INTERIM PROTOCOL FOR MEASURING HYDROLYSIS RATE
CONSTANTS IN AQUEOUS SOLUTIONS
by
J. Jackson Ellington, Frank E. Stand!, Jr.,
Environmental Research Laboratory,
U.S. Environmental Protection Agency
Athens, Georgia, 30613
William D. Payne, Cheryl D. Trusty,
Technology Applications Inc.,
Environmental Research Laboratory
Athens, Georgia, 30613
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
ATHENS, GEORGIA 30613
-------
DISCLAIMER
The information in this document has been funded wholly or in part
by the United States Environmental Protection Agency. It has been subject
to the Agency's peer and administrative review, and it has been approved
for publication as an EPA Document. Mention of trade names or commerical
products does not constitute endorsement or recommendation for use by the
U.S. Environmental Protection Agency.
11
-------
FOREWORD
As environmental controls become more expensive and penalties for
judgement errors become more severe, environmental management requires
more precise assessment tools based on greater knowledge of relevant
phenomena. As a part of this Laboratory's research on occurrence,
movement, transformation, impact, and control of chemical contaminants,
the Measurements Branch determines the occurrence of unsuspected organic
pollutants in the aquatic environment and develops and applies techniques
to measure physical, chemical, and microbial transformation and equilibrium
constants for use in assessment models and for development of property
reactivity correlations.
Mathematical models are widely used in predicting the fate of organic
chemicals in lakes and streams and in soil systems. Application of these
models requires as input the second-order and first-order hydrolysis rate
constants for chemicals containing hydrolyzable functional groups. In
measuring these rate constants, some means is needed to ensure that the
measurements are reliable and reproducible. This protocol specifies
complete step-by-step procedures for hydrolysis measurement to produce
the required level of reproducibility and reliability among measurements
by different investigators.
Rosemarie C. Russo, Ph.D.
Director
Environmental Research Laboratory
Athens, Georgia
in
-------
ABSTRACT
A detailed protocol was developed to measure first- and second-order
hydrolysis rate constants for organic chemicals for use in predicting
persistence in aquatic systems. The protocol delineates theoretical
considerations, laboratory experiments, and calculation procedures.
Repetitive application of the protocol to measure hydrolysis rate
constants for four standard reference compounds over a period of 2 years
yielded coefficients of variation of less than 12% in the measurements.
This report covers a period from October 1985 to March 1988 and work
was completed as of January 1988.
-------
INTRODUCTION
Under the Toxic Substances Control Act (PL 94-469) of 1976, the
Office of Toxic Substances (OTS) screens new chemicals proposed for
manufacture and reviews the safety of existing chemicals already on the
market. To assess potential risk to human health and the environment OTS
must evaluate both effects and exposure potential. Transport and
transformation characteristics in ambient environments are major
considerations in assessing potential exposure. Essential to transport
and transformation assessments are physical and chemical data that permit
estimation of chemical fate either by use of mathematical models or other
techniques. To obtain necessary data, OTS either requests it from
manufacturers or estimates values by comparing the chemical to analogous
chemicals whose properties are known. In either case, reliable data are
necessary. A major transformation process for many chemicals is chemical
hydrolysis; therefore, measurements of hydrolysis rate constants are often
required.
In the measurement of hydrolysis rate constants, some means is needed
to ensure that the measurements are reliable and reproducible. Suggested
laboratory protocols for measuring hydrolysis as a function of pH and
temperature have been published (1-3 ); however, these previously published
protocols fail to specify some of the step-wise procedures in sufficient
detail to enhance reproducibility of measurements made by different inves-
tigators. This report, on the other hand, provides specific guidance
in a protocol for deriving hydrolysis rate constants for use in mathematical
models to predict the fate of a chemicals in aquatic systems. The protocol
has evolved over the past several years at the Environmental Research
-1-
-------
Laboratory, Athens GA, and has been found to provide reproducible rate
constants as a function of pH and temperature.
KINETICS OF HYDROLYSIS
HYDROLYSIS MECHANISM
The importance of hydrolysis as a transformation process for chemicals
in water can be determined from data on rate constants and half-lives
coupled with data describing environmental conditions. Hydrolysis of
organic compounds refers to reaction of the compound with water in which
bonds are broken and new bonds with HO- and H- are formed. A typical
example is the reaction of an alkyl halide with water resulting in the
formation of halide ion (X-):
RX + HOH > ROH + HX (or H+, X~) (1)
The rate of hydrolysis may be promoted by the hydronium ion (H+,
H30+) or the hydroxyl ion (OH~). The former is referred to as specific
acid catalysis and the latter as base mediated hydrolysis. These two
processes together with the pH independent reaction with water are the
only mechanisms considered in this protocol. The H30+ activity is
measured directly and the OH" activity is calculated from accurate determi-
nation (calibration between secondary buffers) of solution pH.
Some chemicals undergo an elimination reaction:
H X
I I H+, OH- v '
_ C - C > C = C + HX (2}
i ' H20 / \
In this protocol, only the disappearance of substrate is monitored with no
attempts to identify mechanisms or reaction products. Reactions represented by
equations 1 and 2 are included in a broad definition of hydrolysis.
-2-
-------
RATE LAWS
In both processes referred to in the discussion of hydrolysis mechanisms,
the rate of disappearance of the organic compound is given by the equation,
d[C]
----- = kh[C] = KA[H+][C] + kB[OH-][C] + kN'[H20][C] (3)
dt
where [C] is the concentration of organic and k^ is the observed pseudo-first-
order rate constant at a specific pH and temperature; k/\ and kg are second-
order rate constants; and k^1 the neutral hydrolysis rate constant for the
acid, base and neutral promoted processes, respectively. The water concen-
tration, because of the large excess, does not change during the reaction,
thus kf^'Ch^O] is a constant (k^).
Equation 3 assumes each individual rate process is first-order in
substrate, thus k^ can be defined as:
kh = kA[H+] + kB[OH-] + kN (4)
Using the autoprotolysis equilibrium expression
KW = [H+][OH-] (5)
equation 4 may be rewritten as
kh = kA[H+] + -— + kN
h A N (6)
Equation 6 shows the dependence of kn on hydronium ion concentration (pH)
and on the relative values of kA, kg, and k^|.
When the disappearance rate constants are determined at pHs 3, 7,
and 11, the second-order rate constants for acid hydrolysis and for base
-3-
-------
hydrolysis can be calculated by dividing the pseudo-first-order rate
constant obtained at the appropriate pH by the hydronium ion or hydroxyl
ion activity, respectively. The neutral contribution is determined by
solving equation 4 for kfj and substituting the observed rate (kn) at pH 7
together with values for k/\ and kg. The half-life of a compound at a
given pH and temperature can be calculated from equation 7, where kn is
[C0J
the observed rate at the given pH and temperature and 0.693 = In
[CtJ
where [C0] equals concentration at time zero and [C^] equals concentration
at 50% reaction.
0.693
ti/2 = (7)
kh
CONTRIBUTING FACTORS TO HYDROLYSIS RATES
Temperature
The effect of temperature on the rate constant for a specific
hydrolysis process (k/\, k^, and kg) can be expressed in several ways.
The familiar Arrenhius or absolute rate theory equation was used in this
protocol development
k = A exp(-Ea/RT) or In k = In A - Ea/RT (8)
where A is the collision frequency term, Ea is the activation energy, and R
is the gas constant (8.314 J/deg mol). To determine Ea and A for each
hydrolytic process (acid, neutral, base), one must know the rate constants
for the three processes k/\, kfj, and kg at two or more temperatures (preferably
separated by 20°C each). A and E are determined for each process by plotting
In kx (X = A, N, or B) versus 1/T (T in °K) and calculating the slope of
-4-
-------
the straight line through the data points (temperatures) or by fitting
the data by regression analysis to the equation In k = In A - Ea/RT.
Between any two temperatures, the following relationship is applicable
Ea (T2 -
In ----- = ------ --------- (9)
kT2 R
where TI and T2 are the respective temperatures and Ea is the activation
energy (E/\, EN, and Eg) for the particular process (k/\, k^, and kg).
Equations 4 and 9 are used to calculate rate constants for hydrolysis
at temperatures and pH values of interest for environmental assessment.
A second effect of temperature must be considered when using equation
5 to calculate [OH~]. The equilibrium constant KW increases with temperature
and with it the activity of [OH~], When determining [OH~] activities, the
Kw at the temperature of interest must be taken into account. In practice,
the pH is measured at the experimental temperature and, from the measured
pH, a value for [OH"] is calculated. The KW at a given temperature is
calculated by the equation given by Harned and Owen (4)
-6013.79
log Kw = -------- - 23.6521 log T + 64.7013 (10)
T
where T(°K) = T(°C) + 273.2
The [OH-] ion activity is then calculated using equation 5 and the calculated
value for kw at the experimental temperature. The [OH"] calculated by
equation 5 is used to calculate kg by dividing the measured pseudo-first-order
rate constant obtained at pH 11 by the hydroxide ion activity at the
measured temperature.
-5-
-------
pH, Buffer Catalysis
The observed rate of hydrolysis (k^) at any pH is described by
equation 4, where k/\ and kg correspond to the second-order specific
acid and base rate constants and k^ corresponds to the first-order neutral
water hydrolysis rate constant. Because of this possible pH dependence,
accurate pH data must be available for each buffer solution at each
temperature. The most practical way to obtain these data is to heat a
standard buffer solution to the desired reaction temperature and, then, to
measure the pH of the solution using a temperature-compensating meter and
probe designed to operate in the temperature ranges of interest.
The effects of ionic strength and buffer salts on hydrolysis reactions
are difficult to predict: rates can be retarded or accelerated depending
on the compound. Generally, buffer concentrations are 5E-3P4 and compound
concentrations 1E-5M. Dilute buffers and the 500:1 ratio of buffer to
compound minimizes buffer catalysis. As a check for catalysis, hydrolysis
rates can be measured over a 100-fold concentration range. A change in k^
greater than experimental error indicates catalysis by the buffer.
Solvent Composition, Metal Ions
Cosolvents can alter the rate of hydrolysis and are to be avoided.
If methanol or acetonitrile are used to prepare stock solutions,
the final concentration of either solvent in samples should be less than
1%. Deionized water equivalent to ASTM Type I should be used to make all
solutions to minimize introduction of metal ions.
-6-
-------
COMPILATION AND DEVELOPMENT OF HYDROLYSIS INFORMATION
LITERATURE SEARCHES
Several things should be considered prior to laboratory measurement of
the hydrolysis rate of any compound. First, the molecular structure of the
compound of interest must be determined. Is the compound an optical
isomer or stereoisomer? What is the correct Chemical Abstract System
(CAS) number for the specific chemical compound? What are the common,
trade, and IUPAC names for the compound?
A search of the literature and computerized databases should be a
prerequisite to any laboratory determinations. The most optimistic
result from the literature search would be to find a thorough rate study
of the compound of interest enabling prediction of a hydrolysis rate constant
over a wide range of pH and temperature. Unfortunately, literature values
from persistence studies or laboratory mechanism studies are often obtained
under conditions different from general environmental temperatures and
pH. The literature values may be useful, however, in setting pH, temperature,
and sampling times in the preliminary screening test.
If literature values are not available, linear free energy relationships
(LFERs) can be used to estimate the hydrolysis rates of closely related
compounds. With well characterized classes of compounds, LFER calculations
will permit choosing experimental conditions for the preliminary screening tests
that will result in reaction times appropriate for precise measurements. The
literature search should be used as a source for other information about the
compound such as chemical and physical properties (bp, mp, UV, IR, and solubility)
and methods of chemical analysis. Examples of sources of information
include:
-7-
-------
1. Manual
a. Merck Index
b. CRC Handbook of Chemistry and Physics
c. Kirk-Othmer Encyclopedia of Chemical Technology
d. Chemical Abstracts Service (CAS)
2. Computerized
a. CHEMFATE
Syracuse Research Corporation
Merrill Lane
Syracuse, NY 13210-4080
b. CAS On-Line (1967 to present)
STN International
c/o Chemical Abstracts Service
2540 Olentangy River Road
P.O. Box 2228
Columbus, OH 43202
c. CIS
Chemical Information System, Inc.
7215 York Road
Baltimore, MD 21212
d. DIALOG
Dialog Information Service Inc.
3460 Hi 11 view Ave
Palo Alto, CA 94304
STANDARD REFERENCE COMPOUNDS
Chemical standards of known concentration have long been used for
assuring reliability of quantitative chemical analyses, calibrating
instruments, and measuring recoveries of analytes from various matrices.
Analogous to using chemicals of known concentration as standards for
concentration measurement, chemicals whose hydrolysis constants have been
measured with established precision by one experimenter or group can be
-8-
-------
used as standard reference compounds (SRCs) by other experimenters in
establishing and maintaining quality control in rate measurements.
Precise measurement of established hydrolysis rate constants for SRCs
interspersed with other rate constant measurements will help assure
reliability and comparability of the measured constants.
Standard reference compounds are used as quality assurance standards
and as references in inter-laboratory generation of hydrolysis data.
Repetition of rate constant measurements in our laboratory for these
compounds over the course of 2 years has established baseline information
for evaluating experimental techniques and for all aspects of quality
assurance (5,6). Four compounds were selected, one each for acid and
neutral hydrolysis, and two for basic hydrolysis (Table 1).
Reproduction of the hydrolysis rate constants of the SRCs at the
established concentrations, pHs, and temperatures ensured that the experi-
mental condidtions were reproducible and helped evaluate the accuracy and
precision of measurements for other compounds. Tables 2 through 5 contain
SRC rate constant data generated during laboratory determinations of hydrol-
ysis rate constants of other cheimcals of interest. Pseudo-first-order hydrol-
ysis rate constants for all SRCs at various temperatures and pH and second-order
rate constants for the acidic and basic reference compound were established
from these determinations.
-9-
-------
Table 1. Standard Reference Compounds
Name
DL-trans-4-chlorostilbene oxide (CSO)
Benzyl chloride
Methyl -2, 4-dichlorophenoxy acetate
(2,4-DME)
Lindane
pH Range
2-5
Neutralb
8 - 9.5
9.5 - 11
Ea(kJ/mol )a
84.7±13.2
84.1±5.8
40.1±4.9
65.3±1.9
In A
37.1±5.30
45.3±2.21
22.7±1.91
27.5±0.75
a. The activation energy (Ea) and collision frequency (In A) were determined by
plotting the mean values of either k/\, k^, or ks at each temperature in Tables
2-5 versus 1/T (as illustrated for lindane in Appendix J).
b. Hydrolysis of benzyl chloride is independent of pH in the pH range 2-12.
-10-
-------
Table 2. Hydrolysis Data for DL-trans-4-chlorostilbene Oxide
Temp. 103 kha kAb
(°C) pH (min -1) (M -1 min -1)
23.Oc 3.13d 10.le 13.6f
3.12 15.5 20.4
2.99 12.9 12.6
24.3 3.03 11.1 11.9
25.0 2.89 29.5 22.9
2.89 35.8 27.8
3.05 24.4 27.4
3.05 22.8 25.6
3.05 22.8 25.6
3.02 17.4 18.2
3.02 16.1 16.8
3.02 19.0 19.9
3.10 16.9 21.2
2.96 18.5 16.9
25.3 2.95 24.2 21.6
2.95 19.4 17.3
28.0 3.06 14.4 16.5
3.06 16.9 19.4
3.06 14.3 16.4
3.06 16.9 19.4
3.01 21.1 21.6
3.01 23.7 24.2
3.01 20.8 21.3
3.07 14.6 17.1
3.07 14.3 16.8
3.10 14.9 18.8
3.10 14.4 18.2
3.13 17.4 23.5
38.2 3.63 17.0 72.3
3.59 23.5 91.4
3.59 24.5 95.3
a. Pseudo-first-order rate constant (kn): the slope of the line from
plot of 1n % chemical remaining versus time.
b. Second-order rate constant (k/\).
c. Error of temperatures: <75°C±0.1°C (water bath); >75°C±1°C (oil bath).
d. Error ±0.02 units all measurements.
e. Standard deviation of the slope (kn) <10% for each determination.
f. The averages of the k/\ values at each temperature were used to calculate
the Ea and In A in Table 1 (as illustrated for lindane in Appendix J).
-11-
-------
Table 3. Hydrolysis Data for Benzyl Chloride (pH 7.00)a
Temp. 104khb
28. Oc 10.4d,e
12.2
11.1
9.8
36.4 31.9
33.9
42.7 70.0
45.0 72.7
72.2
65.8
55.5
63.9
60.6
69.0
69.0
78.0
66.9
69.4
46.0 67.0
49.0 98.9
98.6
52.9 203.0
191.0
211.0
216.0
53.4 140.0
136.0
53.5 154.0
a. Error ±0.02 units all measurements.
b. Pseudo-first-order rate constant (kn): the slope of the line
from plot of 1 n % chemical remaining versus time.
c. Error of temperatures: <_750C±0.1°C (water bath); >75°C±1°C (oil bath),
d. Standard deviation of the slope (kn) <10% for each determination.
e. The averages of the values at each temperature were used to calculate
the Ea and In A in Table 1 (as illustrated for lindane in Appendix 0).
-12-
-------
Table 4. Hydrolysis Data for Methyl Ester of 2,4-D
Temp
23. Oc
25.0
28.0
31.0
45.0
45.3
48.5
70.3
PH
8.81d
8.81
8.81
8.87
8.87
9.10
9.10
9.38
9.45
9.38
9.45
9.06
9.06
9.65
9.65
9.10
9.14
8.72
8.75
8.54
8.74
8.55
8.55
8.00
8.00
7.11
7.11
104 kha
(min-1)
29. 9e
33.0
36.0
57.1
41.8
95.0
79.0
230.0
249.0
224.0
224.0
80.7
70.4
262.0
278.0
118.0
103.0
79.2
91.2
350.0
340.0
412.0
415.0
103.0
86.0
100.0
114.0
D
(M~l min~l)
541f
596
650
770
563
754
627
958
883
933
794
560
489
467
495
681
593
966
1038
2520
1547
2847
2867
2079
1731
4775
5451
a. Pseudo-first-order rate constant (k^): the slope of the line from plot of
In % chemical remaining versus time.
b. Second-order rate constant (kg)
c. Error of temperatures: <75°C±0.1°C (water bath); >75°C±1°C (oil bath).
d. Error ±0.02 units all measurements.
e. Standard deviation of the slope (k^) <10% for each determination.
f. The averages of the kg values at each temperature were used to calculate
the Ea and In A in Table 1 (as illustrated for lindane in Appendix 0).
-13-
-------
Table 5. Hydrolysis Data for Lindane
Temp
22. 8C
37.0
40.0
45.0
45.3
46.0
PH
11.60d
11.29
10.92
10.01
11.20
10.31
10.45
10.37
10.37
10.37
10.71
10.71
11.08
10.98
10.98
103 kha
9.9e
40.2
23.6
22.2
40.2
18.4
20.0
14.5
14.6
14.8
33.1
36.0
83.4
78.0
71.5
!<• b
KB
(M-l min-1)
2.9f
8.7
9.8
7.4
8.7
22.5
17.7
15.4
15.6
15.8
15.8
17.2
16.3
19.2
17.6
85.0 8.73 51.6 272.0
a. Pseudo-first-order rate constant (kn): the slope of the line from plot of
In % chemical remaining versus time.
b. Second-order rate constant (kg)
c. Error of temperatures: <_75°C±0.1°C (water bath); >75°C±1°C (oil bath).
d. Error ±0.02 units all measurements.
e. Standard deviation of the slope (k^) <10% for each determination.
f. The averages of the kg values at each temperature were used to calculate
the Ea and In A (Appendix J, Table 1).
-14-
-------
TECHNIQUES AND REAGENTS
STANDARD REFERENCE COMPOUNDS (SRCs)
The SRC hydrolysis rate constants should be determined before analysis
of any compounds of interest in order to establish operator proficiency.
Determinations of SRC values should then be interspersed with rate constant
measurements of the chemicals of interest. Details of chemical analysis
and data workup are presented in subsequent sections.
TEMPERATURE CONTROL
Reaction rates are strongly dependent on temperature; therefore,
the temperature should be minimized during a run and be accurately measured.
A rule of thumb is that rates double for each 10°C rise in temperature,
increase by 7% for a 1°C rise, and by 0.5% for a 0.1°C rise. This corresponds
to an activation energy of approximately 20 kcal/mol. Constant temperature
baths should be used that can maintain the temperature within ±0.02°C. A
refrigerated bath is necessary for 0 to 30°C measurements. An oil bath is
required for temperatures greater than 75°C. Temperatures should be
measured with American Society for Testing and Materials (ASTM) thermometers,
calibrated by NBS procedures and NBS certified masters, or equivalents.
pH MEASUREMENT
Because hydrolytic reactions can be catalyzed by hydronium or hydroxide
ions, accurate pH data must be available for each buffer solution at each
temperature. The most practical way to accomplish this is to heat the
standard buffer solution to the desired reaction temperature, and then to
measure the pH of the solution. This requires temperature compensation,
either automatic or manual, and a pH probe stable to varying temperatures
and accurate to ±0.02 units in the pH range 3 to 11. The pH of the reaction
-15-
-------
buffer solution should be remeasured upon completion of the hydrolysis
experiment. If the pH has changed more than 0.03 pH units the run should
be discarded. When the observed hydrolysis rate (kn) of a chemical is
due solely to acid catalyzed or base mediated reaction (pseudo-first-order
kinetics), the value of k^ will decrease by a factor of ten for every
change of one pH unit toward neutrality. An excellent treatise on pH
is found in Determination of pH Theory and Practice by Roger G. Bates (7).
BUFFERS
Autoclaved (C02-free) water is used to prepare stock solutions of
buffer and subsequent dilutions. In sealed containers, the 0.005 M_ buffers
hold the pH within (±0.03 pH units) for up to 25 days. Standard buffers
(0.005N[) for pH 3, pH 7, and pH 9 to 11 are prepared in the following
manner.
pH 3.00 Dilute 5-ml of 0.1 M potassium hydrogen phthalate to 100
ml with water, adjust to pH 3 with 0.1'M_ sodium hydroxide
or 0.1 M^ hydrochloric acid.
pH 7.00 Dilute 5-ml of 0.1 hi potassium dihydrogen phosphate to
100-ml with water, adjust to pH 7 with 0.1 hi sodium
hydroxide or 0.1 _N phosphoric acid.
pH 9.00/pH 11.00 Dilute 5-ml of 0.1 M_ dipotassium hydrogen phosphate to
100-ml with water, adjust to pH 9 or pH 11 with 0.1 M
sodium hydroxide.
Water used in preparing buffers is deionized and autoclaved as described
in the following section. pH is measured with a pH meter accurate to
±0.02 units and equipped with a probe capable of operating accurately to 85°C.
-16-
-------
WATER
Water must be sterile and of known electrical conductivity
(<_ 0.06 ymho or less) or electrical resistivity (_> 16.67 mega ohms),
corresponding to ASTM Type I water described in ASTM D-1193-77, "Standard
Specification for Reagent Water." Water meeting these requirements can
usually be attained by passing previously deionized water through ion
exchange resins to remove metal ions and a 0.2 \im membrane filter
to remove bacteria. The water is Autoclaved for 30 min/liter and allowed
to cool before use. Sterile water is stored in a sterile-cotton-plugged
container until used. Sterility of water and buffer solutions before and
after hydrolysis experiments is determined from pour bacterial plate
counts on TGE agar (8).
SAMPLING CONSIDERATIONS
Two methods are preferred for conducting the hydrolysis rate constant
measurements.
Removal of A1 iquots
In this method, the reaction mixture is contained in one reaction
vessel and aliquots are withdrawn at timed intervals for analysis by a
predetermined method. It often is convenient to stop the reaction in the
aliquot removed by some quenching technique (pH adjustment, or cooling to
2°C). The quenched aliquot may be analyzed at the convenience of the
analyst. If the analysis must be performed immediately, it is necessary
that the time required for sampling and analysis be the same for all
samples. This is especially true for reactions with half-lives and
analysis times of only a few minutes. Care must be taken to ensure that
the removal of aliquots does not contaminate the reaction mixture or cause
losses of the reactant.
-17-
-------
Ampule or Test Tube Technique
In this method, aliquots of the reaction mixture are placed in
separate vessels. The vessels can be sealed ampules or test tubes with
Teflon-lined screw caps. Hydrolysis studies of volatile chemicals, long
term studies (>l week), and elevated temperature studies (M5°C) should
always be performed with flame-sealed ampuls. Screw-capped tubes are
adequate for less volatile chemicals with a run duration of
-------
final concentration of 1E-5M is 500-fold less than the 5E-3M buffer
solution. The final concentration must not exceed 50% of its
water solubility. In the case of compounds soluble only in the low
parts-per-billion (ppb) range (10"6 to 10~7 _M), methanol or acetonitrile,
not to exceed 1% of the final solution, can be added to the buffer to
enhance solubility. If the concentration of substrate must be increased
to greater than 1E-5M to achieve an analyzable concentration, the concen-
tration of the buffer is increased accordingly to maintain the 500-fold ratio.
The chemicals must not be heated when they are being dissolved.
HYDROLYSIS EXPERIMENTS
SCREENING TEST (LEVEL I)
Laboratory experiments should be divided into three levels. In
Level I experiments, the hydrolysis protocol is tested in the laboratory
by selecting and determining the hydrolysis rate constants established
for the SRCs in Tables 2-5. Data generated in hydrolysis experiments on
the SRCs are converted to rate constants using the computation techniques
discussed below in the "Data and Reporting" section and the Appendices.
The SRC rate constant measurements are repeated until the desired precision
is attained in all phases of rate constant measurement.
SCREENING TEST (LEVEL II)
Level II experiments are screening tests to determine the approximate
half-life and dependence of hydrolysis on pH, of chemicals of interest
at pHs 3, 7, and 11 at a selected temperature. Results from Level II
experiments are then used to set pH and temperature for Level III experiments.
Level II experiments are intended to quantify the effects of temperature
and pH on the hydrolysis rate of the chemical of interest.
-19-
-------
Buffer solutions at pHs 3, 7, and 11 are prepared by following
instructions in the section above entitled "Buffers." For each chemical,
reaction mixtures should be prepared in each of the three buffer solutions
without the use of heat. The chemical concentration should be less than
one-half its water solubility and at less than 1E-5M (see section on "Test
Chemical Solution"). The test chemical solution is transferred to sealed
ampules or test tubes with Teflon-lined screw caps (15-ml tubes, 10-ml
solution). A minimum of six tubes are prepared at each pH. Then based on a
"best guess" half-life, the tubes are placed in either a 25, 45, 65, or
85°C constant temperature bath. After sufficient time to equilibrate
(30 to 60 min), one sample is taken to determine the time zero concen-
tration. The screening test decision tree (Figure 1) is then used to
determine sampling times for subsequent tubes. Data are calculated
according to procedures given in the "Data and Reporting" section.
DETAILED TESTS (LEVEL III)
The objective of this set of experiments is to determine k/\, kfj,
and kg (if all three processes are operative) at two or more temperatures
(separated by 20°C or more) such that activation energies for each process
can be calculated and used to predict hydrolysis rate constants at other
temperatures and pHs. The preliminary values of k/\, k^, and kg calculated
according to instruction in the "Treatment of Results" section and the
screening test are used to design the Level III detailed tests. Assuming
the kf| values at pH 3 and 11 are due solely to acid- and base-catalyzed
reactions (pseudo-first-order kinetics), the value of kn will decrease by
a factor of 10 for every change of pH unit toward neutrality. If the k^
values at pH 3 and 11 are the same as the pH 7 value, then hydrolysis is
independent of pH and controlled only by temperature. Level III experiments
-20-
-------
for processes independent of pH are conducted at pH 7 and at temperatures
that result in 50-80% hydrolysis between one day and two weeks. If
either or both acid catalysis or hydroxide ion mediated hydrolysis is
indicated in the screening test, then Level III experiments are conducted
at pHs and temperatures (including pH 7) such that rate constants (first-
and second-order) and activation energies can be calculated for the
processes (k/\, k|\|, KB)- Ideally rate constants would be determined at a
constant temperature and at two or more pHs on each side of neutrality.
Establishment of a pH versus first-order disappearance rate constant
curve would allow prediction of rates at other pHs.
To quantify the effect of temperature and pH on the disappearance
rate constant for hydrolysis, two objectives must be attained: (1) expertise
must be established in rate constant measurements by reproducibly measuring
values for the SRCs and (2) rate constants must be replicated (minimum of
three) for the compounds of interest at two or more temperatures separated
by 20°C. Level III experiments are set up and conducted similar to Level
II measurements. Water, buffers, and test solutions are prepared in the
same manner as for the screening tests (6 to 8 tubes or ampules). The
temperatures and pHs are adjusted after consideration of the screening
test results to yield experimental conditions that allow 50-80% reduction
in concentration of the chemical in two weeks or less. The tubes are
removed at regular intervals and the percent remaining of the test chemical
is determined by a method of established accuracy and precision (±5% acceptable),
Two concentrations of chemical, differing by a factor of ten, can be
used as a test to support the first-order kinetics hydrolytic mechanism.
If plots of In % chemical remaining versus time are linear and have the
same slope within experimental error, then first-order kinetics are assumed.
-21-
-------
Rate constants and activation energies are calculated by methods
outlined in the following section.
DATA AND REPORTING
TREATMENT OF RESULTS
Screening Tests
An observed rate constant (kn) for the pH 3, 7, and 11 runs is
calculated by the appropriate method described in the Appendices (G-l,
G-2, H, or I). At constant temperature, the k^ values for pHs 3 and 11
are compared to the value calculated at pH 7 for evidence of H+ or OH~
enhancement of rates. If the observed rates are within experimental
error, hydrolysis is considered to be independent of pH in the range 3 to 11
and Level III experiments are conducted at pH 7. The rate studies at pH
7 should be conducted at two or more temperatures to allow calculation of
the activation energy (Ea) and collision frequency (A) by the method
outlined in Appendix J.
If kn at pHs 3 and 11 are greater than experimental error from the
pH 7 rate, then second-order rate constants (k/\ and kg) can be calculated
from each first-order rate constant at each temperature by dividing the
measured kn at the particular pH by the hydronium ion activity (k/\,
pH 3) or hydroxide ion activity (kg, pH 11). This calculation assumes
hydrolysis at pHs 3 and 11 are second-order reactions (first-order in
compound and first-order in hydronium or hydroxide activity). The
effect of temperature on Kw is taken into account when using equation 5
to calculate hydroxide ion activity.
The results of the screening tests are used to estimate the hydroysis
rate at other pH values, and are used to determine the pHs and temperatures
-22-
-------
for the Level III hydrolysis rate determinations. Assuming that the k^
values at pH 3 and 11 are due solely to acid catalyzed and base mediated
reactions the value of kn will decrease by a factor of 10 for every
change of pH unit toward neutrality, and vary approximately by a factor
of 10 for each 20°C change in temperature (Ea = 84 kJ/mol).
Calculation of Rate Constants and Activation Energies from Level III
Experiments
Conditions of the Level III determinations are set (huge excess
of water, constant pH) such that the rate constant at a given pH and
temperature should be pseudo-first-order. From equation 11 a concentration-
time profile can be expressed where [C0] is the concentration of the test
substrate at time zero and [C] is the concentration at a given time, k is
the first-order rate constant and t is time.
ln[C] = ln[C0] - kt (11)
Several methods can be used to calculate the pseudo-first-order disappearance
rate constant for a compound at a set pH and temperature. The first two
(Appendicies G-l and G-2) consist of plotting either log % chemical
remaining or 1 n % chemical remaining on the Y-axis versus time of sampling
(time) on the X-axis. The slope of the best straight line drawn through
the data points is used to derive the pseudo-first-order rate constant
also called kn (observed rate constant). A third method used to calculate
kn is illustrated in Appendix H using the rearranged log form of equation 11.
The last method (Appendix I) consists of processing data using the Lotus
1-2-3 software with the IBM PC/XT or equivalent. Software programs for
slope are based on the linear least squares analysis of the In of %
chemical remaining versus time. Outputs from the linear regression program
-23-
-------
are (1) slope (pseudo-first-order rate constant), (2) Y-intercept,
(3) variance, (4) standard deviation (SD) of Y-intercept, (5) standard
deviation (SD) of the slope, and (6) the correlation coefficient (r2).
Marked curvature in a plot of 1n % chemical remaining versus time
or a low correlation coefficient indicates that a non-first-order process
is influencing results and that additional experiments are needed. When
acid catalyzed or base mediated hydrolysis is indicated by the screening
test, the kf\ and/or kg are calculated by dividing the measured kn at the
particular pH by the hydronium ion activity (k/\, pH 3) or hydroxide ion
activity (kg, pH 11). This assumes that, at pHs 3 and 11, hydrolysis is
100% dependent on the hydronium and hydroxide ions, respectively. The
value for k^ is calculated using equation 4 and the kn value measured for
the pH 7 hydrolysis.
Under ideal conditions, the Level III hydrolysis studies are conducted
at three temperatures (separated by 20°C each) and the three pHs 3, 7,
and 11. Values of k/\, k^, and kg are calculated at each temperature.
Regression analysis using equation 8 on the three sets of three constants
at three temperatures yields values for E/\, EN> Eg, In A/\, Ln AN and Ln
Ag. These values of E and A can be used to calculate values of kx (X =
A, N, B) at temperatures of interest. The calculated kx values at a
particular temperature are used in equation 4 to calculate kn at a
chosen pH (1^ at the particular temperature is used when calculating [OH~]
by equation 5). Values of A and E for each process can be estimated by
plotting log kx versus 1/T (Arrhenius plot) and taking the best straight
line through the data points. The slope will equal -Ea/R with intercept
of In A. A two point Arrhenius plot can be used when kinetic data are
available at two temperatures only.
-24-
-------
Extrapolation of k^, kA. k|\i, and kp Using Approximate Activation Energies
In instances where k^, kA, k^, and ke are known/measured at
only one temperature and E and A are not known, the rates can be approximated
at other temperatures using equation 9 and assuming a value for the
activation energy (Ea). Activation energies for the majority of compounds
fall in the range of 62.7 - 104.5 kJ/mol. Using an Ea value of 83.6 kJ/mol
in equation 9 to extrapolate a measured k to a new temperature is a
reasonable approximation. The k value obtained using 83.6 kJ/mol will be
within a factor of three of the values obtained using 62.7 kJ/mol or
104.5 kJ/mol (1 kcal = 4.18 kJ). The above values correspond to 15, 20, and
25 kcal/mol.
ACKNOWLEDGMENTS
This work was conducted at the Athens Environmental Research Laboratory
through the combined efforts of EPA, Technology Applications, Inc. (TAI),
and University of Georgia (UGA) personnel. The technical assistance of
Miss Sarah Patman (UGA) is gratefully acknowledged. The assistance of
Drs. Lee Wolfe and William Steen throughout the project and including
review of this report is also gratefully acknowledged. Discussions with
Mr. William Donaldson were always fruitful and so acknowledged. Mrs. Karin
Blankenship's effort in typing the draft and subsequent revisions was exemplary.
REFERENCES
1. Mill, T.W., W.R. Mabey, D.C. Bomberger, T.W. Chow, D.G. Hendry,
and J.H. Smith. 1982. Laboratory Protocols for Evaluating the
Fate of Organic Chemicals in Air and Water. U.S. Environmental
Protection Agency, Athens, GA. EPA/600/3-82/022.
2. Federal Register 50:39283-39285, Hydrolysis as a Function of pH at
25°C. Section 796.3500.
-25-
-------
3. Suffet, I.H., C.W. Carter, and G.T. Coyle. 1981. Test Protocols
for the Environmental Fate and Movement of Toxicants: Proceedings
of a Symposium of the Association of Official Analytical Chemists
(AOAC), October 21, 1980, Washington, DC. G. Zweig and M. Beroza
(eds.). Association of Official Analytical Chemists, Washington, DC.
4. Harned, H.S. and R.B. Owen. 1958. The Physical Chemistry of
Electrolytic Solutions. Reinhold Publishing Corporation, New York,
pp. 638 and 645.
5. Ellington, J.J., F.E. Stancil Jr., and W.D. Payne. 1987. Measurement of
Hydrolysis Rate Constants for Evaluation of Hazardous Waste Land
Disposal: Volume I. Data on 32 Chemicals. U.S. Environmental
Protection Agency, Athens, GA. EPA/600/3-86/043.
6. Ellington, J.J., F.E. Stancil Jr., W.D. Payne, and C.D. Trusty.
1987- Measurement of Hydrolysis Rate Constants for Evaluation
of Hazardous Waste Land Disposal: Volume II. Data on 54
Chemicals. U.S. Environmental Protection Agency, Athens, GA.
EPA/600/3-87/019.
7. Bates, Roger G. Determination of pH Theory and Practice. 1972.
John Wiley and Sons, New York.
8. Franson, Mary Ann H., Managing Ed. Standard Methods for the
Examination of Water and Wastewater. 1985. American Public
Health Association. Washington, DC, 16 ed., Part 907, pp. 864-865.
-26-
-------
SCREENING TEST DECISION TREE
START RUNS, PULL T(0)
CONDITIONS:
pH 3, 7, 11
85 Degrees C
PULL T(1) at +1 HR
ANALYZE T(0), T(1)
REPEAT RUN USING
LOWER TEMPERATURE
PERCENT
REMAINING
PULL T(2) at +2 HRS
PULL T(3) - T(5) at
1 HR INTERVALS
PULL T(2) at +1 DAY
ANALYZE T(0) - T(5)
REPEAT - PULL
2 SAMPLES PER DAY
PERCENT
REMAININ
PULL T(3) - T(5) at
1 DAY INTERVALS
PULL T(3) - T(5)
at 3 DAY INTERVALS
ANALYZE T(0) - T(5)
ANALYZE T(0) - T(5)
-------
CONTENT
Si.mmary of Appendices
Appendix A .Chemical Information Sheet
Appendix B Linearity Data Sheet
Appendix C Hydrolysis Data Sheet. - GC
Ap!"l'idix !) Hydrolysis Data Sheet - HPLC
Appendix E Hydrolysis Data Sheet - UV
Appendix F . . . Summary Sheet for Rate Data
Appendix G-l and G-2 Graphical Determination of
Hydrolysis Rate Constant
and Half-1 ife
Appendix H Computation of Hydrolysis Rate
Constant and Half-life
Appendix I Using a Linear Regression Program
To Determine Hydrolysis Rate
Constants
Appendix J .................... .Determination of Act'vation Energies
-------
APPENDICES
Example Sheets for Generation of Data and Calculation of Rate
Constants and Activation Energies.
-------
SUMMARY OF APPENDICES
The exhibits are intended as aids in obtaining, calculating, and
compiling data. They are not intended as replacements for laboratory
notebooks, but yield quick access to the status of the particular chemical
under study. The Chemical Information Sheet (Appendix A) should be
completed before any laboratory work begins. This particularly includes
the CAS number-structure match and the purity-identity analysis of the
analytical standard used in subsequent hydrolysis studies. The Linearity
Data Sheet (Appendix B) is completed after the method of analysis is
determined. Hydrolysis studies are then performed in the range of
concentrations where response versus amount of compound is linear.
Appendices C, D, and E are used to record and summarize data for the
three major methods of analysis used in hydrolysis rate constant
measurements. In Appendix F the observed rate constants and calculated
second-order rate constants are compiled according to temperature.
Actual determinations of rate constants from disappearance data is
detailed in appendices G, H, and I.
In Appendicies G-l and G-2, either log or In % chemical remaining
versus time is plotted and the negative of the slope of the "best fit"
line is used to generate the rate constant. If there is random scatter
in the points, a good method for estimating the best slope is to first
adopt an intentional bias toward a high slope and to draw the steepest
line that could legitimately be fitted to the points. Second, one should
adopt the opposite bias, and draw the line with the least steep slope
that could be fitted to the points. Finally, one should take the average
of the two extreme slopes as the best slope of the plotted points.
-------
By means of equation A-l (integrated rate equation), the value of k
is computed between tg and tj, between t^ and t2, and so on, and the
average of the resulting k values is taken as the rate constant (Appendix H)
The interval between to
k
log (a-x-j) - log(a-xj) - (tj - tj) (A.I)
2.303
and tj can be neglected if there is any doubt about the accuracy of the
initial concentration (a) or determination of zero time (t0). In many
instances, the k value calculated between tg and t^ is much higher than
succeeding k values.
In Appendix I, the % chemical remaining versus time data were processed
on a Lotus 1-2-3/IBM PC-XT using a data entry/linear regression program.
Values obtained from the linear regression program include the slope
(pseudo-first-order rate constant), Y-intercept, variance, SD of Y-
intercept, SD of slope and the correlation coefficient (r?). The second-
order alkaline elimination rate constant was calculated by dividing the
pseudo-first-order rate constants by the calculated hydroxide ion
activity at 40°C and pH 11.2.
Appendix J is the output from a computer program to fit temperature
dependent rate constant data to a line and obtain the Arrhenius parameters
[collision frequency (A) and activation energy (Ea)]. The Arrhenius
plot requires plotting In k versus the reciprocal of the corresponding
temperature in degrees Kelvin. The slope of the line is equal to
Ea/(R) and In A is the Y-intercept.
-------
APPENDIX A
Chemical Information Sheet
Chemical Abstract Service (CAS) Number: 58-89-9
CAS Name: Cyclohexane, l,2,3.4,5,6-hexach1oro-,(la, 2a, 3g, 4a, 5a,
Common and Trade Names: Lindane, Hexachlorocyclohexane, gamma-BHC
Source: Research Triangle Park Repository
Structure:
Property:
Molecular Weight
Melting Point, °C
Boiling Point, °C, torr
Water Solubility, °C
Vapor Pressure
Octanol-water, Kow
Value
291
112.5-113
6.8-7.8 ppm at 25°C
20°C, 0.03mm Hg
5,250
Source
Purity Determination:
GC, GC-MS
-------
RS:
Analyst:
Compound (A-j )
APPENDIX B
Linearity Data Sheet
Date:
1. Stock Solution
Weight of A]_:
Purity of Aj:
Adjusted cone, of stock:
Date Prepared:
_mg dissolved in
_(solvent)
Suppl ier:
2. Standard for Retention Check
PP_ of
PP of
Solvent:
ml of
PP
.(AT)
.(is)
Method of Analysis: GC
Other
HPLC
Date Prepared:
UV
Attach copy of method to data sheet.
RS = Run series number
Ai = Analyte of interest
pp_ = parts per thousand, million, etc.
-------
(Appendix B cont.)
3. Linearity Standards
Solvent:
Standard
1
2
3
4
5
6
7
8
Cone.
RS:
Extraction performed? Yes No
Dilution Required
-------
(Appendix B cont.)
Data
Std. No. Cone.
1
2
3
4
5
6
7
8
Conclusions:
Response
-------
APPENDIX C
Hydrolysis Data Sheet - GC
RS: 2126 Date: 11-3-87
Analyst: HDP
Compound (A-j): Lindane
1. Stock Solution
Weight of A-j : 10.3 mg dissolved in 10 ml
of acetonitrile (solvent)
Purity of A-J: 99.4% Supplier: RTP
Adjusted cone, of stock: 1024 ppm
Date prepared: 10-6-87
2. Standard For Retention Check
205 ppb of Lindane (A-j)
206 ppb of 1,2,3- and 1,2.4-Trichlorobenzene (IS)
Solvent: Isooctane Date prepared: 10-7-87
GC = Gas Chromatography
RS = Run Series Number
Ai = Analyte of interest
PP_ = parts per thousand, million, etc.
IS = Internal Standard
-------
(Appendix C cont.)
3. Buffer Solution
Buffer strength (cone):
Type of buffer used:
Cone, of buffer stock:
Date-water Autoclaved:
pH of buffer before addi
RS: 2126
0.005 M
KpHPOa
0.10 M. Date prepared/Autoclaved: 10-26-87
10-26-87
tion of Ai : 11.71 @ 22.5 °C
pH of buffer after addition of A, : 11.20 @ 40.0 °C
pH meter standardized at
Cone, of Ai in buffer
@ °C
pH: 7 and 11
205 ppb
Dilutions required:
0.02 ml (stock) > 100 ml (buffer)
-------
(Appendix C cont.)
4. Extraction RS: 1019-1023
Extraction solvent: Isooctane
Internal Std.(IS) 2,4-D methyl ester
Cone, of IS in extraction solvent: 5.25 pprn
Extract 5 ml of buffer with 1 ml of extraction solvent
concentration in extraction solvent before dilution
1.0 ppm A-j
5.25 ppm IS
Is buffer neutralized before extraction? Yes X No
Dilutions: 0.1 ml > 5
Final Cone.: 20 ppb_ Aj
105 ppb IS
-------
(Appendix C cont.)
5. GC Procedure
GC No.
RS: 2126
A. Column (capillary): J and W, DB-5
"(Supplier and code name)
Length: 15M
Dia:
0.53mm
Coating: DB-5
Film thickness: 5.0 micron
B. Oven
Isothermal - oven temp:
Program - Draw program below:
C. Injector
Split/Splitless:
Injector temp.:
,.100°C
'O-mi n "
On Column:
260 °C
280°C, 3 min
15°C/min (Column compensation was used)
Injection volume (size) 1.0
Septum purge: N? Flow =
•v-1.2
mi croliter
ml/min
D. Detector
ECD: X
NPD:
Detector temp: 325 °C
Make-up gas: M? Flow:
Attenuation: 2^2 >2T4
FID:
27
ml/min
Hydrogen flow:
Ai r flow:
Filter:
ml/mi n
ml/min
E. Carrier Gas
Head pressure: 3 psi
Linear velocity: M5 cm/sec
Flow: ^5 ml /min
Comments on GC technique:
-------
(Appendix C cont.)
6. Sample Data*
RS: 2126
pH: 11.20 @ 40.0 °C
Is Aj/IS ratio used for responses? Yes No X
Method of measurement: Area X Height Both
Raw Data:
Samp. No.
Time(min)
Run Temperature =
Response X
40.0
^Remaining
1.
2.
3.
4.
5.
6.
7.
8.
10:40,11-3
0 min
10.2 min
34.8
49.2
60.0
73.8
100.2
44181
40662
38384
27041
30242
10218
9807
5111
5032
3195
3270
2546
2141
746
41,076
28,641
10,012
5,072
3,232
2,344
749
100.0
69.7
24.3
12.3
7.9
5.7
1.8
Were any points discarded Yes
List discarded points:
No X
Give reason for discarding points:
If run was aborted, give reason for aborting the run:
Analyst:
WDP
Date: 11-3-87
Attach computer printout
K = 0.0402
min"
K2= 8.75
min"1
T 1/2 = 17
mi n
*Data used in Aooendices G-l, G-2, H, and I
-------
APPENDIX D
Hydrolysis Data Sheet - HPLC
RS: Date:
Analyst:
Compound (Ai):
1. Stock Solution
Weight of A-j: mg dissolved in ml of
(solvent)
Purity of A-j : Supplier:
Adjusted cone, of stock: _ pp_
Date prepared: _
2. Standard for Retention Check
_ PP_ of
pp_ of _ (IS)
Solvent: Date Prepared:
HPLC = High Performance Liquid Chromatography
RS = Run Series Number
A-j = Analyte of interest
pp = Parts per thousand, million, etc.
IS~~ = Internal Standard
-------
(Appendix D cont.)
3. Buffer Solution RS:_
Buffer Strength (cone.): M
Type of Buffer used:
Cone, of Buffer Stock: _ M. Date Prepared/Autoclaved: _
Date-Water Autoclaved: _
pH of buffer before addition of A-j : _ @ _ °C
pH of buffer after addition of A-j : _ @ _ °C
pH meter standardized at pH: _ and
Cone, of Ai in buffer: _ pp_
-Dilutions required:
Final Cone, of A-j for injection pp_
Is sample neutralized before injection? Yes No
-------
(Appendix D cont.)
4. 1C Procedure RS: LC No.
A. Column:
(Supplier and code name)
Length: cm Dia: mm
Packing: Particle size: m
B. Detector UV: Fluorescence: Electrochemical
Wavelength: nm Attenuation:
Potential: V Range:
Excitation Wavelength: nm
Emission Wavelength: nm
Inj. Vol. ml Flow Rate:
Pressure:
Mobile Phase:
Gradient: Yes No
Draw Gradient:
Comments on LC Procedure:
-------
(Appendix D cont.)
Sample Data
RS:
PH:
Is Aj/IS ratio used for responses: Yes No_
Method of Measurement: Area Height
Both
Raw Data: Run Temperature = °C
Samp. No. Time Response
1.
2.
3.
4.
5.
6.
7.
8.
X %Remaining
Were any points discarded: Yes No_
List discarded points:
Give reason for discarding points:
If run was aborted, give reason for aborting the run:
Analyst:
Date:
Attach. Computer Printout
hr
,-1
T 1/2 =
days
K2 =
-------
APPENDIX E
Hydrolysis Data Sheet - UV
RS: Date:
Analyst:
Compound (A-,-):
1. Stock Solution
Weight of A-j : mg dissolved in ml
of (solvent)
Purity of A-,-: Supplier:
Adjusted cone, of stock: pp_
Date Prepared:
2. Standard For Retention Check
PP_ of
Solvent: Date Prepared:
UV = Ultraviolet
RS = Run Series Number
Ai = Analyte of interest
-------
(Appendix E cont.)
3. Buffer Solution RS:
Buffer strength (cone): M
Type of buffer used:
Cone, of buffer stock: _ M. Date prepared/Autoclaved: _
Date-water Autoclaved: _
pH of buffer before addition of Aj : _ @ _ °C
pH of buffer after addition of A^ : _ @ _ °C
pH meter standardized at pH: _ and
Cone, of A-j in buffer _ pp_
Dilutions required:
Final cone, of Aj for measurement: pp_
Is sample neutralized before measuring? Yes No
-------
(Appendix E cont.)
4. UV Procedure RS:
Reference:
Attach spectrophotometer readout and absorption curve:
Maxima found:
Comments:
-------
(Appendix E cont.)
Data
Run Temp. °C RS:
Absorbance Readings at Maxima
Run No. Time
1.
2.
3.
4.
5.
6.
7.
8.
pH @ ° C
-------
(Appendix E cont.)
Were any points discarded? Yes No
List discarded points:
Give reason for discarding points:
If run was aborted, give reason for aborting the run:
Analyst: Date:
Attach computer printout
Kj = hr'1
K2 = M^hr'1
T 1/2 = days
-------
APPENDIX F
Summary of Data:
Compound: .
RS T pH ^(hr1) kA(M'
Ea =
Method: GC LC UV Other
kfo is the pseudo-first-order rate constant measured at a particular
pH and temperature.
-------
2.0
AHPhNUIX 6-1
RS 2126, pH = 11.20, 40 C
n — i — i — PI — r
-S
V
y
--/
-~l
1
• • r
i ' '
-tf4-
\
*• L.JV •' 4
-*-(-¥ •, ,.P -)
V *
V
rvh
V
A
^
\
_ . ... —
. _
i
1
t •
i:iV-
V
V
— \-
_...
-
—
- - ..
• T
\
\
-
—
\
\
.... .
- - .
— ._
--
\ .
i
—
__.
---
—
—
. _
-
-
-
~
-
-
"""I
-| H — f-
J— — if-i
Y2 - Y-
X2 - X]
^1 nno - n HI 7C
__j_j__c :
:S±._::::::::::
0.44 - 1.76
88 - 14
>
w. i u (j \_ V.UX/CJ
From Equation 11:
log[C] = log[C0
-k
Slooe =
-
"X
-
-
-
_
-
—
4--
^
V
\
— . .
.1
V
- -
—
.
2.303
-k = (2
kh - o.(
s
" X
• \ "
•,
• — \ — —
V
/v
— _ '
\
\
. - -
__. __._ _ _
. .
Kt
2.303
.303) (-0.0178)
140 Q mi n~l
- - - • — .......
-------------
- - - ... —
X V-
2.
\ j_
\ . . —
\ • i
i i
._ ._„.... _
.]_. .
— ._
• i •
1.5
o>
C
(D
OJ
o:
oi
I 1.0
C
C)
O
0.5
60X. . . . 80
1 i mo 1 mi n I
100
120
-------
5.0
APPENDIX 6-2
RS 2126, pH = 11.20, 40°C
y
^
f. >>
i,
4
1
--
--
--
--
-!••
-
-
I
-
-
--
1
--
--
--
T
--
i
y
V
V
-"
--
....
--
--
1
--
1
r;
• -i
, L/ J
. 1
"*•
__]"-.
-T- -
— -
I
Y
\
^
\
"\
- -IJ--
-- - -
-----
- - - -
— — .
--i.
t ~
\
- - — -
-,r
2U 4(
—
\
-V-
\
- --
—
--
\
.
--
--
--
^
. _ _
I
'..._
-•
"
c
to
E
-------
APPENDIX H
Equation 11 can be rearranged to yeild:
2.303
k = (log[C0] - log[C])
t
This equation is often written as:
2.303
k = [log a - log(a-x)]
At
Where the symbol a is used to replace C0, x is the decrease of concentrati
in time t, and a-x = C the concentration at time t.
on
Log % Chemical
Time(min) Remaining k(min~l)
0.0 2.00
10.2 1.84 0.036
34.8 1.38 0.043
49.2 1.09 0.046
60.0 0.90 0.040
73.8 0.76 0.027
100.2 0.25 0.044
0.039±0.007
2.303
k = [2.00 - 1.84] = 0.036
10.2
2.303
k = [1.84 - 1.38] = 0.043
24.6
2.303
k = [1.38 - 1.09] - 0.046
14.4
The remaining three k values are calculated in like manner.
-------
feO
c
CO
£
V
u
0)
d
CO
T3
C
•r-H
-J
0.5
Appendix I
Plot of In % Lindane Remaining vs Time
S^i.
RS «2126 Lindane pH=11.20 at 40 °C
i 1 1 1 1 1 1 1
Time
Minutes
0.0
10.2
34.8
60.0
73.8
100.2
In %
Remaining
4.605
4.244
3.190
2.509
2.066
0.587
Time (minutes)
RS 2126 Lindane pH = 11.20 at 40°C
Number of observations
SD of Slope
Slope of Regression Line
Regression Coefficient
SD of Y-intercept
Y-intercept of Regr. Line
R SQUARED
Psuedo-Kl
120
0.00106
-0.04023
-0.99827
0.060419
4.59417
0.99685
0.04023 min-1
Kw = [OH-][H+L Kw at 40°C = 2.919 X 10'14
Kw 2.919 X ID'14
[OH-] = ---- = ............. = 0.0046
[H+] 6.30 X 10-12
i
0.04023
= 8.745
[OH-] 0.0046
The second-order alkaline elimination rate constant was deter-
mined at other temperatures and used in Appendix J to determine the
activation energy and collision frequency.
-------
4 -
3 -
2 -
1 -
0.0027
Temperature
Appendix J
Arrhenius Plot
Lindane
1
0.0029
0.0031
0.0033
I/Temperature (K)
from Table 5
Number of points
Avg. of Y values
Std. Dev. of Y
Intercept
Coef. of Var. of Y
SSYY1
Std. Dev. of Slope
Slope
Regression Coef.
Y-Intercept (In A)
R Squared
****Energy of Activation****
65.279 ± 1.968 kj/mol
15.617 ± 0.471 kcal/mol
for Arrhenius Equation only
The above plot is a graphical representation of Equation 8
In k = In A - Ea/RT
22.8
23.6
37.0
40.0
45.0
45.3
46.0
85.0
2.90
2.70
8.70
8.60
16.10
16.50
17.40
272.00
8
2.5521
0.75
52.725
14.486
237
-7859.65
-0.997
27.494
0.9945
------- |