Table I. RQValues for Several Hexosamines RG (pure") RG(mixture*) Compound Glucosamine 1.00 . . . 1.00 . . . Galactosamine 0.85 3~0.01 0.85 1 0 . 0 3 1.07 0.00 1.05 +0.01 N-Acetylgalactosamine N-Acetylglucosamine 1.16 iz0.01 1.17 zkO.01 Values from single components developed simultaneously with a mixture. b Values from mixtures developed simultaneously with the individual components.
Figure 1 shows a photograph of a typical chromatogram prepared according to the procedure outlined. A tabulation of the mean Ru values for the four components is listed in Table I. RG values are defined as the ratio of the distance travelled by the unknown divided by the distance travelled by glucosamine.
Q
RECEIVED for review September 23,1970. Accepted January 20, 1971. Work supported by the National Institute of Dental Research through Contract No. Ph-43-67-1172.
Gas-Liquid Chromatography of the Optical Isomers of Threonine and Allothreonine J. C. Dabrowiak' and D. W. Cooke Department of Chemistry, Western Michigan University, Kalamazoo, Mich. 49001
A VERY SENSITIVE and rapid procedure using gas-liquid chromatography for the separation of R and S isomers of many of the common amino acids has been developed by B. Halpern and J. W. Westley(l,2). In this method the amino acid esters are converted to N-trifluoroacetyl-S-prolyl-amino acid derivatives which are diastereomers and are easily separated on the column. However, no attempt was made to determine the relative amounts of R and S amino acid which occurred in a sample mixture. One of the problems in such a determination is the synthesis of optically pure N-trifluoroacety1-Sprolyl chloride. The preparation of this compound is described by Weygand et a(. (3) who assumed that no racemization occurred during synthesis but gave no evidence demonstrating the optical purity of the product. Furthermore, the commercially available reagent ( 4 ) contains significant amounts (3-12%) of the R isomer which unnecessarily complicates quantitative determinations. Using a modification of Weygand's method we have succeeded in synthesizing the R and S isomers of N-trifluoroacetylprolyl chloride with 99 % optical purity. In addition we have used both isomers to determine the relative amounts of R- and S-threonine and R- and S-allothreonine in a mixture (where R and S designate the asymmetric carbon atom alpha to the carboxylic acid function of the amino acid). These two distereomeric amino acids are very similar and their separation presents special problems to other analytical methods (5-7). EXPERIMENTAL
Apparatus. An F and M (Hewlett-Packard, Skokie, Ill.) Model 402 dual column flame ionization detector chromato1
Present address, The Ohio State University, Columbus, Ohio
43210
(1) B. Halpern and J. W. Westley, Tetrahedron Lett., 21, 2282 (1966). (2) B. Halpern and J. W. Westley, Biochem. Biophys. Res. Cornmum, 19,361 (1965). (3) F. Weygand, P. Klinke, and I. Eigen, Chem. Ber., 90, 1896 (1957). (4) Regis Chemical Company, Chicago, Ill. ( 5 ) T. Seki, J. Biochem. (Tokyo),47,126 (1963). (6) A. T. Shulgin, 0. G. Lien, E. M. Gal, and D. M. Greenberg, J. Amer. Chem. SOC.,14,2427 (1952). (7) T. Furuyarna, Bull. Chem. SOC.Jap., 36,126 (1963).
graph was used. The chromatographic columns were glass, 1.82 m X 4 mm i.d., and packed with 10% diethyleneglycolsuccinate (DEGS) on acid washed chromosorb W from Sepelco, Bellefonte, Pa. The separations were carried out isothermally at 200 "C with the injection port at 210 "C and the detector at 230 "C. Gas flows were: nitrogen, 60 ml/min; hydrogen, 25 ml/min; and air, 300 ml/min. The mass spectrum was obtained using an LKB-9000 gas liquid chromatograph-mass spectrograph (LKB Instruments, Inc., Stockholm, Sweden). Here the column was 3 % SE-30 on chromosorb W with a 1.82 m x 4 mm i.d. glass column. The separation was carried out isothermally at 100 "C. Reagents. All solvents were reagent grade and dried using molecular sieves or other standard drying agents. The amino acids S-threonine, racemic threonine, and racemic allothreonine were purchased from Nutritional Biochemical Corp., Cleveland, Ohio. The allothreonine was recrystallized twice from 15% ethanol to remove threonine. R-threonine and S-proline were obtained from Sigma Chemical Co., St. Louis, Mo. and R-proline from Calbiochem, Los Angeles, Calif. A sample of R-allothreonine was generously supplied by Calvin Stevens, Wayne State University, Detroit, Mich. The bis-N(trimethylsily1)trifluoroacetamide silylating agent (BSTFA) was purchased from the Regis Chemical Co., Chicago, Ill. under the name of Regisil. Preparation of N-Trifluoroacetyl R- and S-prolyl Chloride. These compounds were prepared following a modification of Weygand (3). Fifteen ml of trifluoroaceticanhydride was added with rapid stirring to 1.9 g (0.017 mole) of dry finely powdered R- or S-proline suspended in 20 ml of dry ether at dry ice-acetone temperature. (Before use, the S-proline was twice recrystallized from boiling absolute ethanol, using Norite-A in the final recrystallizations.) Immediately after this addition, the bath was removed and the stirred solution allowed to warm to room temperature. After one hour, the ether and unreacted anhydride were removed by vacuum distillation at room temperature, leaving behind a faintly yellow colored oil. The material was converted to the acid chloride by the addition of 25 ml of a solution containing 15 ml of dry benzene and 10 ml of distilled thionyl chloride and stirring at room temperature for 2.5 hr. The thionyl chloride was distilled (bp 84 "C) from a 3 :1 thionyl chloride-linseed oil solution and stored under NP at -20 "C until needed (8).
(8) L. F. Fieser, "Experiments in Organic Chemistry," 1st ed., D.C. Heath and Co., New York, N. Y., 1941, p p 381-82.
ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971
791
Table I. Gas-Liquid Chromatography of Threonine-Allothreonine-TMS-N-trifluoroacetylR-, S-prolyl Methyl Estersa Added S-proline chromatogram R-proline chromatogram Rt Ra St Sa Rt Ra Sa St St Sa Rt Ra 32.7 30.4 26.8 10.2 32.2 30.9 36.9 26.9 10.1 62.9 39.0 30.8 30.2 39.2 30.8 30.2 20.3 69.8 50.0 50.0 49.7 50.3 50.0 50.0 50.0 50.0 50.5 49.5 100 99.2 0.8b 0.4O 99.6d 0 All values are in relative mole per cent and the average from at least two injections. See the text for abbreviations. As RRt. c As SRt. As RRt.
+
+
Figure 1. Structure of prolythreonine - allothreonine dipeptides Threonine has asymmetric centers with R,S and S, R configurations Allothreonine has S, S and R, R arrangements about the asymmetric centers The benzene and unreacted thionyl chloride were removed by vacuum distillation at room temperature leaving a light yellow viscous oil. Working in a dry nitrogen atmosphere, the oil was quantitatively taken up in 100 ml of dry chloroform and the solution stored in 25 five-ml vials with polyethylene pushon caps. Each vial was inserted into a large bottle, sealed, and stored in a freezer (-20 “C) until needed. Samples prepared and stored in this manner exhibited no loss of activity or detectable racemization after 6 months. Preparation of Amino Acid Derivatives. The amino acid mixture, containing threonine and/or allothreonine (30-50 mg) was esterified using 6 ml of a thionyl chloride-methanol solution (1 :9) and refluxed for 4.5 hr (9). (The solution was prepared by the dropwise addition of 10 ml of distilled SOClz to 90 ml of dry methanol at dry ice-acetone temperature.) After cooling, a volume containing about 10 mg of the amino acids was placed in a 120-mm culture tube and the solvent removed by heating on a steam bath under a stream of dry nitrogen gas. One ml of methanol and 3-5 drops of triethylamine were added to the residual solid or oil which remained and the liquid was again evaporated to dryness on a steam bath under nitrogen. Acetonitrile, 0.5 ml, was added to dissolve the material followed by addition of 0.5 ml of BSTFA. The sealed tube was placed on a steam bath for 2 min, then cooled under a stream of cold water for a few minutes. A 2-ml portion of N-trifluoroacetyl R- or S-prolyl chloride in chloroform was added followed by 10 drops of triethylamine. A 2- to 5 - ~ portion l of this solution was injected into the gas chromatograph and the response analyzed as discussed in the text. When this solution was allowed to stand a t room temperature, its color turned dark red-brown. However, a check o n the quantitative nature of the derivatives a day later showed only a slight loss of response.
Time ( m i d
Figure 2. Chromatogram of mixture containing equal amounts of racemic threonine and allothreonine
+
Peaks are: (a) RSt or SRt; (6) RSa or SRa; (c) SSt SSa or RRt RRa. See text for abbreviations used. Conditions: F and M Model 402 flame ionization chromatograph; Sample: 30-50 mg amino acid in 3.5 ml; 2-5 pl injected; column: 1.8 m X 4 nun i.d., glass with 10% W/W% DEGS on Chromosorb W/AW 80-100 mesh: 200 “Cisothermal
+
Using the procedure described in the Experimental Section, threonine and allothreonine can be quantitatively converted
to dipeptides having the general structure shown in Figure 1. Since these amino acids possess an alcohol function, it was advisable to convert it to the less polar trimethylsilyl (TMS) ether before separation by reaction with a silylating agent such as BSTFA. Although the location of the TMS group is not critical to the following discussion, mass spectral data of the separated dipeptides strongly suggests that it is positioned on the amide function of the molecule and not the hydroxyl function as expected (IO). However, further study would be necessary to conclusively show the location of this group. When a mixture containing equal amounts of all four of the isomers, R- and S-threonine and R- and S-allothreonine is reacted with N-trifluoroacetyl S-prolyl chloride four diastereomeric dipeptides are produced. Separation of the resulting mixture using GLC yields three peaks instead of the expected four with relative areas of 1 :1 :2 (Figure 2). The peaks correspond to S-proline-R-threonine (SRt), S-prolineR-allothreonine (SRa) and S-proline-S-threonine (SSt) plus S-proline S-allothreonine (SSa) with retention times of 7.2, 8.1, and 9.2 min respectively. The peak assignment was made using authentic samples of each of the amino acid iso. mers in separate derivatizations with the exception of the
(9) H. Seik, K. Koga, H. Matsuo, S . Ohki, I. Matsuo, and S . Yamada, Chem. Pharm. Bull., 13,995 (1965).
(10) A. E. Pierce, “Silylation of Organic Compounds,” 1st ed., Pierce Chemical Co., Rockford, Ill., 1968, pp. 58-59,63-71.
RESULTS AND DISCUSSION
792
ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971
peak for S-allothreonine which was assigned by enrichment of racemic allothreonine with R-allothreonine. The response of the four diastereomeric dipeptides to the flame ionization detector were found to be identical. The areas of the peaks were calculated from the products of the height of the peak and the peak width at half height. Attempts to separate the third composite peak using SE-30 and propyleneglycoladipate ( I ) as liquid phases failed. The relative amounts of these two isomers, St and Sa were ultimately determined by using N-trifluoroacetyl R-prolyl chloride in the peptide formation. Reaction of the threonine-allothreonine mixture with the R-proline compound instead of its mirror image results in a chromatogram identical to that shown in Figure 2 except that all of the peptides synthesized are enantiomeric to the previous set. That is the derivatives in order of increasing retention times are R-proline-S-threonine (RSt), R-proline-S-allothreonine (RSa) and R-proline-R-threonine (RRt) plus Rproline-R-allothreonine (RRa). The relative areas of the first peaks of this chromatogram represent the amounts of
St (as RSt) and Sa (as RSa) present in the original mixture. By combining the information obtained from the two chromatograms, the composition of the mixture can be correctly found as 25% Rt, 25% Ra, 25% St, 25% Sa. Using the above procedure, the analysis of mixtures of various composition was carried out and the results are presented in Table I. The purity of N-trifluoroacetyl S-prolyl chloride was determined by reacting it with optically pure R-threonine. The separation of this solution showed a major peak at a retention time of 7.2 min and a minor one at 9.2 min. The former corresponded to SRt and the latter to RRt since its mirror image SSt was not possible because no St was present in the sample. The areas of the two peaks provide a direct measure of the optical purity of N-trifluoroacetyl S-prolyl chloride which was found to be 9 9 . 2 z and 0.8z R. The same analysis was applied to the R-proline derivative (Table I). RECEIVED for review October 22, 1970. Accepted January 18, 1971. This work was supported by the National Institutes of Health Grant No. 5 R01 AM12262.
Potentiometric Determination of Potassium Theodore S. Prokopov Department of Chemistry, Upper Iowa College, Fayette, Iowa 52142
MORETHAN a score of analytical procedures (gravimetric, volumetric, spectrophotometric, and flame-photometric) have been proposed and used for a direct or indirect determination of potassium (I-6). All gravimetric procedures, no matter how accurate, are laborious and tedious. Volumetric and indirect spectrophotometric methods at best offer little or no advantages over the gravimetric procedures (7). The flame-photometric method, which is regarded as a simple one, is not simple at all. Nevertheless, it is now widely used as being more advantageous when many routine analyses should be made of samples of similar composition. However, many sources of interference, particularly the presence of sodium, greatly lower the accuracy of potassium determination, bringing the relative error to i3-4 % and higher. It was, therefore, highly desirable to develop a potentiometric method of potassium determination with accuracy not less than *0.3% and with the convenience and speed which potentiometric titration usually offer. For this purpose, a study was undertaken to investigate the possibility of potass ium determination by its precipitation with a measured excess of sodium tetraphenylboron, by consumption of this excess with a known excess of AgN03 in acidic water-ethanol medium and back titration of AgN03 with a NaCl solution. (1) H. Tollert, “Analytik des Kaliums,” Enke, Stuttgart, 1962. (2) S. Kallman, “Treatise on Analytical Chemistry,” I. M. Kolthoff, and P. J. Elwin, Ed., Vol. I, Part 11, Wiley-Interscience, New
York, 1961. (3) E. N. Archibald, W. G. Wilcox, and B. G. Buckley, J. Amer. Chem. Soc., 30, 747 (1908). (4) G. F. Smith and T. F. Ross, ibid. 47, 1020 (1925). (5) M. Kohler 2.A m / . Chem., 138, 9 (1953). (6) J. Dean, “Flame Photometry,” McGraw Hill, New York, 1960. (7) I. M. Kolthoff, E. B. Sandell, E. J. Meehan, and S. Bruckenstein, “Quantitative Chemical Analysis,” 4th ed., Macmillan, London, 1969, p 666.
Table I. Titration of a Mixture of 2 Milliliters of 0.0927M KN03, 3 Milliliters of 0.1M Sodium Tetraphenylboron, 3 Milliliters of 0.1006M AgN03, and 0.5 Milliliter of 6M HNOI with 0.1011M NaCl Solution. Mixture Diluted to 30 Milliliters with Absolute Ethanola AEjAV A2EjAV2 Cl-, ml E , mV 1.70 35 20 1.80
325
55
345 1.90
2.00
400
3 25
20
420
+ 325
aVol. = 1.80 - X 0.1 = 1.85 ml. meq NaCl 650 meq K+ = 0.1852 us. actual 0.1854. Error = -0.11%. 0.1854/30 = 6 X IO-aM.
=
0.1870
[K+] =
EXPERIMENTAL
Apparatus. A Fisher pH meter Model 210 was used. A Beckman billet-type silver electrode and a calomel electrode in which a saturated solution of N a N 0 3 was substituted for solution of KC1, were employed. A magnetic stirrer was used, and the titrant was delivered from a 5-ml microburet. Reagents. All chemicals used (KN03, Na[B(CeH&], AgN03, “03, C2HhOH) were of analytical reagent grade. The solid sodium tetraphenylboron is stable for months, especially if stored in a cold place, protected from light. In solution, however, it is less stable and may develop a turbidity and phenolic odor. No special treatment is needed to prepare a solution sufficiently stable for a week. It is best stored in a refrigerator, and may be used as long as it shows no turbidity. Procedure. The size of sample depends upon the amount of sample available and upon the expected concentration of ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971
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