EPA-600/2-76-072
March 1976
Environmental Protection Technology Series
TECHNICAL MANUAL FOR ANALYSIS OF
ORGANIC MATERIALS IN PROCESS STREAM!
Industrial Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 2771
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application of
environmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL PROTECTION
TECHNOLOGY series. This series describes research performed to develop and
demonstrate instrumentation, equipment, and methodology to repair or prevent
environmental degradation from point and non-point sources of pollution. This
work provides the new or improved technology required for the control and
treatment of pollution sources to meet environmental quality standards.
EPA REVIEW NOTICE
This report has been reviewed by the U. S. Environmental
Protection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect the
views and policy of the Agency, nor does mention of trade
names or commercial products constitute endorsement or
recommendation for use.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/2-76-072
March 1976
TECHNICAL MANUAL FOR ANALYSIS OF
ORGANIC MATERIALS IN PROCESS STREAMS
by
P. W. Jones, A. P. Graffeo,
R. Detrick, P. A. Clarke, and R. J. Jakobsen
Battelle-Columbus Laboratories
505 King Avenue
Columbus, Ohio 43201
Contract No. 68-02-1409, Task 20
ROAP No. AAS-090
Program Element No. EHB-524
EPA Project Officer: L. D. Johnson
Industrial Environmental Research Laboratory
Office of Energy, Minerals, and Industry
Research Triangle Park, NC 27711
Prepared for
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Research and Development
Washington, DC 20460
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TABLE OF CONTENTS
Page
LEVEL 1 - SURVEY METHODS 1
INTRODUCTION 1
SAMPLE EXTRACTION AND RECOVERY 3
Filter Extraction 3
Cyclone, Impactors, and Probe Rinse 3
Porous Polymer Adsorbent Trap Extraction 4
SAMPLE SEPARATION BY LIQUID CHROMATOGRAPHY 5
ANALYTICAL COSTS 8
LEVEL 2 - QUALITATIVE AND QUANTITATIVE ANALYSIS 9
1. INTRODUCTION 9
(a) Rationale 9
(b) Overview 11
2. SAMPLE EXTRACTION AND RECOVERY 12
(a) Filters 13
(b) Cyclones, Impactors, and Probe Rinse 13
(c) Porous Polymer Adsorbent Traps 13
(d) Granular Solids 15
(e) Liquids 15
(f) Preparation of Extracts for Analysis 15
3. SEPARATION BY HIGH PERFORMANCE
LIQUID CHROMATOGRAPHY 17
(a) Separation by Size 18
(i) Column Packings 19
(ii) Mobile Phase 20
(iii) Temperature 21
(iv) Sample Size 21
(v) Column Calibration 21
(b) Separation by Class 22
(i) Gradient Elution 23
(ii) Column Packings 24
(iii) Mobile Phases 25
(iv) Temperature 26
(v) Functional Group Separation 26
iii
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TABLE OF CONTENTS (Continued)
Page
(c) Detector Systems 27
4. SPECTROSCOPIC SCREENING TECHNIQUES 31
(a) Introduction 31
(b) Nuclear Magnetic Resonance Spectroscopy 31
(i) NMR as a Screening Technique 31
(ii) Limitations of NMR as a Screening Technique .... 34
(iii) Experimental Details 36
(c) Infrared Spectroscopy ..... 44
(i) Introduction 44
(ii) Fourier Transform Infrared Spectrometry 45
(iii) Experimental Details 46
(d) High Resolution Mass Spectrometry 51
(e) Evaluation of Screening Data 53
5. ADDITIONAL SEPARATIONS BY HIGH PERFORMANCE
LIQUID CHROMATOGRAPHY 54
(a) Bonded Phase Chromatography 54
(b) Liquid Solid Chromatography 55
(c) Ion Exchange Chromatography 56
(d) Evaluation of Additional Separations 57
6. IDENTIFICATION AND QUANTIFICATION 57
(a) Gas Chromatography (GC) and High Performance
Liquid Chromatography (HPLC) 57
(b) Gas Chromatographic-Mass Spectrometric (GC-MS) 65
(c) Gas Chromatographic-Infrared Spectroscopic Analysis ... 73
(d) Nuclear Magnetic Resonance Spectroscopy 75
7. ANALYTICAL COSTS , 79
8. REFERENCES 80
iv
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LIST OF FIGURES
Page
Figure 1. Adsorbent Sampling System 5
Figure 2. Continuous Extraction Assembly for Adsorbent
Sampler 6
Figure 3. Overall Analytical Strategy 15
Figure 4. Sequential Analysis by HPLC 21
Figure 5. Principles of GPC 23
Figure 6. Sequential Separation - GPC 25
Figure 7. Sequential Separation - Reverse Phase HPLC 33
Figure 8. Typical Elution Order by Class on Reverse
Phase HPLC 34
Figure 9. Reconstructed Ion Chromatograms of Residual
Oil Combustion Effluents 78
Figure 10. Mass Spectrum of Benzfluoranthenes 79
Figure 11. Mass Spectrum of Benzpyrenes 80
LIST OF TABLES
Table 1. Classes of Organic Compounds Eluting in Each
Liquid Chromatography Fraction, and Their
Approximate IR Detection Limits 9
Table 2. Approximate Proton Chemical Shifts of
Representative Functional Groups . . 41
Table 3. Chemical Shifts of Deuterated Solvents 46
Table 4. Relative Resonance Frequencies for Proton
Reference Compounds 49
Table 5. Organic Species Separable by Various GC
Column Types 67
Table 6. Approximate Costs of Individual Analytical Steps ... 90
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TECHNICAL MANUAL FOR ANALYSIS OF
ORGANIC MATERIALS IN PROCESS STREAMS
by
Peter W. Jones, Anthony P. Graffeo, Ruthanne Detrick,
Pauline A. Clarke, and Robert J. Jakobsen
BATTELLE
Columbus Laboratories
LEVEL 1 - SURVEY METHODS
INTRODUCTION
One of the major problems associated with the analysis of
potentially hazardous organic emissions is the very large number of
organic species which may be present in an emission sample. Additionally,
the task of deciding what priority should be given to which emission
source is by no means a trivial question. If comprehensive analytical
methods were applied to every emission source which was suspected of
being hazardous, costs would become wholly unreasonable and much effort
would be wasted.
However, it is important to ensure that important emission
problems do not go undetected. A very simple yet informative analytical
strategy designed to address this problem is presented in this document.
This strategy will provide a cost-effective survey technique which can
reliably characterize emission sources, and provide input into effluent
prioritization strageties.
In order that large numbers of Level-1 analytical surveys can
be carried out, possibly at the same time, the procedures chosen are
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deliberately simple, and can be carried out by technical staff with
limited previous experience in the field. Despite their simplicity, the
procedures are nevertheless highly effective at indicating whether a
problem area exists.
The objective of the Level-1 analytical strategy is to provide
a semi-quantitative estimation of the predominant classes of organic
compounds present in a Level-1 sample. To achieve this result, the
extracted sample will be subjected to liquid chromatography using step-
wise solvent gradient elution in order to obtain separation of the sample
into eight fractions containing the different organic classes which are
present. Each fraction will subsequently be subject to gravimetric
analysis in order to estimate the weight of material present. In
addition, an infrared analysis of each fraction will be performed.
Infrared analysis will enable the major classes of organic compounds in
each of the eight fractions to be identified. Thus the ultimate result
of the Level-1 analysis will be a qualitative and semi-quantitative
analysis of the industrial emission sample.
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SAMPLE EXTRACTION AND RECOVERY
While it is presently anticipated that Level-1 sample acquisition
will involve the same basic sampling train as Level-2 sampling, the
following extraction procedures may be applied to any chosen organic
sampling system. Thus there is no reason why the sample extraction
procedures should not be basically similar.
The extracts of the filter, cyclones, or impactors, and the
porous polymer adsorbent trap may be combined or analyzed separately
after extraction. The combined or separate extracts should be reduced
to a volume of about 0.5 ml by rotary evaporation.
Filter Extraction
Extraction of filter material should be carried out with
methylene chloride, which is likely to extract most organic particulate
matter. Extraction may be carried out using a Soxhlet apparatus for 24
hours, or by ultrasonic agitation of the filter with solvent in a sealed
glass container for 1 hour, followed by conventional filtration of the
shredded filter and insoluble particulate matter.
Cyclone, Impactors, and Probe Rinse
Particulate material which has been removed from a cyclone,
impactor, or rinsed from a probe should be solvent extracted in a similar
manner to a filter. Sohxlet extraction may be carried out in a cellulose
thimble plugged with glass wool, or in a fritted (fine) glass thimble with
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a glass wool plug. Ultrasonic extraction of particulate may be carried
out by agitation with solvent in a sealed glass container followed by
conventional filtration to remove the insoluble material.
Porous Polymer Adsorbent Trap Extraction
Porous polymer adsorbtion traps for organic species are coming
into widespread use as a replacement for impinger collection because of
their higher efficiency. A typical adsorbent trap is shown in Figure 1,
which schematically depicts the Battelle Adsorbent Sampler. A larger
version of this system will be used for sampling at flow rates up to 5
cfm or more.
Solvent extraction of chromatographic traps should be carried
out by continuous solvent extraction of the adsorbent for a period of 24
hours. Such an extraction apparatus is shown in Figure 2, which depicts
the continuous extraction assembly developed for the Battelle Adsorbent
Sampler; the condenser and solvent reservoir are not shown. It is
recommended that a similar apparatus be used for the extraction of all
chromatographic adsorbent traps.
The choice of solvent for extracting a chromatographic adsorbent
trap depends partly upon the nature of the adsorbent. It is important
that the solvent chosen does not affect the adsorbent in any way, yet
is still an effective solvent for the removal of collected material.
For chromatographic adsorbent traps utilizing Tenax, extracting with
pentane is recommended. The disadvantage of using a relatively inefficient
solvent such as pentane is readily overcome by using the continuous extrac-
tion mode, when even polar compounds are readily extracted by the solvent.
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FLOW DIRECTION
GLASS WATER
JACKET
RETAINING SPRING -i
8-MM GLASS
COOLING COIL
GLASS FRITTED
DISC
FRITTED STAINLESS STEEL DISC
15-MM SOLV-SEAL JOINT
FIGURE 1. ADSORBENT SAMPLING SYSTEM
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TO CONDENSER
t
SOLVENT
RETURN
TUBE
TO SOLVENT
FLASK
FIGURE 2. CONTINUOUS EXTRACTION ASSEMBLY FOR ADSORBENT SAMPLER
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SAMPLE SEPARATION BY LIQUID CHROMATOGRAPHY
Each sample extract should be separated into eight fractions
by liquid chromatography on a silica gel column. The extracts will
consist of separate or combined extracts from the probe, cyclone, filter,
and porous polymer trap. A supply of activated silica gel (> 200 mesh)
should be prepared by freshly heating in an oven at 200 C for 24 hours.
Standard 1 x 25 cm liquid chromatography columns should be
prepared as needed by partially filling the column with methylene
chloride and slowly adding silica gel to a height of 23.5 cm through a
funnel while agitating the column with an electric vibrator. As the
column fills, sufficient methylene chloride should be added to keep the
liquid level above the silica gel. When the column is filled, agitation
should be continued for at least ten minutes in order to remove any air
bubbles. The column should now be prepared for analytical separation
by eluting the following solvents (distilled in glass) through it:
(1) 100 ml methyl alcohol
(2) 25 ml methylene chloride
(3) 25 ml 60/80 petroleum ether
The level of solvent remaining in the column should be 0.5 cm above the
top of the silica gel after each solvent elution.
Sample separation into eight fractions is achieved by carefully
transferring the 0.5 ml sample extract to the top of the liquid chromato-
graphy column with a disposable pipette. The column is then sequentially
eluted with the following eight solvent mixtures, each being collected in
a separate vial.
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8
(1) 25 ml 60/80 petroleum ether
(2) 25 ml 20% methylene chloride in 60/80 petroleum ether
(3) 25 ml 50% methylene chloride in 60/80 petroleum ether
(4) 25 ml methylene chloride
(5) 25 ml 5% methyl alcohol in methylene chloride
(6) 25 ml 20% methyl alcohol in methylene chloride
(7) 25 ml 50% methyl alcohol in methylene chloride
(8) 25 ml methyl alcohol.
Table 1 lists the classes of organic compounds which are expected
to be found in each of the above eight fractions, if these species were
originally present in the collected sample, together with their estimated
minimum detection limits by infrared spectroscopy.
Each collected fraction is preliminarily reduced to about
0.25 ml using a Kuderna-Danish evaporator. The samples are then separately
transferred to preweighed aluminum micro-weighing pans and the solvent
allowed to evaporate in air. The weighing pans are reweighed as necessary
until a marked decrease in the rate of weight loss indicates that the
solvent is sufficiently removed to permit a reasonably accurate sample
fraction weight determination to be made. The weights of each of the
sample fractions should be tabulated.
In order to carry out infrared analysis, each of the samples is
redissolved in a minimum quantity of methylene chloride and an infrared
spectrum is obtained with a film of the sample placed between polished
sodium chloride windows, following evaporation of the solvent. The in-
frared spectrometer should be a grating instrumoit. Many suitable models
are available from a large number of manufacturers. Experience indicates
that acceptable instruments for Level-1 analysis cost at least $6,000,
examples are Perkin Elmer 457, Beckman IR-8, or an equivalent instrument
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TABLE 1. CLASSES OF ORGANIC COMPOUNDS ELUTING IN EACH LIQUID CHROMATOGRAPHY
FRACTION, AND THEIR APPROXIMATE IR DETECTION LIMITS
*
Fraction Compound Type Approximate IR Sensitivity
1 Aliphatic hydrocarbons 1-10 ^,g
Aromatic hydrocarbons 1-10
POM
PCB
Halides
Esters 0.1-1 ug
Ethers
Nitro compounds
Epoxides
Phenols 0.1-1 Uig
Esters
Ke tones
Aldehydes
Phthalates
Phenols 0.1-1
Alcohols
Phthalates
Amines
Amides 0.1-1
Sulfonates
Aliphatic acids
Carboxylic acid salts
Sulfonates 0.1-1
Sulfoxides
Sulfonic acids
8 Sulfonic acids 0.1-1 ug
* Using Perkin Elmer 521 (or equivalent) when used by a professional IR
Spectroscopist.
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10
from any other manufacturer. The manufacturer's instructions for
operating the infrared spectrometer should be carefully followed.
No infrared absorbtion band should be less than 10 percent trans-
mission (no greater than 90 percent absorbtion) for a satisfactory
spectrum to be obtained.
The tabulated weights of each of the eight LC fractions,
together with the IR spectra of each fraction should be transmitted
to a Level 2 laboratory for appraisal, at the direction of the
cognizant Project Officer.
ANALYTICAL COSTS
In view of the low level of analytical effort which is
required in this analytical strategy, the analytical costs are
accordingly modest and are tabulated below:
Sample extraction $50
LC fractionations, 8 fractions 100
Solvent removal, weighing 100
Obtaining IR spectra, 8 180
Total Costs $430
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LEVEL 2 - QUALITATIVE AND QUANTITATIVE ANALYSIS
1. INTRODUCTION
The increasing awareness and concern regarding the emission of
potentially hazardous organic materials from industrial processes, has
resulted in a variety of analytical procedures being devised for their
identification and quantification. Once a potential hazard is recognized,
the cognizant Government agency can then implement emission measurement
programs with an ultimate view to emission control. Chemical analysis
of complex mixtures of unknown organic species is a challenging prospect
for a knowledgeable and well equipped analyst. While opinions frequently
differ regarding the choice of technique and the suitability of analytical
methods for known pollutants, the possibility of unsuspected chemical
hazards remaining undetected reiterates the need for a unified analytical
approach which will maximize the probability that all chemical species
will be detected and measured. This manual presents an optimal compre-
hensive analytical scheme for the measurement of organic compounds
collected by established methods (see for example, Technical Manual for
Process Sampling Strategies for Organic Materials - Monsanto Research
Laboratory (1976).
(a) Rationale
In order that organic emission measurement programs may be
carried out in a consistent, coordinated, and comprehensive manner, it
is desirable that a moderately detailed procedural guide should be
established for this purpose. This manual is intended for use by
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12
experienced analytical chemists who have access to a wide range of state-of-
the-art analytical techniques. It is not intended to consist of detailed step-
by-step instructions, but rather will guide the user in a manner which is
intended to ensure that the maximum of analytical data may be obtained from any
complex organic mixture, irrespective of its source or method of collection.
In previous analytical studies of industrial emissions, various
techniques have been used by different workers, with the result that
differing data have sometimes been obtained for the same pollution sources.
Furthermore, it is unfortunate that many techniques have previously been
employed which have a limited capability for the determination of widely
differing classes of organic compounds, with the result that much potentially
valuable information relating to the emissions was not obtainable. It is
not cost-effective to embark upon an analytical strategy for a complex
emission source, without a reasonable probability that the maximum quantity
of analytical data which is accessible to all modern state-of-the-art
techniques will be obtained. If a unified and comprehensive analytical
approach is not taken, much unnecessary cost and duplication of effort
will be inevitable.
Thus, the ensuing analytical scheme has been developed with a
view to meeting the analytical demands of any complex mixture of organic
compounds. The high performance liquid chromatography (HPLC) separations
proposed are equally suitable for a low molecular weight hydrocarbon, a
high molecular weight ionic compound, or any other organic species falling
between these extremes. HPLC separation techniques have a significantly
superior efficiency to acid-base extraction methods. The qualitative and
quantitative methods discussed include the most powerful techniques available
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13
to the modern analytical chemist. Many species are more particularly
suited to individual analytical techniques such as infrared spectroscopy,
nuclear magnetic resonance spectroscopy, mass spectrometry, and chromato-
graphy, but a combination of more than one of these techniques assisted
by suitable dedicated computer routines constitutes a most powerful re-
source for the analytical chemist. It is envisaged that if the following
analytical strategy is applied to any complex organic emission sample, the
data obtained will represent the maximum information reasonably attainable
by present organic analytical techniques.
(b) Overview
The initial step of the analytical scheme (See Figure 1) described
in this manual consists of a sequential separation by HPLC resulting in a
high resolution fractionation of complex mixtures of differing classes of
organic compounds. It is anticipated that of the order of twenty or more
HPLC fractions will be obtained for subsequent analysis.
The preliminary analysis will consist of screening each HPLC
fraction by a combination of techniques such as infrared spectroscopy,
nuclear magnetic resonance spectroscopy and high resolution mass spectro-
metry. Having thus identified the classes of organic compounds which are
present in each of the fractions, qualitative and quantitative analysis
will be carried out by gas chromatographic-mass spectrometry (GC-MS), gas
chromatographic-infrared spectroscopy (GC-IR), gas chromatography (GC),
nuclear magnetic resonance (NMR), and high performance liquid chromatography
(HPLC). HPLC in combination with NMR, IR, and mass spectrometry (MS) will
be utilized exclusively for identification and quantification of very polar
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14
and ionic compounds, since these species are not amenable to separation by
gas chromatography and have been largely ignored in earlier studies.
As can be seen in Figure 3, the separation into fractions and
analytical screening will be iterative procedures, in so far as separation
techniques will be reapplied as extensively as necessary until the screen-
ing techniques indicate that the complexity of each HPLC fraction is
sufficiently reduced to facilitate more straightforward qualitative and
quantitative analysis.
All of the techniques described in this manual have been avail-
able to the analyst for several years, and all of the equipment discussed
is commercially available. The most recent analytical innovations are
discussed where appropriate, and alternative methods are suggested only
when these represent effective alternatives.
The final section of the manual presents an estimated cost
breakdown for each phase of the analysis.
2. SAMPLE EXTRACTION AND RECOVERY
In most instances, samples of organic effluent will have been
collected by means of a sampling train which may consist of various
combinations of filters, cyclones, impactors, and porous polymer adsorbent
traps. However, samples may also be provided which consist of liquids or
granular solids other than particulate from impactors or cyclones.
Additionally, solvent rinses from probes and filter holders may be provided
for analysis; it is recommended that any such solvent rinses should be
made with solvents such as methyl alcohol followed by methylene chloride,
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15
DISCARD
SAMPLE
EXTRACTION
SIZE SEPARATION
CLASS SEPARATION
IDENTIFICATION
QUANTITATION
ADDITIONAL
SEPARATION
FIGURE 3. OVERALL ANALYTICAL STRATEGY
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16
in order to ensure complete recovery of organic species. Procedures for
sample extraction, and volume reduction are described below.
(a) Filters
Extraction of filter material should be carried out initially with
methylene chloride followed by a reextraction of the residue and filter with
methyl alcohol in order to ensure that all polar and non polar organic
material is efficiently recovered. Extraction may be carried out using a
Soxhlet apparatus for 24 hours, or by ultrasonic agitation of the filter
with solvent in a sealed glass container for one hour, followed by con-
ventional filtration of the shredded filter and insoluble particulate
matter. We have found both methods of extraction to be satisfactory,
although recent reports (1) suggest that ultrasonic agitation may be
slightly superior.
(b) Cyclones, Impactors, and Probe Rinse
Particulate material which has been removed from a cyclone,
impactor or rinsed from a probe may be solvent extracted in a similar
manner to a filter. Soxhlet extraction may be carried out in a cellulose
thimble plugged with glass wool, or in a fritted (fine) glass thimble
with a glass wool plug. Ultrasonic extraction of particulate may be
carried out by agitation with solvent in a sealed glass container followed
by conventional filtration to remove the insoluble material.
(c) Porous Polymer Adsorbent Traps
Porous polymer adsorbent traps are coming into widespread use as
a replacement for impinger collection in EPA Method 5 type sampling trains
on account of their significantly higher efficiency. A typical adsorbent trap
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17
is shown in Figure 1, which schematically depicts the Battelle Adsorbent
Sampler (2,3). Collected sample may be recovered from these sampling
devices by thermal desorbtion (4,5) or solvent extraction (2,3).
The preferred method of solvent extraction involves continuous
solvent extraction of the adsorbent for a period of 24 hours (2,3). Such
an extraction apparatus is shown in Figure 2, which depicts the continuous
extraction assembly developed for the Battelle Adsorbent Sampler; the
condenser and solvent reservoir are not shown. We would recommend that a
similar apparatus should be used for the extraction of all porous polymer
adsorbent traps.
The choice of solvent for extracting a porous polymer adsorbent
trap depends partly upon the nature of the adsorbent. It is important that
the solvent chosen does not affect the adsorbent in any way, yet is still an
effective solvent for the removal of collected material. For porous polymer
adsorbent traps utilizing Tenax, we would recommend extracting with a hydro-
carbon such as pentane, since more polar solvents readily dissolve the
adsorbent. The disadvantage of using a relatively inefficient solvent such
as pentane is readily overcome by using the continuous extraction mode, when
even polar compounds are extracted by pentane. Care should however be taken
to ensure that the very polar compounds are extracted; such compounds may be
anticipated on the basis of Level-1 studies, or from prior knowledge of the
source. It is possible that extraction with a more polar solvent compatible
with the adsorbent material may be required.
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(d) Granular Solids
Granular solids should be crushed and ground to mesh size 200 or
finer and then subjected to ultrasonic solvent extraction in the same manner
as particulate samples. Alternatively the solid material should be suspended
in the extraction solvent and subjected to high frequency dispersion (6).
(e) Liquids
Aqueous solutions may be solvent extracted by means of conventional
liquid-liquid extraction in a separatory funnel; methylene chloride will
frequently prove to be a suitable solvent. Alternatively when the organic
compounds sought are believed to have a reasonable vapor pressure (most
compounds except the very polar), vapor displacement and adsorbtion may be
used. Inert gas is bubbled through the liquid, and is then passed through
a trap containing chromatographic adsorbent where the volatilized vapors are
trapped (7). Sample recovery from the adsorbent may be made by extracting
it with a suitable solvent, as discussed previously.
A further aqueous extraction procedure involves percolating the
solution through a column of chromatographic adsorbent or resin (8), again
followed by solvent extraction or elution of the solid to recover the organic
sample.
Organic oils should not be extracted but should be directly subjected
to high performance liquid chromatography as described in the next section.
(f) Preparation of Extracts for Analysis
The manner in which the various sample extracts are combined depends
upon the information that is required from the emission source. It is possible
that particulate and vapor may need to be analyzed separately on account of data
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19
required for an emission control strategy, for example. In any case, each
extract or combination of extracts should be reduced to a volume of about
0.5 ml by a Kuderna-Danish evaporator, the sample is then ready for
separation into fractions by high performance liquid chromatography. While
it is true that some of the more volatile species may be partly lost during
this procedure, the sampling techniques utilized for organic materials from
process streams are generally not effective for the most volatile species
in any case. Much of the sample loss is likely to be volatile hydrocarbons,
which are generally regarded as less important from a health effect point of view.
It is usually desirable to add the prerequisite internal standards
to the sample extract before volume reduction and liquid chromatography
separation (3). However, the analysis of a grossly complex mixture as
envisioned here precludes addition of internal standards to the HPLC
fractions until qualitative analysis has been carried out, since it is
impossible to prejudge the nature of the compounds present. The present
analytical scheme incorporating high performance liquid chromatography
separation techniques does however offer the significant advantage of
truly quantitative sample fractionation. This largely obviates the usual
necessity of adding internal standards before sample fractionation, since
in the GPC and HPLC separations described, the sample integrity is expected
to remain above 90%, whereas with the older acid-base-neutral fractionation
techniques it was not surprising to lose well over 5070 of the sample during
the extraction procedures.
HPLC fractionation steps are not expected to introduce an un-
certainty of greater than +107o, and thus when utilizing the quantification
techniques described in Chapter 6, a high degree of analytical accuracy is
expected to be achieved for even complex mixtures of organic compounds.
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3. SEPARATION BY HIGH PERFORMANCE
LIQUID CHROMATOGRAPHY
In order to successfully separate and analyze a complex mixture,
the use of multiple chromatographic techniques, or sequential analysis,
is necessary (Figure 4). The use of high performance liquid chromatography
(HPLC"> allows sequential analysis to be performed more quickly and
efficiently than conventional column chromatographic techniques.
Significant improvements have come to liquid chromatography
over the past ten years both in terms of column design and instrumentation.
Presently, small diameter columns (2-5 mm) packed with supports of particle
diameters down to 5 ^ are being used. The eluent is pumped through the
column at higher linear velocities than classical liquid chromatography
(0.1 to 5 cm/sec), which results in larger pressure drops (200-5000 psi)
across the column. This has resulted in efficiencies 100-1000 times
higher than in classical methods, and thus the name, high performance
liquid chromatography.
For this reason, high performance liquid chromatography is rapidly
replacing older column and thin layer chromatographic techniques, and has
taken a position alongside gas chromatography as a highly efficient, highly
sensitive separation tool. Chromatographic columns with greater than
20,000 theoretical plates per meter are currently being used to affect
high resolution separations.
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21
MIXTURE
o
o
SIZE SEPARATION
CLASS SEPARATION
FIGURE 4. SEQUENTIAL ANALYSIS BY HPLC
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22
As a result of the significant improvement in chromatographic
columns, the ancilliary equipment required for their success has
necessarily become more sophisticated. Although the foundation of high
performance liquid chromatography has as its base the improvement in the
conventional liquid chromatographic column, its overall success depends
on precision instrumentation, such as constant flow, high pressure
pumping systems, and detectors with micro-flowthrough cells to prevent
remixing of resolved peaks. For a further discussion of high performance
liquid chromatography, the following books (9-12) and review articles
(13,14) should be consulted.
a) Separation by Size
The first step in the separation of a complex mixture is to
separate by size or molecular weight, using gel permeation chromatography
(GPC). Gel permeation chromatography is an exclusion technique in which
retention is based on a molecule's ability to penetrate the pores of the
chromatography support. Figure 5 illustrates the principles of gel permeation
chromatography. Large molecules elute with the solvent front while small
molecules totally permeate the support and elute later on. Within the
range of elution volume, molecules can selectively permeate the support and
thus be separated. Supports must be chosen for the exclusion limits that
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EXCLUSION
MOLECULAR
SIZE
OPTIMUM
MOLECULAR
WT
SELECTIVE PERMEATION
V
m
TOTAL PERMEATION
ELUTION VOLUME
FIGURE 5. PRINCIPLES OF GPC
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24
correspond to the molecular weight range that one desires to separate.
For more detailed information, the following books and review articles
should be consulted (9,12,15).
There are several advantages in using GPC as an initial
separation method. A knowledge of the molecular weight of an individual
fraction can dictate the choice of further separation steps such as gas
or liquid chromatography as discussed subsequently. Narrow molecular
weight ranges also allow simpler interpretation of the class separations
that follow. Finally, information concerning molecular weight is useful
for final identification purposes.
Figure 6 shows a fractionation by GPC which arbitrarily assumes
that all the compounds of molecular weight under 500 are of interest.
The number of fractions actually obtained as well as the molecular weight
range of each fraction will depend on the individual samples analyzed.
Since we are assuming the most complex possible case, all fractions will
be further analyzed. With modern high performance liquid chromatographic
columns, molecular weight differences of 15 percent can be distinguished
and therefore the number of fractions collected during the first separation
depends on the sample and the analysis goals.
(i) Column Packings
Separation in gel permeation chromatography is controlled by
the type of packing used. The region of selective permeation depends
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25
EXTRACTED SAMPLE
GPC
MOL.
WT.
>500
350-500
50-200
<50
CLASS SEPARATION
FIGURE 6. SEQUENTIAL SEPARATION - GPC
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26
on the pore size of the support and therefore the choice of the packing
material for a given separation is most important. Since we are interested
in the selective permeation of small molecules (molecular weights under
500) supports with small pore sizes (100-500 A) should be used. The
GPC packing most often used for sequential analysis is the semi-rigid
type and is based on a styrene-divinyl benzene polymer (Styrogel, Biobeads,
or equivalent). For complex mixtures, small particle GPC columns packed
with micro Styrogel can be obtained with efficiencies of 10»000 plates
per meter allowing the separation of compounds with differences of only
15 percent in molecular weight. For a good, although somewhat dated
review of GPC packing materials, see Reference 16.
When using packings of the styrene-divinylbenzene type, the
columns should not be allowed to dry out since channeling will occur
which is deleterious to column efficiency. For the same reason, air bubbles
should be avoided when connecting and disconnecting columns.
(ii) Mobile Phases
The mobile phase in GPC does not participate in the separation
process. Therefore it should be chosen for its ability to dissolve the
sample, its low viscosity, and its compatibility with the support and
the detection system.
High viscosity solvents should be avoided since diffusion of the
solutes is restricted and resolution is decreased. Some solvents cause
excess swelling which is deleterious to the support material; acetone
and alcohols are not used with styrene-divinylbenzene packings for this
reason.
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27
When choosing solvents for GPC, the detection system must
always be considered. If UV is used the solvents must be transparent
at the wavelength chosen. When refractive index detection is used,
solvents with low refractive index allow more sensitive detection.
Since air can be detrimental to the packing materials, the solvents
chosen should always be degassed before using. Common solvents used for
GPC include tetrahydrofuran, toluene, or methylene chloride.
(iii) Temperature
GPC is often run at elevated temperatures. High temperatures
allow increased sample solubility and lowers the solvent viscosity.
Since low molecular weight compounds are of interest, elevated temperatures
are not expected to be necessary.
(iv) Sample Size
Sample size in GPC is limited mainly by sample viscosity and
volume. A rough guide is that the sample solution should have a viscosity
no greater than twice that of the mobile phase. With samples of low
molecular weight, usually about 20 mg of sample per 100 ml of column
volume can be injected.
(v) Column Calibration
Most GPC calibrations are based on units of molecular length
or molecular hydrodynamic diameter, since size is related to the hydro-
dynamic volume of the molecules (15). For our purposes, a rough molecular
weight calibration is sufficient. This can be done by measuring the
elution volumes of a series of known compounds (preferably compounds of
interest) within the molecular weight range of interest. This procedure
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28
will define the limits of exclusion and permeation and also indicate
approximate regions of molecular weight.
Gel permeation chromatography is the simplest form of high
performance liquid chromatography. Its use will divide a complex multi-
component sample into manageable fractions. Once Step 1 of sequential
analysis is completed, samples can now be further separated by class.
b) Separation by Class
In the previous section, we have seen how narrow molecular
weight fractions could be obtained from a complex mixture by separation
on GPC columns. The GPC fractions can now be further separated according
to polarity by bonded phase liquid chromatography.
Bonded phase liquid chromatography is a form of liquid partition
chromatography in which the stationary liquid is permanently chemically
bonded to the support. Although the mechanism of retention is no longer
strictly partition, bonded phases possess distinct advantages over liquid-
liquid systems. Since the stationary phase is bonded to the support, it
cannot be stripped off by the eluent and therefore precolumns are not
necessary. More importantly, gradient elution, an extremely important
technique which will be discussed shortly, can be performed with a wide
choice of solvents. Finally, a wide range of selectivity can be obtained
by the proper choice of chemically bonded phase.
In this second step of sequential analysis, a reverse phase
bonded support should be used. It has been shown that for similar solutes,
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29
a plot of the log of retention time versus water solubility yields a
straight line, when using reverse phase columns with water-methanol
mixtures as the mobile phase (17). Since we have already obtained
fractions of narrow molecular weight, it can be assumed that retention
will be roughly a function the particular functional groups attached
to the molecule. We can therefore obtain a rough class separation by
performing gradient elution on a high performance reverse phase column.
(i) Gradient Elution
Since individual GPC fractions will usually contain components with
widely different chemical structures, isocratic solvent conditions (constant
strength) cannot be employed for the elution of such mixtures. Since
the relative migration rates of individual components in the mixture
will vary widely, early eluting peaks are poorly resolved while excessively
retained peaks require long analysis times. Therefore the technique of
gradient elution would be used extensively during the class separations.
In gradient elution, the mobile phase composition is continuously
changed from a weak to a strong eluent during a chromatographic run. In
this way, compounds of widely different polarity can be eluted from the
column in a reasonable length of time. Quantitative recovery of the
injected sample is therefore possible when a strong enough eluent is used.
As we shall see, this technique allows a total class separation of widely
different compounds.
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30
The instrumentation used to perform gradient elution can vary
widely. Modern liquid chromatography usually employs two high pressure
pumps which meter solvents from their reservoirs into a mixing chamber
under pressure and to the chromatographic column.
Solvent composition can thus be changed continuously during a
chromatographic run by differentially varying the flow rates of the two
pumps. This can be accomplished electronically with the use of a.
gradient programmer. Since the flow rates of the two pumps can be
precisely controlled, reproducible gradients can be formed giving
retention times with precisions less than 1 percent relative standard
deviation.
An important consideration when using gradient elution is
column regeneration. After a chromatographic run, the mobile phase must
be returned to initial conditions and reequilibrated with the stationary
phase before attempting another run. Also the solvents chosen for
gradient elution must be compatible with the detection systems used to
monitor the eluent. This will be discussed in more detail in a later
section. It is important to reemphasize the importance of gradient
elution in the analysis of complex mixtures. This technique is the basis
of the class separations to be discussed.
(ii) Column Packings
Many different column packing materials can be used to further
develop a sequential analysis. Reverse phase packings are recommended
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31
for the following reasons. Firstly as we have mentioned, a rough
separation by class is possible using this support material. Secondly,
one gradient run allows the elution of a wide range of compounds from
ionic and very polar to nonpolar species. This means that the entire
sample can be eluted from the column without losses. Silica gel, which
has been used previously as a second column in sequential analysis,
suffers in this regard. Finally, column reequilibration after a
gradient run is extremely fast using reverse phase packings. Since we
are interested in obtaining as much resolution as possible, columns
containing microparticle (5-10 MO reverse phase packing should be chosen.
These columns can generate about 10,000 plates per meter, and are
available from a. variety of different manufacturers. When large samples
are to be chromatographed preparative columns should be used. These
columns are capable of handling over one gram of material without over-
loading.
(iii) Mobile Phases
Typical gradient solvents in reverse phase chromatography are
water modified with methanol, isopropanol, or acetonitrile. The choice
of solvents is dictated by the type of detection used. When using
refractive index detection, gradient elution is difficult, but not
impossible, to run since large changes in baseline occur. There is
usually no problem using a UV detector. Detection will be discussed
further in the next section.
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32
Civ) Temperature
Elevated temperatures are sometimes used in reverse phase
HPLC, since lower viscosities and therefore higher efficiencies can
be obtained. Retentions are usually lowered at higher temperatures.
With the use of high performance micro-particle columns, elevated
temperatures are not necessary on a routine basis, but certainly should
be used if the added efficiency can solve a particular separation
problem.
(v) Functional Group Separation
The fractions obtained from GPC can be further separated by
reverse phase HPLC using gradient elution. Figure 7 shows, once again,
a rough guide to the kind of separation one might expect. Very polar
compounds will elute first and very nonpolar compounds will elute last.
The fractions which are collected during this run may contain 1 or 2
components in a simple case or 100 components in an extremely complex
mixture. However, these fractions should be sufficiently homogeneous
to be screened by spectroscopic techniques (see Chapter 4) for classes
of organic compounds. If screening techniques indicate that a fraction
contains compounds amenable to GC analysis, this route should be taken.
Complex mixtures or mixtures containing compounds which cannot be run
by GC should be further separated by HPLC. The choice of columns for
further separation by HPLC is discussed in Chapter 5. Figure 8 is an
illustration of the wide variety of compounds that can be eluted during
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33
GPC FRACTION
REVERSE PHASE LC
VERY NONPOLAR
NONPOLAR
SCREENING
GC ANALYSIS
POLAR
VERY POLAR
SCREENING
HPLC ANALYSIS
FIGURE 7. SEQUENTIAL SEPARATION - REVERSE PHASE HPLC
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34
ELUTION
VOLUME
H2O
HYDROCARBONS
OLEFINS
POM'S
THIOPHENES
-I PCB'S
!CARBAZOLES
ETHERS
ESTERS
^ ALDEHYDES & KETONES
H AMINES
-I PHENOLS
POLYFUNCTIONALS
ACIDS. BASES
CH3OH
FIGURE 8. TYPICAL ELUTION ORDER BY CLASS ON REVERSE PHASE HPLC
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35
a gradient run from water to methanol. Large ranges in solvent composition
are given to account for the molecular weight differences in a given
class of compounds. For example, naphthalene and coronene might be
expected to elute towards the two extremes of the POM range. Relative
retentions for standard compounds of interest should be determined prior
to unknown analysis so that narrow fractions can be obtained from the
gradient run.
After step 2 of sequential analysis a considerable separation
of the samples has been accomplished and effective screening techniques
should now be used to determine further cause of action.
c) Detection Systems
The ability to continuously monitor the column effluent is yet
another advantage of modern HPLC. With the advent of small volume
(8-20 M-l) flow-through cells, highly resolved chromatographic peaks can
be detected without significant remixing in the detector cell. Both
universal, or bulk property, detectors and specific, or solute property,
detectors are presently available, but unfortunately, there is no HPLC
equivalent to the flame ionization detector in GC for both universal and
sensitive detection. Nonetheless, valuable information can be obtained
by HPLC detection methods and frequently quantitation can be achieved.
The two universal detectors in common use are the refractive
index detector and the solute transport detector. The refractive index
(RI) detector measures the difference in refractive index between the
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36
mobile phase and the mobile phase containing dissolved solute. Since it
responds to all sample components it is often the choice when non UV
active compounds are analyzed. Its major limitation is low sensitivity,
and mobile phases should be chosen to enhance differences in refractive
index between solvent and solutes. Gradient elution is difficult to
perform using RI detection since large baseline changes occur when the
mobile phase is changed. Refractive index matching of the initial and
final solvents is essential if gradient elution is to be performed.
This can be accomplished when reverse phase chromatography is being used
(class separation) by an appropriate mixture of methanol and acetonitrile
to match the refractive index of water. RI detectors have a large linear
range but unfortunately lower limits of detection are about 1 |i gram for
favorable solutes.
The second universal detector used in HPLC is the solute transport,
or moving wire detector. The chromatographic eluent is dripped over a
moving wire depositing some sample. The wire is then fed to an oven
which evaporates the mobile phase, and then to a flame ionization detector
(FID). The advantages of this system are twofold. Detection is not
dependent on the solvent and thus gradient elution can be used, and secondly,
the sensitivity is potentially better than RI detection. However, the
detector is limited to relatively nonvolatile solutes, and since this is
a destructive detection technique collection and further analysis of the
sample is impossible. Finally, although FID is a sensitive detection
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37
device, only about 1-2 percent of the sample to be analyzed is deposited
on the wire and therefore overall detection at present is only slightly
better than RI. For these reasons RI detection is recommended as a
universal detector for the present analytical strategy.
The two specific detectors most commonly used are UV and
fluorescence. UV detection is the most widely used in HPLC today, and
is the first detection choice for compounds that absorb in the UV.
High sensitivity and specificty is obtained using UV detection and
lower limits of detection for favored samples is in the nanogram range.
The first detectors used a mercury lamp and monitored the eluent at
354 run, but more recently multiwavelength detectors have become available
in which eluent monitoring from 200-800 nm can be obtained. Although
multiwavelength detectors suffer a slight loss in sensitivity, the
increased specificity and ability to monitor wavelength maxima of com-
pounds make them valuable detection systems.
The second specific detector used for HPLC monitoring is the
fluorescence detector, which is receiving a great deal of interest
recently due to its high sensitivity and high selectivity. Fluorescent
compounds can be detected and quantitated in the presence of coeluting
compounds. As an example of its usefulness in the analysis of organic
eluents, POM's may be selectively detected during the class separation.
A combination UV, fluorescence detector is presently commercially
available and could prove valuable in monitoring organic effluents during
sequential analysis. When using fluorescence detection, care should be
taken to degass the solvents used for the mobile phase and to avoid
halogenated solvents, since these conditions quench fluorescence.
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38
For the analysis of a complex multicomponent mixture, a
combination of detectors in series is an effective way to monitor the
HPLC eluent. Valuable information about individual fractions or
peaks can be obtained and correlated with the screening techniques
used in Chapter 4 for compound or functional group identification.
Furthermore, quantitative analysis can be performed as discussed in
more detail in Chapter 6.
We have referred more than once to the importance of solvent
detector compatibility; it is important to emphasize this point again,
since transparent solvents must be used with specific detectors and
if RI detection is used to monitor a gradient, refractive index matching
is essential.
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39
4. SPECTROSCOPIC SCREENING TECHNIQUES
a) Introduction
Separation of the sample into organic classes by HPLC will
provide fractions which can be screened for complexity and/or types of
compound which are present. Identification of individual components
is not intended in a screening technique, but rather the ability to
discern the presence or absence of interferring compounds in a given
class, and to judge the overall complexity of the mixture. In the case
of the most complex mixture, screening will provide an indication of
the type of separation needed to further fractionate the sample. For
simple mixtures, screening will be a step toward qualitative and
quantitative analysis.
The techniques used for screening include infrared spectroscopy,
nuclear magnetic resonance spectroscopy, and high resolution mass spectrometry.
4 b) Nuclear Magnetic Resonance Spectroscopy
(i) NMR as a Screening Technique
Nuclear magnetic resonance spectroscopy (NMR) is a powerful screening
technique for mixtures of organic compounds. Although not used extensively
in the past for analysis of mixtures, recent progress in instrumentation
to improve resolution, stability, and sensitivity has made the observation
of components of a mixture practical. The types and relative amounts
of various functional groups in the mixture can be determined, and types
of hydrocarbons can be identified. Asa screening technique, NMR not only
characterizes the general nature of the fraction, but also provides sufficient
information to determine the types of further separations required. For
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40
example, a typical liquid chromatography fraction submitted for screening
might be expected to contain only polynuclear aromatic hydrocarbons. Their
presence can immediately be confirmed by observation of the aromatic region
of the spectrum. Other compounds with similar polarity eluted at the same
time would also be observed, and can be classified as to functionality.
Since the types of compounds to be separated are identified, a scheme for
their separation can be devised. Thus, obtaining the NMR spectrum of a
sample has become a logical step in the analysis of organic components of a
mixture.
The presence of various functional groups and identification of
the types of hydrocarbons is determined from the chemical shift of peaks in the
spectrum. This is due to the fundamental principle of NMR that the same
nucleus in a different chemical environment resonates at a different frequency.
A partial list of the approximate chemical shifts for protons in various
functional groups is provided in Table 2. A more comprehensive listing
of chemical shifts for protons and for other nuclei as well as a general
review of NMR in the analysis of organic compounds is available from a
number of sources (18-22).
The recent advances in instrumentation have extended NMR
such that less abundant nuclei can now be observed. The greatest growth
13
has been in C NMR. Carbon is of extreme importance due to its fundamental
role in the structure of organic compounds. The carbon backbone of a
compound and nonprotonated functional groups containing carbon, such as
carbonyls or nitriles, can now be observed directly. Proton NMR and
13
C NMR together cover almost all organic compounds, making the combined
methods a powerful screening technique for mixtures of organic components.
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41
TABLE 2. APPROXIMATE PROTON CHEMICAL SHIFTS
OF REPRESENTATIVE FUNCTIONAL GROUPS
Type of
Cyclopropane
Primary
Secondary
Tertiary
Vinyl ic
Acetylenic
Aromatic
Benzyl ic
Allylic
Fluorides
Chlorides
Bromides
Iodides
Alcohols
Ethers
Esters
Esters
Acids
Carbonyl cmpds .
Aldehydic
Hydroxylic
Phenolic
Enolic
Carboxylic
Amino
gem-dichlorides
Proton
RCH3
R2CH2
R3CH
C=C-H
C=C-H
Ar-H
Ar-C-H
C = C-CH3
HC-F
HC-C1
HC-Br
HC-I
HC-OH
HC-OR
RCOO -CH
HC-COOR
HC-COOH
HC-C = 0
RCHO
ROH
ArOH
C=C-OH
RCOOH
RNH2
HC12
Chemical Shift (a)
0.2
0.9
1.3
1.5
4.6-5.9
2-3
6-8.5
2.2-3
1.7
4-4.5
3-4
2.5-4
2-4
3.4-4
3.3-4
3.7-4.1
2-2.2
2-2.6
2-2.7
9-10
1-5.5
4-12
15-17
10.5-12
1-5
5.8
(a) ppm from TMS.
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42
NMR has an additional advantage over proton NMR. Since the
shift range for carbon is 600 ppm compared to less than 20 ppm for
proton nuclei,there is an enhancement in effective resolution. Broad
envelopes of overlapping peaks in proton NMR may many times be
resolved into individual carbon resonances. This allows for ready
identification of specific functional groups. For a review and further
13
discussion of C NMR see references 23-26.
(ii) Limitations of NMR in a Screening Technique
The primary limitation of NMR is sample size. For conventional
continuous wave (CW) NMR, milligram quantities are needed for proton
13
NMR, and natural abundance C NMR is essentially impossible. This has
largely been overcome by the use of Fourier Transform NMR. Unlike CW
NMR, which slowly sweeps the radio frequency (RF) applied in a fixed
magnetic field, Fourier Transform (FT) methods use short bursts of RF
power at a discrete frequency for rapid data acquisition. The observation
of spectra is made after the RF power is turned off. In this mode, data
are generated as free induction decay patterns (signal intensity versus
time). The Fourier transform of this is then mathematically determined,
which is a conventional NMR spectrum (signal intensity versus frequency).
This multichannel excitation and detection results in a hundredfold
improvement in sensitivity. For proton NMR in the pulse Fourier transform
13
mode, a practical minimum is 10 M>g of sample. For C NMR, milligram
quantities are required. Spectra of smaller amounts may be obtained by
using micro-techniques discussed later. The availability of a dedicated
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43
computer also provides the opportunity for data handling such as spectral
arithmetic. The spectra can be adjusted in size and unwanted components
can be removed by subtraction without the necessity of chemical separation.
For a detailed discussion of FT-NMR, see reference 27.
The only practical alternative to operating in the Fourier
transform mode is to operate in the continuous wave mode while using a
time averaging computer (computer of average transients, CAT). Even
here, unavoidable instability of the system sets the upper limit over
which signal averaging can be carried out without loss of effectiveness
or degradation of resolution to 20-30 hours. Since the signal to noise
ratio (S/N) increases with only the square root of the number of scans, the
time required per sample to achieve a suitable S/N is approximately two
orders of magnitude greater than time averaging of FT scans. This would be
prohibitive due to both time and cost unless an unusually large amount
of sample is available.
Another limitation of NMR is interference from solvent peaks.
Large solvent peaks may not only obscure regions of interest, but also
degrade the general quality of the spectra. This is due to introduction
of spurious resonances (beat frequencies) into the spectrum, difficulties
in phasing the spectrum, and loss of accuracy due to limitations of
analog-to-digital conversion. Several techniques have been developed
to suppress an unwanted solvent signal (28). However, for routine proton
NMR, using an unprotonated solvent, such as CCl^ or CS2, or using a
minimum amount of deuterated solvent for systems with few exchangeable
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44
protons is usually satisfactory. This will limit the observation of
those amines, acids, or other compounds with exchangeable hydrogens
which have low solubility in unprotonated solvents.
(iii) Experimental Details
Preparation of Sample. Each sample fraction from HPLC is
likely to be in a solvent unsuitable for NMR, such as protonated solvents.
Should this be the case, the sample must first be separated from the
extraction and separation solvents. Some of the methods which might
be used include: lyophilization (freeze-drying); evaporation with a
Kuderna-Danish concentrator; evaporation with a stream of inert gas;
removal under a vacuum at room temperature. In any of these methods,
the primary problem is the possibility of loss of volatile sample com-
ponents. If heating is used there is also the possibility of decomposition.
The actual choice of a method will depend on the nature of the solvent.
For example, for more volatile solvents such as the hydrocarbons,
evaporation using an inert gas (nitrogen, helium, argon) is preferable.
For more polar, less volatile solvents, such as water, lyophilization is
suggested. Removal of the solvent under a vacuum at room temperature
should be avoided unless the components are known to be nonvolatile, and
the solvent is unable to be removed adequately by other methods.
Choice of Solvent. After the separation solvents are removed
(when needed), the sample is redissolved in a solvent suitable for NMR.
The choice of a suitable solvent depends on a number of factors. It
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45
must first of all be of appropriate polarity to redissolve the sample.
This can be determined from the nature of the HPLC fraction and the
elution solvent. The solvent must also allow a sufficient window for
the observation of the sample. Ideally for proton NMR the solvent should
contain no protons, and for carbon NMR it should contain no carbon, thus
providing no solvent obstructions in the spectrum. Typical nonprotonated
solvents include carbon tetrachloride and carbon disulfide. These com-
pounds may not be desirable, however, due to low solubility of the
sample fraction. Therefore, as a second choice, deuterated solvents
may be used for proton NMR, and a solvent with one type of carbon may
be used for carbon NMR. Deuterated solvents should be chosen such that
the resonance of residual protons is outside the region of probable
interest (See Table 3). If interferences are unavoidable, two solvents
with differing residual resonances may be used in series to observe the
entire spectrum. In any case, a minimum amount of solvent should be
used to avoid a large solvent peak which can degrade the spectrum.
Finally, the ease of removal of the solvent should be considered, since
the sample may need to be further separated and characterized following
the determination of the NMR spectrum. Solvents such as dimethylformamide,
or dimethyl sulphoxide, which are very difficult to remove, should
be avoided.
Standards. The chemical shifts for a given nucleus are extremely
small compared to the resonance frequencies of the nuclei. While it is
possible to measure frequencies in the megahertz range to 0.1 Hz, there
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46
TABLE 3. CHEMICAL SHIFTS OF DEUTERATED SOLVENTS
Solvent
Acetic Acld-d^
Acctone-dj
Acetonitrile-dj
Benzene-dfc
Chloroform-d
Cyclohexane-d j^
Deuterium Oxide
1 ,2-Dichloroe thane-d4
Diethyl-d10 Ether
Diglyme-d^
Dimethylf ormamide-d 7
Diraethyl-dg Sulphoxide
p-Dioxane-dg
Ethyl Alcohol-d6 (anh)
Glyroe-d10
Hexafluoroacctone Deuterate
H>ffT-d18
Methyl Alcohol-d^
Methylene Chloride-d2
Nitrobenzene-dj
Nitroracthane-d3
Isopropyl Alcohol-dg
Pyridine-dj
Tetrahydrofuran-dg
Toluene-dg
Trifluoroacetic Acid-d
2,2,2-Trifluorocthyl Alcohol-d3
Chemical Shlft(a)
Residual i'rotons
11.53(1)
2.03(5)
2.04(5)
1.93(5)
7.15(br)
7.24(1)
1.38(br)
4.63(DSS)
4.67(ISP)
3.72(br)
3.34(m)
1.07(m)
3.49(br)
3.40(br)
3.22(5)
8.01(br)
2.91(5)
2.74(5)
2.49(5)
3.53(m)
5.19(1)
3.55(br)
l.ll(m)
3.40(ni)
3.22(5)
5.26(1)
2.53(2 x 5)
4.78(1)
3.30(5)
I
5.32(3)
8.11(br)
7.67(br)
7.50(br)
4.33(5)
5.12(1)
3.89(br)
1.10(br)
8.71(br)
7.55(br)
7.19(br)
3.58(br)
1.73(br)
7.09(m)
7.00(br)
6.98(in)
2.09(5)
11.50(1)
5.02(1)
3.88(4 x 3)
fnu.lt^b>
Carbons
178.4(br)
20.0(7)
206.0(13)
29.8(7)
118.2(br)
1.3(7)
128.0(3)
77.0(3)
26.4(5)
43.6(5)
65.3(5)
14.5(7)
70.7(5)
70.0(5)
57.7(7)
162.7(3)
35.2(7)
30.1(7)
39.5(7)
66.5(5)
56.8(5)
17.2(7)
71.7(5)
57.8(7)
122.5(4)
92.9(7)
35.8(7)
49.0(7)
53.8(5)
148.6(1)
134.8(3)
129.5(3)
123.5(3)
62.8(7)
62.9(3)
24.2(7)
149.9(3)
135.5(3)
123.5(3)
67.4(5)
25.3(br)
137.5(1)
128.9(3)
128.0(3)
125.2(3)
20.4(7)
164.2(4)
116.6(4)
126.3(4)
61.5(4 x 5)
(a)
(b)
Ppm relative to IMS.
The multiplicity of the peak; br indicates a broad peak.
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47
is no independent means to measure the magnetic field to the accuracy of
8 9
1 part in 10 or 10 . Thus absolute measure of the resonance is
impossible, so chemical shifts are reported relative to a standard or
reference.
There are two types of references used in NMR: internal and
external. An internal reference is a compound that is dissolved directly
in the sample solution. The reference is uniformly distributed at a
molecular level through the sample, such that the magnetic field acts
equally on the sample and reference molecules. The only serious problem
with an internal reference is the possibility of intermolecular inter-
action which would influence the resonance frequency of the reference.
The most common reference, now generally accepted for both proton and
13C, is tetramethylsilane (TMS). It is relatively inert, highly volatile,
gives a single peak in both proton and carbon NMR, and resonates at a
field higher than most common nuclei. TMS, however, is not soluble in
aqueous solutions. The common reference compound for aqueous solutions
is sodium 2,2-dimethyl-2-silapentane-5-sulfonate, (CH3)3Si(CH2)3SC>3Na, (DSS) .
This is used at a low concentration so the methyl singlet is observed
but the spin coupled methylene groups do not interfere appreciably.
Shifts measured with respect to TMS in chloroform will be within a few
hundredths of a ppm for the same peaks measured with respect to DSS in
water.
An external reference is a compound placed in a separate con-
tainer from the sample. This may be in a sealed capillary tube inside
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48
the sample tube, or in the annulus with the sample inside the capillary
tube, When the sample is rotated rapidly the reference signal appears
as a sharp line superimposed on the spectrum of the sample. An external
reference removes the problems of intermolecular interaction, chemical
reaction, and insolubility in the sample solution. However, there is a
difference in the bulk magnetic susceptibility between sample and
reference. The susceptibility correction is normally < 1 ppm. For
13
nuclei with large chemical shifts, as C, the correction may not be
necessary. For proton resonances, when accurate chemical shifts are
needed, a correction for the difference in susceptibility should be made.
For dilute solutions of the sample, the conversion to internal TMS may
be made by means of measured frequency differences for TMS in different
solvents. (See Table 4). This is particularly crucial for the identi-
fication of specific compounds from reference spectra, but may be
ignored for many spectra of mixtures if only general classification of
the types of compounds is desired.
NMR data are usually measured in frequency units (hertz) from
the chosen reference. However, the chemical shift is dependent on the
value of the magnetic field. Therefore, it is customary to report
chemical shifts in the dimensionless unit of parts per million (ppm),
which is independent of the rf frequency or magnetic field strength.
The chemical shift in ppm (6) is
6 = VS - VR X106,
vR
where vs and vR are the resonance frequencies of the sample and reference,
respectively.
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49
TABLE 4. RELATIVE RESONANCE FREQUENCIES FOR PROTON
REFERENCE COMPOUNDS
Compound
Internal TMS
TMS in CCl4a
TMS in CDCl3a
t-Butanol
Cyclohexane
Acetone
Dioxane
Water
Benzene
Chemical Shift (ppm)
0
0.43
0.52
1.39
1.63
1.87
3.80
5.14
6.95
(a) 1 percent by volume
(b) Temperature dependent
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50
For this work, a dilute solution of TMS in carbon tetrachloride
in the annulus of an NMR tube is recommended. This allows for the use
of micro techniques as discussed in the next section and removes the
problem of solubility in aqueous solutions. The minimum amount of TMS
required to observe a peak should be used to avoid degrading the
spectrum. All chemical shifts should be reported in ppm relative to
TMS.
Micro Techniques. One method used routinely to improve sensitivity
recommended for this work is the use of micro techniques. The goal is a small
volume of sample at the maximum concentration such that the entire sample
can be detected by the instrument. This can increase the effective
sensitivity by as much as 100 times. Commercially available accessories
include microcells, which confine a small amount of sample to the region
of the receiver coil in the probe, and mini-probes, which are designed
specifically for small sample volumes by using specially would receiver
coils. Further details and instructions as to the use of these techniques
can be found in reference 29 and the manufacturers' literature.
An effective microcell can also be easily constructed by
sealing approximately 20 ul of the sample solution in a 2 mm O.D.
capillary tube slightly longer than a standard NMR sample tube. The
capillary tube is inserted into the sample tube and held in place by
protruding through a tight-fitting hole in the tube cap. One or more
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51
Teflon spacers around the capillary tube to hold it firmly in the center
of the sample tube are also helpful. Before this technique is first
used with a particular probe in a given instrument, the required amount
of sample and the optimum position of the capillary tube must be determined.
This is done by using a known compound in the capillary tube and adjusting
the position of the tube and the sample amount until the resonance peak
is maximized. To run a spectrum, a dilute solution of IMS (and lock
compound if the instrument has an internal lock) in carbon tetrachloride
is placed in the annulus. A solution of the sample is placed in the
capillary tube.
In summary, nuclear magnetic resonance spectroscopy is an
important tool for classifying mixture components. NMR readily identifies
functional groups and estimates the complexity of a mixture. A combination
of proton and carbon-13 NMR now allows observation of almost all organic
compounds, making it applicable to all types of mixtures. The only major
problem is sensitivity, which is to a large extent overcome by the use of
Fourier transform NMR and micro-techniques.
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52
4. c) Infrared Spectroscopy
(i) Introduction
Infrared spectroscopy has been, and still is, the most widely
used tool for identification of organic compounds. One of the major
uses of infrared spectroscopy (IR) has been during the separation of
complex organic mixtures. It has provided functional group (especially
on polar groups) identification in such mixtures, it has been used to
monitor or screen the course of separation of the mixture, and ultimately
it has been used to identify the compounds present when the separation
has proceeded as far as required. As with most analytical tools, less
information is obtainable when the mixture was very complex as compared
to when the mixture was separated into smaller fractions. Now, however,
the use of Fourier Transform infrared systems (FT-IR), has clearly demon-
strated that a major increase in sample information can be obtained - even
on complex mixtures - through the use of the FT-IR dedicated computer to
do spectral arithmetic.
Thus, as separation techniques are applied, IR should be
used to monitor the separation and identify the separated classes of
species. Spectral subtraction should be used to magnify the differences
between the separated fractions. Whenever possible, this spectral
arithmetric will be used to minimize the need of chemical separations.
Used in this manner, IR will provide a major source of
structural information to evaluate the HPLC separation and
to determine whether or not more such separations are needed. When the
IR data are combined with NMR data even more information can be
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53
obtained. Combining IR and NMR with good separation techniques
and subsequently with GC-MS and GC-IR, utilizes the most powerful tools
available for organic compound identification.
(ii) Fourier Transform Infrared Spectroscopy
Fourier Transform infrared systems (30) differ from conventional
dispersive infrared spectrophotometers in that conventional infrared
Spectroscopy uses a monochrometor to generate the spectral information
whereas an interferometer is used for this purpose in Fourier Transform
infrared Spectroscopy (FT-IR). The use of an interferometer to generate
spectral information in the form of an interferogram (light intensity
versus time) necessitates a second difference between the two types of
infrared Spectroscopy. This difference is that FT-IR systems use a
dedicated digital computer to obtain the Fourier Transform of the inter-
ferogram, converting it to a conventional infrared spectrum (light intensity
versus wavelength or frequency). These two differences lead to the fol-
lowing two major advantages of FT-IR over conventional infrared Spectroscopy:
o Using an interferometer results in a substantial gain
in energy or light throughout as compared to a mono-
chromotor. This gain in energy results from the fact
that all wavelengths of light are examined simultaneously
in an interferometer and no energy is lost (as in a dis-
persive instrument by examining the light one wavelength
at a time). This additional energy can be used in one
of several ways: (a) for faster scan speeds (as fast as
0.6 sec.), (b) for up to a 30-fold increase in signal-to-
noise ratio, or (c) for 10^-10^ greater sensitivity.
• The availability of a dedicated computer offers several
major data-handling advantages. Not only can spectra be
ratioed against each other to remove absorption bands due
to background materials, but the computer can be used to
perform spectral arithmetic. Thus, spectra can be added
or subtracted from each other and also multiplied or
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54
divided. In this way, the spectra can be adjusted in
size, and unwanted components can be removed from the
spectra without the necessity of chemical separation.
This ability to utilize a computer is not unique to Fourier
Transform spectroscopy, i.e., in theory a computer could be
attached to a conventional dispersive infrared spectro-
photometer. However, in practice, this is rarely done, whereas
whereas all Fourier Transform systems use a computer.
Thus, from a practical standpoint, the use of a computer
is a major advantage in FT-IR systems.
(iii) Experimental Details
Most experimental details (except for some instrumental
parameters) are equally applicable to either Fourier Transform infrared
systems or to conventional dispersive infrared spectrophotometers.
Therefore except where noted, all of the following experimental sections
apply to both types of infrared spectroscopy.
Method of Running Samples. Whenever possible (the vast majority
of the time), the spectra should be obtained on a sample prepared as a
film on an infrared transmitting crystal. In a few instances, the
sample may be a highly light-scattering solid which is difficult to run
as a film. In these cases the spectra should be obtained on a sample
prepared by the pressed disk (KBr) technique. Details of both the film
and pressed disk techniques can be found in Reference 31.
Preparation of Samples. As for NMR (See Section 4b, Prepara-
tion of Sample), the sample will come from HPLC fractionation as a dilute
solution, which therefore will necessitate removal of the solvent. The
same methods of solvent removal as given in Section 4b are applicable here
with one small exception. For infrared studies it is only necessary to
concentrate the solvent-sample system rather than to take the sample to
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55
dryness. After the solution is concentrated to a few drops, the remainder
of the solution is placed on the infrared crystal and the remaining solvent
allowed to evaporate. If at this point, usuable infrared spectra can
not be obtained, the sample should be removed from the infrared crystal
and the pressed disk (alkali halide) technique used to prepare the sample.
Since some of the HPLC solvent systems contain water, it will
be necessary to then use water resistant infrared crystals such as AgCl
or Irtran. For all other solvents the common infrared crystals (NaCl,
KBr) can be used.
If time is not a problem, it will be beneficial to use the
solutions used for NMR rather than the HPLC solutions to prepare the infrared
samples. The NMR samples will be either in the same solvent as used for
HPLC or in a better solvent (for infrared purposes of evaporation) than
the solvent used for HPLC. In addition the NMR solution will require less
concentration than the HPLC solution.
Micro Sampling Techniques. During the separation procedures
most of the samples will be of reasonable size and conventional infrared
sampling techniques (as described above) can be used to prepare the
sample for the screening process. However, as the separations proceed,
the separated fractions can approach a size where micro sampling techniques
will be needed. It is much more difficult to select a standard technique
for micro samples than for conventional-sized samples. Not only does the
handling of micro samples and micro sampling equipment require experi-
ence, but there are large differences in the micro techniques used by
various laboratories. Therefore, while some micro techniques will be
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56
suggested, experience in handling micro samples is so important that each
individual will probably get the best results from the technique most
familiar to him.
For practically all solids and many liquids, the micro pressed
disk technique is recommended. It is the most universal technique and
generally greater sensitivity is achieved than with a micro film
technique. This micro-pressed disk technique requires the use of a beam
condenser, micro-disk holders, and a press for producing the micro-pressed
disks. All of these are commercially available.
For liquids where the micro-pressed disk technique is not
applicable, a micro film technique can be employed. Here the film is
constrained to an area on the infrared crystal which is the size of the
infrared beam at the focal point of the beam condenser. This can easily
be done by shaping the infrared crystal to this size or by digging a
groove of this size in the infrared crystal.
It should be emphasized here that one of the main reasons for
recommending FT-IR as opposed to dispersive IR spectroscopy is the
greater sensitivity of FT-IR systems. In general a midrogram of sample
is needed to obtain a reasonable dispersive infrared spectrum, while
only 10-100 nanograms is needed for an equivalent spectrum using an
FT-IR system.
Instrumental Parameters. While there is basically only one
mid-infrared Fourier Transform system available commercially, there are
numerous commercially available dispersive infrared spectrophotometers.
Thus it can be difficult to define meaningful instrument parameters
since even the parameter nomenclature can vary from instrument to
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57
instrument. However, some general remarks can be made and the details
of good spectrophotometer conditions and operation can be obtained from
Reference 31.
First it is necessary to consider whether or not all of the
available instruments will give satisfactory spectra. Certainly all
Fourier Transform infrared systems will yield high performance spectra.
FT-IR systems differ from dispersive spectrophotometers in that inter-
ferometers give constant resolution over the entire wavelength range.
In addition, FT-IR systems have greater wavelength accuracy than
dispersive spectrophotometers. For these reasons, coupled with the
FT-IR advantages previously listed, it is strongly recommended that
FT-IR systems be used for all IR studies.
However, at the present time there are only approximately 75
FT-IR systems in the world as compared to many thousands of dispersive
infrared spectrophotometers. Thus not all laboratories have access to
FT-IR systems and some of the screening work undoubtedly will utilize
spectrophotometers rather than FT-IR systems. While this may be
necessary, it must be remembered that using a dispersive spectrophotometer
will result in a definite sacrifice of sample information.
When using a dispersive infrared spectrophotometer care must
be exercised in choosing an instrument which is capable of achieving the
necessary performance specifications. In general most of the instruments
costing less than $6,000 do not meet the necessary requirements because
they lack the flexibility needed for the varied screening samples.
Obviously, the spectrophotometers costing over $20,000 give the highest
quality spectra (of the dispersive instruments) and their use is recom-
mended when FT-IR systems cannot be used.
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58
In general the instrument parameters used for spectrophotometers
should be those given in Reference 31, while good operating conditions
for FT-IR systems are listed in Reference 30, FT-IR spectra should be
run at a resolution of 4 cm"-'- (boxcar apodization) , while dispersive
IR spectra should be obtained at a slit width which yields an average
resolution of 4 cm"1 over the spectral range (at least 3800-600 cm"-'-).
Sample thickness should be adjusted so that the strongest absorption
band gives about 10% transmission. In many cases several sample thick-
nesses (both thicker and thinner) will be desirable (especially in FT-IR
where spectra are to be subtracted). Noise levels should not exceed
2% peak to peak. The infrared instruments should be purged by dry gas
or evacuated so that atmosphere absorption should not exceed the allowable
noise level.
It is certainly desired that the wavelength accuracy approach
+10 cm"1 above 2000 cm"1 and be less than that below 2000 cm"1. The
wavelength readibility should be better (for sharp peaks) than 10 cm"1
at wavenumbers greater than 2000 cm"1 and better than 5 cm"1 below
2000 cm" . To do this it is necessary to use charts greater than 8-1/2
by 11 inches.
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4. (d) High Resolution Mass Spectrometrv (HRMS)
High resolution mass spectrometry is periodically used as a
screening technique for organic effluent samples, but in this instance
the "Screening" is of a rather different nature to that generally in-
tended in this manual. HEMS screening is most useful in searching for
specific trace components, but is not readily able to give useful data
regarding the general nature of organic species present in a complex
mixture. For example, if the objective of an industrial effluent analysis
is to determine whether any of several predetermined hazardous materials
are present in the emission, then HRMS screening will provide very use-
ful data, and will readily tell the analyst whether the selected com-
pounds are absent in the sample, subject to the recognized detection
limits of HRMS techniques. This type of HRMS screening makes use of
peak matching (32) to determine exact fragment masses, and thus if frag-
ment ions of the correct empirical formulae for fragment ions of a sought
compound are absent, then the sought compound is judged to be absent. How-
ever, if fragments of the correct empirical formulae for the sought compound
are present, this does not necessarily mean that the sought compound itself
is present, since the apparently correct fragments could possibly have
arise through the presence of an isomer or analogue of the sought compound.
Thus, this type of HRMS screening will indicate whether a particular com-
pound is absent, but is unable to definitely indicate that a compound is
present. Screening studies of this type might more properly be termed
"negative screening".
We would not anticipate that negative screening by HRMS would
be a very widely applied technique, since many analytical studies are
predominantly concerned with determining what species are present, which
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60
precludes negative screening. Nevertheless, occasions will arise when
the appropriate Government agency will require to know whether a specific
effluent is being emitted from an industrial facility, and in such in-
stances HRMS negative screening will be the screening procedure of choice
to determine whether the specific effluent is absent or not, before
resorting to other powerful analytical techniques, such as GC-MS and
GC-IR, in order to ascertain whether the presence of the compound can
be confirmed, and then to carry out quantification if necessary.
Because suitable standards for general chemical ionization
HRMS are not available at the present time, HRMS is limited to electron
impact ionization in most instances. The detection limits of HRMS is thus
strongly dependent upon the ease of ionization of the compound being
sought. For a relatively polar compound with large charge separations
and ease of fragmentation, a reasonable detection limit may be well over
a hundred nanograms, possibly a few hundred nanograms. For compounds
that fragment with difficulty, such as highly aromatic species, and
especially POM, the detection limit will be appreciably lower and might
typically fall in the 10 to 30 nanogram range. It is possible to optimize
sensitivity for HRMS negative screening by reducing the ionizing potential
in order to reduce the initial excess energy of the molecular ion. How-
ever, this process must not be taken too far, when the ionization efficiency
of the species sought would begin to fall rapidly.
Sample should be introduced into the HRMS by probe insertion; a
portion of the organic extract will first be carefully evaporated to remove
excess solvent in the glass micro-vial which is situated at the tip of the
probe. The sample probe may be maintained at a constant temperature, but
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is generally more useful to use a thermally programmed probe, which will
provide some degree of separation of the sample, which can make inter-
pretation of the data a little simpler. Several recent studies have
described the utility of thermally programmed sample inlet probes (33).
In summary, it is expected that HEMS negative screening will
find limited usefulness, except in instances where only specific compounds
are being sought. Even in this case, the generally rather high detection
limits of HRMS makes the technique somewhat unattractive as an individual
screening technique.
4. (e) Evaluation of Screening Data
Determination of the complexity of a given HPLC fraction by the
above-mentioned spectroscopic screening techniques leads to a decision-
making point regarding whether the next step should be further separation,
identification and quantification, or discard the sample due to lack of
compound presence.
If the sample is of sufficient complexity or of mixed classes,
further HPLC separations, as discussed in Chapter 5, must be performed
prior to identification and quantification. Such a step would result in
a recycling of the samples through HPLC separation and spectroscopic
screening techniques until one class, a simple mixture of classes or a
single component, suitable for identification and quantification, can
be isolated.
If the screening procedures show the presence of only one class
of compounds, a very simple mixture, or a single component, specific
identification and quantification by one of the techniques discussed in
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Chapter 6 can be performed immediately.
In the case of a simple effluent sample mixture, several of
the HPLC class separation samples may not contain any compounds. If
spectroscopic screening procedures show a blank sample, then the fraction
should be thus noted and the sample discarded without further analysis.
5. ADDITIONAL SEPARATIONS BY HIGH
PERFORMANCE LIQUID CHROMATOGRAPHY
In chapter 3, we discussed the power of sequential analysis
using HPLC to affect a separation of a complex mixture. Frequently,
this may prove entirely successful for compounds of interest. In
dealing with complex multicomponent fractions however, further separation
is often essential before identification and quantitation can be achieved.
Fortunately, there are additional modes of HPLC available to the analyst
in his quest for total analysis. In this chapter, these additional chromato-
graphic modes will be discussed, their selection will depend on the results
of screening and prior separation and therefore a coordinated analytical
effort is a necessity.
(a) Bonded Phase Chromatography
We have used bonded phase chromatography in step two of sequential
analysis to obtain a initial class separation. The same reverse phase
column can be used either isocratically or with gradient elution, to
further separate the sample. Optimum solvent conditions can be estimated
from the class separation already performed. For example, if a fraction
containing multiple peaks was collected when the composition of the mobile
phase was between 20 and 2570 methanol, that fraction can be chromatographed
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isocratically at 20% methanol or a gradient performed from 15 to 30%
methanol. This second separation will vastly improve the resolution
of the mixture. Since the column and solvent consitions are available,
this is probably a good first step in additional separation.
Normal bonded phase chromatography in which the stationary
phase is polar (alkyl amino or cyano groups are bonded to the surface)
can also be used as an additional separation mode. Different selectivities
can be obtained using these phases since the interactions of various
functional groups with the surface is markedly different. When employing
these phases, usually non polar eluents are used; gradients can be run
using heptane and isopropanol, for instance, as solvent extremes.
When very polar or ionic compounds have been diagnosed by
screening, reverse phase bonded chromatography or ion exchange (see
later) is the separation mode of choice.
Selectivity can be largely controlled by the pH of the mobile
phase when ionic compounds are chromatographed on a reverse phase column,
and therefore difficult separations can be achieved. Very acidic or basic
compounds are best separated by ion exchange.
(b) Liquid Solid Chromatography
Liquid solid, or adsorption, chromatography is the oldest and
most widely used separation mode. In adsorption chromatography, an ad-
sorbant, such as silica gel or alumina, is used as a polar stationary
phase, and non polar to polar mobile phases are used to elute components.
One of the big advantages in using this mode is that a wealth of information
can be obtained from thin layer chromatographic literature that can be
applied to HPLC separations. Only slight changes in conditions are necessary
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to transfer TLC data directly to a column separation.
Another fact which has contributed to the wide use of silica
gel as an HPLC stationary phase is that the first high performance micro-
particle columns which were successfully slurry packed were of silica gel.
Liquid solid chromatography is best used for non-ionic compounds
that are not amenable to G C. Compounds of different chemical type or
differing numbers of functional groups are easily separable by adsorption.
The best resolution of chromatographic peaks are achieved on adsorption
columns, so if total separation is necessary, LSC should be tried, LSC has
difficulty in separating homologous series or compounds differing in the
extent of aliphatic substitution. Very polar or ionic compounds are also
difficult to elute using adsorption and are best done by reverse phase
or ion exchange chromatography0
(c.) Ion Exchange Chromatography
Ion exchange chromatography has always been the established method
of separating ionic species. It has therefore been used extensively in
biochemical separations, but its usefulness in treating general complex
samples is limited. However, after sequential analysis has been performed
and an ionic fraction isolated, ion exchange chromatography can be used to
affect a separation.
Ion exchange chromatography is carried out with stationary
phases which contain charge-bearing functional groups. The mechanism is
frequently simple ion exchange as follows:
X~ 4- R+Y~ "• Y~+ R+X" (anion exchange)
X + + R~Y+ * Y++ R~X+ (cation exchange)
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65
where X = sample ion, Y = mobile phase ion and R = ionic sites on the
exchanger. The solute ion competes with the mobile phase ion for the
ionic sites on the stationary phase. For this reason changes in the pH
or ionic strength of the eluent have a dramatic effect on retention for
further discussions concerning retention on cationic or amonic exchange
support see Reference 12.
Both reverse phase liquid chromatography and ion exchange can
be used to separate ionic compounds. With proper control of pH, reverse
phase chromatography is capable of many separations of weak to moderally
ionic compounds. Compounds which are strongly ionic can be
chromatographed by ion exchange.
(d) Evaluation of Additional Separations
Evaluation of the additional separations will be carried out
by the spectroscopic procedures described in Chapter 4. The objective
of this evaluation is fully described in Chapter 4, and is to determine
what classes of organic compounds are present in the further separated
fractions in order to facilitate qualitative and quantitative analysis
described in Chapter 6.
6. IDENTIFICATION AND QUANTIFICATION
(a) Gas Chromatography (GC), and High Performance
Liquid Chromatography (HPLC)
Introduction
Gas chromatography is a powerful tool for separations of complex
organic mixtures. In combination with selective detectors, particularly
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mass spectrometry, it is among the most powerful instruments available to
the analyst. The principles of HPLC have been extensively discussed in
Chapter 3, and thus only limited additional comments on its role in quanti-
tative analysis will be made in this chapter.
There are a number of gas chromatographs commercially available
which are suitable for complex organic mixtures. It is important that
the column oven is programmable at various rates up to about 400 C. The
facility for dual columns and dual detectors is sometimes useful, especially
when column effluent is split between two detectors such as a general pur-
pose flame ionization detector (fid) and specific detectors such as a
flame photometric detector (fpd) or electron capture detector (ecd).
The most important part of a gas chromatograph is the chromato-
graphic column. There are various types of column ranging from packed,
through support coated open tubular (SCOT), to high resolution capillary
columns. Column packings include silica gel, alumina and polymers such
as the Poropak and Chromosorb series, but the coated diatomaceous earth
type are very commonly used. There are a great number of column packings
commercially available; many of these have been developed in response to
specific problems, whereas others have a more general usefulness. The listing
given below in Table 5 does not pretend to be a complete inventory of column
packings and coatings, but is presented to serve as a preliminary guide.
Useful data may be frequently found in chromatographic supply manufacturers
catalogues, in addition.
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TABLE 5. ORGANIC SPECIES SEPARABLE BY VARIOUS GC COLUMN TYPES
Compound Type
Acids C-C
Crci8
Alcohols
Crci8
Polyalcohols
Aldehydes C^
C5-
Amines
Amides
Esters
Ethers
Freons
Glycols
Halides
Hydrocarbons
Aromatic
Olefins
POM
Ketones
Pesticides
Phenols
Column Type
Chromosorb 101
FFAP
Poropak Q, Chromosorb 101
Silar 5CP, Carbowax 20M, FFAP
FFAP
Poropak N, DC-550, Ethofat
Carbowax 20M, Silar 5CP
Poropak Q/PEI, Poropak R
Chromosorb 103, Pennwalt 223
Versamid 900, Igepal CO-630
Poropak Q, Dinonylphthalate
Chromosorb 101 or 102
Carbowax 20M, Silar 5CP
Poropak Q, Chromosorb 102
Chromosorb 107
OV-210, FFAP
OV-101, SE-30
Silar 5CP, Carbowax 20M
DC-550, DC-703
Dexsil 300, OV-101, SE-30
Poropak Q, Chromosorb 102, FFAP
OV-101, OV-225, OV-1, OV-17, SE-30
OV-17, Silar 5CP, Carbowax 20M
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68
A significant proportion of GC separations may frequently be
achieved using conventional packed columns. Capillary columns are be-
coming more widespread but their use requires greater skill than packed
columns. It is anticipated that the use of capillary columns in the
analytical strategy presented here will be restricted to the separation
of isomers and other groups of similar compounds, since many of the
difficult separations will already have been accomplished by means of
quantitative high pressure liquid chromatography, which has been described
earlier.
Gas chromatography can frequently provide good resolution of
complex mixtures of organic compounds, and for this reason it is commonly
used in organic analysis. An obvious limitation of the technique is that
involatile materials are not amenable to this method of analysis. In-
volatile materials are most expediently analyzed by liquid chromatography,
as described earlier.
A typical organic extract should normally be concentrated to a
few hundred microliters, before subjecting one or two microliters to GC
analysis. When an extract has been separated by high pressure liquid
chromatography, it is usual to concentrate this extract to about fifty to
a hundred microliters prior to GC analysis. The choice of solvent for GC
is not generally critical; commonly used solvents such as methylene chloride,
acetone, or benzene generally elute significantly before any of the compounds
of interest. If interference between solvent and sample peaks is observed
it may be necessary to change solvents or GC columns.
The sensitivity of GC with conventional fid depends to a large
extent upon peak sharpness and the level of noise or other interfering
peaks. In the absence of appreciable interference, a sensitivity of 50 ng/(j,l
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may reasonably be obtained for hydrocarbons using fid. With fpd or ecd,
for sulfur compounds or chlorinated pesticides for example, a sensitivity
or between one and two orders of magnitude better than this is reasonably
obtainable in most instances.
Screening Studies with GC
While GC is generally most useful for quantification, it is
nevertheless possible to obtain some useful preliminary screening data in
addition. The use of GC with fid detection as a preliminary screening
tool is somewhat limited by a lack of selectivity, nevertheless GC/fid
screening can provide some useful data regarding the general nature of
compounds which may be present in a sample. The most useful approach
to GC screening is to use a generally applicable column such as 370 OV-17
or OV-101, and program at about 6 C min from 100 to 300 C. See earlier
GC column listing. Few commonly encountered organic compounds of moderate
to zero polarity will not give reasonably good peaks under such conditions.
However, all that GC/fid screening studies will be able to ascertain is
whether or not organic compounds are likely to be present in a given sample,
and whether they may be reasonably resolved by GC without use of additional
HPLC separation. Rather more information can be gained from GC screening
by the use of selective detectors such as fpd or ecd, but the use of such
selective detectors rather presupposes that only specific information is
required from the sample. For example, if we only wish to know whether a
sample contains sulfur compounds or not, the obvious way to approach this
problem is by use of an fpd with a sulfur filter. It is a useful practice
when doing screening of this nature to obtain a simultaneous fpd and fid
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output which will permit the specific fpd peaks to be related back to the
fid or mass spectrometric ion current trace (through the fid trace).
The nature of the GC column can give only limited information
regarding the nature of compounds present, and it is not recommended that
an appreciable effort should be devoted to such a study. For example,
compounds eluting only at relatively high temperatures on a relatively
non-polar column such as OV-17 or OV-101, are probably highly polar
materials such as dialdehydes or other nonfunctional compounds, or large,
bulky compounds such as POM. Compounds which are only resolved with
difficulty on relatively polar columns such as Silar 5CP or Carbowax 20M
are likely to be relatively small molecules of moderate polarity.
Perhaps the most valuable data obtained from GC screening,
however, is simply to determine whether the HPLC separations previously
carried out have provided a sample in which the individual compounds are
sufficiently well resolved for further qualitative and quantitative analysis
on the most appropriate GC column. If this is not found to be the case,
additional HPLC separations must be carried out before further analysis can
be attempted.
Quantification with GC and HPLC
Gas chromatography is probably the most commonly used tool for
quantification in organic analysis. High performance liquid chromatography
has recently been used more extensively for quantification on account of
significant improvements in instrumentation. In order to carry out quanti-
tative analysis by either GC or HPLC, it is necessary that the compounds of
interest should be reasonably well resolved from other peaks in the chromato-
gram. Furthermore, it is highly desirable that pure samples of the compounds
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71
sought should be available; if the components sought are not available,
an approximation may be made through use of their analogues. An internal
standard, or standards, must be chosen which will elute reasonably close
to the peak(s) of interest, and will itself be free from interference from
other chromatographic peaks.
Calibration is carried out by preparing a known mixture of
internal standard(s) and compounds of interest, and obtaining a chromato-
gram for several different amounts of the mixture. The response factor
for each peak may be determined by measuring height, area, or preferably
by use of an automatic integrator or computer integration routine.
Sufficient calibration chromatograms are run until a consistent response
ratio between the internal standard(s) and the chromatographic peaks of
interest is obtained. When response ratios between every compound of
interest and an internal standard have been satisfactorily determined,
the appropriate internal standards are added to the unknown mixture in
amounts which are estimated to be of the same order of magnitude as the
peaks which are being determined. GC analysis of the complex mixture
plus internal standards is now carried out using the same chromatographic
conditions at which the response ratios were originally obtained; from
the response factors for the compounds sought in the complex mixture, and
for the known amounts of internal standards together with the previously
determined response ratios, the absolute quantity of each of the compounds
sought in the complex mixture may be readily determined.
When it is possible to use specific detectors, such as GC-ecd for
halogenated pesticides, GC-fpd (sulfur specific filter) for sulfur compounds,
or HPLC-fluorescence for POM compounds, it is often possible to carry out
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72
quantification without obtaining good peak resolution on the chromatogram.
This may be feasible because the sensitivity of the specific detector is
very low for all but the compounds of interest, which permits previously
interfering peaks to be neglected on the specific detector chromatogram.
The internal standards chosen for GC and HPLC quantification
are usually similar in nature to the compounds which they are being
used to determine, although this is not always mandatory. For example
when carrying out GC quantification of POM species in combustion emissions
an uncommon alkylated or phenylated POM compound is often chosen as an
internal standard; it is of course important that the standard chosen
does not occur in the complex mixture under analysis. In the GC analysis
of GI? to C?, hydrocarbons from an oil-spill sample, it is not uncommon
to add an absent C_? hydrocarbon to serve an internal standard for the
mixture. When the highest accuracy is not sought, it is an accepted
practice to assume that the chromatographic response for each member of a
class of compounds is the same. Thus the POM compound 9-phenylanthracene
could be used as an internal standard for all other POM compounds, each
being assumed to have the same response as the internal standard, and the
response ratio of each compound to the internal standard equal to unity.
The accuracy and reproducibility of GC and HPLC quantification
using internal standards is generally better than +W7*. The reproducibility
may be readily determined while obtaining the response ratio calibration for
each compound sought.
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73
6. b) Gas Chromatographic-Mass
Spectrometric Analysis (GC-MS)
Gas chromatographic-mass spectrometry is presently a widely
used and powerful tool for organic analysis. It has become common
practice to interface a GC-MS system with a dedicated mini-computer, and
a variety of output units such as a CRT teletype, or XY plotter, and a
high-speed line printer, it is probably true to say that the GC-MS
analysis contemplated in this manual could not be accomplished without
an interfaced mini-computer.
Electron Impact and Chemical lonization
Mass Spectra may be obtained by electron impact ionization (El)
or by chemical ionization (CI); in the latter mode, sample ionization is
accomplished by means of an ionized reagent gas. In CI GC-MS analysis,
the reagent gas, such as methane or isobutane, is most commonly introduced
through its use as the GC carrier gas, no molecular separator being used
at the interface between the GC and MS. The MS source pressure for CI
with methane, for example, is typically as high as 600 microns, and thus
the far higher concentration of methane than sample in the source ensures
that sample ionization will occur exclusively by collision with ionized
methane. Chemical ionization results in a rather different mass spectrum
to electron impact in view of its being a much lower energy process; CI
spectra are characterized by less extensive fragmentation of the molecular
ion, and the fragmentation which occurs generally proceeds through loss of
neutral molecules and appreciably more stable fragments than is the case
with El (34). It is usual to observe a protonated molecular ion in CI,
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74
and this is frequently accompanied by two adduct ions at M+29 and M+41,
• *
in the case of methane CI, caused by the addition of C«H,_ and C H radicals
in the source of the mass spectrometer. Such adduct ions are generally
diagnostic for the protonated molecular ion, and thus it is frequently
possible to quickly assign a molecular weight during CI GC-MS analysis.
Since CI analysis is still relatively new, El analysis main-
tains a significant advantage which is well suited to the analysis of
complex organic mixtures. Over the past few years extensive data files
of El spectra have been built up, many of these giving particular empha-
sis to toxic and hazardous substances. As yet, no comparable files exist
for CI spectra. It is not unusual to possess a data file of 25,000 or
more El spectra with which to carry out spectral matching as an aid to
identifying unknown compounds. Spectral matching is often carried out
by gaining access to spectral files in a central computer, several lab-
oratories sharing the same data file. In our experience, it is consider-
ably more convenient to store a spectral matching data file on disk, and
by means of a dual disk drive assembly interfaced with the GC-MS mini-
computer it is then possible to carry out instantaneous spectral matching
in the GC-MS laboratory. In any case, spectral matching is a very
useful aid to the interpretation of unknown El mass spectra, although
it will often fail to suggest an immediately satisfactory spectral fit.
Spectral matching often provides a useful guide which when coupled with
other analytical data, such as a molecular weight from a CI mass spectrum,
liquid chromatography separation data, and NMR and IR screening studies;
correlation of such combined data can frequently lead to a reasonable
interpretation of the mass spectrum.
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75
Correlation with Separation and Screening Data
GC-MS analysis does of course presuppose that the complex
mixture subject to analysis is capable of separation by gas chromatography
and an obvious limitation is compounds which are too polar to be satis-
factorily separated by GC. Such compounds may be routinely analyzed by
HPLC, as discussed earlier. A great deal of valuable data on an unknown
sample will be obtained by screening studies even before GC-MS analysis
is attempted, and the complexity of the mixture will have been signifi-
cantly reduced by the HPLC separation scheme which has been discussed
previously.
HPLC will initially be used to separate a complex mixture into
three to five fractions by GPC on the basis of molecular weight, and
then each of these size fractions will be separated into as many fractions
as appears desirable by silica gel or reverse phase HPLC in order to pro-
vide relatively simple mixtures for the subsequent stages of analysis.
Following the complete HPLC separations, probably into about 20 fractions,
screening studies using IR, NMR, HRMS, and GC will have been carried out
as appears appropriate.
Each fraction of the complex mixture of a given molecular weight
range will be separated by HPLC on the basis of polarity into a number of
well resolved fractions. The very non polar fractions will consist of
aliphatic hydrocarbons and will not be subject to GC-MS analysis, since
these species are more efficiently analyzed by GC alone. The most polar
fractions from the HPLC separation scheme will be unsuitable for GC
separation on account of their high polarity and involatility, and will
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76
be characterized by further HPLC separation together with IR, NMR, and low
resolution MS as required. However, the HPLC fractions in between these
two extremes will range from non polar POM species to polar polyfunctional
compounds, and these will be identified by GC-MS (and possibly GC-IR)
preceeded by extensive screening of each HPLC fraction by the other
analytical techniques as discussed earlier.
Whether CI or El mass spectra, or both, are obtained will depend
to a large extent on the information available from the preliminary
screening and HPLC separation data. For fractions containing the POM
species, there is little point in obtaining both El and CI spectra, since
fragmentation is almost negligible and uninformative in both cases.
However, the preliminary screening data will make it clear that POM
species are present, and CI mass spectra will provide molecular weights
which when coupled with chromatographic data will permit unambiguous
compound identification.
For HPLC fractions containing monofunctional compounds, such as
aldehydes and phenols, CI mass spectra may frequently provide sufficient
confirmatory data, especially when coupled with the information from the
screening analysis. For example, if an HPLC fraction is known from
screening to contain primarily aromatic aldehydes, it should be relatively
straightforward to assign benzaldehyde, tolualdehydes, and higher alkyl
benzaldehydes from the CI spectra, since the only prominent fragmentation
will be loss of 28 mass units for CO, to leave the carbon skelton. The
mass spectra are unlikely to give guidance regarding isomers, but this
information may be inferred from chromatographic data, or possibly GC-IR
data.
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77
For HPLC fractions which contain polyfunctional compounds, or a
mixture of compounds of different functionality, the most expedient
approach in GC-MS analysis would be to initially run El spectra, and
initially rely upon spectral matching, and fragmentation interpretation,
to suggest some possible structures for the compounds present. The
available screening data will greatly assist in narrowing the choice from
the list of spectral matches. It may not at this stage be possible to
make a good assignment of some of the compounds present, and it would
probably be most helpful to additionally obtain CI mass spectra. The CI
spectra would further narrow the possibilities by providing a probable
molecular weight for the compounds of interest, and the CI fragmentation
may give further valuable data necessary to arrive at a structural assign-
ment. CI fragmentation for moderately polar species can be rather more
informative than for less polar compounds, since the greater charge separa-
tion in the molecule may be more likely to promote fragmentation; for the
same reason, El fragmentation of very polar species may often be too
extreme to be of much value.
GC-MS Quantification, and Other
Useful Computer Routines
The basic routines available with all commercial mini-computers
for GC-MS are 'RGC' (reconstructed gas chromatogram) and a mass spectrum
printing routine. It is highly desirable that a CRT teletype unit should
be available for instantaneous display and manipulation of data; we will
assume in this discussion that a CRT unit is available, although the
routines are almost identical when used with a more time consuming XY
plotter.
-------
TOTAL ION
CHROMATOGRAM
203 ION
OVERLAY
253 ION
OVERLAY
331 ION
OVERLAY
00
0 13 23 33 t(3 53 63 7(3 83 30 130 113 123 133 1.1C 1S3 163 173 180 133 23C
SPECTRUM NUMBER
FIGURE 9. RECONSTRUCTED ION CHROMATOGRAMS OF RESIDUAL
OIL COMBUSTION EFFLUENTS
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79
SPECTRUM 167-162
8
GC-MS (CH4) OF RESIDUAL OIL COMBUSTION EFFLUENTS
8.
;o
Wo
oio _
HI
R-<
(M + 1)
(M + 29)
(M + 41)
230 210 2S8 260 278 280 290 308 310
M/E
FIGURE 10. MASS SPECTRUM OF BENZFLUORANTHENES
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80
SPECTRUM 179-169
8
8.
;P_
U.O
OLO .
V
?_
R-
GC-MS (CH4) OF RESIDUAL OIL COMBUSTION EFFLUENTS
(M + 1)
|nM|llll|i.M|MM,M..|l...|Mlnllll|l,Mi.||||..l.|.Mt|
(M + 29)
(M + 41)
'«r'"l ""!' (""i""!-"!""!""!"'!"-^'^"!""!""!
230 2K5 2SO 260 270 290 290 300 310
«/£
FIGURE 11. MASS SPECTRUM OF BENZPYRENES
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81
Modifications of the RGC routine are invaluable for the loca-
tion of minor chromatographic peaks, and also for performing rapid
quantitative analyses. The normal RGC plot consists of a reconstructed
chromatogram which contains ions of all mass numbers, see for example the
'total ion chromatogram' in Figure 9. In order to locate the GC peak for
a compound whose mass spectrum is known, RGC plots containing prominent
ions in the mass spectrum of this compound may be made. These RGC's
should be overlaid upon the original total ion RGC, maxima in the RGC
specific ion overlays will occur at the spectrum number corresponding to
the compound of interest in the total ion RGC.
For example, Figure 9 shows a portion of the GC-MS analysis of
residual oil combustion effluents. In this case, the sample extract was
subjected to liquid chromatography on silica gel to isolate the hydro-
carbon and POM species. Several individual ion overlays are shown super-
imposed upon the total ion chromatogram. The 253 ion overlay shows
maxima at spectrum numbers 166 and 179, although it is apparent that no
peaks are visible on the original total ion chromatogram. If the spectra
at spectrum numbers 166 and 179 are displayed or printed out, it is
evident that these two peaks are benzfluoranthene or benzpyrene isomers;
the spectra are both characterized by a base peak at m/e = 253 (M+l) and
adduct ions (M+29, M+41) at m/e = 281 and m/e = 293 as shown in Figures
10 and 11. In practice, spectrum 167 minus spectrum 162, and spectrum
179 minus spectrum 169 would be displayed in order to subtract any
spurious background ion peaks due to column bleed, and the tailing from
other chromatographic peaks. It is important that the presence of the
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82
compound sought should be confirmed by printing out its mass spectrum in
this manner, since spurious 'hits' sometime occur due to interfering
fragments from other compounds.
Specific ion current integration is the basis for a very rapid
quantification routine (3). Some mass spectroscopists use the total ion
current for quantification purposes, and while this procedure may work
well for well resolved peaks at high concentrations, the procedure unfor-
tunately neglects the high specificity and sensitivity which the mass
spectrometer is capable of providing. In order to obtain the specific
ion currents due to minor peaks which are confused and overlaid by other
major peaks, the precise position of the minor peak is firstly deter-
mined by means of the RGC overlay technique. For example, in Figure 9,
the location of pyrene (spectrum 64) and fluoranthene (spectrum 56) is
established by overlaying the 203 ion on the total ion chromatogram;
203 is the mass number for the protonated molecular ion of both these
compounds. Peak width limits are then read off from the chromatogram,
or located with a CRT cursor, and in this case are seen to be spectra 54
to 61 and spectra 61 to 69 for fluoranthene and pyrene respectively.
Having established the peaks limits, another computer routine is used to
sum the ion currents due to all of the prominent ions in the mass spectrum
of the compounds of interest. The ion integration procedure is then
repeated for an internal standard which was previously added in a precisely
known quantity to the complex mixture. In this example, the internal
standard used was 9-phenylanthracene, whose position is indicated by the
255 ion overlay in Figure 9, as spectrum 100. Quantification of the
compound of interest is then achieved by ratioing the ion current of
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83
of the compound of interest to that of the internal standard, and applying
a previously determined calibration factor which allows for the difference
in ionization efficiencies of the compound sought and the internal standard.
Interference by fragmentation from other unwanted compounds can almost
invariably be avoided by using CI (which minimizes fragmentation), and by
careful choice of the fragment ions used for quantification. This quanti-
fication procedure has repeatedly been demonstrated to have an accuracy
and reproducibility of better than +15%; with care this figure may readily
be reduced to below +10%. The above quantification procedure has simi-
larities to the widely used specific ion monitoring technique, which of
course is not suitable for the analysis of large numbers of compounds in
environmental samples on account of unavailability of isotopically
labelled reference materials.
The choice of internal standards for GC-MS quantification should
be made with greater care than for GC internal standards, since not only
do we require a compound that elutes conveniently near to the compounds to
be measured, and is not itself present in the sample, but we also require
a compound which will give simple fragmentation in order to obviate
interference from fragment ions from other materials during ion current
integration.
The sensitivity for GC-MS quantification, and for GC-MS analy-
sis in general, will always depend upon the nature of compounds being
studied. For a compound such as an aliphatic dialdehyde with extensive
fragmentation, and whose mass spectrum is necessarily weak, a sensitivity
of 100 ng/yl or poorer would not be surprising. For compounds showing
little or no fragmentation, such as POM species, a sensitivity of 1 to 10
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84
ng/yl can routinely be obtained, sensitivities of an order of magnitude or
more higher than this may be obtained if mass spectrometer conditions are
optimized.
One of the most useful analytical routines is spectral matching,
as previously mentioned. We consider that it is preferable to use a dual
disk drive whereby the bank of reference spectra are stored on a separate
disk which is readily accessible during mass spectral analysis. Ideally,
the RGC of a fraction from HPLC separation should be displayed on the
CRT, and a queue of mass spectra corresponding to RGC peaks should be
stored in the computer memory by command from the teletype. When the
queue is established, the matching routine may be activated and a chosen
number of compound matches (with correlation coefficients) for each RGC
peak may be printed by a high-speed line printer.
6. c) Gas Chromatographic-Infrared Spectroscopic Analysis
While infrared spectroscopy (IR) is the most widely used
analytical tool for the identification of organic compounds, it has
given way to mass spectrometry (MS) for the identification of organic com-
pounds in vaporizable, complex mixtures. The reason for this was that
the characteristics of mass spectrometry were well suited to coupling
with a gas chromatograph (GC) so that GC could be used to separate the
mixture and MS used to identify the separated compounds. The sensitivity
and speed of MS was such that this could be done "on-line" or "on-the-fly".
GC-IR studies used to require trapping and collection of each GC peak, and
not only was this time consuming, but often there was not enough
material available for this purpose.
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85
With the advent of Fourier Transform infrared systems (FT-IR),
"on-line" GC-IR became a reality. The extra energy throughput of the
interferometer of the FT-IR system could be used to give complete
infrared scans in as little as 0.5 sec. Thus scanning speed was certainly
fast enough for "on-line" GC-IR without the necessity for trapping and
collection of the GC peaks. While the sensitivity level is not as low as
desired, it is adequate for many samples. In addition designs are avail-
able for modifying the infrared light pipe used for GC-IR and this coupled
with the use of a liquid nitrogen cooled infrared detector has been
demonstrated to lower the sensitivity to 400 nanograms per GC peak. This
sensitivity range makes it possible to obtain infrared spectra "on-the-fly"
of most gas chromatographic peaks. Not only would such modifications be
fairly easy to make, but a complete unit incorporating these changes
should soon be commercially available.
Thus GC-IR joins GC-MS as a routinely used instrument for the
analysis of complex mixtures and will be an invaluable analytical tool
for identification of the components separated by gas chromatography.
It is important to appreciate that GC-MS and GC-IR techniques frequently
complement each other. Since the bases for GC-MS and GC-IR identifi-
cations are fundamentally different, analyses using both techniques will
provide substantially more compound identification data than either
technique used independently.
The same remarks concerning the use of separation techniques to
prepare the sample for GC-MS (Section 6b) will apply to the samples for
GC-IR and need not be repeated here. Since the GC-IR system is fully
automated, the sample is merely injected into the gas chromatograph and
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86
the FT-IR computer automatically scans, collects, and stores the interf-
erogram for each GC peak. The operator then has to manually instruct
the computer to plot out each spectrum. Identification is made by standard
infrared procedures, that of matching the unknown spectrum with a reference
spectrum from available reference libraries of up to 150,000 spectra.
This can be aided by computer search systems. When an exact match (with
a reference spectrum) can not be found, the functional group information
(available from the IR spectrum) can often be coupled with the MS data
to uniquely identify an unknown compound.
6. d) Nuclear Magnetic Resonance Spectroscopy
Nuclear magnetic resonance spectroscopy will be used for
quantification of effluent samples in only two cases: (1) a single
component isolated by the HPLC separation scheme, or (2) the very polar
or ionic compounds which cannot be quantified easily by the other
available methods.
It is desirable that quantitative work should be carried out
by FT-NMR, if possible, in view of the advantages of this technique
discussed in Chapter 4. Quantification in NMR terms means either a
relative ratio of the resonances present in the sample or addition of a
known amount of a standard whose resonance will not interfere with those
of the sample. For C spectra, quantification also means running
nuclear Overhauser enhancement (NOE) experiments to ascertain the true
spectral intensities. In this document, quantification will be discussed
in general terms applicable to either H or C spectra.
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87
Relative Ratios
In the case of relative ratios, a sharp resonance peak such as
a -CH singlet is chosen as a known. The intensities of the integrals
for the other resonances are then compared to the known . In this way
the relative number of protons can be determined for a single peak as a
chemical shift region (i.e., aromatic, olefinic, aliphatic). Sample
preparation and experimental details for this procedure are those given
in Chapter 4. The spectral integration is carried out using the computer
program package provided with the instrument in the case of FT-NMR, or
by linear summation of the integration trace with conventional NMR. The
limit of accuracy for this method is +57° provided no exchangable protons
or interfering groups are present in the spectrum. It must be again
cautioned that this is a relative ratio; one which does not take into
account such factors as relaxation times which can mask the true peak
13
intensities especially in C NMR.
Absolute Quantification
In the case of absolute quantification, the spectrum of the
HPLC fraction to be quantified is run according to the guidelines given
in Chapter 4. Once the appearance of the spectrum and its suitability for
quantification have been determined, the spectrum can be scrutinized for
a blank area or window in which no sample resonances appear or are sus-
pected, and a suitable internal standard chosen with a resonance in this
area. A good compound for consideration as an internal standard would be
relatively unreactive with the class of compounds in the sample and would
contain a sharp singlet, or several singlets which are easily integrated.
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88
Integration is carried out by the computer program of each instrument,
in the case of FT-NMR, or by linear summation of the integration trace.
Quantification to + 570 can be accomplished by comparing the
integral of peak of known concentration to those for the compound.
Specific Problems in the Analysis
of Mixtures
The general approach to running NMR spectra as outlined in
Chapter 4 will normally produce spectra which provide sufficient informa-
tion for screening HPLC separation fractions, or identification of specific
compounds in simple mixtures. Occasionally, however, the interpretation
of the NMR spectrum of a mixture may be complicated by overlapping peaks
or line-broadening. Depending on the particular problem and the nature
of the sample components, several approaches can >e used to simplify the
spectrum.
In simple mixtures what appears to be the spin-spin coupling
system of a single compound, may in actuality be peaks from two or more
components. Spin decoupling techniques should be used to determine the
integrity of the miltiplet. If the system collapses to a single
resonance, the multiple peaks are part of one spin system.
The presence of paramagnetic materials in a sample can cause
significant line broadening, and in some cases a low resolution spectrum.
The effect is significantly increased if the particle is ferromagnetic.
These interferences, from the original sample or the result of contamination
during sample handling (i.e., steel spatulas), may be removed by using a
permanent magnet and decanting the solution.
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89
To resolve overlapping in compounds which have one or more
Lewis base groups, especially hydroxy groups, carbonyls, and amines, a
shift reagent may be used. Shift reagents are normally compounds containing
a metal from the lanthanide series which complex with the Lewis base.
The perturbation of the shift reagent on the proton magnetic resonance
spectra of the ligands results in spectral simplification. For an
excellent review of rare earth shift reagents, references 34-37 should
be consulted.
Finally, as stated before, the use of FT-NMR with a dedicated
computer provides the additional opportunity for special data handling,
such as spectral arithmetic. Although not currently available on all
commercial spectrometers, spectral arithmetic is a powerful tool for the
similification and interpretation of spectra. For example, suspected
components may be subtracted, both simplifying the resulting spectrum and
confirming the component's presence.
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90
7. ANALYTICAL COSTS
The following analytical cost estimates are presented in order to
serve as a guide in planning emission measurement programs in a cost
effective manner. The costs quoted for each operation (see Table 6) make
the assumption that the analytical operation being carried out involves
a complex mixture of organic compounds. In our experience, the individual
costs given are reasonably representative of those experienced by the
major research laboratories in this country.
TABLE 6. APPROXIMATE COSTS OF INDIVIDUAL ANALYTICAL STEPS
_
Extraction 50 (per emission)
GPC 110 (per extract)
HPLC 110 (per fraction)
IR Screening 60 (per fraction)
NMR Screening 100 (per fraction)
HRMS Screening 150 (per fraction)
GC Quantification 300 (per fraction)
HPLC-NMR Quantification 250 (per HPLC Peak)
HPLC-IR Quantification 125 (per HPLC Peak)
GC-MS Qualitative 600 (per El or CI run)
GC-MS Quantitative 600 (per El .or CI run)
GC-IR qualitative 500 (per run)
LC Quantification 50 (per LC Peak)
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91
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Environmental Analytical Chemistry (1975).
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(20) N. S. Bhacca, and D. H. Williams, Applications of NMR
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science Publication, John Wiley and Sons, Inc, New York, N.Y., 1975.
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Pollution Control Assoc., 23, 704 (1973).
(34) B. Munson, Analytical Chemistry 43 (13) 28A (1971).
(35) A. F. Cockerill, G. L. 0. Davies, R. C. Harden, and D. M. Rackham,
Chem. Rev., 73 (6), 553 (1973).
(36) P. V. Demarco, Lloydia, 3_5 (4), 362 (1972).
(37) M. P. Peterson, Jr., and G. H. Wahl, Jr., J. Chem. Ed., 49 (12),
790 (1972).
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93
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
I. REPORT NO.
EPA-600/2-76-072
2.
3. RECIPIENTS ACCESSION-NO.
4. TITLE AND SUBTITLE
Technical Manual for Analysis of Organic Materials
in Process Streams
5. REPORT DATE
March 1976
6. PERFORMING ORGANIZATION CODE
7. AUTHORS p.w.Jones, A.P.Graffeo, R. Detrick,
P. A. Clarke,and R.J.Jakobsen
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Batte lie-Columbus Laboratories
505 King Avenue
Columbus, Ohio 43201
10. PROGRAM ELEMENT NO.
EHB-524; ROAP AAS-090
11. CONTRACT/GRANT NO.
68-02-1409, Task 20
12. SPONSORING AGENCY NAME AND ADDRESS
EPA, Office of Research and Development
Industrial Environmental Research Laboratory
Research Triangle Park, NC 27711
13. TYPE OF REPORT AN.D PERIOD COVERED
Tech Manual; 7/75-1/76
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES project officer for this manual is L.D.Johnson, Mail Drop 62,
Ext 2557.
16. ABSTRACT Tne manua;i presents a very simple, yet informative, analytical strategy
for the purpose of initial survey, to ensure that important emission problems do not
go undetected. It also presents a more complex and detailed scheme for use on
samples given high priority by the initial survey analysis. The manual was developed
because of a major problem associated with the analysis of potentially hazardous or-
ganic emissions: a very large number of organic compounds may be present in a
given industrial sample. If exhaustive analytical methods were applied to every
emission source to be assessed, costs would become completely unreasonable, and
much effort would be misdirected on samples of little concern. A comparison
volume, concerned with sampling of organic substances, is available, and has been
fully coordinated with the technical manual to ensure compatibility.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Air Pollution
Chemical Analysis
Analyzing
Sampling
Surveys
Organic Compounds
Industrial
Processes
Hazardous Ma-
terials
b.IDENTIFIERS/OPEN ENDED TERMS
Air Pollution Control
Stationary Sources
Analytical Strategy
Process Streams
c. COSA7I Held/Group
13B
07D
14B
07C
13H
13L
B. DISTRIBUTION STATEMENT
Unlimited
19. SECURITY CLASS (Tin's Report)
Unclassified
21. NO. OF PAGES
98
20. SECURITY CLASS (Tillspage)
Unclassified
22. PRICE
EPA ron-,1 2220-1 (D-73)
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