Preparation of Methyl Esters of Amino Acids for Gas Chromatography Using Dimethyl Dodecanedioate as an Internal Standard Mildred Gee Western Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Albany, Gal$ 94710
THEUSE of gas chromatography for amino acid analyses has been extensively explored, Reports of the use of this method for routine studies have not been widespread. More often amino acid data have been obtained by the Spackman, Stein, and Moore (1) method of ion exchange chromatography using automatic recording equipment. The need to form derivatives of the amino acids and the desire for quantitative results have probably deterred some workers from using gas chromatography for amino acid investigations. Recently a detailed description of the use of gas chromatography for amino acid analysis has been reported by Gehrke and Stalling (2) using the butyl ester trifluoroacetylated amino acid derivatives. These workers have also reviewed the literature on the application of other esters for amino acid studies. The methyl esters, however, are suitable for gas chromatographic examination (3-7)) because they are more volatile than the higher esters and easier to prepare. This paper describes an improved method for making the methyl esters of amino acids in 30 minutes and the gas chromatography of the methyl ester trifluoroacetates of amino acids using dimethyl dodecanedioate as an internal standard for quantitative evaluations. EXPERIMENTAL Gas Chromatography. Results reported were obtained using a dual hydrogen flame ionization detector gas chromatograph (F & M, Series 810, Avondale, Pa.). Amino acid derivatives were separated on a 1-meter, l/*-inch o.d., 0.075-inch i d . aluminum column packed with 5 neopentyl glycol succinate on Chromosorb W, HMDS, 100/120 mesh. The column oven was temperature programmed from 64" to 210" C at 4" rise/minute; injector and detector temperatures were held at 250' C ; gas flow of helium was maintained at 50 ml/minute and hydrogen at 31 ml/minute. Reagents. Standard amino acid samples were taken from the Amino Acid Reference Collection for Chromatography (BDH, Poole, England). Absolute methanol, Baker Analyzed Reagent and Eastman Grade thionyl chloride were used as received. Dodecanedioic acid was obtained from Harchem Division of Wallace and Tiernan, Inc., Belleville, N. J. Trifluoroacetic anhydride was obtained from Matheson, Coleman and Bell Division. Solutions of 5-10 hydrogen chloride in methanol were prepared by bubbling anhydrous hydrogen chloride into a chilled container of methanol. This reagent was usable for about 1 month when stored in the refrigerator. Method. Studies of peak areas of individual amino acids were made using 5 mg of each amino acid plus 5 mg of dodecanedioic acid. The mixed acids were refluxed with 10 ml of hydrogen chloride in methanol and 0.1 ml of thionyl
(1) D. H. Spackman, W. H. Stein, and S. Moore, ANAL.CHEM., 30, 1190 (1958). (2) C. W. Gehrke and D. L. Stalling, Separufion Science, 2, 101 (1967). (3) P. B. Hagen and W. Black, Can.J. Biochem., 43, 309 (1965). (4) P. A. Cruickshank and J. C. Sheehan, ANAL.CHEM.,36, 1191 (1964). (5) A. Darbre and K. Blau, J. Chromatog., 17,31 (1965). (6) H. A. Saroff and A. Karmen, Atzal. Biochem., 1, 344 (1960). (7) N. Ikekawa, J . Biochem., 54,279 (1963).
chloride for 30 minutes. Excess esterification reagents were removed by rotary evaporation at water aspirator pressure. The hydroxyls and amino groups were trifluoroacetylated with 2 ml of trifluoroacetic anhydride at reflux for 10 minutes. Aliquots of 2-4 pl were taken from this reaction mixture for gas chromatography. The peak area relationships were made by cutting out the individual peaks from the chromatographic record and weighing each peak. These relationships can also be determined from integrator results or planimeter measurements, The procedure for the separation of amino acids from foam-mat dried tomato and orange powders was described in an earlier publication (8). RESULTS AND DISCUSSION The method of methyl esterification of amino acids was adapted from the one previously reported for nonamino plant acids (9). Application of this procedure to amino acids produced esterification results similar to those obtained by Cruickshank and Sheehan ( 4 ) on amino acids. These workers reported the quantitative aspects of the esterification. The dimethyl sulfite used by these workers was prepared by refluxing methanolic hydrogen chloride and thionyl chloride and isolation of dimethyl sulfite before addition to the esterification reaction of the amino acids. Though the proportions reported here are not the same to produce maximum yield of dimethyl sulfite, the esterification route is believed similar. The method of Brenner and Huber (IO), for preparing methyl esters of amino acids using larger proportions of thionyl chloride in anhydrous methanol, required 2 hours for esterification. The method described here is an improvement in time and convenience over earlier reports. No special treatment of reagents such as redistillation or drying was necessary for high esterification. The 30-minute reflux reaction period was chosen to achieve maximum esterification of all the amino acids present in a mixture though some amino acids esterified in a shorter period of time, The method reported by Gehrke and Stalling ( 2 ) takes at least 3 hours to prepare the butyl esters of the amino acids. The workers Darbre and Blau (5) favored the higher boiling esters over methyl esters as they lost some material when they evaporated excess diazomethane from the esterification reaction. No loss of methyl esters is possible by this procedure when excess reagent is evaporated as the esters are in the form of the hydrochloride salt and would not be volatile at low temperatures at water aspirator pressure. The use of the trifluoroacetylation reaction mixture for the gas chromatographic injection sample would also prevent loss of the more volatile amino acid derivatives. In order to obtain peak area data, a series of 18 amino acids were methyl esterified and trifluoroacetylated in two batches. Using the GLC operating conditions described, some overlap of peaks occur. The two mixtures of amino acids per(8) M. Gee, R. P. Graham, and A. I. Morgan, Jr., J . Food Sci., 32, 78 (1967). (9) M. Gee, ANAL.CHEM.,37, 926 (1965). (10) M. Brenner and W. Huber, Hela. Chim. Acfa., 36, 1109 (1953). VOL. 39, NO. 13, NOVEMBER 1967
a
1677
n
Standards
132O
165"
203O
Figure 1. Gas-liquid chromatogram of standard amino acid samples (5 mg each) for peak area response ratios 1. Alanine, 2. isoleucine, 3. threonine, 4. norleucine, 5. proline, 6. aspartic acid, 7. glutamic acid, 8. dodecanedioic acid, 9. ornithine
64'
Stand a rds
89O
106O
165" 1 7 5 O
132O 145O
204'
Figure 2. Standard amino acids for peak area measurements 1. Valine, 2. glycine, 3. serine, 4. -/-aminobutyric acid, 5. methionine, 6. phenylalanine, 7. dodecanedioic acid, 8. tyrosine, 9. lysine Tomato powder
I n mitted resolution of peaks. These separations are shown in Figures 1 and 2. Equal amounts of each acid ( 5 mg) were used for the study of standards, Though equal responses are not obtained using the hydrogen flame ionization detector, this was believed due to variations in functional groups and structure of the amino acids studied. 117'
Table I. Ratios of Peak Areas of Methyl Ester Trifluoroacetate Derivatives of Amino Acids Relative to the Peak Area of Dimethyldodecanedioate Obtained by Gas Liquid Chromatography Peak Amino acids area ratiose Range 0.91 1.0 -0.86 Alanine 0.90 1 . 2 -0.8 Valine 0.73 0.80-0.65 Isoleucine 0.63 0.66-0.52 Glycine 0.77 0.78-0.70 Threonine 1.16 1.4 -1.0 Norleucine 0.95 1.0 -0.87 Proline 0.68 0.79-0.57 Serine y-Aminobutyric acid 0.71 0.70-0.80 0.69 0.70-0.66 Aspartic acid Methionine 0.43 0.63-0.42 0.43 0.39-0.46 Glutamic acid 1.13 1.30-1.04 Phenylalanine Dodecanedioic acid 1.o ... 0.88 0.89-0.78 Tyrosine 0.35 0.38-0.33 Ornithine 0.52 0.55-0.42 Lysine Average of 6 to 8 determinations. 0
Table 11. Calculated Data for Amino Acids Found in Foam-Mat Dried Tomato and Orange Powders Amino acids (mg/gram of powder) Tomato Orange 2.3 0.6 Alanine 0.6 0.3 Valine 0.5 0.5 Isoleucine Threonine, glycine 0.9 0.6 0.6 ... Leucine 0.5 0.3 Norleucine 0.3 10 Proline 1.5 2.7 Serine 6.0 3.5 Aspartic acid y-Aminobutyric acid 13 5.0 Glutamic acid 27 2.8 0.4 0.4 Phenylalanine 3.0 1.7 Arginine Lysine 0.8 1.2
e
ANALYTICAL CHEMISTRY
154O
Figure 3. Free amino acids found in foam-mat dried tomato powder 1. Alanine, 2. valine, 3. isoleucine, 4. threonine, 5. leucine, 6. norleucine, 7. proline, 8. serine, 9. aspartic acid, 10. 7-aminobutyric acid, 11. glutamic acid, It. phenylalanine, 13. dodecanedioic acid (added standard), 14.arginine, 15. lysine.
The relationship of peak areas of individual amino acids relative t o dimethyldodecanedioate are shown in Table I. Dodecanedioic acid was chosen as an internal standard because of the ease of esterification of the carboxyl groups and the fact that the ester derivative has a retention time different from the amino acids studied. In practice, when an unknown mixture of amino acids is being studied, an aliquot of a known concentration of dodecanedioic acid in methanol is added to the amino acids before esterification. The amount of dodecanedioic acid added should be chosen to approximate the response of one of the larger quantities of an individual amino acid in the mixture. If all the peaks are in the same scale, quantitative relationships can be directly determined. As the known amount of dodecanedioic acid is added to the total sample of starting material, the amino acid values in Orange powder
I
56'
6
115O
132'
164O
Figure 4. Distribution of free amino acids found in foam-mat dried orange powder 1. Alanine, 2. valine, 3. isoleucine, 4. threonine, glycine, 5. norleucine, 6. proline, 7. serine, 8. aspartic acid, 9. y-aminobutyric acid, 10. glutamic acid, 11. phenylalanine, 12. dodecanedioic acid (standard),
13. arginine, 14. lysine
1678
132"
the starting material can be obtained by peak area relationship as follows: Wt. dodecanedioic acid added X peak area of amino acid Peak area of dimethyldodecanedioate X peak area ratio wt. of amino acid in sample (1) Thus regardless of range selection or sample siLe injected into the gas chromatograph, the known amount of dodecanedioic acid added to an unknown mixture before derivative formation can be used to obtain quantitative relationships. The freshly prepared amino acid derivatives should be examined as soon as possible as some of these derivatives are not stable after several days.
Examples of amino acids extracted from foam-mat dried tomato powder and foam-mat dried orange powder are shown in Figures 3 and 4. Quantitative data of amino acids from these gas chromatographs are compiled in Table 11. It is possible by using this rapid method for ester preparation of amino acids to examine eight samples by gas chromatography in an average working day, including the time required to prepare the ester trifluoroacetylated derivatives. RECEIVED for review May 15, 1967. Accepted August 28, 1967. Reference to a company or product name does not imply approval or recommendation o f the product by the U. S. Department of Agriculture to the exclusion of others that may be suitable.
Double-Path Quartz Micro-Cell for Solutions of Unknown Absorbance Denis M. Abelson Department of Medicine, Graduate Hospital of the Unioersity of Pennsyhania, Philadelphia, Pa. I9146 MANYBIOCHEMICAL OPERATIONS involve the spectrophotometric analysis of compounds in solution in unknown concentrations. If the optical density at the relevant wavelengths is above the range of accurate measurement for the instrument, it is necessary to dilute the solution appropriately and repeat the measurement, or alternatively to employ a shorter path length by using a special cell or inserting spacers. A doublepath, fused quartz cell was accordingly designed to obviate these difficulties (Figure 1). The internal length of the cell compartment is 10 mm. The internal width is also accurately known. The author has worked mainly with cells 4 mm in width but, with proper preliminary adjustment of the pin-hole light beam, the lower practicable limit is probably 2 mm or less. The short sides of the cell are prolonged laterally as flanges so that an overall square cross-section is preserved, and the cell can be accommodated in standard carriages. It should be noted that in some carriages the springs automatically position all the cells nearer one side than the other. Care should always be taken to insert the carriage in the cell compartment so that the cells are closer to the light beam, which may be slightly divergent. It is also advisable to mark each cell so that it can always be oriented in the same direction in the cell carriage. The height of the cell is 46 mm, the lower 12 mm of which is a quartz block. With the cell inserted in the standard carriage, the floor will be located just beneath the light beam, thus permitting the use of small volumes of solution. The BesseLowry slide used with the Beckman DU spectrophotometer is provided with three pinholes, o f which the middle-sized is probably the most suitable. A preliminary check should be carried out in a darkened room to ensure that the beam does not touch the walls of the cell when the longer path is being used. With the photocell housing removed, the slide is positioned so that the beam passes through the center of each cell in turn as it is placed in position. The slide is then locked in place with the screw provided. When the optical density of a solution is too great to be measured accurately, the sample and reference cells are both turned 90 degrees, a new reading is obtained, and the result
t e
I
N
1 Figure 1.
Double-path quartz micro-cell
10 10 in our case -. A useful short path length’ 4 check of accuracy is also provided by the fact that, if the expected ratio between the readings in the two directions is not obtained, then one reading must be in error, e.g., because of inadequately cleaned optical surfaces. multiplied by
ACKNOWLEDGMENT
Thanks are due to Precision Cells, Inc., New York, N. Y . (Ross Scientific Co., Hornchurch, Essex, England), who constructed the cell according to the above specifications. RECEIVED for review June 20, 1967. Accepted August 16, 1967. This work was supported by the John A. Hartford Foundation.
VOL. 39, NO. 13, NOVEMBER 1967
1679
