Gas chromatographic procedure to analyze amino acids in lake waters

Aug 1, 1973 - Wayne S. Gardner and G. Fred. Lee. Environ. Sci. Technol. , 1973, 7 (8), pp 719–724. DOI: 10.1021/es60080a007. Publication Date: Augus...
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Gas Chromatographic Procedure to Analyze Amino Acids in Lake Waters Wayne S. Gardner' and G. Fred Lee2 Water Chemistry Program, University of Wisconsin, Madison, Wis. 53706

A gas chromatographic procedure for the determination of free amino acids in lake water samples or hydrolyzates has been developed. A combination of ligand exchange and ion exchange chromatography was used to isolate and concentrate the amino acids from water samples. A semimicro derivative preparation technique was developed where the N-trifluoroacetyl methyl ester derivatives were formed. Recovery of the standard amino acids ranged from 53-9370. Poor recoveries were obtained for phenylalanine and lysine.

Amino acids constitute a substantial portion of the organic nitrogen in natural water, but until recently the amino acid composition of lake systems has been largely unknown. Previous studies on the composition of amino acids in natural waters have been reviewed by Gardner (1971). Data on dissolved free amino acids (DFAA) have been particularly limited because of problems caused by low concentrations of DFAA, the presence of interfering substances in natural water and the high biological activity of amino acids. Analytical techniques used to differentiate and detect amino acids from natural waters include paper, thin layer, ion exchange chromatography, and gas chromatography. This paper presents a description and evaluation of the use of gas chromatography to analyze amino acids isolated from lake water by ion exchange and ligand exchange techniques. The described method has been used to determine the amino acid composition of Lake Mendota, a eutrophic lake in Madison, Wis. (Gardner, 1971). Experimental

The procedures evaluated to isolate and analyze amino acids from lake water are described below. Isolation Procedures. Dissolved Free A m i n o A c i d s ( D F A A ) . The water samples were membrane filtered (0.45 p pore size) shortly after collection. The filtrates ( 2 liters) were passed through 10 ml of Biorad AG-50 or Dowex 50 (H+ form, 100-200 mesh) contained in glass columns (12.5 cm x 2.2 cm i.d.). A liquid head of about 35 cm was maintained above the columns with a siphon arrangement. The amino acids were eluted from the cation exchange columns with 2 N ammonia solution (60 ml) (Gee et al., 1967). Prior to ligand exchange cleanup, ammonia was removed from each eluate by rotary evaporation a t 60°C. The ligand exchange system consisted of a semimicro modification of the columns introduced by Siege1 and Degens (1966). A schematic diagram of the ligand exchange column apparatus is shown in Figure 1. The columns were assembled by placing plugs of glass wool above the restricted ends of disposable transfer pipets and covering each plug with 1 cm of Chelex-100-NH3 resin and 6 cm of Chelex-100-Cu-NH3 resin. Glass tubes were connected to Present address, Skidaway Institute of Oceanography, Savannah, Ga. To whom correspondence should be addressed.

the lower ends of the columns (with silicone rubber gas chromatograph septums) to maintain the liquid level above the resin surfaces. The samples were introduced through glass funnels attached to the upper ends of the columns to provide liquid heads of about 20 cm. After the solutions had passed through, the S-tubes were removed and the tips of the columns were capped (with closed septums) until the amino acids were eluted with 3N ammonia solution. The first ml of each eluate was discarded and the amino acids were collected in the following 3 ml in a 5-ml graduated cylinder. The contents of each cylinder were mixed and equal portions of the eluates were transferred (0.5 ml a t a time) into two respective reflux ampuls (described below). Water was removed with dry nitrogen and volatile derivatives were formed. Dissolved (Free Combined) Amino Acids (Filtered Hydrolyzed Fraction). Lake water filtrate (200 ml) acidified with four drops of concentrated HC1 (to prevent CaC03 precipitation) and concentrated to about 1.5 ml by rotary evaporation a t 60°C. The final concentrate was transferred first into a 5-ml graduated cylinder for exact volume measurement, and then into a constricted borosilicate glass test tube. Sufficient concentrated HC1 plus 0.1 ml (to compensate for the concentrate left in the flask) was measured into the graduate cylinder to make the final concentrate 6N in HCl when the acid was added to the test tube. Portions of this concentrated HC1 were used to rinse the round-bottom flask used in the final concentration step. After the acid was transferred into the test tube, the air over the hydrolysis solution was displaced with nitrogen and the tube sealed. The samples were hydrolyzed by heating the sealed tubes in a steam bath (100°C) for 24 hr. The hydrolyzates were quantitatively

+

r

GLASS FUNNEL

1

TYGON

CONNECTER

22cm-

8D

CHE-EX-100-CU-NH,

RESIN-

GLASS

WOOL

P-JG

SEPTUM

Figure 1. Schematic diagram of semimicro ligand exchange column apparatus Volume 7 , Number 8, August 1973

719

transferred into respective 500-ml Erlenmeyer flasks and the p H of each solution was adjusted to 9.5-9.7 with aqueous NaOH and HCl solutions. Each solution was diluted to approximately 400 ml with glass-distilled water and passed through a semimicro ligand exchange column. A liquid head of approximately 40 cm plus the height of the liquid in the flask was maintained over each column with a siphon. The ligand exchange columns were attached to funnels, and glass-distilled water (20 ml) was passed through each column to remove residual salts from the hydrolysis procedure. The amino acids were eluted with 3N ammonia, derivatized and analyzed by gas chromatography. Derivative Preparation. During the last decade, several procedures have been developed to derivatize and analyze amino acids by gas chromatography (Gehrke and Takeda, 1972). The volatile amino acid derivatives used in this investigation were the N-trifluoroacetyl methyl esters. A micromodification of the derivative preparation procedure suggested by Gee (1967) was utilized. The methyl esters were prepared by refluxing the dried amino acids with acidified methanol containing thionyl chloride as a catalyst. The methylation reagent was prepared by carefully reacting acetyl chloride (0.1 ml) with anhydrous methanol (2 ml) (which resulted in the formation of HCl and methyl acetate in methanol) and adding thionyl chloride ( 2 drops) (Gas Chromatograph N e w s l e t t e r , 1970). Trifluoroacetylation of hydroxyl and amino groups was accomplished by refluxing with 10 p1. of a mixture of trifluoroacetic anhydride and methylene chloride (1:4). A schematic diagram of the aluminum block apparatus designed to form the N-trifluoroacetyl methyl ester amino acid derivatives is shown in Figure 2. Microreaction vessels ("reflux ampuls") were constructed from disposable transfer pipets. The pipets were severed 2 cm above the enlargements and the large ends were sealed with an oxygen flame and blown to round uniform ends wih a rubber bulb assembly. Holes in the aluminum block were positioned to hold the ampuls in appropriate positions for solvent removal and refluxing. The aluminum block was

I1

REFLUX

AMPUL

POSITIONED FOR

REFLUX AMPUL

SOLVENT

POSITIONED

FOR K (100'CI

diagram of aluminum block apparatus for preparing amino acid derivatives

Figure 2. Schematic

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Environmental Science & Technology

maintained at a temperature of 100 f 2°C with a hot plate. After the ligand exchange eluates were added, the contents of each respective ampul were taken to dryness. Dry nitrogen was passed slowly into each ampul through drawn-out glass tubes attached to the manifold by rubber tubes. Complete drying of lake water samples was assured by redrying each sample after adding methylation reagent. (The methylation reagent was added to the ampuls with a drawn-out glass tube attached to a syringe.) A second portion of methylation reagent (50 pl.) was added to each sample. The ampuls were sealed with a flame, placed (small end down) into the holes in the block, and allowed to reflux for 10 min with the methylation reagent. After cooling, the contents of each ampul were centrifuged to the large end. The sealed tip was severed and the excess reagent removed using the aluminum block apparatus. The large end of the ampul was suspended into an ice bath (from a cut-off rubber stopper). Acylation reagent (10 pl.) was added with a Hamilton syringe and the ampul was immediately sealed. The samples were refluxed with the aluminum block system for 5 min. Prior to gas chromatographic analysis, the resulting solution was centrifuged to the small end of the reflux ampul and mixed with a vortex mixer. Gas Chromatograph Analysis. The gas chromatograph used in this investigation was a Varian Aerograph 1520 B equipped with a matrix temperature programmer and a flame ionization detector. A modification of the column suggested by Darbre and Islam (1968) was used to separate and identify the amino acid derivatives. Glass columns (4 m x 3 mm 0.d.) were packed with 0.7% XE-60 plus 0.5% OV-101 plus 0.2% QF-1 on Diatoport S (80-100 mesh). The approximate gas chromatographic conditions for sample analysis were as follows: flow rate of nitrogen carrier gas, 30 ml per minute; injector temperature, 125°C; detector temperature, 250°C. The temperature program used was: isothermal at 54"C, 5 min; 5.6"C per minute, 10 min; isothermal, 5 min; 4.8"C per minute, 15 min; isothermal, 10 min. Generally, 5 pl. of the final reaction mixture from each sample was injected into the gas chromatograph, but smaller quantities could be injected when high concentrations were expected in the samples. A chromatogram of a standard mixture of amino acids is presented in Figure 3. Also shown is a chromatogram of dissolved free amino acids isolated from Lake Mendota. Two internal standard compounds were utilized to quantitate amino acid levels. Diphenylmethane (0.5 pg per ampul) was dissolved in the acylation reagent and added during the last step of derivative preparation. 6 Amino valeric acid (3 pg per ampul) was mixed directly into the ligand exchange eluate and carried through the sample manipulations of final evaporation, derivative formation, and gas chromatographic analysis. Calibration curves were obtained for each amino acid by plotting peak height ratios (peak height amino acid/ peak height internal standard) against the micromoles of amino acid. Average relative peak heights obtained from various volumes of a standard amino acid mixture were utilized to obtain standard calibration curves. Separate sets of calibration curves were prepared for each of the two internal standards used. The procedures used to analyze dissolve amino acids from lake water are summarized in Figure 4. Evaluation of Precision and Accuracy of Procedures. Estimates of precision for the respective procedures were made by determining the recoveries of known amounts of amino acids added to lake water filtrates.

01

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0 0 ll C 0

C c

21

I-

I

ll 0

I 0

I

I

I

1

IO

20

30

40

T I M E (minutes) Figure 3. Chromatograms of amino acids (N-trifluoroacetylmethyl ester dervatives) and internal standards Each chromatogram represents 5 pl. of derivative preparation a Derivative preparation (10 pl.) contained 0.15 pmol of each amino acid b. Derivative preparation (10 PI.) contained dissolved free amino acids isolated from 1 liter of Lake Mendota water sample (sampled January 27, 1970)

T o estimate the precision and accuracy of the DFAA isolation and analysis procedure, six 2-liter samples from a common filtrate of Lake Mendota water (sampled August 25, 1970) were fortified with standard amino acid mixtures containing 0.05 pmol of each amino acid. Two samples from the filtrate were not spiked. All of the samples were passed through the DFAA isolation and analysis procedures. Diphenylmethane was used as the internal standard. The mean concentrations, standard deviations, and coefficients of variation for the respective amino acids

from the six fortified samples are presented in Table I. Also given are the mean concentrations of amino acids from the two unspiked samples and the net recoveries and percent recoveries of the various amino acids. It can be seen that the coefficients of variation range from 6 to 20%. The percent recoveries for the different amino acids ranged from a low of 36% for Lys to a high value of 97% for Ileu. The other recoveries were all in the interval of 51-78%. A similar experiment was conducted to evaluate the

Table I . Statistical Evaluation of DFAA Procedure (Lake Mendota, August 25, 1970)a

memhrane f i l t e r

Internal standard: diphenylmethane Unfortified (N = 2) Fortified (N = 6) Amino acid

Ala Val

Ileu Thr Leu Ser Pro

Phe Try LY s

X

20.2 22.8 28.8 22.2 23.8 22.2 23.0 18.8 19.0 13.0

s

1.1

1.5 3.1 3.3 1.9 3.4 1.6 1.6 2.2 2.6

CV, %

6 7 11 15

8 15 7

8 12 20

2

7.5 8.5 4.5 2.5 5.5 5.0 5.0 2.5 3.0 4.0

Net recovery 12.7 14.3 24.3 19.6 18.3 17.2 18.0 16.3 16.0 9.0

Percent recovery

51 57 97 78 73 69 72 65 64 36

Concentration expressed in nmol/liter. N = number of replicates. X = mean concentration. s = standard deviation = [ Z ( X - x ) ' / ( N 1\11 2 . CV = coefficient of variation = 100 s i x . a

I

Isolate and concentrate DFAA w~. ith

c o n c e n t r a t e samo1es by r o t a r y e v a p o r a t i o n

ion exchanoe

Hydrolyre w i t h "21

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( E l u t e With

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Dilute h y d r o l y s a t e t o 400 m l with glass d i s t i l l e d Water and a d J u S t pH to 9 . 5 w i t h

i

Demove a m o n l a

from e l u a t e by r o t rv e v a p o r d t l o "

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I s o l a t e amino a c i d s w i t h semi-micro l i g a n d exchange [ E l u t e With ? Y

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Prepare N - t r i f l u o r o a c e t y l "ethyl e s t e r d e r i v a t i v e s of amino a c i d s ,

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separate and m e a s u r e i n d i v i d u a l a m i n o acids b y temnerature proqrdmed a a s ciromatooraphv

Figure 4. Sample processing scheme for amino acid analysis

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Table I l l . Statistical Evaluation of DFAA Procedure (Lake Mendota, January 30, 1971)"

Table II. Statistical Evaluation of DFAA Procedure (Lake Mendota, January 30, 1 9 7 1 ) " Internal standard: drphenylmethane

Internal standard: &amino valeric a c i d

Unfortified IN = 5 ) Fortified (h = 5) Amino acid

Ala Val I leu Thr Leu Ser Pro Phe TYr LYS

1

s

c v , Yo

26.2 24.8 25.0 21.8 30.0 28.4 16.4 20.4 21.6 15.2

1.3 1.3 1.6 1.6 1.4 1.9 0.9 1.5 1.6 2.5

5 5 6 7 5 7 6 7 7 16

i

7.2 4.6 4.0 2.0 3.2 7.2 2.0 1.6 2.0 1.7

Net rec:overy

Percent recovery

19.0 20.2 21.0 19.8 26.8 21.2 14.4 19.4 19.6 13.5

76 81 84 79 107 a5 58 78 78 54

Unfortified (N = 5)' Fortified (N = 5) Amino acid Ala Val I leu Thr Leu Ser Pro Phe TY r LYs

i

s

CV,%

33.4 37.2 39.4 30.2 33.6 33.2 20.4 24.4 28.2 18.6

2.4 1.9 1.5 1.1 0.9 2.7 0.9 0.9 0.8 3.0

7 5 4 4 3 8 4 4 3 16

X

110.0 7.4 7.0 4.6 7.0 9.0 5.0 4.6 3.2 2.2

Net recovery

Percent I.ecovery

23.4 29.8 32.4 25.6 26.6 24.2 15.4 19.8 25.0 16.4

94 119 130 102 106 97 61 79 100 66

Concentration expressed in nmol/liter. N = number of replicates. - i)'/'(N t)]"*. CV = coefficientof variation = 1 O O s / X .

a Concentration expressed in nmol/liter. h = number of replicates. i = mean concentration. s = standard deviation = [Z(X i)2/(N 1) 1 ' ' z . CV = coefficient of variation = 100 s/X.

P = mean concentration. s = standard deviation = [Z(X

precision and recoveries of amino acids with the DFAA procedure using both internal standards. A filtrate of Lake Mendota water (sampled January 30, 1971) was divided into ten 2-liter fractions. Five of the samples were fortified with standard amino acid mixtures containing 0.05 pmol of each amino acid. The remainder of the samples were not fortified. The samples were subjected to the DFAA isolation procedure. 6-Amino valeric acid (6 pg per two ampuls) was added to each ligand exchange eluate. Diphenylmethane (0.5 pg) was added directly with the acylation reagent during derivative formation. The results obtained with the two respective internal standards are presented in Tables I1 and 111. Comparison of values in the two tables for the respective amino acids indicates that higher values of amino acid concentrations were generally obtained with 6-amino valeric acid than with diphenylmethane which may reflect losses of 6-amino valeric acid during evaporation, transfer, and derivative preparation. Diphenylmethane was not added until the last step in derivative preparation. The percent recoveries with &amino valeric acid ranged from 66% for Lys to 130% for Ileu, as compared to 54% for Lys to 107% for Ileu with diphenylmethane. Higher recoveries were obtained with diphenylmethane for the samples of January 30, 1971 (Table 11) than for the samples of August 24, 1970 (Table I). The procedures used for hydrolyzed samples were evalu-

ated for precision and recovery by dividing a filtrate of Lake Mendota water (taken December 2, 1970) into ten 200-ml fractions and fortifying five of these fractions with a standard amino acid mixture containing 0.05 pmol of each amino acid. The other five fractions were not fortified. 6-Amino valeric acid was used as the internal standard. The mean concentration, standard deviation, and coefficient of variation for each amino acid from the fortified and unfortified samples, respectively, are presented in Table IV. The net recovery and percent recovery for each amino acid is also indicated in Table IV. Pro was not evaluated because of unsatisfactory precision and recovery. It can be seen that the percent recoveries were quite variable for different amino acids. Recovery was very low for Phe (15%) and Lys (34%) but somewhat improved for the remainder of amino acids (5593%). The lower recovery observed for amino acids from the hydrolyzed samples (as compared to the DFAA fractions) suggests that the amino acids were destroyed or removed from solution during hydrolysis or possibly, that ligand exchange efficiency was reduced for the neutralized hydrolyzates. The precision of the derivative formation and gas chromatographic analysis was checked by adding a portion of standard amino acid mixture (containing 0.15 pmol of each amino acid) and 6-amino valeric acid ( 3 pg) to each of six ampuls. The ampuls were dried, derivatives were prepared and each sample was analyzed by gas chroma-

-

a

-

Table I V . Statistical Evaluation of Procedure Used for Hydrolyzed Samples (Lake Mendota, December 2, 1970)" Internal standard: &amino valeric acid Unfortified (N = 5 )

Fortified ( N = 5 ) Amino acid Ala Val I leu Thr Leu Ser Phe TY r

LY S

P

S

cv, %

66.4 40.0 28.6 49.4 36.4 53.6 9.6 28.6 13.4

6.7 6.3 3.2 3.4 3.6 4.6 1.8 2.5 3.3

10 16 11 7 10 9 19 9 25

X

52.6 18.8 11.0 30.6 15.2 38.4 5.8 5.4 4.8

S

cv, %

7.6 1.9 0.7 1.8 1.6 3.5 0.4 0.5 2.1

14 10 6 6 10 9 7 9 44

Net I'ecovery

13.8 21.2 17.6 18.8 21.2 15.2 3.8 23.2 8.6

Percent recovery 55 85

70 75 85 61 15 93 34

aConcentration expressed in nmo1/100 ml. h = number of repiicates. X = mean concentration. s = standard deviation = [ Z ( X - P)*,'(N - l ) ] ' *. CV = coefficient of variation = 100 s / X . --

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Environmental Science,& Technology

Table V I . Comparative Precision of Ligand Exchange and Cation Exchange Isolation Proceduresa

Table V. Precision of Derivative Formation and Gas Chromatographic Procedurea

Internal standard: &amino valeric acid

Internal standard: &amino valeric acid

Cation exchange and Semimicro ligand exchange (N = 5)

N=6

Amino acid

Ala

Val lleu Thr

Leu Ser Pro

Phe TY LY s

X

13.2 11.8 12.7 12.7 13.3 12.5 12.5 13.0 12.5 13.3

S

1 .o 1.2 0.8 0.8 0.8 0.8 0.8 0.8 1.4 1.6

cv, % 8 10 6

6 6 6 6 6 11 12

QQuantity expressed in nmol. N = number of replicates X = mean quantity of amino acid. s = standard deviation = [ B ( X - ,?)z/(N 1 ) l ’ z. CV = coefficient of variation = lOOs/ic.

tography. The mean concentration (relative to the standard calibration curve), standard deviation, and coefficient of variation for each amino acid are presented in Table V. The coefficients of variation ranged from 6-12%. A series of samples was processed for DFAA to compare the precision of ligand exchange isolation to cation exchange isolation of DFAA. Lake Mendota water (20 liters, sampled October 28, 1970) was membrane filtered and divided into ten 2-liter portions. Each of the samples was processed through the cation exchange isolation procedure. After ammonia removal, five of the cation exchange eluates were passed through semimicro ligand exchange columns. The other five ammonia free eluates were passed through semimicro columns containing cation exchange resin for final concentration and clean up. The ligand exchange resins were eluted with 3 N ammonia. The cation exchange columns were eluted with 2N ammonia and the amino acids were collected in the third through fifth ml of eluate. The mean concentrations, standard deviations and coefficients of variation for each amino acid with both procedures are presented in Table VI. With the exception of Lys and Pro, the coefficient of variation for all the amino acids was lower for the samples processed by semimicro ligand exchange. T h e mean concentrations were not greatly different for the two isolation procedures indicating that the average recovery was similar. The effect of freezing lake water filtrates on DFAA recovery was observed by freezing two fortified portions (2 liters each) of Lake Mendota filtrate (sampled January 30, 1971). After approximately two weeks, the filtrates were thawed and analyzed for DFAA. &Amino valeric acid was used as the internal standard. The average concentrations of the respective amino acids were compared to the average concentrations obtained from the unfrozen spiked filtrates of January 30, 1971 (Table 111). The average concentrations recovered from the unfrozen and frozen filtrates, respectively, are shown in Table VII. The observed concentrations of most amino acids were considerably lower in the frozen samples than in the samples analyzed directly. It is possible that a portion of the amino acids was removed by a precipitate (apparently inorganic salts) which formed during freezing and failed to redissolve when the filtrates were thawed. Discussion

G a s Chromatographic Analysis. Prior to this investigation gas chromatographic techniques had not been per-

Amino acid

X

Ala Val

29.2 23.6 24.0 20.8 23.0 26.4 21.4 17.2 16.8 13.0

lleu Thr

Leu Ser Pro

Phe TY r LYs

s

cv. %

3.3 3,4 3.7 2.6 2.9 4.7 1.5 1.9 1.9 0.7

11 14 15 12 13 18 7 11 11

5

Cation exchange and semimicro cation exchange (N = 5 ) X

29.4 22.2 23.4 20.6 22.2 24.2 21.0 16.6 16.0 17.6

5

CV.%

2.9 2.8 2.1 1.9 1.8 3.4 1.7 0.5 0.7 1.3

10 12 9 9 8 14 8 3 4 8

aQuantity expressed in nmol. N = number of replicates. X = mean concentration. 5 = standard devlation = [Z(X - X ) 2 ; ( N - l ) ] ’ z . CV = coefficient of variation = 100 s / X .

Table V I I . Comparison of Recovery from Unfrozen and Frozen Spiked Filtrates (Lake Mendota, January 30. 1 9 7 1 ) a Amino acid

Ala Val lleu Thr

Leu Ser Pro

Phe TY LYs

Spiked ( N = 5 ) unfrozen X 33 37 39 30 33 33 20 24 28 18

4 2 4 2 6 2 4 4 2 6

Spiked ( N = 2) frozen x 25 22 22 17 24 21 9 14 17 9

5 0 5 5 0 0 5 0 0 0

Concentrations expressed in nmol per liter N = number of replicates X = mean concentration

fected for measurement of low levels of amino acids from natural waters. Gas chromatographic analysis of amino acids has been slower in developing than analysis of other classes of compounds because of the need to prepare suitable volatile derivatives before analysis. Recently, other investigators have used gas chromatography to measure amino acids isolated from seawater (Pocklington, 1972) and river water (Peaks et al., 1972). The development of the described reflux ampuls allowed simple and rapid derivative preparation without concentration of the final derivative preparation. The amino acids were concentrated in their relatively stable natural forms before forming the N-trifluoroacetyl methyl ester derivatives. Derivatives were formed from quantities of amino acids similar to the quantities needed for gas chromatographic analysis. The total volume of the acyla. sample, which eliminated tion reagent was only 10 ~ 1 per the need to concentrate the final reaction mixture before gc injection. If less sensitivity was desired for a sample, a larger volume of acylation reagent could be used to form the volatile compounds. A single reflux ampul was used for solvent removal and for both steps of derivative formation, so possible sample loss from transfer of the microsample was prevented. The design of the ampuls helped to prevent sample contamination. The gas chromatographic procedure used in this investigation was not suitable for the analysis of Met, Trp, His, Cystine, or Arg. Ligand Exchange Isolation. The ligand exchange techVolume 7, Number 8, A u g u s t

1973

723

nique was originally designed for use with an amino acid analyzer (Siegel and Degens, 1966). A difficulty in adapting the procedure to gas chromatography was the quantitative volume reduction and transfer of the ligand exchange eluates into the reflux ampul. Also, a residue was eluted from the ligand exchange resin which was undesirable for derivative preparation. These problems were eliminated by the semimicro ligand exchange technique. The substantially reduced quantity of resin allowed the amino acids to be contained in a volume of eluate which could be added directly to the reflux ampuls without prior concentration. In this investigation, the ligand exchange method was not found to be satisfactory for the quantitative isolation of Gly, Glu, and Asp. A compound eluted from the resin interfered with Gly analysis and the recoveries were low for Asp and Glu. Ligand exchange is particularly applicable to analyzing seawater for amino acids because it does not retain salts from seawater as does cation exchange. With eutrophic lake water samples, interfering organic substances present a greater problem than high salt content. Neither ligand exchange nor cation exchange proved to be adequate to isolate the amino acids from Lake Mendota water in a form sufficiently pure for derivative formation and gas chromatagraphic analysis. However, a combination of the two isolation procedures allowed the isolated mixtures of amino acids to be successfully derivatized and analyzed by chromatography. It is possible that the cation exchange step eliminated natural organic substances which may have interfered with derivative formation. Other Sources of Error. Aside from experimental errors the greatest potential sources of problems affecting amino acid data are contamination and bacterial activity during sample processing. Appropriate precautions prevent contamination but the potential problem of bacteria affecting the amino acid concentrations is more difficult to evaluate. Preservatives were not added to the samples because killing the organisms caused release of amino acids by autolytic processes. For example, Brehm (1967) found that large quantities of amino acids were released from algae suspended in a dilute phenolic solution. T o

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Environmental Science & Technology

minimize bacterial effects as much as possible, the samples were processed shortly after collection. Summary and Conclusions Studies were made with fortified water samples to evaluate the precision and percent recovery of amino acids obtained with gas chromatographic procedures. The coefficients of variation generally were found to range between 5 and 20% for the various amino acids in the different procedures. The percent recoveries varied considerably among the respective amino acids. A brief experiment with frozen lake water filtrates suggested that freezing lowered the recovery of DFAA. It was concluded that gas chromatography could be used to analyze lake water for selected amino acids after appropriate purification of the samples. The relative speed of analysis offers a distinct advantage over other available procedures, but more work is needed to improve the precision and accuracy of the described procedures and t o permit analysis of the remaining common protein amino acids. Literature Cited Brehm, J., Arch. Hydrobiol. Suppl., 32,313-435 (1967). Darbre, A., Islam, A., Biochem. J., 106,923-5 (1968). Gardner, W. S., “A Study of Selected Amino Acids in Lake Mendote,” PhD Thesis, Water Chemistry, University of Wisconsin, Madison, Wis., 1971. Gas Chromatograph Newsletter (Applied Science Laboratories, Inc., State College, Pa.), 11, 2 (July-August, 1970). Gee, M., Anal. Chem., 39,1677-9 (1967). Gee, M., Graham, R. P., Morgon, A. I., J. Food Sci., 32, 78-80 (1967). Gehrke, C. W., Takeda, H., “Gas-Liquid Chromatographic Studies on the 20 Protein Amino Acids: A Single Column Separation,” J. Chromatog., submitted, August 1972. Peake, E., Baker, B. L., Hodgson, G. W., Geochim. Cosmochim. Acta, 36,867-83 (1972). Pocklington, R., Anal. Biochem., 45,403-21 (1972). Siegel, A., Degens, E. T., Science 151,1098-1101 (1966).

Received for reveiw September 11, 1972. Accepted March 30, 1973. This study was supported by EPA Training Grant No. 5T02-WP-00184 and EPA Research Grant No. 16010-EHR. I n addition, support was given this project by the University of Wisconsin Department of Civil and Enuironmental Engineering and the Engineering Experimental Station.