. Use of a set of calibrated weights is
recommended at the start of each weighing session to insure that the
balance has been properly calibrated.
Samples should not be taken in wet or dry soils. In wet soils,
friction along the sides of the sampler and vibrations due to hammering
are likely to result in viscous flow of the soil and thus in compression
of the sample. When this occurs the sample obtained is unrepresentative,
being denser than the body of the soil. Compression may occur in dry
soils if they are very loose.
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Vhenever a sample is taken, one should carefully observe whether the
soil elevation inside the sampler is the same as the undisturbed surface
outside the sampler. One can roughly estimate in this manner whether the
density of the sample is changing because of sampling.
In dry or hard soils, another problem arises. Hammering the sampler
into the soil often shatters the sample, and an actual loosening during
sampling may occur. Samplers pressed into the soil usually avoid the
vibration which causes this shattering. Close examination of the soil
sample usually allows one to estimate whether serious shattering occurred.
And, as in the case of wet soils, soil level inside and outside the
sampler must remain the same if the sample is to be considered
satisfactory.
4.4.7.2. DATA QUALITY OBJECTIVES
Repeated Measurement
Measurement
Reporting
Error
at
Accuracy
Variable
Units
Lower Limit
Upper Limit
Tolerance
Soil Bulk
Mg/m3
N/A
N/A
N/A
Density
4.4.7.3. COMPUTER DATABASE CODES
Variable
Code
Soil Bulk Density
SCBD
4.4.8. REFERENCES
Blake, G.R. 1965. Bulk Density. In: Methods of Soil Analysis. Part 1.
C.A. Black (ed.). ASA MonograpE No. 9. American Society of Agronomy,
Inc. Madison, WI.
Fernandez, I. 1983. Field Study Program Elements to Assess the
Sensitivity of Soils to Acidic Deposition Induced Alterations in
Forest Productivity. National Council of the Paper Industry For Air
and Stream Improvement, Inc. (NCASI) Technical Bulletin No. 404. New
York, New York.
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Hillel, D. 1971. Soil and Water. Physical Principles and Processes.
Academic Press, New York. p.49-77.
U.S. Department of Agriculture. 1954. Diagnosis and improvement of
saline and alkali soils. USDA Handbook 60.
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4.5. STANDARD OPERATING PROCEDURE FOR MEASUREMENT OF FIELD VATER CONTENT
(GRAVIMETRIC APPROACH)
4.5.1. SCOPE AND PURPOSE
This procedure is designed to determine the vater content of a given
volume of soil as it exists in the field at a given point in time.
Field measurement of water available for plant growth requires the
direct or indirect measurement of the water content of the surrounding
soil. Described in this standard operating procedure is a direct method
of measuring water content by gravimetry. The procedure requires a
minimal investment of equipment and labor and is easy to perform.
However, it is a destructive procedure, requiring removal of soil from the
test area each time a measurement is made. Therefore, repeated use of
this procedure over long periods of time can have a substantial impact on
relatively small test areas.
Soil water content alone does not yield direct information as to the
amount of plant-available water in a soil. The amount of plant-available
water is related to soil water content through a soil water characteristic
curve (sometimes referred to as a soil moisture release curve; Hillel,
1971). Thus, this standard operating procedure assumes that suitable soil
water characteristic curves have been determined for the soil under study.
Standard operating procedures for determining soil water characteristic
curves are available in- the literature (Richards, 1949; Black, 1965).
4.5.2. MATERIALS AND SUPPLIES
4.5.2.1. EQUIPMENT
° analytical balance (+ 0.01 g)
0 oven capable of maintaining 105°C + 2CC
0 thermometers (0°C to 200®C)
° stainless steel soil corer (1 inch in diameter)
° aluminum containers with tight fitting lids
0 desiccators
0 spatula
4.5.2.2. CHEMICALS/REAGENTS
• Desiccant
4.5.3. PROCEDURES
4.5.3.1. SAMPLE PREPARATION
1. Number aluminum containers. Use water-proof ink or metal dies.
Use of metal dies is recommended.
2. Place aluminum cans with lids into oven (1059C) for 24 hours.
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3. Remove from oven, let cool, and record the veight of each core
and can to the nearest 0.01 g.
Field Sampling
4. Remove surface accumulation of organic matter before sampling.
5. Insert stainless steel soil corer into soil to desired depth and
remove from soil.
6. Immediately transfer soil from corer into a numbered aluminum
can and secure with tight fitting lid. Record number of
aluminum can and sampling position in field notebook.
7. Place in suitable shipping container to prevent possible
spillage during transit.
Laboratory Measurements
8. Record weight of capped aluminum cans as soon as possible after
receipt of samples in the laboratory. If weighing is delayed,
store in an environment where evaporation will be negligible
(e.g., 4°C).
9. Remove lids from weighed cans and place in oven (105°C+2°C) for
24 hours.
10. Remove cans from oven and place in desiccators to cool. Weigh
cans plus lids and record weight to nearest 0.01 g.
11. Select approximately 20% of the samples weighed and place back,
into oven for an additional 24 hour drying period.
12. Remove from oven, allow to cool in desiccator and reweigh to the
nearest 0.01 g. If first and second weighings do not agree
within + 0.05 g, place entire sample set in oven and dry for
another 24 hour period.
13. Continue steps 11 and 12 until weighings agree within 0.05 g.
14. Discard soil in can and rinse cans and lids with running tap
water and rinse with distilled water. Dry cans as detailed in
step 2.
15. Follow guidelines detailed in Good Laboratory Practices section
for recording of all raw data. Note that raw data include the
following: (a) analytical observations necessary to calculate
the final results, (b) calibration data, (c) calibration checks,
and (d) quality control checks. Deviations from standard
operating procedures during sample preparation, calibration, or
actual analyses are to be fully documented and initialed by
laboratory personnel. Samples suspected of being in error or
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outside of the calibration range are to be 'flagged', and this
notation carried through all records to the final report.
Retain all vritten materials (graphs, tables, etc.) generated as
part of an analysis. Do not discard portions of laboratory
notebooks or any other information directly related to
calculation of the final result for a set of samples.
4.5.3.2. EQUIPMENT OPERATION
Follow instruction manual procedures for proper operation of
convection ovens.
4.5.4. PREVENTIVE MAINTENANCE
Check temperature of oven daily and after loading with samples. Note
any prolonged delays in reaching the desired temperature setting after
filling the oven vith samples.
Record maintenance operations in maintenance log.
Prevent contamination of samples from oven vails or racks if samples
are to be used for other analyses. Replace all metal parts having
corroded surfaces.
If samples are kept on trays during time in oven, check to see that
the bottom of each tray is clean and does not contain material that could
contaminate samples beneath the tray.
4.5.5. CALIBRATION PROCEDURES
Check oven settings vith several thermometers of the appropriate
range to ensure selection of correct temperature (105°C).
4.5.6. CALCULATIONS/UNITS
Calculate % moisture content using the folloving formula:
f A-T)
7=-=^ x 100 = X moisture content on an oven dry weight basis
where A = vet veight of sample plus can and lid, B = oven dry veight of
sample plus can and lid, and T = tare veight of can and lid.
Percent moisture content is often expressed on a % water content per
unit bulk volume basis. To convert £ vater content on an oven dry veight
basis to a per unit volume basis, multiply by the bulk density of the
soil.
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4.5.7. ERROR ALLOWANCE AND DATA QUALITY
4.5.7.1. CONSIDERATIONS
Each sample core represents a unique measurement; thus use of
duplicates to monitor quality control is limited. Duplicate cores
obtained from the field vill reflect both laboratory and field variation
in the results.
Preliminary sampling studies should be carried out to establish how
many individual cores are required in each sampling set to account for
field variation. Laboratory quality control should focus on carrying out
procedures exactly as written.
A major source of laboratory variation in this procedure is failure
to obtain a constant oven-dry weight. When using a convection oven, it is
recommended that at least 24 hours should be allowed for drying. Care
should be taken that cojnvection ovens are not opened during the final 12
hours of drying. Also, guidelines should be established as to the load
limit for each oven in use. Overloading an oven will increase the time
necessary to reach constant weight.
Accuracy in water content measurements is also directly related to
establishing a reproducible oven-dry weight. Therefore, the actual oven-
drying procedures used should exactly duplicate those used to generate the
soil water characteristic curve. Normally, drying at 105°C is selected,
but alternative drying procedures do exist.
Balance error will influence the accuracy of the final calculations.
Influence of balance error on final calculations is discussed in detail by
Gardner (1965); however, the error will not exceed 0.1% water content (up
to 100% water content) if the ratio of balance error to oven-dry weight of
sample does not exceed 0.001 (e.g., 100 mg/100 g.,10 mg/10 g., or 1 mg/1
g.).
Improper adjustment of the balance can also result in a constant bias
in the final calculations. Use of a set of calibrated weights is
recommended at the start of each weighing session to insure that the
balance has been properly calibrated.
4.5.7.2. DATA QUALITY OBJECTIVES
Repeated Measurement
Measurement
Reporting
Error
at
Accuracy
Variable
Units
Lower Limit
Upper Limit
Tolerance
Field Water
X (w/w)
N/A
N/A
N/A
Content
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4.5.7.3. COMPUTER DATABASE CODES
Variable
Code
Field Vater Content
SFVC
4.5.8. REFERENCES
Black, C.A. 1965. Methods of Soil Analysis. Part I. ASA Monograph No.
9. American Society of Agronomy, Inc., Madison, VI.
Gardner, V. H. 1965. Vater Content. In: Methods of Soil Analysis.
Part 1. ASA Monograph No. 9, C.A. Hack (ed.). American Society of
Agronomy, Inc., Madison, VI.
Hillel, D. 1971. Soil and Vater. Physical Principles and Processes.
Academic Press, New York. p.49-77.
Richards, L.A. 1949. Methods of measuring soil moisture tension. Soil
Sci. 68:95-112.
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4.6. STANDARD OPERATING PROCEDURE FOR PARTICLE SIZE ANALYSIS (HYDROMETER
METHOD)
4.6.1. SCOPE AND PURPOSE
This procedure is designed to determine the percent sand (2.00 - 0.05
mm), silt (0.05 - 0.002 mm), and clay (<0.002 mm) fraction in whole soil.
This standard operating procedure describes the hydrometer method for
determining the particle size fractions in a whole soil sample. Results
from this analysis can be used to determine the textural class of the soil
sample. Several procedures are available for determining the particle
size fraction of whole soils. The reader is referred to the work, of Day
(1965) for a review of the various procedures in use, including a review
of the theory on which the hydrometer method is based.
4.6.2. MATERIALS AND SUPPLIES
4.6.2.1. EQUIPMENT
° oven (50-200°C + 2°C)
° thermometer (20-100°C)
0 thermometer (0-50°C)
0 mortar with rubber tipped pestle
0 analytical balance (+0.01 g)
° standard hydrometer, ASTM No. 152H, with Bouyoucos scale in g per
liter
0 electric mixer with replaceable stirring paddle (ASTM stirring
apparatus A)
° glass cylinder marked at the 1000 ml level 36 + 2 cm from the
bottom (inside)
° Pasteur-Chamberlain filter candles, fineness "F" (store in
distilled water)
° brass plunger which consists of a circular brass plate 1/16 inch
thick by 2 1/2 inch diameter with a 3/16 by 20 inch brass rod
fastened normal to the plate at its center
° pH meter
° 250 ml glass beakers and watch glasses
° 2 mm sieve
° hot plate
° desiccator
° chronometer
4.6.2.2. CHEMICALS/REAGENTS
° Distilled water
° Sodium metaphosphate
° Sodium carbonate (Na2C03)
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° Dispersing agent - dissolve 100 g of sodium metaphosphate plus
approximately 3 g of sodium carbonate in 1 liter of distilled
water. Adjust pH to 8.3 with additional sodium carbonate.
0 Hydrogen peroxide, 30% (H202)
4.6.3. PROCEDURE
4.6.3.1. SAMPLE PREPARATION
1. Dry and crumble sample and pass through a 2 mm sieve. Break up
aggregates remaining on sieve with mortar and rubber-tipped
pestle.
2. Discard debris remaining on 2 mm sieve.
3. Mix sieved soil veil. Place a 40 g subsample into a tared 250
ml beaker, and record weight to the nearest 0.01 g.
4. Dry sample overnight at 105°C. Cool in desiccator and record
weight to the nearest 0.01 g.
5. Add 30 ml of distilled water and mix contents by swirling
beaker.
6. Cautiously add 1 ml of 30% H202 , cover with glass beaker, and
gently swirl beaker. This step is designed to remove soil
organic matter. If the sample is suspected to have substantial
amounts of organic matter, reduce amount of 30% B202 added to
prevent excessive foaming. Swirl beaker to help reduce foaming.
7. When initial reaction subsides, add additional 30% H202 and heat
for 1 hour on a hotplate (90°C). Repeat as necessary to
complete the reaction.
8. When reaction is complete, remove excess liquid with Pasteur-
Chamberlain filter-candle and aspirator with liquid trap.
9. When outer surface of filter-candle becomes coated with several
mm of soil during filtration, remove coating by appling positive
pressure to the filter-candle while at the same time gently
tapping filter-candle against the side of the glass beaker.
10. Repeat this procedure as needed to facilitate filtration.
11. Wash soil by adding distilled water. Suspend soil with jet of
distilled water directed at the sediment in the bottom of the
beaker. Remove excess water using filter-candle.
12. Continue washing soil several times to remove dissolved
minerals.
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13. During final washing, remove soil from filter-candle vith rubber
policeman and fine jet of distilled water. Place beaker in oven
(105°C) and dry until constant weight.
14. Record constant oven-dry weight to nearest 0.01 g. Retain
beaker and soil for particle size analysis.
Calibration of Hydrometer
15. Add 50 ml of dispersing agent to a 1 liter cylinder and bring to
volume vith distilled water. Mix contents thoroughly with brass
plunger.
16. Lower hydrometer into solution and determine scale reading at
the upper edge of the meniscus surrounding the stem. Record
this value for use in calculations. This value will be referred
to as the value of RL. (Note; Normally only one RL value is
used in the final calculations, but use of one RL value is only
valid if all measurements are made at the same temperature. An
alternative is to take an RL reading each time a sample reading
is made, but this approach increases the uncertainty in the
calculations and should be avoided if possible.)
Particle Size Analysis
17. Add 50 ml of dispersing agent to 250 ml beaker with soil. Add
an additional 150 ml of distilled water and let stand for 10
minutes.
18. Transfer contents to mixing vessel and mix contents with
electric mixer for 5 minutes. Transfer contents to 1 liter
cylinder and bring to volume with distilled water. Be sure both
transfers are quantitative, using a rubber policeman and jets of
distilled water as necessary.
19. Mix contents gently with brass rod. Remove rod and measure
temperature of suspension. Record temperature to the nearest
degree C.
20. Insert brass rod and mix vith up and down movement to mix
contents of cylinder thoroughly. Hold cylinder with one hand to
prevent tipping over of cylinder while mixing.
21. Use strong upward strokes to suspend soil at the bottom of the
cylinder. However, restrict movement near the top of the
cylinder so as not to spill part of the suspension during the
mixing.
22. When all the soil is suspended in column, finish mixing with two
or three smooth strokes and quickly remove brass rod, tipping it
slightly at the top of the cylinder to remove drops of
suspension adhering to the brass plate.
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23. Record the time as soon as the rod is removed from the cylinder.
It is recommended that two technicians work together at this
part of the procedure.
24. As soon as the brass rod is removed, insert the hydrometer into
the suspension. DO NOT RELEASE HYDROMETER! Carefully insert
hydrometer until it is evident that hydrometer is approximately
at an equilibrium point in the suspension. Sudden release of
the hydrometer will cause the hydrometer to bob up and down in
the cylinder. Such motion essentially is a mixing action and
will bias the results. There is also the possibility that the
hydrometer will break if it hits the bottom of the glass
cylinder.
25. At the thirty (30) second mark record the reading on the
hydrometer at the upper edge of the meniscus surrounding the
stem. This reading and others specified in step 30 will be
referred to as R values in calculations.
26. Since it is not possible to see through the suspension, the
position of the meniscus is best seen by viewing the hydrometer
from an angle of 10 to 20 degrees above the plane of the liquid.
A light placed above the head, but shielded from the eyes, may
help in noting the exact position of the mensicus on the stem of
the hydrometer.
27. Leave hydrometer, in cylinder and take another reading at sixty
(60) second mark. Record reading.
28. Remove hydrometer slowly from the cylinder, rinse with distilled
water, and wipe dry.
29. Let cylinder stand and do not disturb cylinder or subject to
mechanical vibrations.
30. Insert the hydrometer again for subsequent readings at 3, 10,
30, and 90 minutes and after 6 and 12 hours. As before, use
care in inserting the hydrometer so as not to mix the column of
suspended soil particles. Record reading, remove hydrometer and
record the temperature of the suspension to the nearest degree
C.
31. If it is suspected that insertion of hydrometer has caused undo
mixing of column, restart analysis at step 19.
32. After recording 24 hour reading and temperature, discard
suspension. Rinse all glassware in tap water followed by
distilled water and air dry. Note: pouring of suspension down
sink is NOT recommended. A calcium salt is usually added at the
end of the analysis to flocculate the suspended clay particles.
The clear supernatant is then decanted off and the remaining
suspension transferred to a waste bucket for suitable disposal.
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33. Follow guidelines detailed in Good Laboratory Practices section
for recording of all raw data. Note that raw data include the
following: (a) analytical observations necessary to calculate
the final results, (b) calibration data, (c) calibration checks,
and (d) quality control checks. Deviations from standard
operating procedures during sample preparation, calibration, or
actual analyses are to be fully documented and initialed by
laboratory personnel. Samples suspected of being in error or
outside of the calibration range are to be 'flagged', and this
notation carried through all records to the final report.
Retain all written materials (graphs, tables, etc.) generated as
part of an analysis. Do not discard portions of laboratory
notebooks or any other information directly related to
calculation of the final result for a set of samples.
4.6.3.2. EQUIPMENT OPERATION
Follow instruction manual procedures for proper operation of ovens.
4.6.4. PREVENTIVE MAINTENANCE
Check temperature of oven daily and after loading with samples. Note
any prolonged delays in reaching the desired temperature setting after
filling with samples.
Record maintenance operations in maintenance log.
Prevent contamination of samples from oven walls or racks if samples
are to be used for other analyses. Replace all metal parts having
corroded surfaces.
If samples are kept on trays during time in oven, check to see that
the bottom of each tray is clean and does not contain material that could
contaminate samples beneath the tray.
4.6.5. CALIBRATION PROCEDURES
1. Constant temperature throughout a series of measurements with
hydrometer is necessary to the accuracy of the procedure.
Analysis should be carried out in constant temperature room or in
a room with minimum temperature fluctuation.
2. If temperature of suspensions varies by more than + 2°C, consider
moving analysis to another facility or using constant temperature
baths.
4.6.6. CALCULATIONS/UNITS
1. Calculation of particle size fractions requires construction of
summation percentage versus effective particle diameter chart,
using semi-log paper. Summation percentage and effective
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particle diameter values are calculated using a series of
formulas and charts (Day, 1965).
2. For each R reading at a given time t, calculate the suspension
concentration in g/1 using the equation:
C = R - RL
where C = suspension concentration in g/1, R = hydrometer
reading at time t (30, 60 seconds, 3, 10, 30 90 minutes, and 6,
12, and 24 hours), and RL = calibration hydrometer reading.
3. Calculate summation percentage for each time t using the
following equation:
P = 100 x —
Co
where P = summation percentage at time t (30, 60 seconds; 3,
10, 30, and 90 minutes; and 6, 12 and, 24 hours), C = suspension
concentration in g/1 at time t, and Co = is oven-dry weight of
sample after treatment with H20, in g/1.
4. Calculate the effective particle diameter for each time t using
the following equation:
d = 0
7t
where d = effective particle diameter in ym, 0 = constant
corresponding to the R reading at time t (Charts of 0 versus R
are available in published texts dealing with particle size
analysis; (e.g., see Day, 1965), and t = time in minutes.
5. Published charts of 0 versus R values assume a sedimentation
temperature of 30°C. Multiply each value of d by the square
root of the ratio of the viscosity of water at the temperature
of measurement to the viscosity of water at 30°C.,
6. Plot P vs d on semi-logarithmic paper, using the log scale for
d. Draw a smooth curve through the plotted points.
7. Percent clay in the sample can be read directly from the graph
by interpolating the summation percentage at 2 ym (0.002 mm).
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8. Interpolate the summation percentage reading at 50 ym (0.050
mm). Subtract the value for percent clay to arrive at the
percent silt in the sample.
9. Subtract the interpolated value for 50 ym from 100 percent to
arrive at the percent sand in the sample.
10. Report values of % sand, silt, and clay to the nearest whole
percent.
4.6.7. ERROR ALLOWANCE AND DATA QUALITY
4.6.7.1. CONSIDERATIONS
Replicate analyses can be used as a measure of precision; however,
the large sample size used in this technique precludes lack of precision
in sample preparation unless sample is not mixed after sieving.
Since the procedure is essentially nondestructive once the sample has
been placed in the 1 liter cylinder, precision can also be estimated by
repeated measurements on the same sample. This latter approach is
recommended due to the critical steps involved in actually carrying out
the analyses. This is especially true for the first two readings at the
30 and 60 second marks. These measurements could be repeated several
times without noticeably adding to the overall time of the analyses. It
is recommended that readings be repeated on every tenth sample.
Bias can be introduced into the final results because of poor
dispersion, presence of minerals such as gypsum or calcium carbonate, or
the presence of easily disintegrated rock or mineral fragments. Failure
to disperse the sample will underestimate the clay and silt fraction.
However, overmixing may lead to disintegration of sand-sized particles and
overestimating of the silt and clay-size fraction.
Each soil sample from different areas should be evaluated as to
potential problems that may result in a bias in the final calculations.
Comparing results with soil mapping descriptions with a similar soil
series can serve as one check on the accuracy of the determination.
Exchange of samples with other laboratories is also recommended.
Consideration of precision and accuracy should also take into account
the intended use of the final data. The readings obtained from this
procedure are intended primarily for soil texture analysis. The various
soil textural classes recognized have rather wide tolerances, ranging from
10 to 40 percent. Thus, accuracy and precision of approximately + 2%
particle size content should be sufficient for most uses of the data.
Analysis of soil samples can be repeated when the results fall on the
border of two textural classes; however, such occurrences tend not to be
the norm.
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4.6.7.2. DATA QUALITY OBJECTIVES
Repeated Measurement Measurement
Reporting Error at Accuracy
Variable Units Lover Limit Upper Limit Tolerance
Particle X particle 10% (cv) 10% (cv) N/A
Size size content
4.6.7.3. COMPUTER DATABASE CODES
Variable Code
Percent Sand
SPSN
Percent Silt
SPSL
Percent Clay
SPCL
4.6.8. REFERENCES
Day, P.R. 1965. Particle Fractionation and Particle-Size Analysis. IN
C.A. Black. Methods of Soil Analysis. Part 1. ASA Monograph No. 9.
American Society of Agronomy, Inc., Madison, VI.
Hillel, D. 1971. Soil and Water. Physical Principles and Processes.
Academic Press, New York. p.49-77.
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5. SOIL CHEMICAL ANALYSIS
5.1. STANDARD OPERATING PROCEDURE FOR MEASUREMENT OF pH (DI WATER, 0.01M
CaCl2, IN KC1 - AIR-DRY SOILS)
5.1.1. SCOPE AND PURPOSE
The purpose of this procedure is to determine the hydrogen-ion
activity in soil solution. Results are commonly expressed as the negative
logarithm of the hydrogen-ion activity, abbreviated as 'pH'. Actual
measurement is based on the NBS standard pH scale using calibrated
standard buffer solutions.
Soil pH measurements are a function of the potential acidity of the
soil and the concentration of neutral salt cations in the soil solution.
Use of 0.01M CaCl2 salt'has been recommended as approximating the ionic
strength in soil solutions. Use of the IN KC1 salt has been recommended
to overcome the salt effect and to provide an estimate of the potential
acidity of the soil.
5.1.2. MATERIALS AND SUPPLIES
5.1.2.1. EQUIPMENT
0 digital pH meter capable of measuring pH to + 0.01 pH unit
and temperature to + 0.5°C with automatic temperature
compensation: capability.
° high quality, ldw sodium glass pH and reference electrodes. Gel
type reference electrodes must not be used. A combination
electrode is, recommended, and the procedure is written assuming
one is used.
0 disposable stirrer
° disposable cups;
° chronometer
° rinsing apparatus
5.1.2.2. CHEMICALS/REAGENTS
0 NBS traceable pH buffers (pH = 3, 4, 6, 7, 10).
0 Distilled deionized (DI) water.
0 Stock. 3.6M CaCl2 solution prepared as follows:
Dissolve 799.10 g of reagent grade anhydrous CaCl2 in DI water
and dilute to 2.000 L.
0 0.01M CaCl2 solution prepared as follows: Dilute 50 mL of stock
3.6M CaCl2 in 18 L with DI water. Adjust pH to between 5 and
6.5 with Ca(0H)2 or HC1. Discard and remake if electrical
conductivity is not between 2.32 + 0.08 mmho/cm at 25°C.
0 IN KC1, dissolve 74.56 g of reagent grade KC1 in 1 liter
of DI. Adjust pH to between 5 and 6.5 with K0H or HCl.
0 pH quality control check sample (QCCS) of pH 4.0. This source
may not be from the same containers or lot as the NBS standards
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used for electrode calibration. A 0.05M potassium biphthalate
(KHP) source can be purchased or prepared as follows: Dry KHP
for 2 hours at 110°C. Dissolve 10.21 g KHP in DI water and
dilute to 1.000 L. Add 1.0 mL chloroform or a single thymol
crystal per liter for preservation. The pH of this solution as
a function of temperature is:
PH = 3.999 4.002 4.008 4.015 at
T (°C) = 15 20 25 30
5.1.3. PROCEDURES
5.1.3.1. SAMPLE PREPARATION
1. Samples should be air-dried and passed through a 2 mm stainless
steel sieve.
2. Prepare soil (g): solution (mL) ratios for mineral or organic
soil samples as follows:
Mineral soil (< 402 LOI) - Add 10 ml of DI, 0.01M CaCl2, or
IN KC1 to 5.0 g of soil.
Organic soil <> 40* LOI) - Add 20 ml of DI, 0.01M CaCl2, or
IN KC1 to 2.00 g of soil.
The amounts of soil listed in this step are considered the
minimum to be used with this procedure. More sample can be used
with either measurement, provided solution to solid ratio of
either 2:1 for mineral soils, and 10:1 for organic soils is
maintained.
3. Stir the soil solution mixture for 1 minute with a disposable
stirrer at 0, 15, 30, 45, and 60 minutes after addition of DI or
salt solution.
4. Allow suspension to settle for 30 minutes.
5. Position electrode so that the glass membrane is partly settled
into the suspension and the ceramic junction is immersed just
deep enough into the clear supernatant solution to establish good
electrical contact.
6. Record reading to nearest 0.01 pH unit after 30 seconds.
Properly functioning pH electrodes should produce stable readings
within 30 seconds. Record second reading after 2 minutes.
Readings at 30 seconds and 2 minutes should not differ by more
than + 0.05 pH units.
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7. Rinse electrode with DI water between each sample. Do not rub or
blot dry. For samples with substantial amounts of organic matter
(> 20% LOI) it may be necessary to rinse electrode with IN HCl
then with DI water between samples to maintain desired response.
Otherwise electrode response may deteriorate after only several
samples. Final rinse with DI water is necessary to prevent
contamination of next sample or standard. Daily treatment with
mild bleach solution is also recommended when working with these
samples.
8. Follow guidelines detailed in Good Laboratory Practices section
for recording of all raw data. Note that raw data include the
following: (a) analytical observations necessary to calculate the
final results, (b) calibration data, (c) calibration checks, and
(d) quality control checks. Deviations from standard operating
procedures during sample preparation, calibration or actual
analyses are to be fully documented and initialed by laboratory
personnel. Samples suspected of being in error or outside of the
calibration range are to be 'flagged', and this notation carried
through all records to the final report. Retain all written
materials (graphs, tables, etc.) generated as part of an
analysis. Do not discard portions of laboratory notebooks or any
other information directly related to calculation of the final
result for a set of samples.
5.1.3.2. EQUIPMENT OPERATION
Consult operating manual for proper operation of digital pH meter.
Set isopotential point for the pH of the buffer actually in use.
Leave instrument on standby and verify combination electrode is
connected and the reference filling solution is at least 3 cm above sample
line.
5.1.4. PREVENTIVE MAINTENANCE
It is critical that the internal filling solution be maintained at
near full condition to prevent the possible diffusion of sample solution
into the electrode. Flushing of the internal solution is also recommended
on a regular basis to prevent accumulation of KC1 crystals in the bottom
of the reference electrode. Formation of crystals will block the entrance
to the ceramic junction, reducing the diffusion of KC1 to the external
solution. Reduction in diffusion of KCl to the sample solution will be
reflected in erratic response and drift by the electrode.
Storage solutions are available for pH electrodes. In general the
electrode should not be stored in DI water. This is especially true for
electrodes using Ag/AgCl reference electrodes. Storage in DI or dilute
salt solutions leads to the rapid formation of a AgCl precipitate in the
ceramic junction because of the difference in ionic strength between the
internal filling solution and DI water. Discoloration of the ceramic
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junction is a visual indication of the formation of the precipitate. This
precipitate will eventually plug the junction, leading to erratic response
or drift by the electrode.
Various techniques can be used to remove organic films and clay films
from the surface of the glass electrode. Hovever, these often cause wear
on the glass membrane which gradually leads to a deterioration in
response. Replacement of the electrode should be considered as being
normal after several months usage.
Never allow glass membrane to air-dry. If necessary to wipe glass
membrane, allow time for electrode to equilibrate before using. Rinsing
with IN HC1 will aid in rewetting the membrane surface.
Record maintenance operations in maintenance log.
5.1.5. CALIBRATION PROCEDURES
1. Calibrate for temperature by measuring room temperature of
solution with thermometer.
2. Calibrate with buffer solutions above and below all sample
values. Stir buffer solution for 30 seconds. Cease stirring and
read and adjust pH meter.
5.1.6. CALCULATIONS/UNITS
Report results as pH measured in DI, 0.01M CaCl2 or IN KC1, using a
1:2 or 1:10 solid to solution ratio.
5.1.7. ERROR ALLOWANCE AND DATA QUALITY
5.1.7.1. CONSIDERATIONS
Electrode response is the most critical parameter to be monitored.
Stable readings at the calibrated pH values should be obtained within 30
seconds of contact with the buffer solutions. The 2 minute reading should
agree with the 30 second reading by + 0.05 pH units. Slower response
times indicate fouling of the glass membrane or plugging of the ceramic
junction. A similar response time should be evident for salt pH values
for most soils. Soils having near neutral pH values may drift slightly
when measuring DI pH. Various pH electrodes are available. It is
recommended that the cheapest electrode be used that meets the
requirements outlined in this procedure. Electrodes should be replaced as
soon as there is an irreversible decline in response time.
Calibration of the pH meter should be checked after every 10 samples.
This is done using standard buffers or the quality control calibration
sample. Values for the quality control calibration sample should be
checked with every 20 samples. Record readings for buffer and quality
control calibration sample. Failure to obtain a reading of 4.00+0.05
units within 30 seconds should be taken as an indication of measurement
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process out of control. Failure of the 30 second and 2 minute readings to
agree within + 0.05 pH units should also be taken as an indication of
measurement process out of control.
If failure in quality control is detected, check, electrode response
with fresh quality control calibration sample. If failure is still
detected, stop analyses and correct error. Replace electrode if
necessary, recalibrate electrode and continue analyses from last data
point in control. Repeat samples if delay in correcting error is greater
than 2 hours.
Include 3 replicates with every 30 samples to check"precision.
Samples selected as replicates should also be run on different days
to estimate day-to-day precision.
Record temperature during pH analysis. Record temperature of sample
solutions and buffer solutions, not room temperature. Monitor effects of
changes in room temperature on sample solution temperature by recording
periodically temperature of aliqout of DI in a sample container.
Critical ranges for repeated measurement error and accuracy will be
set by Quality Assurance Officer in the Data Quality Objectives (DQO)
table.
5.1.7.2. DATA QUALITY OBJECTIVES
Variable
Reporting
Units
Repeated Measurement
Error at
Lower Limit Upper Limit
Measurement
Accuracy
Tolerance
pH
0.01 pH units
+ 0.01 pH
units
+ 0.01 pH
units
N/A
5.1.7.3.
COMPUTER DATABASE
CODES
Variable
Code
pH in DI water ( SWPH
pH in 0.01M CaCl2 SCPH
pH in IN KC1 SKPH
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5.1.8. REFERENCES
Blumet Louis J. Chemical and Physical Characterization of Soils.
Statement of Work. National Acid Deposition Survey. IFB No. WA 85-
566. Environ. Mon. Sys. Lab. U.S. Environmental Protection Agency.
Las Vegas, NV 89114.
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5.2. STANDARD OPERATING PROCEDURE FOR MEASUREMENT OF EXCHANGEABLE BASES
(Ca, Mg, Na, K) BY IN NH^Cl
5.2.1. SCOPE AND PURPOSE
This procedure is designed to determine the quantity of exchangeable
base cations attached to the clay and organic constituents of a soil.
Exchangeable bases are defined as those base cations that can be exchanged
with other positively charged ions in the soil solution.
Extraction of exchangeable base cation depends to some extent on the
extracting solution selected. Unbuffered salts are preferred in order to
minimize changes in soil pH. This procedure uses IN NH^Cl as an
extractant.
5.2.2. MATERIALS AND SUPPLIES
5.2.2.1. EQUIPMENT
c 5.5-cm Buchner funnels
0 no. 40 or 42 Vhatman filter paper
0 250 mL Erlenmeyer flasks
0 250 ml side-arm suction flasks
0 aspiration vacuum apparatus
0 volumetric cylinder or repipette (25 ml)
° 60 mL polyethylene bottles
0 open pan balance (+ 0.01 g)
0 automatic Diluter/Dispenser
5.2.2.2. CHEMICALS/REAGENTS
0 Distilled-deionized (DI) water
° IN NH^Cl extracting solution (unbuffered):
(Dissolve 53.49 g crystalline NH^Cl in one liter of DI
water.)
0 Lanthanum Oxide (La203) (99.99% pure)
5.2.3. PROCEDURES
5.2.3.1. SAMPLE PREPARATION
1. Prepare soil (g): solution (ml) ratios for mineral or organic
soil samples as follows:
Mineral soil (< 40% LOI) - Add 100 ml of extracting solution
to 5 g of soil in a 250 ml Erlenmeyer flask or suitable
container.
Organic soil (> 402 LOI) - Add 100 ml of extracting solution
to 2 g of soil in a 250 ml Erlenmeyer flask or suitable
container.
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The amounts of soil listed in this step are considered the
minimum to be used with this procedure. More sample can be used
with either measurement, provided solution to solid ratio of
either 20:1 for mineral soils, and 50:1 for organic soils is
maintained.
2. Cap Erlenmeyer flasks with parafilm and shake vigorously for 1
hour on a mechanical shaker. Be sure shaking action is
sufficient to have extracting solution in contact with entire
sample. Failure to meet precision guidelines in the Data Quality
Objectives table may result because of poor shaking technique.
Shaking procedure should be evaluated if precision tests fail
quality control guidelines.
3. Filter the suspension, vith light suction, by transferring to a
Buchner funnel with no. 40 or 42 filter paper and collecting a
portion of filtrate in a 60 ml polyethylene bottle.
4. Add several drops of 12M BCl to acidify filtrate (pH < 1).
Addition of acid is necessary to preserve sample and prevent
microbial growth. Failure to add acid will result in microbial
grovth within filtrate within 24 hours.
5. It is recommended that the acidified filtrate not be stored for
longer than 2 weeks before analysis.
6. Follow guidelines detailed in Good Laboratory Practices section
for recording of all raw data. Note that raw data include the
following: (a) analytical observations necessary to calculate the
final results, (b) calibration data, (c) calibration checks, and
(d) quality control checks. Deviations from standard operating
procedures during sample preparation, calibration, or actual
analyses are to be fully documented and initialed by laboratory
personnel. Samples suspected of being in error or outside of the
calibration range are to be 'flagged', and this notation carried
through all records to the final report. Retain all written
materials (graphs, tables, etc.) generated as part of an
analysis. Do not discard portions of laboratory notebooks or any
other information directly related to calculation of the final
result for a set of samples.
5.2.3.2. EQUIPMENT OPERATION
It is assumed that either emission or atomic absorption spectroscopy
will be used to analyze contents of filtrates. Consult operating manuals
for detailed instructions on operation of instruments.
If alternative methods of elemental analysis are selected, precision
of the chosen methods must be equal to that available using emission or
atomic absorption spectroscopy.
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5.2.4. PREVENTIVE MAINTENANCE
Carry out all maintenance procedures as recommended by the
manufacturer. Record maintenance operations in maintenance log.
5.2.5. CALIBRATION PROCEDURES
1. Four standards plus a reagent zero should be used to calibrate
instrumentation for the analysis of each element. Standards
should define linear operating range of the instrument.
Standards should be run at the beginning and end of each sample
group and after a set number of samples within each group.
2. Prepare standards from certified commercial stock sources or from
primary reagen.ts.
3. Standard solutions should be prepared using class A volumetric
glassware and transferred to cleaned (IN HN03) polyethylene
bottles for storage between analyses. Special care should be
taken that temperature of solutions during preparation of
standards meets that stated for the calibration of the glassware.
4. Standard solutions are to be made up in IN NH^Cl extracting
solution. Dilution of filtrates and addition of matrix modifiers
will be necessary for analysis by flame atomic absorption
spectrophotometry. Lanthanum chloride (prepared from lanthanum
oxide, 5000 mg/1 in 0.5N HC1) is recommended as a matrix modifier
for determination of Ca and Mg by flame AAS.
5. Aspiration of IN NH^Cl will probably clog most burner assemblies
on emission and atomic absorption spectrometers. Dilution with
ammonium acetate is recommended as a matrix modifier to reduce or
eliminate problems from clogging of aspirators during analysis.
6. Establish typical diluting ranges for filtrates and prepare
standard solutions of suitable concentration in order to yield a
linear working curve after dilution. Use of an automatic
diluter-dispehser is highly recommended to ensure accurate and
rapid dilution.
7. Record all problems or other observations regarding calibration
in raw data notebook. Flag all samples that fall outside of
calibration range of instrument.
5.2.6. CALCULATIONS/UNITS
Standard curves should be fitted by the method of least squares after
hand graphing to check for continuity.
The following is an example of how to calculate the final
concentration (mg/kg oven-dried soil) of base cation extracted per sample:
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(AxDFxB) £ , ... . „ ,
- ^ - = yg of a base cation extracted
per g of soil = mg/kg
where A = pg of a base cation per ml of diluted filtrate aliquot, DF =
dilution factor, B = total volume of filtrate, and C - equivalent oven-
dried weight of sample.
Convert final concentration values to meq M+ per 100 g of soil, where
M+ represents the base cation determined.
Conversion to meq M+ per 100 g of soil can be done using the
following formula:
Y
2 x 0.1 o meq M+ per 100 g of soil
where Y = concentration of base cation M (mg/kg oven-dried soil) and Z =
equivalent weight of base cation M.
Equivalent weights for the exchangeable bases are as follows:
1 meq Ca = 20.OA mg Ca
1 meq Mg. = 12.156 mg Mg
1 meq K. =¦ 39.102 mg K
1 meq Na = 22.9898 mg Na.
5.2.7. ERROR ALLOWANCE AND DATA QUALITY
5.2.7.1. CONSIDERATIONS
Blanks, replicates, and in-house secondary standards will be included
in analyses of all samples. One replicate sample and one in-house control
will be included with every 10 soil samples extracted. Samples will be
chosen at random for replicates, but the operator should ensure that
samples of both mineral and organic soils are part of the quality control
procedure.
Samples selected as replicates should also be run on different days
to estimate day-to-day precision.
Replicate samples will be used to monitor precision. Percent
coefficient of variation will be calculated using the industrial statistic
I, where XCV = 200I//2 and I=|A-B|/A
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Critical ranges for repeated measurement error and accuracy will be
set by Quality Assurance Officer and listed in the Data Quality Objectives
(DQO) table.
Accuracy will be evaluated through the use of interlaboratory sample
exchanges. These exchanges will be coordinated by the Quality Assurance
Specialist assigned to the project.
Accuracy of the procedure can also be checked with use of spiked
samples. Spiked samples will be prepared such that the spike will contain
an appropriate amount of Ca, Mg, K and Na to approximately equal that
normally extracted from soil samples. The spike will be prepared as a
separate extracting solution and used in place of the extracting solution
in Section 5.2.3., step 1.
Percent recovery (%P) will be calculated using results from the
spiked samples using the following formula:
%P = 100 x A-B
S
where A = yg of Ca, Mg, K, or Na found for sample plus spike, B = Mg of
Ca, Mg, K, or Na found for sample, and S = ug of Ca, Mg, K, or Na added in
spike.
Note: Use of spikes with this procedure is a test of the procedure
itself and does not directly reflect upon the ability of laboratory
personnel to perform the analyses.
5.2.7.2. DATA QUALITY OBJECTIVES
Repeated Measurement
Measurement
Reporting
Error at
Accuracy
Variable
Units
Lower Limit Upper Limit
Tolerance
cmole(+)/kg
Ca
cmole(+)/kg
+ .05 + 1.00
N/A
= meq/lOOg
Mg
If
+ .03 + .50
N/A
K
If
+ .05 + .30
N/A
Na
11
+ .05 + .05
N/A
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5.2.7.3. COMPUTER DATABASE CODES
Variable
Code
Exchangeable Ca
Exchangeable Mg
Exchangeable K
Exchangeable Na
SXCA
SXMG
SXEK
SXNA
5.2.8. REFERENCES
Fernandez, I. 1983. Field Study Program Elements to Assess the
Sensitivity of Soils to Acidic Deposition Induced Alterations .in
Forest Productivity. National Council of the Paper Industry For Air
and Stream Improvement, Inc. (NCASI) Technical Bulletin No. 404. Nev
York, NY.
Johnson, D.V., D.tf. Cole, F.V. Horng, H. Van Miegroet, and D.E. Todd.
1982. Chemical Characteristics of Two Forested Ultisols and Two
Forested Inceptisols Relevant to Anion Production and Mobility. Oak
Ridge Nat. Lab. Environmental Sciences Division Publication No.
U.S. Environmental Protection Agency. 1979. Handbook for Analytical
Quality Control in tfater and Wastewater Laboratories. Environ. Mon.
and Sup. Lab. U.S. EPA. Cincinnati, OH. 45268. EPA-600/4-79-019.
1670.
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5.3. STANDARD OPERATING PROCEDURE FOR MEASUREMENT OF EXCHANGEABLE ACIDITY
5.3.1. SCOPE AND PURPOSE
This procedure is designed to determine exchangeable acidity content
(meq / 100 g of oven-dried sample) of soil samples. Exchangeable acidity,
sometimes referred to as potential acidity, is typically defined as the
sum of exchangeable A1 and exchangeable B removed from a soil by an
extracting solution. This procedure uses an unbuffered salt extracting
solution (IN KC1).
The procedure as presented is a modification of the standard method
in which total exchangeable acidity is determined by titration of the
unbuffered salt extract to the phenolphthalein endpoint (Thomas, 1982).
Also included is a procedure recently published by Logan et al. (1985).
Titration to the phenolphthalein endpoint overestimates the total
exchangeable acidity in. a soil sample and underestimates exchangeable Al
(Logan et al.t 1985). the phenolphthalein titration procedure is included
in this standard operating procedure, however, because it is the method by
which past determinations of exchangeable acidity have been measured.
Investigators may wish to try both procedures to determine the magnitude
of error in their results using the phenolphthalein endpoint titration
versus the approach of Logan et al. (1985).
Use of IN KC1 to extract exchangeable Al and exchangeable H is
largely based on work dealing with agronomic (mineral) soils. Use of this
extractant with organic soils should be approached with caution because
the pH of the extractant in equilibrium with organic soils often drops
below 3.0. Such low pH values may result in dissolution of non-
exchangeable Al, thus overestimating the amount of exchangeable Al in the
sample.
5.3.2. MATERIALS AND SUPPLIES
5.3.2.1. EQUIPMENT
0 5.5-cm Buchner funnels
° no. 40 or 42 Vhatman filter paper
0 125 & 250 mL Erlenmeyer flasks
° 250 ml side-arm suction flasks
0 aspiration vacuum apparatus
° volumetric cylinder or repipette (25 ml)
° 125 polyethylene bottles
0 open pan balance (+ 0.01 g)
0 Mettler memotitrator (or equivalent) fitted with
a Pt ring electrode
0 automatic Diluter/Dispenser
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5.3.2.2. CHEMICALS/REAGENTS
° Distilled-deionized (DI) water
0 IN Potassium Chloride (KC1) solution - transfer 74.56 g of KCL to
a 1 liter flask and bring to volume vith DI water.
° Potassium acid phthalate
0 Aluminum complexing solution, IN potassium fluoride (KF): titrate
58.1 g of KF in 1 liter of DI water to the phenolphthalein
endpoint with NaOH.
° 0.02N Sodium hydroxide (NaOH), standardized
0 0.1N Sodium hydroxide (NaOH), standardized (protect standardized
base from exposure to atmosphere)
° 0.1N Hydrochloric acid (HC1), standardized
0 Phenolphthalein solution: 1 g of phenolphthalein dissolved in 100
ml of ethanol.
5.3.3. PROCEDURES
5.3.3.1. SAMPLE PREPARATION
5.3.3.1.1. IN KCl Hethod
1. Veigh 5 g of <2 mm air-dried soil into a 250 ml Erlenmeyer
flask. Add 50 ml of IN KCl solution and cap tightly with
parafilm.
2. Shake 250 ml Erlenmeyer flasks vigorously for 1 hour on a
mechanical shaker. Be sure shaking action is sufficient to have
extracting solution in contact with entire sample. Failure to
meet precision guidelines in the Data Quality Objectives table
may result because of poor shaking technique. Shaking procedure
should be evaluated if precision tests fail quality control
guidelines.
3. Filter the suspension, with light suction, by transferring to a
Buchner funnel with no. 40 or 42 filter paper and collecting a
portion of the filtrate in a 125 ml polyethylene bottle.
4. Rinse soil filter cake in Buchner funnel with four 25 ml
aliquots of IN KCl.
4. Filtrate can be stored in polyethylene bottles until analysis.
Natural acidity of the filtrate will retard microbial growth,
but samples should be analyzed as soon as possible.
5.3.3.1.2. Phenolphthalein Endpoint Titration
1. Add 50 ml of filtrate to a 125 ml Erlenmeyer flask. Add 4 to 5
drops of phenolphthalein solution.
2. Titrate with 0.1N NaOH to the first permanent pink endpoint.
Add titrant slowly near endpoint to allow reaction to come to
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equilibrium. Pink endpoint should remain stable approximately
2-3 minutes. A deep pink color indicates titration has been
carried too far. Analysis should be restarted with fresh sample
aliquot.
3. Drift in color as one nears the pink endpoint is due to the
formation of an aluminum precipitate in solution. For samples
with substantial amounts of exchangeable Al, the precipitate
should be readily visible near the endpoint. Addition of base
must proceed slowly to allow base to diffuse into precipitate to
insure 100% neutralization of all trivalent Al in the extract.
4. Excess amounts of precipitate, as indicated by a translucent
suspension at the endpoint, are an indication that smaller
aliquots of filtrate should be used.
5. When pink endpoint is stable for 2-3 minutes, record the amount
of base added to the sample.
6. Add 10 ml of aluminum releasing solution (IN KF). Mix and
titrate with 0.1N HC1 until the pink color in solution
disappears. The pink color is due to the high pH of the solution
after addition of IN KF. The fluoride anion displaces hydroxyls
from the aluminum precipitate formed with addition of NaOH.
7. As with addition of NaOH, approach clear endpoint slowly to
allow dissolution of precipitate by IN KF. Solution should
remain clear for at least 30 minutes at the endpoint.
8. When at the clear endpoint, record the amount of acid added to
the sample.
9. Determine blank for IN KC1 extracting solution by titrating 50
ml (or equivalent volume) with 0.1N NaOH.
10. Discard all solutions when finished. Rinse with tap water.
Then treat all glassware with 0.1N HC1. Rinse with DI water.
5.3.3.1.3. Differential Potentiometric Titration (Logan et al., 1985)
1. Titrate 50 ml of IN KCl extract with 0.02N NaOH using a Mettler
memotitrator (model DL40) fitted with a Pt ring electrode. Use
the following instrumental settings as a guide in titrations:
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Parameter
Med to Blanks and
High Acidity Lov acidity
stir time(s)
increment for titrant (ml)
DeltaE end (mV)
Delta! (s)
DeltaE x trend (mV)
15
0.1
18
1
- 0.3
5
1
- 0.1
15
0.01
where stir time = equilibration time before starting titration,
DeltaE end = is the value that DeltaE must exceed to record
endpoint, DeltaT = time interval over which measurements of emf
are made, and DeltaE x trend = rate of change in emf readings
before addition of next increment of titrant.
2. Record emf readings for each increment of titrant added till
reaching endpoint.
3. Steps 1 and 2 are the steps to follow in determining total
acidity in the filtrate. The A1 content of the filtrate must be
determined using either atomic absorption or emission
spectroscopy or colorimetry (Alizarin Red S) (Logan et al.,
1985; Lancaster and Balasubramanian, 1974).
5.3.3.1.A. Documentation
Follow guidelines detailed in Good Laboratory Practices section for
recording of all raw data. Note that raw data include the following: (a)
analytical observations necessary to calculate the final results, (b)
calibration data, (c) calibration checks, and (d) quality control checks.
Deviations from standard operating procedures during sample preparation,
calibration, or actual analyses are to be fully documented and initialed
by laboratory personnel. Samples suspected of being in error or outside
of the calibration range are to be 'flagged', and this notation carried
through all records to the final report. Retain all written materials
(graphs, tables, etc.) generated as part of an analysis. Do not discard
portions of laboratory notebooks or any other information directly related
to calculation of the final result for a set of samples.
5.3.3.2. EQUIPMENT OPERATION
It is assumed that either emission or atomic absorption spectroscopy
will be to determine the Al content of the filtrates when using the
procedure of Logan et al., (1985). Consult operating manuals for detailed
instructions on operation of instruments.
If alternative methods of Al analysis are selected, precision of the
chosen methods must be equal to that available using emission or atomic
absorption spectroscopy.
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5.3.4. PREVENTIVE MAINTENANCE
Carry out all maintenance procedures as recommended by the
manufacturer. Record maintenance operations in maintenance log.
5.3.5. CALIBRATION PROCEDURES
1. Standardize base using potassium acid phthalate. Standardize
acid using standardized base. Record all readings when preparing
standardized acid and base.
2. Blank readings for IN KC1 extracting solution are required and
should be performed during titration of samples.
3. Prepare standards for determination of A1 via atomic absorption
spectroscopy or emission spectroscopy in IN KC1. Make dilutions
as needed to remain within the linear calibration range.
5.3.6. CALCULATIONS/UNITS
Phenolphthalein Endpoint Titration
Total acidity can be calculated using the following formula:
((A-B) x DF x N x 100) r „ „ n
- p - = meq of total acidity per 100 g
where A = volume of base added to reach phenolphthalein endoint, B =
volume of base for IN KC1 blank, DF = dilution factor (ratio of total
extraction volume to volume of aliquot used in titration), N = normality
of base, and C = equivalent oven-dry weight of sample.
Exchangeable Al can be calculated using the following formula:
(A x DF x N x 100) , , ,, *,
^ ¦= = meq of exchangeable Al per 100 g
v
where A = volume of acid added to reach clear endpoint, DF = dilution
factor (ratio of total extraction volume to volume of aliquot used in
titration), N = normality of acid, and C = equivalent oven-dry weight of
sample.
Exchangeable acidity can be calculated as the difference between
total exchangeable acidity and exchangeable aluminum. The results are
expressed as meq of exchangeable acidity per 100 g of oven-dry soil.
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Differential Potentiometric Titration
(Logan et al., 1985)
Determine the equivalence point for titration with 0.02N NaOH by
plotting recorded emf values against the volume of titrant added.
Record volume of titrant added at equivalence point.
Total exchangeable acidity in the sample ( meq / 100 g oven-dried
soil) can be calculated as follows:
^ p ^ X x 100 = meq / 100 g oven-dried soil
where A = ml of base for sample at equivalence point, B = ml of base for
blank .(IN KCl) at equivalence point, C = ratio of total extraction volume
of IN KCl for a sample to titration aliquot( N = normality of base
(0.02N), and D = equivalent oven-dried weight of sample.
Exchangeable Al (meq AL/ 100 g of oven-dried soil) can be calculated
as follows:
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Samples selected as replicates should also be run on different days
to estimate day-to-day precision.
Replicate samples will be used to monitor precision. Percent
coefficient of variation will be calculated using the industrial statistic
I, where %CV = 200I//2 and I=|A-B|/A+B, A and B being the results for the
replicate samples (U.S. EPA, 1979).
Precision will also be monitored using Shewhart control values for R
(range) values, if the total number of sample groups to be analyzed will
generate enough points to make use of such charts worthwhile.
Critical ranges for repeated measurement error and accuracy will be
set by Quality Assurance Officer and listed in the Data Quality Objectives
(DQO) table.
Accuracy will be evaluated through the use of interlaboratory sample
exchanges. These exchanges will be coordinated by the Quality Assurance
Specialist assigned to the project.
Accuracy of the procedure can also be checked with use of spiked
samples. Spiked samples will be prepared such that the spike will contain
an appropriate amount of acidity (Al +H) to approximately equal that
normally extracted from soil samples. The spike will be prepared as a
separate extracting solution and used in place of the extracting solution
in Section 5.3.3.1.1.
Percent recovery (%P) will be calculated using results from the
spiked samples using the following formula:
XP = 100 x ^
where A = meq of H or Al found for sample plus spike, B = meq of H or Al
found for sample, and S = meq of H or Al added in spike.
Note: Use of spikes with this procedure is a test of the procedure
itself and does not directly reflect upon the ability of laboratory
personnel to perform the analyses.
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5.3.7.2.
DATA QUALITY
OBJECTIVES
Repeated Measurement
Measurement
Reporting
Error at
Accuracy
Variable
Units
Lover Limit Upper Limit
Tolerance
Exchangeable cmole(+)/kg
Acidity
Exchangeable cmole(+)/kg
Al
Exchangeable cmole(+)/kg
H
cmole(+)/kg
+ .05 + 1.0
+ .05 + 1.0
+ .05 + 1.0
N/A
N/A
N/A
5.3.7.3. COMPUTER DATABASE CODES
Variable Code
Exchangeable Acidity STXA
Exchangeable Al SXAL
Exchangeable H SXEH
5.3.8. REFERENCES
Logan, K.A.B., M.J.S. Ploate and A.D. Ironside. 1985. Determination of
exchangeable acidity and exchangeable aluminum in hill soils. Part 1.
Exchangeable acidity. Commun. in Soil Sci. Plant Anal. 16:301-308.
Lancaster, L.A. and R. Balasubramaniam. 1974. An automated procedure for
the determination of aluminum in soil and plant digests. J. Sci. Fd.
Agric. 25:381-386.
Thomas, G.W. 1982. Exchageable cations. In: Methods of Soil Analysis,
Part 2. Chemical and Microbiological Properties. Agronomy Monograph
No. 9 (2nd Edition). ASA-SSSA, 677 S. Segoe Rd., Madison, VI.
U.S. Environmental Protection Agency. 1979. Handbook for Analytical
Quality Control in Water and Wastewater Laboratories. Environ. Mon.
and Sup. Lab. U.S. EPA. Cincinnati, OH. 45268. EPA-600/4-79-019.
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5.4. STANDARD OPERATING PROCEDURE FOR MEASUREMENT OF EXTRACTABLE METALS
(Fe, Zn, Cu, Pb, Cd, Ni, Mn)
5.4.1. SCOPE AND PURPOSE
This procedure is designed to determine the quantity of extractable
metal cations attached to the clay and organic constituents of a soil.
Extractable metals are defined as those metal cations that can directly
interact with the soil solution.
Extraction of metal cation depends on the extracting solution
selected. The extractant selected for this standard operating procedure
is 0.1N HC1 (Neuhauser and Hartenstein, 1980; Baker and Amacher, 1982).
5.4.2. MATERIALS AND SUPPLIES
5.4.2.1. EQUIPMENT
0 mechanical shaker
° analytical balance (0.01 gm)
0 250 ml Erlenmeyer flasks
0 60 ml polyethylene bottles with screw caps
0 No. 42 filter paper
0 Parafilm
0 filter funnels (9 cm)
° filter funnel racks
5.4.2.1. CHEMICALS/REAGENTS
0 Distilled-deionized (DI) water
0 15N Nitric acid (HN03)
° 6N Hydrochloric; acid (HCl) (It is recommended that HC1 be
redistilled in an all-Pyrex still before use. However, pure
HCl solutions (approximately 3M HCl) can be obtained using
isopiestic distillation, and redistilling of HCl is not
necessary.)
0 Extracting solution (0.1N HCl) - dilute 1 volume of redistilled 6N
HCl with 59 volumes of DI water. Alternatively, dilute
appropriate volume of standardized 3N HCl (isopiestic
distillation) to yield 0.1N HCl solution.
5.4.3. PROCEDURES
5.4.3.1. SAMPLE PREPARATION
1. Weigh 5.0 g of <2 mm air-dried soil into a 250 ml Erlenmeyer
flask.
2. Add 100 ml of .0-.IN HCl extracting solution.
3. Cap Erlenmeyer flasks with parafilm and shake vigorously for 1
hour on a mechanical shaker. Be sure shaking action is
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sufficient to have extracting solution in contact vith entire
sample. Failure to meet precision guidelines in the Data Quality
Objectives table may result because of poor shaking technique.
Shaking procedure should be evaluated if precision tests fail
quality control guidelines.
4. Remove flasks from shaker and let stand for 30 minutes to allow
suspension to settle.
5. Filter through No. 42 filter paper and collect in acid washed (IN
HNOj) polyethylene bottle. Note: All calculations will be based
on 100 ml extracting solution volume. Quantitative recovery of
extracting solution in filtration step is not necessary.
6. Filtrate can be stored at room temperature, but it is recommended
that analyses be completed as soon as possible.
7. Follow guidelines detailed in Good Laboratory Practices section
for recording of all raw data. Note that raw data include the
following: (a) analytical observations necessary to calculate the
final results, (b) calibration data, (c) calibration checks, and
(d) quality control checks. Deviations from standard operating
procedures during sample preparation, calibration, or actual
analyses are to be fully documented and initialed by laboratory
personnel. Samples suspected of being in error or outside of the
calibration range are to be 'flagged', and this notation carried
through all records to the final report. Retain all written
materials (graphs, tables, etc.) generated as part of an
analysis. Do not discard portions of laboratory notebooks or any
other information directly related to calculation of the final
result for a set of samples.
5.4.3.2. EQUIPMENT OPERATION
It is assumed that either emission or atomic absorption spectroscopy
will be used to analyze contents of filtrates. Consult operating manuals
for detailed instructions on operation of instruments.
If alternative methods of elemental analysis are selected, precision
of the chosen methods must be equal to that available using emission or
atomic absorption spectroscopy.
5.4.4. PREVENTIVE MAINTENANCE
Carry out all maintenance procedures as recommended by the
manufacturer. Record maintenance operations in maintenance log.
5.4.5. CALIBRATION PROCEDURES
1. Four standards plus a reagent zero should be used to calibrate
instrumentation for the analysis of each element. Standards
should define optimum operating range of the instrument.
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Standards should be run at the beginning and end of each sample
group and after a set number of samples within each group.
2. Prepare standards from certified commercial stock sources or from
primary reagents.
3. Standard solutions should be prepared using class A volumetric
glassware and transferred to cleaned (IN HN03) polyethylene
bottles for storage between analyses. Special care should be
taken that temperature of solutions during preparation of
standards meets that stated for the calibration of the glassware.
4. Establish typical diluting ranges for filtrates and prepare
standard solutions of suitable concentration. Use of an
automatic diluter-dispenser is highly recommended to ensure
accurate and rapid dilution.
5. Record all problems or other observations regarding calibration
in raw data notebook. Flag all samples that fall outside of
calibration range of instrument.
5.4.6. CALCULATIONS/UNITS
Standard curves should be fitted by the method of least squares after
hand graphing to check for continuity.
The following is an example of how to calculate the final
concentration (mg/kg oven-dried soil) of metal cation extracted per
sample:
(A x DF x B) _ '0.1N HC1 extractable' metal/kg
where A = concentration of metal in aliquot as determined from standard
curve, DF = dilution factor if required, B = total volume of extracting
solution (100 ml), and C = equivalent oven-dried weight of sample.
5.4.7. ERROR ALLOWANCE AND DATA QUALITY
5.4.7.1. CONSIDERATIONS
Blanks, replicates, and in-house secondary standards will be included
in analyses of all samples. One replicate sample and one in-house
secondary standard will be included with every 10 soil samples extracted.
Samples will be chosen at random for replicates, but the operator should
ensure that samples of both mineral and organic soils are part of the
quality control procedure.
Samples selected as replicates should also be run on different days
to estimate day-to-day precision.
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Replicate samples will be used to monitor precision. Percent
coefficient of variation will be calculated using the industrial statistic
I, where %CV = 200I//2 and I=|A-B|/A+B, A and B being the results for the
replicate samples (U.S. EPA, 1979).
Precision will also be monitored using Shewhart control values for R
(range) values, if the total number of sample groups to be analyzed will
generate enough points to make use of such charts worthwhile.
Critical ranges for repeated measurement error and accuracy will be
set by Quality Assurance Officer and listed in the Data Quality Objectives
(DQO) table.
Accuracy will be evaluated through the use of interlaboratory sample
exchanges. These exchanges will be coordinated by the Quality Assurance
Specialist assigned to the project.
Accuracy of the procedure can also be checked with use of spiked
samples. Spiked samples will be prepared such that the spike will contain
an appropriate amount of metals to approximately equal that normally
extracted from soil samples. The spike will be prepared as a separate
extracting solution and used in place of the extracting solution in
Section 5.4.3.1.
Percent, recovery (£P) will be calculated using results from the
spiked samples using the following formula:
XP = 100 x
where A = yg of metal ion found for sample plus spike, B = yg of metal ion
found for sample, and S = yg of metal ion added in spike.
Note: Use of spikes with this procedure is a test of the procedure
itself and does not directly reflect upon the ability of laboratory
personnel to perform the analyses.
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5.4.7.2. DATA QUALITY OBJECTIVES
Repeated Measurement
Measurement
Reporting
Error at
Accuracy
Variable
Units
Lover Limit Upper Limit
Tolerance
mg/kg
Fe
1.0 + 1.0
+
5.0
N/A
Zn
0.1 + 0.1
+
5.0
N/A
Cu
0.1 + 0.1
+
0.5
N/A
Pb
0.1 + 0.1
+
1.0
N/A
Cd
0.1 + 0.1
+
0.2
N/A
Ni
0.1 + 0.1
+
0.1
N/A
Mn
1.0 + 1.0
+
5.0
N/A
>.7.3.
COMPUTER DATABASE CODES
Variable
Code
Extractable Fe
SEFE
Extractable Zn
SEZN
Extractable Cu
SECU
Extractable Pb
SEPB
Extractable Cd
SECD
Extractable Ni
SENI
Extractable Mn
SEMN
5.4.8. REFERENCES
Baker, D.E. and M.C. Amacher. 1982. Nickel, Copper, Zinc, and Cadmium.
In: Methods of Soil Analysis. Part 2. Chemical and Microbiological
Properties. 2nd edition, A.L. Page (ed.). ASA Monograph No. 9. ASA-
SSSA. Madison, VI.
Burau, R.G. 1982. Lead. In: Methods of Soil Analysis. Part 2. Chemical
and Microbiological Properties. 2nd edition, A.L. Page (ed.). ASA
Monograph No. 9. ASA-SSSA. Madison, VI.
Gambrell, R.P. and V.H. Patrick,Jr. 1982. Manganese. In: Methods of
Soil Analysis. Part 2. Chemical and Microbiological Properties. 2nd
edition, A.L. Page (ed.). ASA Monograph No. 9. ASA-SSSA. Madison, VI.
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Neuhauser, E.F. and R. Hartenstein. 1980. Efficiencies of extractants
used in analyses of heavy metals in sludges. J. Environ. Qual. 9:21-
22.
Olson, R.V. and R. Ellis,Jr. 1982. Iron. In: Methods of Soil Analysis.
Part 2. Chemical and Microbiological Properties. 2nd edition, A.L.
Page (ed.). ASA Monograph No. 9. ASA-SSSA. Madison, VI.
U.S. Environmental Protection Agency. 1979. Handbook for Analytical
Quality Control in Water and Wastewater Laboratories. Environ. Mon.
and Sup. Lab. U.S. EPA. Cincinnati, OH. 45268. EPA-600/4-79-019.
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5.5. STANDARD OPERATING PROCEDURE FOR MEASUREMENT OF EXTRACTABLE
PHOSPHORUS ('BRAY 1')
5.5.1. SCOPE AND PURPOSE
This procedure is designed to measure extractable phosphorus.
Determination of phosphorus in soils requires careful consideration of
various methods of extraction as well as detection. Phosphorus extractions
may yield total P, including organic and inorganic forms, organic P alone,.
or "available" P, that amount which is soluble in acid and of immediate
significance to tree growth. The procedure described in this section
yields an estimate of "available" P in acid soils.
5.5.2. MATERIALS AND SUPPLIES
5.5.2.1. EQUIPMENT
° UV/Vis spectrophotometer
° 1 cm flow thru cell for spectrophotometer
° analytical balance (0.001 g)
° mechanical shaker
° volumetric flasks
0 repipettes (5-10 ml capacity)
° 20 ml plastic centrifuge tubes
0 50 ml Erlenmeyer flasks
° 60 ml polyethylene bottles with screw caps
° No. 42 filter paper
° filter funnels (9 cm)
° filter funnel racks
° Parafilm
0 vortex mixer
5.5.2.2. CHEMICALS/REAGENTS
° 12M Hydrochloric acid (HC1)
° 15N Nitric acid (HN03)
0 Distilled-deionized (DI) water
° Stock phosphate solution: dissolve 0.4393 g of oven dry potassium
dihydrogen phosphate in DI water. Dilute to 1 liter total
volume. One ml of stock solution = 100 ug of P.
0 IN Ammonium fluoride (NH^F): dissolve 37 g of NH^F in DI water and
dilute to 1 liter. Store in polyethylene bottle.
0 0.5 N Hydrochloric acid: add 20.2 ml 12M HCl to a 500 ml
volumetric flask, bring to volume with DI water..
0 Extracting solution: Add 15 ml of IN NH^F and 25 ml of 0.5N HCl to
460 ml of distilled water.
0 Stannous chloride dihydrate (SnCl2*2H20) stock solution: dissolve
10 g of SnCl2*2H20 in 25 ml of 12M HCl. Store in black, glass
stoppered bottle. Prepare fresh solution every 6 weeks.
0 Ammonium paramolybdate [ (NH^KMo^O-^* ,H„0]: dissolve 15 g of
ammonium paramolybdate in 350 ml or DI water. Add 350 ml of
10N HCL while slowly stirring solution. Allow to cool, then
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bring to 1 liter total volume with DI water. Store in black,
glass stoppered bottle. Prepare fresh solution every 2 months.
0 Stannous chloride dilute solution: mix 1 ml of SnCl2 stock
solution with 333 ml of DI vater. Make fresh daily just before
use. Solution is stable for only 2 hours.
5.5.3. PROCEDURE
5.5.3.1. SAMPLE PREPARATION
1. Weigh 1 g of < 2 mm air-dried soil into a 20 ml centrifuge tube.
2. Add 7 ml of extracting solution, cap tightly and mix for 1
minute.
3. Filter suspension through,No. 42 filter paper into a 20 ml acid
vashed (IN HNO,) plastic tube. Cap with parafilm and store until
analysis.
A. Transfer 2 ml of sample or standard solution into 20 ml plastic
tube. Add 5 ml of DI water (repipette).
5. Add 2 ml of molybdate-vanadate solution (repipette). Mix contents
on vortex mixer. Add 1 ml of SnCl2 dilute solution and mix on
vortex mixer. Final acidity of color solution is critical.
Solution should be 0.36 N to produce optimum results.
6. Allow color to develop for 10 minutes and measure absorbance at
660 nm. Record absorbance reading when readout is stable.
7. Follow guidelines detailed in Good Laboratory Practices section
for recording of all raw data. Note that raw data include the
following: (a) analytical observations necessary to calculate the
final results, (b) calibration data, (c) calibration checks, and
d) quality control checks. Deviations from standard operating
procedures during sample preparation, calibration, or actual
analyses are to be fully documented and initialed by laboratory
personnel. Samples suspected of being in error or outside of the
calibration range are to be 'flagged', and this notation carried
through all records to the final report. Retain all written
materials (graphs, tables, etc.) generated as part of an
analysis. Do not discard portions of laboratory notebooks or any
other information directly related to calculation of the final
result for a set of samples.
5.5.3.2. EQUIPMENT OPERATION
Consult operating manual of spectrophotometer for proper adjustment
of 0XT and 1002T before taking readings.
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5.5.4. PREVENTIVE MAINTENANCE
Perform all maintenance procedures specified in the spectrophotometer
operating manual. Check COT, 100%T and 1 Abs settings weekly during use.
Check these settings daily if the spectrophotometer is a common use
instrument.
Record maintenance operations in maintenance log.
5.5.5. CALIBRATION PROCEDURES
1. Prepare standard working curve using from stock P solution.
Standards should range from 0.1 to 1.0 yg P per 2 ml of
extracting solution.
2. Zero instrument using blank prepared with 2 ml of extracting
solution.
3. Read set of standards at the beginning and end of analyses. Read
individual standards with samples during analysis.
5.5.6. CALCULATIONS/UNITS
Standard curves should be fitted by the method of least squares after
hand graphing to check for continuity.
Blank values are not subtracted from readings because sample blanks
and standard blanks should be nearly identical.
The concentration of "available" P in a sample (mg P/kg oven-dried
soil) will be calculated using the following equation:
^ ^ = mg "available" P/kg oven-dried soil
V
where A = concentration of P in 2 ml aliquot determined from standard
curve, B = ratio of sample aliquot (2 ml) to total volume of extracting
solution (7 ml), and C = oven-dried weight of soil sample.
5.5.7. ERROR ALLOWANCE AND DATA QUALITY
5.5.7.1. CONSIDERATIONS
Blanks, replicates, and in-house secondary standards will be included
in analyses of all samples. One replicate sample and one in-house
secondary standard will be included with every 10 soil samples extracted.
Samples will be chosen at random for replicates, but the operator should
ensure that samples of both mineral and organic soils are part of the
quality control procedure.
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Samples selected as replicates should also be run on different days
to estimate day-to-day precision.
Replicate samples will be used to monitor precision. Percent
coefficient of variation will be calculated using the industrial statistic
I, where £CV = 200I//2 and I=|A-B|/A+B, A and B being the results for the
replicate samples (U.S. EPA, 1979).
Precision will also be monitored using Shewhart control values for R
(range) values, if the total number of sample groups to be analyzed will
generate enough points to make use of such charts worthwhile.
Critical ranges for repeated measurement error and accuracy will be
set by Quality Assurance Officer and listed in the Data Quality Objectives
(DQO) table.
Accuracy will be evaluated through the use of interlaboratory sample
exchanges. These exchanges will be coordinated by the Quality Assurance
Specialist assigned to the project.
Accuracy of the procedure can also be checked with use of spiked
samples. Spiked samples will be prepared such that the spike will contain
an appropriate amount of P to approximately equal that normally extracted
from soil samples. The spike will be prepared as a separate extracting
solution and used in place of the extracting solution in Section 5.5.3.1.,
step 1.
Percent recovery (XP) will be calculated using results from the
spiked samples using the following formula:
XP = 100 x
where A = ug of P found for sample plus spike, B = yg of P found for
sample, and S = yg of P added in spike.
Note: Use of spikes with this procedure is a test of the procedure
itself and does not directly reflect upon the ability of laboratory
personnel to perform the analyses.
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5.5.7.2. DATA QUALITY OBJECTIVES
Repeated Measurement
Measurement
Reporting
Error
at
Accuracy
Variable
Units
Lover Limit
Upper Limit
Tolerance
Extractable
mg/kg
20% (cv)
5% (cv)
N/A
Phosphorus
5.5.7.3. COMPUTER DATABASE CODES
Variable
Code
Extractable P
SEBP
5.5.8. REFERENCES
Olsen, S.R. and L.E. Sommers. 1982. Phosphorus. In: Methods of soil
Analysis. Part 2. Chemical and Microbiological Properties. 2nd
edition, A.L. Page!(ed.). ASA Monograph No. 9. ASA-SSSA. Madison,
WI.
U.S. Environmental Protection Agency. 1979. Handbook for Analytical
Quality Control in Water and Wastewater Laboratories. Environ. Mon.
and Sup. Lab. U.S. EPA. Cincinnati, OH 45268. EPA-600/4-79-019.
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5.6. STANDARD OPERATING PROCEDURE FOR MEASUREMENT OF EXTRACTABLE
SULFATE-S
5.6.1. SCOPE AND PURPOSE
The purpose of this procedure is to measure extractable sulfate-S.
In this procedure tvo extractants will be used to determine the sulfate-S
content of soils: distilled-deionized water and 0.016M sodium phosphate
solution.
Extraction vith distilled-deionized water will estimate the total
amount of soluble sulfate in the soil. This fraction includes sulfate-S
in the soil solution and readily soluble sulfate salts.
Extraction vith Q.016M sodium phosphate vill estimate the amount of
adsorbed sulfate-S retained on clay and iron and aluminum oxide surfaces.
Neither of these extractants vill estimate the amount of relatively
insoluble sulfates (e.g. aluminum sulfate) present in the soil.
This procedure assumes that sulfate-S in the extracts vill be
determined by ion-exchange chromatography.
5.6.2. MATERIALS AND SUPPLIES
5.6.2.1. EQUIPMENT
0 centrifuge (3000 rpm minimum, 50 ml capacity holders)
0 mechanical shaker
0 vortex mixer
0 Ion chromatograph (Dionex or equivalent)
° automatic injection system (optional)
0 analytical balance (0.001 gm)
0 100 ml centrifuge tubes vith screw caps
• 60 ml polyethylene bottles vith screv caps
• volumetric flasks (100 ml, 1 liter)
0 membrane filters (0.2 micron)
° filtration apparatus for membrane filters
° volumetric pipettes
0 repipette
° Parafilm
5.6.2.2. CHEMICALS/REAGENTS
0 Distilled-deionized (DI) water
0 15M Nitric acid (HN03)
0 Monobasic sodium phosphate (NaH-PO.-H-O)
0 Extracting solution - transfer 2.227 g of NaH-PO^'HjO to a 1 liter
volumetric flask. Dissolve in 100 ml of DI water. Bring to
volume vith DI water (0.016M NaH-PO.-H-O, 500 mg/1 P)
0 Magnesium sulfate (MgSO^)
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0 Stock Sulfate-S standard (1000 mg/1 S0^) - transfer 1.2530 g of
MgSO, into a 1 liter volumetric flask. Dissolve in DI water
and Bring to volume with DI water.
5.6.3. PROCEDURES
5.6.3.1. SAMPLE PREPARATION
5.6.3.1.1. Distilled-Deionized Vater Method
1. Weigh 4.00 g of <2 mm air-dried soil into a tared 100 ml
centrifuge tube. Add 80 ml of DI water. Cap and mix contents
with vortex mixer.
2. Place centrifuge tubes on shaker for 1 hour. Remove every 15
minutes and invert tubes to ensure dispersion of total sample.
3. Centrifuge tubes at 1900 rpm for 15 minutes.
4. Filter aliquot of supernatant through 0.2 micron filter and
store filtrate in acid washed (IN HN03) 60 ml polyethylene
bottle until analysis. Refrigerate bottles at 4°C. It is
recommended that sulfate content be determined the same day.
5.6.3.1.2. 0.016M NaHoP0, Method
2—4
1. Weigh 4.00 g of <2 mm air-dried soil into a 100 ml centrifuge
tube.
2. Add 20 ml of 0.016M Na^PO^ extracting solution. Cap and mix
contents with vortex mixer.
3. Place tubes on shaker for 30 minutes. Remove at 15 minute mark
and invert tubes to ensure dispersion of total sample.
4. Centrifuge tubes at 1500 rpm for 10 minutes.
5. Decant supernatant into 100 ml volumetric flask.
6. Repeat steps 2 through 5 three (3) more times.
7. Bring 100 ml volumetric flasks to volume with 0.016M Naf^PO,
extracting solution. Cap volumetric flask with Parafilm ana mix
end-to-end 10 times to mix contents.
8. Filter aliquot from 100 ml volumetric flask through 0.2 micron
filter and collect filtrate in an acid washed (IN HN03) 60 ml
polyethylene bottle. Refrigerate (4°C) until ready for
analysis.
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5.6.3.1.3. Documentation
Follow guidelines detailed in Good Laboratory Practices section for
recording of all raw data. Note that raw data include the following: (a)
analytical observations necessary to calculate the final results, (b)
calibration data, (c) calibration checks, and (d) quality control checks.
Deviations from standard operating procedures during sample preparation,
calibration, or actual analyses are to be fully documented and initialed
by laboratory personnel. Samples suspected of being in error or outside
of the calibration range are to be 'flagged', and this notation carried
through all records to the final report. Retain all written materials
(graphs, tables, etc.) generated as part of an analysis. Do not discard
portions of laboratory notebooks or any other information directly related
to calculation of the final result for a set of samples.
5.6.3.2. EQUIPMENT OPERATION
It is assumed that sulfate-S in extracts will be determined using
ion-exchange chromatography.
Consult instruction manual for proper operation of ion chromatograph.
Different eluents may be required because of the use of the phosphate
anion as an extractant. The high concentration of phosphate anion in the
0.016M Na^PO^ extractant will completely saturate the separator column
and produce a broad output peak. Unless appropriate steps are taken, this
peak will interfere with or completely mask the peak from the sulfate
anion.
One approach to running phosphate extracts is to reduce eluant
concentration to 2.0 millimolar carbonate. Lower eluant concentrations
result in slower peak elution and improved phosphate and sulfate peak
resolution. Another alternative requires use of a buffered eluant with a
pH (e.g., pH = 12.5) greater than the pK of HPO, (pK = 12.37), resulting
in phosphate peak elution after the sulfate peak with improved resolution.
For most situations investigators are strongly urged to refer to
manufacturer recommendations and to communicate with the manufacturer's
technical support division in developing acceptable measurement techniques
for sulfate concentrations in the phosphate extracts.
When using non-suppressed ion-exchange chromatography, presence of
bicarbonate and carbonate anions in the extract may result in negative or
positive peaks which coincide with that from the sulfate anion. Steps
should be taken to remove carbon dioxide from the extract, or to shift the
position of the eluding carbonate peak.
Zero instrument output with aliquot of extracting solution (either DI
water or 0.016M NaH„P0.).
2 4
Prepare standards in extracting solution selected.
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Use a strip chart recorder or integrator to record output. Record
either peak height or peak area for sample and standards. Peak area
measurements generally are linear, whereas, peak height measurements tend
to be non-linear, especially at higher concentrations.
When using a strip chart recorder, set range on recorder to record
output throughout entire width of recorder paper. Avoid recorder settings
that limit the sensitivity of the final readout when using a stripchart
recorder.
5.6.4. PREVENTIVE MAINTENANCE
Carry out all maintenance procedures as recommended by the
manufacturer. Record maintenance operations in maintenance log.
Keep a record of eluted sulfate peaks to determine condition of
separator column over time. Change guard column on a regular basis.
Change separator columns when peak shapes shift and continued elution for
long periods of time do not restore peak shape.
5.6.5. CALIBRATION PROCEDURES
Four standards plus a reagent zero should be used to calibrate ion
chromatograph. Several analytical ranges are possible because of the wide
linear response of the detector. Standards will be run at the beginning
and end of each sample group and after a set number of samples within each
group.
Standard solutions should be prepared using class A volumetric
glassware and transferred to cleaned (IN HNOj) polyethylene bottles for
storage between analyses. Special care should be taken that temperature
of solutions during preparation of standards meets that stated for the
calibration of the glassware.
5.6.6. CALCULATIONS/UNITS
Standard curves should be fitted by the method of least squares after
hand graphing to check for continuity.
Distilled-Deionized Water Method
Final concentration (mg/kg oven-dried soil) of sulfate-S in a sample
will be calculated as follows:
CA x C)
-—g-—- = mg sulfate-S/kg oven-dried soil
where A = ug of sulfate-S per ml of extracting solution as determined from
the calibration curve, C = total volume of extractant (80 ml), and B =
equivalent oven-dried weight of sample.
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Multiply by 0.033379 to convert results to mg S/kg oven-dried soil.
Report results as mg extractable S (distilled-deionized water)/ kg
oven-dried soil.
0.016M NaH^PO^ Method
Final concentration (mg/kg oven-dried soil) of sulfate-S in a sample
will be calculated as follows:
= mg sulfate-S/kg oven-dried soil
where A = yg of sulfate-S per ml of extracting solution as determined from
the calibration curve for 0.016M Nal^PO^, C = total volume of
extractant(100 ml), and B = equivalent oven-dried weight of sample.
Multiply by 0.033379 to convert results to mg S/kg oven-dried soil.
Report results as mg extractable S (0.016M NaH-PO,)/ kg oven-dried
soil.
5.6.7. ERROR ALLOWANCE AND DATA QUALITY
5.6.7.1. CONSIDERATIONS
Blanks, replicates, and in-house secondary standards should be
included in analyses of all samples. One replicate sample and one in-
house secondary standard should be included with every 10 soil samples
extracted. Samples should be chosen at random for replicates, but the
operator should ensure that samples of both mineral and organic soils are
part of the quality control procedure. Spiked samples are not appropriate
for use with this extractant.
Samples selected as replicates should also be run on different days
to estimate day-to-day precision.
Replicate samples will be used to monitor precision. Percent
coefficient of variation will be calculated using the industrial statistic
I, where %CV = 200I//2 and I=|A-B|/A+B, A and B being the results for the
replicate samples (U.S. EPA, 1979).
Precision will also be monitored using Shewhart control values for R
(range) values, if the total number of sample groups to be analyzed will
generate enough points to make use of such charts worthwhile.
Critical ranges for repeated measurement error and accuracy will be
set by Quality Assurance Officer and listed in the Data Quality Objectives
(DQ0) table.
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Accuracy will be evaluated through the use of interlaboratory sample
exchanges. These exchanges vill be coordinated by the Quality Assurance
Specialist assigned to the project.
Accuracy of the 0.016M sodium phosphate procedure can also be
checked with use of spiked samples. Spiked samples will be prepared such
that the spike vill contain an appropriate amount of sulfate-S to
approximately equal that normally extracted from soil samples. The spike
will be prepared as a separate extracting solution and used in place of
the extracting solution in Section 5.6.3.1.
Percent recovery (XP) will be calculated using results from the
spiked samples using the following formula:
X? = 100 x
where A = yg of sulfate-S found for sample plus spike, B = yg of sulfate-S
found for sample, and S = yg of sulfate-S added in spike.
Note: Use of spikes with this procedure is a test of the procedure
itself and does not directly reflect upon the ability of laboratory
personnel to perform the analyses.
5.6.7.2. DATA QUALITY OBJECTIVES
Repeated Measurement Measurement
Reporting Error at Accuracy
Variable Units Lower Limit Upper Limit Tolerance
Extractable mg/kg 20% (cv) 5X (cv) N/A
Sulfate
5.6.7.3. COMPUTER DATABASE CODES
Variable Code
Water Extractable S SEWS
Phosphate Extractable S SEPS
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5.6.8. REFERENCES
Blume, L. J. 1985. Statement of Work. National Acid Deposition Soil
Survey. Chemical and Physical Characterization of soils. IFB No. VA
85-566. Environ. Mon. Sys. Lab. U.S. EPA, Las Vegas, NV. 89114.
Tabatabai, M.A. 1982. Sulfur. In: Methods of Soil Analysis, Part 2.
Chemical and Microbiological Properties. ASA Monograph No. 9. 2nd
Edition, A.L. Page (eds.). ASA-SSSA, 677 S. Segoe Rd., Madison, VI.
53711.
Johnson, D.V. and G.S. Henderson. 1979. Sulfate adsorption and sulfur
fractions in a highly weathered soil under a mixed deciduous forest.
Soil Sci. 128:34-40.
U.S. Environmental Protection Agency. 1979. Handbook for Analytical
Quality Control in Water and Wastewater Laboratories. Environ. Mon.
and Sup. Lab. U.S. EPA. Cincinnati, OH. 45268. EPA-600/4-79-019.
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5.7. STANDARD OPERATING PROCEDURE FOR MEASUREMENT OF TOTAL (KJELDAHL)
NITROGEN
5.7.1. SCOPE AND PURPOSE
This procedure is designed to determine the total nitrogen content of
the forest floor and mineral soil.
This standard operating procedure (SOP) is based on the Kjeldahl
method for determining 1 the total N content of soils. The basic procedure
is performed in a matrix of boiling sulfuric acid (H„S0^) with salt and
catalysts added to increase the rate of oxidation. The end product is
conversion of organically bound N to NH^-N. Recovery of N from soils and
forest floor material has been shown to be satisfactory, even though the
Kjeldahl procedure does not give accurate results for N-containing
compounds having N-N and N-0 linkages (Bremner and Mulvaney, 1982). A
pretreatment step added to the original Kjeldahl procedure yields
acceptable recovery of;N from compounds having N-N and N-0 linkages, but
it appears that such compounds do not exist in soils or forest floor
material (Bremner and Mulvaney, 1982). Therefore, the pretreatment step
is not included in this standard operating procedure.
Two digestion mixtures (K„S0,+Se+Cu or K„S0^+Hg+Cu) commonly used for
determining Kjeldahl N are included in this SOP. Both mixtures are often
cited in the peer reviewed literature, and appear to yield essentially
identical results for most soil and forest floor material. Also included
is a manual procedure for determining the NH^-N content of the digestate,
even though it is recognized that use of a colorimetric procedure in
combination with an AutoAnalyzer is the method of choice for doing large
numbers of samples.
5.7.2. MATERIALS AND METHODS
5.7.2.1. EQUIPMENT
0 analytical balance (+ 0.001 g)
0 digestion block with digestion tubes (or equivalent)
0 steam distillation apparatus that will accommodate digestion tubes
0 250 ml Erlenmeyer flasks
0 10 ml buret with reservoir
0 repipette (10 ml)
0 boiling chips
° plastic vials (15 ml) with air-tight caps
5.7.2.2. CHEMICALS/REAGENTS
K^SO^+Se+Cu Digestion Mixture
0 18M Sulfuric acid (H_S0,)
0 Distilled deionized (DIJ water
0 Potassium sulfate (^SO^)
° Selenium powder
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° Copper sulfate (CuS0,*5H„0)
0 Digestion mixture: mix luOO g of 8 CuSO, *5^0 and 5 g
of Se powder together. Use suitable protection vnen preparing
mixture to prevent skin contact or inhalation of dust.
Commercial formulations approximating this formula may be
available and are acceptable to use in place of preparation by
laboratory personnel.
K^SO^+Hg+Cu Digestion Mixture
0 18M Sulfuric acid (H-SO.)
° Distilled deionized (DIJ water
0 Kel-Pak No. 1 digestion catalyst (9.9g F^SO^O^lg Hg0:0.08g CuSO^
) (available from Curtin Matheson Scientific, Inc.)
NH^-N Determination by Distillation and Titration
0 Methyl red (Na salt)
0 Methylene blue
° Distilled deionized (DI) water
° Boric acid (H3B03)
0 15N Sodium hydroxide (NaOH)
° Sodium thiosulfate (Na2S203)
0 Potassium bi-iodate (primary reagent grade) (KH(I03)2)
° Mixed indicator solution: Dissolve 200 mg of methyl red in 100 ml
of DI. water. Dissolve 100 mg of methylene blue in 50 ml of DI
water. Store at 4°C. Solution is stable for 1 month.
0 Boric acid solution: dissolve 20 g of boric acid and 10 ml mixed
indicator solution in 1 liter of DI water. Store at 4°C.
0 15N NaOH solution: dissolve 600 g of NAOH and 40 g of Na2S203 in 1
liter of DI water.
0 Potassium Bi-Iodate titrant: strength of titrant is a function of
N content of samples -
0 0.01N KH(I03)2 - transfer 3.8862 g to 1 liter volumetric flask and
bring to mark with DI water.
0 0.05N KH(I03)j - transfer 19.4310 g to 1 liter volumetric flask
and bring to mark with DI water.
5.7.3. PROCEDURES
5.7.3.1. SAMPLE PREPARATION
This procedure requires samples that are finely ground. The
subsample selected for further grinding must be of sufficient size to
include a representative sampling of the various discrete particles that
make up the total soil sample. Failure to take a large enough subsample
will introduce a bias into the final results that will not be detected
unless a separate subsample is taken, finely ground, and analyzed. It is
recommended that at least 10 g be chosen as the minimum subsample size
from which to prepare finely ground samples.
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Preparation of finely ground subsamples can be done by hand using an
agate mortar and pestle or with a mechanical grinder, ball mill or mixer
mill (such as a Spex mixer mill or equivalent). Use of a Spex mixer mill
(or equivalent) to prepare aliquots of finely ground soil requires
selection of a suitable sample mixer container because the sample will
become contaminated from contact vith container walls during the grinding
process. Tungsten carbide containers are recommended for use with a mixer
mill.
5.7.3.1.1. K^SO^+Se+Cu Digestion Mixture
1. Add approximately 1.5 g of catalyst and 2 or 3 boiling chips to
digestion tubes (Pyrex tubes 25 x 200 mm).
2. Weigh out 0.5 g of finely ground soil or 0.1 to 0.2 g of forest
floor and quantitatively transfer to digestion tube. Note: 0.5
g of forest floor will consume a large amount of f^SO^ and
result in an incomplete digestion.
3. Add 6 ml of 18M sulfuric acid. Cap tubes and let stand
overnight.
4. Remove caps and place tubes on preheated digestion block at
350°C.
5. Continue digestion at 350°C for 1 hour after solution clears.
Note: reference is made to a .color change, not total dissolution
of the mineral matrix of the soil. Solutions may still be
turbid but clear of color.
6. Remove tubes from block and let cool. Dilute with approximately
20 ml of DI water. Mix well and let cool to room temperature.
7. Bring to 50 ml calibration mark with DI water. Mix well, cap
and let stand overnight.
8. Decant off aliquot for analysis (15 ml) and discard remainder of
the digestate. Do NOT pour digestate down sink. Follow
guidelines established by host institution for disposal of toxic
wastes.
9. Store aliquot in air-tight plastic vial until analysis.
5.7.3.1.2. K^SO^+Hg+Cu Digestion Mixture
1. Add approximately 3 g of Kel-pak digestion mixture to a
digestion tube. It is recommended that 250 ml digestion tubes
be used if NH^-N is to be determined by distillation. Most
automatic steam distillation units are equipped to accommodate
250 ml digestion tubes.
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2. Veigh out 0.5 g of finely ground soil or 0.1 to 0.2 g of forest
floor and quantitatively transfer to digestion tube.
3. Add 10 ml of 18M sulfuric acid, using acid to remove traces of
sample from the sides of the digestion tube.
4. Place on a preheated digestion block (385°C) and heat until
solution clears. Continue heating for 0.5 hour after solution
clears. Note: reference is made to a color change, not total
dissolution of the mineral matrix of the soil. Solutions may
still be turbid but clear of color.
5. Remove tubes from block and let cool. While solution is still
lukewarm, add approximately 20 ml of DI vater and mix veil.
Addition of DI water prevents formation of solids when solution
reaches room temperature.
6. Cap tubes with parafilm and store until analysis. It is
recommended that contents of digestion tubes not be transferred
to storage containers if NH^-N content is to be determined using
steam distillation. Quantitative transfer from the digestion
tubes is difficult to perform without using large amounts of DI
water. Excess amounts of DI water will make determination of
NH^-N by steam distillation cumbersome.
7. If analysis of NH^-N is to be done with a colorimetric
procedure, add DI water to give the desired final volume in the
digestion tube and mix well. Let tubes stand overnight.
8. Decant off aliquot for analysis (15 ml) and discard remainder of
the digestate. Do NOT pour digestate down sink. Follow
guidelines established by host institution for disposal of toxic
wastes.
5.7.3.1.3. Documentation
Follow guidelines detailed in Good Laboratory Practices section for
recording of all raw data. Note that raw data include the following: (a)
analytical observations necessary to calculate the final results, (b)
calibration data, (c) calibration checks, and (d) quality control checks.
Deviations from standard operating procedures during sample preparation,
calibration, or actual analyses are to be fully documented and initialed
by laboratory personnel. Samples suspected of being in error or outside
of the calibration range are to be 'flagged', and this notation carried
through all records to the final report. Retain all written materials
(graphs, tables, etc.) generated as part of an analysis. Do not discard
portions of laboratory notebooks or any other information directly related
to calculation of the final result for a set of samples.
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5.7.3.2. EQUIPMENT OPERATION
Digestion Block. - follow manufacturer's manual for start-up, shut-
down, calibration, and maintenance procedures.
Technicon AutoAnalyzer (or equivalent) - follow manufacturer's manual
for start-up, shut-dovn:, and maintenance procedures.
If using an AutoAnalyzer, it is recommended to determine the NH^-N
content of digestates using the salicylate-dichloroisocyanurate reaction
in the presence of nitro-prusside.
NH^-N Determination by Distillation and Titration
1. It is assumed that determination of the NH^-N content of the
digestates will be done using an automatic steam distillation
unit.
2. Place 10 ml of boric acid solution in a 125 ml Erlenmeyer flask.
3. Place 125 ml Erlenmeyer flask with boric acid solution on
receiver side of automatic steam distillation unit.
4. Mount digestion tube containing blank or sample on distillation
unit. Set timers for delivery of sodium hydroxide solution
(containing sodium thiosulfate) and for distillation.
5. Start distillation cycle. Typically three minutes is sufficient
to recover all of the NH^-N contained in the digestate. Boric
acid solution should turn green if NH^-N is present in the
digestate. Failure to see green color indicates no NH^-N present
or not enough base has been added to the digestate to make it
basic. At the end of distillation cycle check pH of solution in
digestion flask with pH paper. If solution is still acid,
replace 125 ml Erlenmeyer Flask with fresh boric acid solution
and restart cycle. Adjustment of timer for base delivery may be
necessary.
6. Remove 125 ml Erlenmeyer flask and set aside for titration.
Remove digestion tube from distillation unit. CAUTION! Digestion
tube will be HOT. Also be careful of contact with concentrated
base. Use of face shield and gloves is required throughout all
phases of this procedure.
7. Do NOT pour solution in digestion tube down sink. Follow
guidelines established by host institution for disposal of toxic
wastes.
5.7.4. PREVENTIVE MAINTENANCE
Carry out all maintenance procedures as recommended by the
manufacturer. Record maintenance operations in maintenance log.
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5.7.5. CALIBRATION PROCEDURES
1. Four standards plus a blank should be used to calibrate
AutoAnalyzer. Standard concentrations will vary depending on the
concentration of N in the original sample and selection of flow
pattern for AutoAnalyzer. Follow manufacturer's guidelines in
selecting appropriate range of standards for analysis. It is
recommended that standards be run at the beginning and end of
each sample group and after a set number of samples within each
group.
2. Calibration of steam distillation procedure is not necessary as
final results are determined by titration with standardized acid.
3. Titrate 125 ml Erlenmeyer flask containing boric acid solution
and NH^-N with either 0.01N or 0.05N KH(I03)2, depending on the
amount of N in the original sample.
4. Boric acid solutions containing NH.-N should be green. Titrate
with acid until purple endpoint. Record ml of acid added to
sample.
5. Titrate blanks with 0.01N KH(I03)2. Record ml of acid added to
sample.
5.7.6. CALCULATIONS/UNITS
Calibration of AutoAnalyzer output is usually set equal to the final
concentration of N (expressed as % oven-dried sample) in sample. Final
calculations are not necessary when this is done.
Use the following equation to calculate the concentration of N in the
sample with results from the titration procedure:
(VS - VB) x N x 14006.7 M/1 £
- —^ = mg N/kg of oven-dried soil
where VS = ml of titrant used for sample, VB = ml of titrant used for
blank, N = normality of titrant (0.01N or 0.05N), and A = equivalent oven-
dried weight of sample.
Report results for N on a X weight basis (1% N by weight = 10000 mg
N/kg).
5.7.7. ERROR ALLOWANCE AND DATA QUALITY
5.7.7.1. CONSIDERATIONS
Blanks, replicates, and in-house secondary standards should be
included in analyses of all samples. Within one group of approximately 40
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samples (20 samples if 250 ml digestion tubes are used) there should be
one blank, two in-house secondary standards, and three replicates.
Certified NBS reference, materials should be included on a monthly basis.
Samples selected as replicates should also be run on different days
to estimate day-to-day precision.
Replicate samples vill be used to monitor precision. Percent
coefficient of variation should be calculated using the industrial
statistic I, where %CV = 200I//2 and I=|A-B|/A+B, A and B being the
results for the replicate samples (U.S. EPA, 1979).
Precision will also be monitored using Shevhart control values for R
(range) values, if the total number of sample groups to be analyzed will
generate enough points to make useage of such charts worthwhile.
Critical ranges for repeated measurement error and accuracy will be
set by Quality Assurance Officer and listed in the Data Quality Objectives
(DQO) table.
Accuracy vill be evaluated through the use of interlaboratory sample
exchanges and use of NBS SRM's. Sample exchanges will be coordinated by
the Quality Assurance Specialist assigned to the project.
5.7.7.2. DATA QUALITY OBJECTIVES
Repeated Measurement
Measurement
Reporting
Error at
Accuracy
Variable
Uni ts
Lover Limit Upper Limit
Tolerance
Kjeldahl N
0.0U (v/v)
lOX (cv) 10% (cv)
i5%
5.7.7.3. COMPUTER DATABASE CODES
Variable
Code
Kjeldall Nitrogen
STKN
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5.7.8. REFERENCES
Bremner, J.M. and C.S. Mulvaney. 1982. Nitrogen - Total. In: Methods of
Soil Analysis. Part 2. Chemical and Microbiological Properties. 2nd
edition, A.L. Page (ed.). ASA Monograph No. 9. ASA-SSSA. Madison, VI.
U.S. Environmental Protection Agency. 1979. Handbook for Analytical
Quality Control in Vater and Wastewater Laboratories. Environ. Mon.
and Sup. Lab. U.S. EPA. Cincinnati, OH. 45268. EPA-600/4-79-019.
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5.8. STANDARD OPERATING PROCEDURE FOR MEASUREMENT OF TOTAL
CARBON/NITROGEN/SULFUR CONTENT USING AN ELEMENTAL ANALYZER
5.8.1. SCOPE AND PURPOSE
This standard operating procedure is designed to measure total
carbon, nitrogen, and sulfur content by using an elemental analyzer. A
number of manufacturers currently produce instruments capable of analyzing
finely ground soil materials for total content of carbon and nitrogen, as
well as for sulfur which often requires the purchase of appropriate
accessories. Because of the uniformity in measurement which can be
obtained using automated elemental analyzers, it is recommended that this
equipment be used for the analysis of carbon, nitrogen, and sulfur in soil
samples where instrumentation is available. Where elemental analyzers are
not available for one or more of these elements, alternative procedures
for element content measurements as outlined in this document should be
employed.
5.8.2. MATERIALS AND SUPPLIES
5.8.2.1. EQUIPMENT
0 elemental analyzer for C-N-S
° analytical balance (0.001 g)
5.8.2.2. CHEMICALS/REAGENTS
As per manufacturer's instructions
5.8.3. PROCEDURES
5.8.3.1. SAMPLE PREPARATION
1. Most elemental analyzers use relatively small sample sizes
compared to the other SOPs in this section (50-100 mg vs. 0.5-
2.0 g). Preparation of sample, therefore, is critical to ensure
acceptable accuracy and precision.
2. Soil samples are essentially collections of discrete particles,
which ultimately limits sample homogeneity because the measured
property is not associated equally with each discrete particle
(Mullins and Hutchison, 1982). A subsample for analysis must be
of sufficient size to include a representative sampling of the
various discrete particles that make up the soil sample.
Chemical analyses requiring less than 1.0 g quantities of sample
should be prepared from larger subsamples than have been finely
ground (< 32 mesh) and mixed. Failure to take a large enough
subsample will introduce a bias into the final results that will
not be detected unless a separate subsample is taken, finely
ground, and analyzed. It is recommended that at least 10 g be
chosen as the minimum subsample size from which to prepare finely
ground samples.
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3. Preparation of finely ground subsamples can be done by hand using
an agate mortar and pestle or with a mechanical grinder, ball
mill, or mixer mill (such as a Spex mixer mill or equivalent).
However, preparation and mixing of finely ground samples requires
a substantial amount of time and should be properly accounted for
vhen designing experiments.
5.8.3.2. EQUIPMENT OPERATION
Several commercial elemental analyzers are currently available for
analysis, of soils. Follow the manufacturer's instructions for proper
operation of particular instrument selected for the analysis.
5.8.4. PREVENTIVE MAINTENANCE
Carry out all maintenance procedures as recommended by the
manufacturer. Record maintenance operations in maintenance log.
5.8.5. CALIBRATION PROCEDURES
Follow the manufacturer's instructions for proper calibration of the
instrument.
5.8.6. CALCULATIONS/UNITS
Need for calculations will depend on instructions for calibration of
instrument. Follow the manufacturer's recommended guidelines.
5.8.7. ERROR ALLOWANCE AND DATA QUALITY
5.8.7.1. CONSIDERATIONS
Blanks, replicates, and in-house secondary standards will be included
in analyses of all samples. The number of samples run per day will vary
depending on the elements measured and equipment used. At a minimum, one
blank, one in-house secondary standard, and one replicate will be included
for every 25 samples. Certified NBS reference materials should be
included on a monthly basis, as appropriate.
Replicate samples will be used to monitor precision. Percent
coefficient of variation will be calculated using the industrical
statistic I, where % CV = 200I//2 and I = a-B/a+B, A and B being the
results for the replicate samples.
Precision vill also be monitored using Shewhart control values for R
(range) values, if the total number of sample groups to be analyzed will
generate enough points to make use of such charts worthwhile.
Critical ranges for repeated measurement error and accuracy will be
set by each project leader in the Data Quality Objectives (DQO) table.
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5.8.7.2.
DATA QUALITY OBJECTIVES
Repeated Measurement
Measurement
Reporting Error at
Accuracy
Variable
Units Lower Limit
Upper Limit
Tolerance
N
0.01% (w/w) 15% (cv)
10% (cv)
15%
S
mg/kg 10% (cv)
10% (cv)
15%
C
mg/kg 15% (cv)
10% (cv)
15%
5.7.7.3.
COMPUTER DATABASE CODES
Variable
Code
Total Elemental N
STTN
Total Elemental S
STTS
Total Elemental C
STTC
5.8.8. REFERENCES
Mullins, C.E., and B.J; Hutchison. 1982. The variability introduced by
various subsampling techniques. J. Soil Sci. 33:547-561.
U.S. Environmental Protection Agency. 1979. Handbook for Analytical
Quality Control in Water and Wastewater Laboratories. Environ. Mon.
and Sup. Lab. U.S. EPA. Cincinnati, OH. 45268. EPA-600/4-79-019.
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5.9. STANDARD OPERATING PROCEDURE FOR MEASUREMENT OF ELEMENTAL CONTENT BT
TOTAL DISSOLUTION: Ca, K, Mg, Al, Fe, S, P, Cu, Mn, Zn, Na, Cd, Ni,
Pb, V
5.9.1. SCOPE AND PURPOSE
This standard operating procedure (SOP) is designed to determine the
total elemental content (mg/kg oven-dried soil) of samples. Three
dissolution procedures are described for preparation of samples for
analysis. Tvo of the dissolution techniques make use of HF acid (Stomberg
et al., 1984; Lim and Jackson, 1982). The third technique is based on
fusion vith lithium borate to dissolve samples (Brenner et al., 1980;
Chase et al., 1985; Duke University Geology Dept., personal
communication). Elemental content of the acid digestates can be
determined using either emission or atomic absorption spectroscopy.
Note: Separate procedures are required for determining total
elemental carbon, nitrogen, and boron content.
5.9.2. MATERIALS AND SUPPLIES
5.9.2.1. EQUIPMENT
0 microwave oven (650 vatt, 1.3 cu. ft., lined vith teflon and
equipped with proper exhaust system for handling corrosive
gases;
0 Teflon digestion vessels
0 muffle furnace (1000°C)
° convection oven (110°C)
c graphite crucibles (20 ml) (available from Ultra Carbon Corp)
0 magnetic stirrers
° volumetric glassware
° analytical balance (0.0001 g)
° polyethylene or polycarbonate screw cap bottles (60 ml)
0 automatic diluter-dispenser
0 agate mortar and pestle or Spex impact grinder (or equivalent)
vith tungsten carbide containers
0 widemouth polypropylene bottles, 250 ml capacity
° flat bed mechanical shaker
0 hot plate
° 15 ml Pyrex centrifuge tubes
5.9.2.2. CHEMICALS/REAGENTS
° Distilled-deionized (DI) water
° 15M Nitric acid (HN03)
° 48% Hydrofluoric acid (HF)
0 Boric acid (H3B03)
° Boric acid saturated solution - place 7 g of boric acid in 1 liter
of DI water. Mix several times and let stand overnight.
0 Lithium metaborate (LiB02)
° Lithium tetraborate (Li^B^)
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Flux mixture - thoroughly mix 1 part of reagent grade powdered
anhydrous li.thium raetaborate with 2 parts of anhydrous lithium
tetraborate. Store mixture in tightly capped bottle.
1M Ammonium carbonate [{NH^KCO^'f^O] solution: dissolve 114.1 g
of ammonium carbonate in I liter of DI water.
Aqua regia: mix 3 ml of 12M HC1 with 1 ml of 15M HN03. Prepare
solution just before use.
5.9.3. PROCEDURES
5.9.3.1. SAMPLE PREPARATION
Rapid total dissolution requires samples that are finely ground. The
subsample selected for further grinding must be of sufficient size to
include a representative sampling of the various discrete particles that
make up the total soil sample. Failure to take a large enough subsample
will introduce a bias into the final results that will not be detected
unless a separate subsample is taken, finely ground, and analyzed. It is
recommended that at least 10 g be chosen as the minimum subsample size
from which to prepare finely ground samples.
Preparation of finely ground subsamples can be done by hand using an
agate mortar and pestle or with a mechanical grinder, ball mill, or mixer
mill, (such as a Spex mixer mill or equivalent). Use of a Spex mixer mill
(or equivalent) to prepare aliquots of finely ground soil requires
selection of a suitable sample mixer container because the sample will
become contaminated from contact with container walls during the grinding
process. Tungsten carbide containers are recommended for use with a mixer
mill.
5.9.3.1.1. Dissolution Using HF and Microwave Oven
1. Place 200 to 500 mg of finely ground sample into teflon
digestion vessel.
2. Add 5 ml of 15M HN03 and 2 ml of 482 HF. Immediately cap and
seal digestion vessels. CAUTION! HF is a highly corrosive acid.
Do all work in a properly designed chemical hood. NEVER touch
acid or sample containers with bare skin. Wear at least two
layers of gloves when handling containers with HF acid. Thin
latex gloves inside heavier duty chemical gloves provide an
extra margin of safety in case of spills. Have solutions of
saturated boric acid solution close at hand to neutralize
spills. In case of contact*with skin, flush affected area with
water (15 minutes minimum) and seek immediate medical attention.
Contact with HF causes internal damage to skin and body tissue
that does not express visual symptoms until several hours after
contact.
3. Place containers in microwave digestion oven and heat for 10
minutes at 25£ full power. Continue heating for another 30
minutes at 35X full pover.
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4. Teflon containers are equipped with pressure vents. If venting
of gas occurs during digestion, lover pover setting.
5. Remove digestion vessels. Rinse with DI vater and allow to
cool. Cooling in tap vater or with ice bath may be necessary to
open vessels.
6. Add approximately 20 ml of saturated boric acid solution to
dissolve metal fluorides. NOTE: this step will generate SiF^
gas. Add boric acid solution in hood. Cap immediately after
adding boric acid solution if content of Si in sample is to be
determined.
7. Allow reaction to proceed for 30 minutes.
8. Transfer contents of digestion vessel to a tared polyethylene or
polycarbonate bottle and swirl to dissolve any precipitate. Use
small jets of DI vater to aid in transfer.
9. Add DI to bring net weight of bottle to 50 g. Cap and mix
contents. Store solutions in bottles until analysis.
5.9.3.1.2. Dissolution Using Lithium Borate
1. Transfer approximately 1.2 g of flux mixture to a vaxed or
plastic-coated veighing paper.
2. Weigh and transfer to the flux mixture 200 mg of finely ground
soil sample as prepared in Section 5.9.3.1.
3. Mix soil and flux mixture by rolling successive corners of the
paper about 10 times, and transfer carefully and completely to a
graphite crucible.
4. Tamp down contents by tapping each crucible several times
against the table surface.
5. Fuse in a muffle furnace at 1000°C for 15 minutes. Remove and
allow to cool.
6. When cool, transfer bead to 50 ml of 10% HN03 in a 250 ml
beaker. Gently mix solution by use of teflon stirring bar in
bottom of beaker.
7. After dissolution of bead, transfer to a 100 ml volumetric flask
and bring to volume with DI water. Mix each flask 10 times and
transfer contents to polyethylene or polycarbonate bottles for
storage until analysis of solutions.
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5.9.3.1.3. Dissolution Using HF and Closed Vessels
1. Place approximately 200 mg of sample in a 15 ml Pyrex centrifuge
tube.
2. Wash sample with 1M (NH.KCO,, solution 5 times, then once with
80% methanol.
3. Dry in convection oven (110°C) for 12 hours. Remove sample and
thoroughly mix to break down any lumps that may have formed
during drying.
4. Heat sample again in oven (110°C) for 1 hour. Remove and place
in a desiccator.
5. Veigh out a 50 mg + 0.1 mg sample onto glazed powder paper.
6. Transfer sample to a tared widemouth bottle by inserting glazed
paper with sample into bottle while the bottle is in a
horizontal position. Holding the edges of the glazed paper
together facilitates insertion of sample into the bottle.
7. Return bottle to vertical position and gently tap powder paper
to quantitatively transfer sample to bottle.
8. Prepare Aqua Regia solution. Add 1 ml to each bottle by letting
solution run down inside of widemouth bottle. Gently swirl
bottle to mix sample and acid. Let stand for several minutes to
allow acid to react with any metal carbonates that may be
present in sample*
9. Add exactly 10 ml of 48£ HF. Immediately cap bottle. Place
bottle on flat bed shaker and gently shake for 2 hours.
10. If the color of the reaction mixture suggests that sample is
still present after 2 hours, heat bottle on a hot plate (surface
temperature 75-100°C) for 30 minutes.
11. An alternative to steps 9 and 10 is to let bottles stand for
several days at room temperature with occasional miying of each
bottle by hand. Dissolution is usually complete in several
days.
12. After dissolution of sample, add 100 ml of saturated boric acid
solution using a graduated cylinder. Immediately cap bottle to
prevent loss of SiF^ gas.
13. Addition of the saturated boric acid solution results in an
exothermic reaction. Heat generated from this reaction should
be sufficient to dissolve metal fluorides formed during
dissolution of the original sample. Dissolution of the metal
fluorides, however, may take from minutes to several hours.
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14. When solution cools, add enough DI to the widemouth bottle to
give a final net weight of 200 g + 0.1 g. Cap bottle and mix
well.
15. Store digestate in bottle for subsequent determination of
elemental composition.
5.9.3.1.4. Documentation
Follow guidelines detailed in Good Laboratory Practices section for
recording of all raw data. Note that raw data include the following: (a)
analytical observations necessary to calculate the final results, (b)
calibration data, (c) calibration checks, and (d) quality control checks.
Deviations from standard operating procedures during sample preparation,
calibration, or actual analyses are to be fully documented and initialed
by laboratory personnel. Samples suspected of being in error or outside
of the calibration range are to be 'flagged', and this notation carried
through all records to the final report. Retain all written materials
(graphs, tables, etc.) generated as part of an analysis. Do not discard
portions of laboratory notebooks or any other information directly related
to calculation of the final result for a set of samples.
5.9.3.2. EQUIPMENT OPERATION
Consult operating manuals for
instruments.
5.9.4. PREVENTIVE MAINTENANCE
detailed instructions on operation of
Consult operating manuals for selected instrumentation for proper
maintenance procedures.
Record maintenance operations in maintenance log.
Store all reagents in plastic ware to prevent contamination from
glassware during storage.
Use of saturated boric acid solution should neutralize HP remaining
after digestion. Monitor glass surfaces in instruments to ensure that
sample mixture is not causing failure of glass components due to the
incomplete neutralization of HF acid.
Graphite crucibles used in Section 5.9.3.1.2., step 3, can be reused
several times. Inspect crucibles after each fusion to ensure complete
removal of flux bead. Crucibles will wear out and cost of replacement
should be included in budget.
5.9.5. CALIBRATION PROCEDURES
1. Four standards plus a reagent zero should be used to calibrate
instrumentation for the analysis of each element. Standards
should define linear operating range of the instrument.
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Standards should be run at the beginning and end of each sample
group and after a set number of samples within each group.
2. Prepare standards from certified commercial stock sources or from
primary reagents.
3. Standard solutions should be prepared using class A volumetric
glassware and transferred to cleaned (IN HN03) polyethylene
bottles for storage between analyses. Special care will be taken
that temperature of solutions during preparation of standards
meets that stated for the calibration of the glassware.
4. Standard solutions should be prepared to match sample matrix
solutions. For example, standards to be used with the
dissolution procedure in closed vessels should contain 0.5X aqua
regia (vol/wt), 5% HF (vol/wt) and 50% saturated boric acid
solution (vol/wt). Dilution of sample solutions and addition of
matrix modifiers will be necessary for analysis by flame atomic
absorption spectrophotometry. Lanthanum chloride (prepared from
lanthanum oxide, 5000 mg/1 in 0.5N HCl) is recommended as a
matrix modifier for determination of Ca and Mg by flame AAS.
5. Establish typical diluting ranges for filtrates and prepare
standard solutions of suitable concentration to yield a linear
working curve after dilution. Use of an automatic diluter-
dispenser is highly recommended to ensure accurate and rapid
dilution.
5.9.6. CALCULATIONS/UNITS
Standard curves should be fitted by the method of least squares after
hand graphing to check for continuity.
Standard blanks and sample blanks may not be identical. Correction of
standard and sample readings for respective blank values is to be carried
out before graphing of standard curves and calculation of final results.
Final concentration of an element in a sample can be calculated as
follows:
(A x DF x C) - , j • j "t
- = - = mg / kg oven-dried soil
where A = ug of element per ml of digestate as determined from the
calibration curve, DF = dilution factor if required, C = total volume of
digestate (assume density of 1 g/cc), and B = total weight of oven-dried
sample.
Final results are expressed as mg/kg or %.
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5.9.7. ERROR ALLOWANCE AND DATA QUALITY
5.9.7.1. CONSIDERATIONS
Blanks, replicates, and in-house secondary standards will be included
in analyses of all samples. Approximately 35 samples will compose each
group of analyses. Vithin one group there will be one blank, tvo in-house
secondary standards, and three replicates. Certified NBS reference
materials will be included vhen appropriate.
Samples selected as replicates should also be run on different days
to estimate day-to-day precision.
Replicate samples will be used to monitor precision. Percent
coefficient of variation will be calculated using the industrial statistic
I, where %CV = 200I//2 and I=|A-B|/A+B, A and B being the results for the
replicate samples (U.S. EPA, 1979).
Precision will also be monitored using Shewhart control values for R
(range) values, if the total number of sample groups to be analyzed will
generate enough points to make use of such charts worthwhile.
Critical ranges for repeated measurement errors and accuracy will be
set by each project leader in the Data Quality Objectives (DQO) table.
Spiked soil samples will not be used because of the small sample size
and number of elements to be determined. The availability of a range of
NBS and Canadian SRM's should preclude the use of spiked samples to ensure
accuracy. However, NBS SRM's should be checked for minimum sample size on
which certification is based before use.
5.9.7.2. DATA QUALITY OBJECTIVES
Repeated Measurement
Measurement
Reporting
Error at
Accuracy
Variable
Units
Lower Limit Upper Limit
Tolerance
—%(v/v)—
% (CV)
Ca
0.1
10.0 10.0
15%
K
0.1
10.0 10.0
15%
Mg
0.1
10.0 10.0
15%
A1
0.1
15.0 10.0
15%
Fe
0.1
10.0 10.0
15%
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DATA QUALITY OBJECTIVES
(cont'd)
Repeated Measurement
Measurement
Reporting
Error at
Accuracy
Variable
Uni ts
Lover Limit Upper Limit
Tolerance
—mg/kg j
—X (cv)
S
1.0
10.0
10.0
15%
P
1.0
10.0
10.0
15%
Cu
1.0
15.0
10.0
15%
Mn
1.0
15.0
10.0
15%
Zn
1.0
10.0
10.0
15%
Na
1.0
10.0
10.0
15%
Cd
0.1
15.0
10.0
.15%
Ni
1.0
20.0
10.0
15%
Pb
1.0
20.0
10.0
15%
V
1.0
20.0
10.0
15%
5.9.7.3.
COMPUTER DATABASE CODES
Variable
Code
Total Ca
STCA
Total K
STTK
Total Mg
STMG
Total Al
STAL
Total Fe
STFE
Total S
STTS
Total P
STTP
Total Cu
STCU
Total Mn
STMN
Total Zn
STZN
Total Na
STNA
Total Cd
STCD
Total Ni
STNI
Total Pb
STPB
Total V
STTV
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5.9.8. References.
Chase, D.S., D.D. Nygaard and D.A. Leighty. 1985. Analysis of geological
materials by ICP. Application Note #187. Instrumentation Laboratory.
Analytical Instrument Division. Andover, MA. 01810.
Brenner, I.B., A.E. Vatson, G.M. Russell and M. Goncalves. 1980. A new
approach to the determination of the major and minor constituents in
silicate and phosphate rocks. Chem. Geol. 28:321-330.
Lim, C.H. and M.L. Jackson. 1982. Dissolution for total elemental
analysis. In: Methods of Soil Analysis, Part 2. Chemical and
Microbiological Properties. Agronomy Monograph No. 9 (2nd edition),
A.L. Page (ed.). ASA-SSSA, 677 S. Segoe Rd., Madison, VI. 53711.
Stomberg, A.L., D.D. Hemphill, Jr. and V.V. Volk. 1984. Yield and
elemental concentration of sveet corn grown on tannery vaste-amended
soil. J. Environ. Qual. 13:162-166.
U.S. Environmental Protection Agency. 1979. Handbook for Analytical
Quality Control in Vater and Wastewater Laboratories. Environ. Mon.
and Sup. Lab. U.S. EPA. Cincinnati, OH. 45268. EPA-600/4-79-019.
187
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Baker, D.E. and M.C. Amacher. 1982. Nickel, Copper, Zinc, and Cadmium.
In: Methods of Soil Analysis. Part 2. Chemical and Microbiological
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Bartlett, R. and B. James. 1979. Behavior of chromium in soils: III.
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Bartlett, R. and B. James. 1980. Studying dried, stored soil samples -
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Beaty, R.D. 1978. Concepts, Instrumentation and Techniques in Atomic
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Bengtson, C., S. Larsson, and C. Liljenberg. 1978. Effects of water
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Birk, E.M. and P.A. Matson. Site fertility affects seasonal carbon
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Black, C.A. 1965. Methods of Soil Analysis. Part I. ASA Monograph No.
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Blume, L. J. 1985. Statement of Vork. National Acid Deposition Soil
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Chase, D.S., D.D. Nygaard and D.A. Leighty. 1985. Analysis of geological
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Dhalquist, R.L. and J.V. Knoll. 1978. Inductively coupled plasma-atomic
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Dux, J.P. 1986. Handbook of Quality Assurance for the Analytical
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Eastin, E.F. 1978. Total nitrogen determination for plant material
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Fernandez, Ivan J. 1983. Field Study Program Elements to Assess the
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Franich, R.A., L.G. Veils, and P.T. Holland. 1978. Epicuticular wax of
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I
Freeman, G., L.G. Albrigo, and R.H. Biggs. 1979. Ultrastrueture and
chemistry of cuticular waxes of developing Citrus leaves and fruits.
J. Amer. Soc. Hort. Sci. 104(6):801-808.
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194
-------
APPENDICES
195
-------
APPENDIX A
Attendees
Laboratory Analytical Techniques Workshop
QA Methods Manual Development
13-14 March
Raleigh, NC
Ruth Alscher
Boyce Thompson Institute
Cornell University
Tower Road
Ithaca, NY 14853
Charles F. Baes III
Oak Ridge National Laboratory
Environmental Sciences Division
P. 0. Box X
Oak Ridge, TN 37830
615/576-2137
Jim Bailey
Texas A&M University
Porest Genetics Laboratory
College Station, TX 77843
409/845-1325
John Duff Bailey
U.S. EPA
200 Stf 35th Street
Corvallis, OR 97330
503/757-4324
Roger Blair
Corvallis Environmental Research
Laboratory
200 SV 35th Street
Corvallis, OR 97333
Richard D'Guilio
School Forest Environment Studies
Duke University
203 A Bioscience Blvd.
Durham, NC 27706
919/684-2135
Ivan J. Fernande2
University of Maine
1 Deering Hall
Dept. of Plant & Soil Science
Orono, ME 04469
207/581-2932
Richard Flagler
N.C. State University
Crop Science Dept.
Raleigh, NC 27695
919/737-3575
Valter W. Heck
Air Quality Research Programs
N.C. State Unviersity
1509 Varsity Drive
Raleigh, NC 27606
919/737-3311
Kim Joyner
NCSU Acid Deposition Program
1509 Varsity Drive
Raleigh, NC 27606
919/737-3520
Jack Jorgensen
USFS
Soil Science
P. 0. Box 12254
RTP, NC 27709
919/541-4217
Janet McFayden
NCSU Acid Deposition Program
1509 Varsity Drive
Raleigh, NC 27606
919/737-3520
Susan Medlarz
Northeastern Forest Experiment
Station
370 Reed Road
Broomall, PA 19008
215/461-3014
Richard A. Reinert
Air Quality Research Program
North Carolina State University
1509 Varsity Drive
Raleigh, NC 27606
919/737-3962
196
-------
Wayne P. Robarge
Department of Soil Science
N. C. State University
Box 7619
Raleigh, NC 27695-7619
919/737-2636
Michele Schoeneberger
USDA Forest Service
Box 12254
3041 Cornvallis Road
Research Triangle Park, NC 27709
919/541-4213
Steven Shafer
Air Quality Program
N.C. State Unviersity
1509 Varsity Drive
Raleigh, NC 27606
919/737-2142
Carol Veils
U.S. Forest Service
P. 0. Box 12254
RTP, NC 27709
919/541-4215
Art Viselogel
Research Associate
Forest Science Dept.
Texas A&M
College Station, TX 77843
409/845-5033
197
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APPEHDIX B: DATA QUALITY OBJECTIVES TABLE
Variables
Techniques
Measuring
Units
Repeated Measurement Error Measurement
Reporting Expected at Accuracy
Units Range Lower Limit Upper Limit Tolerance
Soil
Field Water
Content
Bulk Density
Pa rt i cle size
Extractable
Aniona :
Orthophospate
Sulfate
LOI
pH by K^O
by CaCl
i
by KC1
Exchangeable
Cations:
Ca
Mg
K
Gravimetric
Gravimetric/
volume
Sedimentation
Extraction
Bray I
Double acid
Phosphate
Ext raction
ignition/
gravimetric
mete r
mete r
meter
grams
grams
g/i
pq/al
^g/ml
pg/ml
pH units
pH units
pH units
*(w/w)
Mg/m3
%(w/w)
mg/kg
mg/kg
mg/kg
%
0.01 pH units
0.01 pH units
0.01 pH units
NH Cl extract. //g/ml extractant cmole(+)/kg
4
NH Cl extract. /sg/ml extractant cmole ( +)/kg
4
NH Cl extract. vq/ml extractant cmole(+)/kg
4
0-100
0.7-1.5
0-100
20-300
0-1000
0-1000
1-95
3. 00-7. 00
3.00-7.00
3.00-5.00
0.05-20.00
0.03-9 . 00
0.05-6.00
N/A*
N/A
10%
2 0%
20%
20%
50%
±1
±•1
±1
±.05
±03
±.05
N/A
N/A
10%
5%
5%
5%
5%
±•1
±•1
±-1
±1 .00
±.50
±.30
N/A
N/A
N/A
N/A
N/A
N/A
5%
N/A
N/A
N/A
N/A
N/A
N/A
+ - Absolute values refer to measuring units. Percent values are coefficient of variation (100 x s.d./x).
• - N/A = Not Applicable
-------
Variables
Techniques
Measuring
Units
Reporting
Units
Expected
Bange
+
Repeated Measurement Error
at
Lower Limit Upper Limit
Measurement
Accuracy
Tolerance
Na
NH Cl extract
4
fig/ml extractant
cmole(+)/kg
0 .05-1.00
±.05
±.05
N/A
A1
KC1 extract
meg/ml extractant
cnole(+)/kg
0.10-18.00
±10
±1 . 00
N/A
H
KCL extract
meq/ml extractant
cmole(+)/kg
0.10-18.00
±.10
±1 .00
N/A
Exchangeable Acidity
KCL extract
meq/ml extractant
cmole(+)/kg
0.10-18.00
±.io
±1 . 00
N/A
Extractable Hetals:
re
0.1N HC1
uq/al
og/kg
10-100
±1
±5
N/A
Zn
0.1N HC1
vq/ml
0.1 aq/kq
1.0-10.0
±1
±5
N/A
Cu
0.1N HC1
uq/nl
0.1 ag/kg
1.0-10.0
±¦1
±•5
N/A
Pb
0.1H HC1
v g/ol
0.1 ag/kg
0.5-20.0
±-1
±1.0
N/A
Cd
0 ..IN HC1
pg/ml
0.1 ag/kg
0.1-3.0
±1
±.2
N/A
Hi
0.1N HC1
nq/mX
0.1 aq/kq
0.1-2.0
±¦1
±-1
N/A
Mn
0.1N HC1
u g/ml
aq/kq
10-100
±1
±5
H/A
Elemental Content:
C (organic)
Total
mg/kg
0 .1%
0.1-40.0
15%
10%
15%
N
Total or TKN
pg/ml
0.01%
0.02-2.10
15%
10%
15%
Ca
Total
pg/ml
0.01%
0.1-1.0
10%
10%
15%
K
Total
#/g/ml
0.01*
0.5-2.5
10%
10%
15%
Mg
Total
#»g/nl
0.1%
0.0-1.0
10%
10%
15%
A1
Total
pq/ml
0 .1%
0.2-10.0
15%
10%
15%
Pe
Total
nq/al
0.1%
0.2-5.0
10%
10%
15%
+ - Absolute values refer to measuring units. Percent values are coefficient of variation (100 x s.d./x).
* — N/A = Not Applicable
-------
Variables Techniques
Measuring
Units
Reporting
Units
Expected
Range
+
Repeated Measurement Error
at
Lower Limit Upper Limit
Measurement
Accuracy
Tolerance
s
Total
nq/m 1
mg/kg
100-1000
10%
10%
15%
p
Total
//g/ml
mg/kg
500-3000
10%
i0%
15%
Cu
Total
pg/ml
mg/kg
15-200
15%
10%
15%
Hn
Total
eg/ml
mg/kg
50-1000
15%
10%
15%
Zn
Total
//g/ml
mg/kg
20-200
10%
10%
15%
Na
Total
/ig/ml
mg/kg
500-6000
10%
10%
15%
Cd
Total
//g/ml
mg/kg
0.2-5.0
15%
10%
15%
Ni
Total
//g/ml
mg/kg
10-100
20%
10%
15%
Pb
Total
//g/ml
mg/kg
20-250
20%
10%
15%
V
Total
//g/ml
mg/kg
10-100
20%
10%
15%
Plant Nutrients:
C
Total
VV/k<3
0.1%
30-70
5%
5%
5%
N
Total or TKN
n g/ml
0.1%
0.5-2.5
10%
10%
10%
P
Total
//g/ml
0.01%
0.05-0.50
20%
10%
10%
K
To t a 1
it g/ml
0 .1%
0.1-5.0
10%
10%
15%
Ca
Total
ii g/ml
0 .1%
1000-6000
10%
10%
15%
Mg
Total
//g/ml
mg/kg
400-1000
15%
10%
15%
S
Total
//g/ml
mg/kg
400-1500
10%
10%
15%
Mn
Total
//g/ml
mg/kg
20-1000
10%
10%
15%
Zn
Tota 1
//g/ml
mg/kg
5-100
10%
10%
15%
+ -
* __
Absolute values refer to neasuring
N/A = Not Applicable
units. Percent
values are coefficient of variation (100 x
s.d./x ) .
-------
Variables
Techniques
Measuring
Units
Reporting
Units
Expected
Range
Repeated Mea
Lower Limit
+
surement Error
at
Upper Limit
Measurement
Accuracy
Tolerance
r«
Total
pg/ml
mg/kg
50-1000
10%
10%
15%
A1
Total
v g/ml
mg/kg
0-3000
20%
10%
15%
Cu
Tota 1
jig/ml
mg/kg
o
H
1
H
O
20%
10%
15%
B
Total
p g/ml
mg/kg
0-30
10%
10%
15%
Cd
Tota 1
p<3/ml
mg/kg
0 .0-100.0
20%
10%
15%
Ha
Total
*g/ml
mg/kg
5-30
20%
10%
15%
Ni
Total
jig/ml
0.1 mg/kg
0.0-100.0
20%
20%
15%
Pb
Total
jsg/ml
0.01 mg/kg
0.00-100.00
25%
25%
15%
V
Total
It g/ml
0.1 mg/kg
0
1
o
o
20%
20%
15%
Ba
Total
jig/ml
0.1 mg/kg
O
O
IN
1
O
e
15%
10%
15%
Cs
Total
j/g/ml
0.01 mg/kg
0.10-0.60
10%
10%
15%
Rb
Tota 1
//g/ml
mg/kg
10-60
10%
10%
15%
CI
Ext raction
//g/ml
mg/kg
100-1000
5%
5%
15%
Clorophyl1
Ext rac t ion
Abs .
mg/kg tissue
1-10
10%
10%
N/A
Starch
Extraction
vq/ml
mg/g dry tissue
0.00-100.00
10%
10%
10%
Total Sugars
Ext ract ion
//g/ml
mg/g dry tissue
0 .00-100.00
10%
10%
H/A
Cuticular Wax
gravimetric
mg
mg/sample
0.0-3.0
5%
5%
H/A
H-Alkanes
Analyses
GLC
peak area
pg/sample
0-200
5%
5%
N/A
- Absolute values refer to measuring units.
- N/A = Not Applicable
Percent values are coefficient of variation (100 x s.d./K).
-------
APPENDIX C
VARIABLE CODES
(Computer Database Codes)
VARIABLE NAME CODE
Foliar Inorganic Analyses:
Foliar Kjeldahl-N FTKN
Foliar Extractable CI FECL
Total Foliar Elemental Al FTA1
Total Foliar Elemental B FTTB
Total Foliar Elemental Ba FTBA
Total Foliar Elemental C FTTC
Total Foliar Elemental Ca FTCA
Total Foliar Elemental Cd FTCD
Total Foliar Elemental Cs FTCS
Total Foliar Elemental Cu FTCU
Total Foliar Elemental Fe FTFE
Total Foliar Elemental K. FTTK
Total Foliar Elemental Mg FTMG
Total Foliar Elemental Mn FTKN
Total Foliar Elemental N FTTN
Total Foliar Elemental Na FTNA
Total Foliar Elemental P FTTP
Total Foliar Elemental Pb FTPB
Total Foliar Elemental Rb FTRB
Total Foliar Elemental S FTTS
Total Foliar Elemental V FTTV
Total Foliar Elemental Zn PTZN
Foliar Organic Analyses;
n-Docosane FC22
n-Dotriacontane FC32
n-Eicosane FC20
n-Beneicosane FC21
n-Hentriacontance FC31
n-Heptacosane FC27
n-Hexacosane FC26
n-Hexatriacontane FC36
n-Nonacosane FC29
n-Nonadecane FC19
n-Octacosane FC28
n-Octadecane FC1B
n-Pentacosane FC25
n-Pentatciacontane FC35
n-Tetracosane FC24
n-Tetratriacontane FC34
n-Triacontane FC30
n-Tricosane FC23
202
-------
VARIABLE NAME CODE
Foliar Organic Analyses (cont'd):
n-Tritriacontane FC33
Total Chlorophyll FCHL
Total Sugars FSUG
Extractable Starch FSTR
Culticular Wax FTCW
Soil Physical Analyses;
Field Water Content SFVC
Loss on Ignition SLOI
Percent Clay SPCL
Percent Sand SPSN
Percent Silt SPSL
Soil Bulk Density SCBD
Soil Chemical:
Exchangeable Acidity STXA
Exchangeable Al SXAL
Exchangeable Ca SXCA
Exchangeable H SXEH
Exchangeable K SXEK
Exchangeable Hg SXMG
Exchangeable Na SXNA
Extractable Cd SECD
Extractable Cu SECU
Extractable Fe SEFE
Extractable Mn SEMN
Extractable Ni SENI
Extractable P SEBP
Extractable PB SEPB
Extractable S (Water) SEWS
Extractable S (Phosphate) SEPS
Extractable ZN SEZN
pH in DI Water SWPH
pH in 0.01M CaC12 SCPH
pH in IN KCL SKPH
Soil Kjeldahl N STKN
Total Soil Elemental Al STAL
Total Soil Elemental C STTC
Total Soil Elemental Ca STCA
Total Soil Elemental Cd STCD
Total Soil Elemental Cu STCU
Total Soil Elemental Fe STFE
Total Soil Elemental K STTK
Total Soil Elemental Mg STMG
Total Soil Elemental Mn STMN
Total Soil Elemental N STTN
Total Soil Elemental Na STNA
203
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VARIABLE NAME
CODE
Soil Chemical (cont'd):
Total Soil Elemental Ni STNI
Total Soil Elemental P STTP
Total Soil Elemental Pb STPB
Total Soil Elemental S STTS
Total Soil Elemental V STTV
Total Soil Elemental Zn STZN
204
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