ANALYTICAL CHEMISTRY, VOL. 50, NO. 1, JANUARY 1978 * 55
T h e indirect analytical method here described may have general application in those cases where the molecular weights (and so t h e diffusion rates in t h e atmosphere) of t h e main component and of t h e compound t o be monitored and their stability as well are similar. T h e results of this work show t h a t monitoring of B C M E is possible in t h e atmosphere of chemical plants by gas chromatographic techniques alone. It is obvious t h a t the availability of a GC-MS system equipped with the accessory for mass fragmentography and on-line computing devices makes this type of determination easier and more specific. However, the cost of such apparatus is still much too high t o be available in small chemical plants, where the presence of B C M E should be suspected and monitored in a large number of cases.
LITERATURE CITED (1) S. Laskin, M. Kuschner, R. T. Drew, V. P. Cappiello, and N. Nelsol, Arch. Environ. Health, 23, 135 (1971). (2) G. J. Kallos and R. A. Solomon, Am. Ind. Hyg. Assoc., (November) 469 (1973). (3) K. P. Evans, A. Mathias. N. Mellor, R. Silvester. and A. E. Williams, Anal. Chem., 47, 821 (1975). (4) R. A. Solomon, and G. J. Kallos. Anal. Chem., 47, 955 (1975). (5) L. S. Frankel and R. F. Black, Anal. Chem., 48, 732 (1976). (6) P. Ciccioli, G. Bertoni, E. Brancaleoni, R . Fratarcangeli, and F. Bruner, J . Chromatogr., 126, 757 (1976). (7) F. Bruner, P. Ciccioli, and F. Di Nardo, J Chromatogr., 99, 757 (1976). ( 8 ) A. Di Corcia and A. Liberti, "Gas Liquid Solid Chromatography", in "Advances in Chromatography", R . A. Keller and G. C. Giddings. Ed.. Vol. X I V , Marcel Dekker Inc. New York, N.Y., 1976.
RECEIVED for review August 1, 1977. Accepted October 11, 1977.
Gas Chromatographic Determination of Conjugated Aldehydes in Products of Periodate Oxidation of Pyranose Sugars as Diethyl Dithioacetals Susumu Honda, * Yoko Fukuhara, and Kazuaki Kakehi Faculty of Pharmaceutical Sciences, Kinki University, Kowakae, Higashi-Osaka, Japan
A gas chromatographic method is described for determination of component aldehydes in dialdehyde compounds formed on oxidation of carbohydrates with periodate. A sample of a dialdehyde (10-8-10-s mol) is treated with a 1 O : l mixture of ethanethlol and trifluoroacetic acid at room temperature; this treatment causes direct, quantitative conversion of the component aldehydes to diethyl dithioacetals, and the latter are determined simultaneously by gas chromatography of their trlmethylsilyl derivatives. The relative errors and standard deviations for six model dialdehydes were within 1% and 2.5 YO,respectively. This is a good micro method for analyses of the interglycosidic linkages in oligosaccharides and for studies on details of the mechanism of periodate oxidation.
Dialdehyde compounds formed on oxidation of oligo- and polysaccharides with periodate are composed of a variety of aldehydes mutually bound through hemialdal and hemiacetal bonds, and determination of these conjugated aldehydes provides useful information on the structures of the original carbohydrates, especially t h e positions of attachment of interglycosidic linkages. The methods used in previous studies on this subject include gas chromatography of the hydrolysates, with ( I ) or without (2) their oximation, of borohydride-reduced dialdehydes a n d thin-layer or column chromatography of 2,4-dinitrophenylhydrazonesof component aldehydes ( 3 ) . T h i s paper describes a simpler and quicker method involving quantitative conversion of component aldehydes t o their dithioacetals, and gas chromatographic determination of these dithioacetals. EXPERIMENTAL Material. Ethanethiol, trimethylchlorosilane, and D-xylitol
were obtained from Tokyo Kasei Kogyo Co., Ltd. (Toshima. Tokyo). Trifluoroacetic acid, hexamethyldisilazane, and sodium 0003-2700/78/0350-0055$01 0010
metaperiodate were purchased from Wako Pure Chemical Industries, Ltd. (Doshomachi, Osaka). All these reagents were of the highest grade available. Pyridine was dehydrated by refluxing it with barium oxide and distilled before use. Reducing carbohydrates and methyl glycosides of monosaccharides were obtained from Wako Pure Chemical Industries, Ltd., and Sigma Chemical Co. (St. Louis, Mo.), respectively. Methyl glycoside acetates of disaccharides were synthesized by Konigs-Knorr condensation of the corresponding acetobromo-sugars with methanol in the presence of silver carbonate ( 4 ) . Dialdehyde models, 2 ( 5 ) , 3 ( 5 ) ,5 (6), and 6 (61, shown in Figure 1, were prepared as described previously. Dialdehyde 1 was prepared as a syrup by fractionation on a column of silica gel of the reaction product obtained by periodate oxidation of methyl cu-D-glucopyranoside with three equivalents of sodium metaperiodate for 45 min, and then deionization of the products with resins. This dialdehyde gave a yellow spot on thin-layer chromatography ( R f 0.41, Merck TLC Plate Silica Gel 60 F254, 10:l chloroformmethanol) when sprayed with aniline hydrogen phthalate, and then heated, whereas dialdehydes 4 (Rj 0.741, 5 ( K , 0.54), and 6 (R,0.54) turned red with this reagent. Syrupy dialdehyde 4 was obtained by oxidation of methyl a-D-xylopyranoside for 24 h and deionization of the product. The values obtained by elementary analyses of the model dialdehydes were consistent with the structures given in Figure 1. Authentic specimens of all the diethyl dithioacetals, except hydroxymalonaldehyde, were prepared by mercaptalation of the corresponding aldehydes in cold concentrated hydrochloric acid and purification of the products on a column of silica gel. The diethyl dithioacetal of hydroxymalonaldehyde was obtained by mercaptalation of dialdehyde 1 with a 10:l mixture of ethanethiol and trifluoroacetic acid and subsequent purification of the reaction mixture in a similar manner. All dithioacetals gave single spots cin thin-layer chromatography (7:3 benzene-ethyl acetate, U V light, and concentrated sulfuric acid spray) and single peaks on gas chromatography. Apparatus. Isothermal gas chromatography was carried out on a Shimadzu 4BMPF instrument equipped with a hydrogen flame ionization detector (FID, 240 "C) fitted with a glass column (0.3-cm i.d., 200 em, 180 "C) packed with 3% OV-1 on Chromosorb Z! 1977 American Chemical Society
56
ANALYTICAL CHEMISTRY, VOL. 50, NO. 1, JANUARY 1978
Gas Chromatographic Parameters for t h e TMS Derivatives of Dithioacetals Derivable from t h e Component Aldehydes in Dialdehydes, 1-6 Table I.
Molar response factor Retention Relative reFID FPD time, min tention time (A.s/mol) (Almol)
Dithioacetal Glycolaldehyde diethyl dithioacetal Glyoxal bis-(diethyl dithioacetal) D-Glyceraldehyde diethyl dithioacetal Hydroxymalonaldehyde bis-( diethyl dithioacetal) D-Erythrose diethyl dithioacetal Benzaldehyde diethyl dithioacetal D-Xylitol (internal standard for FID)
1.67 7.07 3.25 19.15 8.06 2.78 4.62
0.36 1.53 0.70 4.14 1.74 0.60 1
0.194 0.218 0.281 0.315 0.323 0.242 0.418
Relative molar response FID FPD
190
0.464
90
0.521
95 43 15
0.641 0.153 0.172 0.578
175 0
1
2.1 1 1.1
0.48 0.17 1.9 0
Scheme I. Possible Types of Dialdehydes Obtainable by Periodate Oxidation of Reducing and Nonreducing Pyranose Residues k&R,
IR.+h) A
11
U-b-
DIALDEHYDE 2 Ri =H. R2=OMe DIALDEHYDE 3 Ri =OMe. Rz=H
R5
R5
m - .
H o a O M OH e DIALDEHYCE L
.hH
C" R i
n a?
a
R. 20
iC RTENTIOIU
TIME
lmlnl
Figure 1. Gas chromatographic analysis of the component aldehydes in model dialdehydes (FID).Peak assignment: ( l ) , TMS derivative of glycolaldehydediethyl dthioacetal: (2), benzaldehyde diethyl dfthioacetal; (3),TMS derivative of D-glyceraldehyde diethyl dithioacetal; (4), TMS derivative of D-xylitol (internal standard): (5), glyoxal bis-(diethyl dithioacetal); (6), TMS derivative of c-erythrose diethyl dithioacetal; (7), TMS derivative of hydroxymalonaldehyde bis-(diethyl dithioacetal).
Internal hemiaklal and hemiacetal bondings are neglected in the structure dialdehyde 1
of
W. The flow rate was 50 mL/min throughout the work. Peaks were integrated with a Shimadzu ITG-2A integrator. Sulfur compounds were also detected selectively on a Shimadzu 4CPP instrument equipped with a hydrogen flame photometric detector (FPD, filter wavelength 394 nm, 240 "C). The column conditions were the same as for analysis with the FID. Procedure for Simultaneous Determination of Component Aldehydes in Dialdehydes. A 1 O : l mixture (by volume, 20 pL) of ethanethiol and trifluoroacetic acid was added to a dialdehyde sample (10-a-104 mol) in a sample tube (0.5-cm id., 5 cm). The tube was tightly closed with a polyethylene stopper, and covered with Parafilm to prevent the stopper from popping out when the inner pressure increased. The sample was dissolved by gentle swirling, and the solution was kept for 30 min at 25 "C. Then a solution (50 pL) of D-xylitol (internal standard) in pyridine was added, followed by hexamethyldisilazane (100 pL) and trimethylchlorosilane (50 pL). The mixture was incubated for 30 min at 50 "C, and then centrifuged and the component aldehydes were analyzed by injecting a 1-pL sample of the supernatant into the gas chromatography column. Oxidation of Carbohydrates with Periodate. A sample of free carbohydrate was dissolved in 1%NaI04 (0.2 mL) and the solution was kept in the dark a t 25 "C for the reaction time shown in Tables I11 and IV. For the oxidation of the methyl glycoside of a disaccharide, an equivalent amount of its acetate was dissolved in 0.05 M methanolic sodium methoxide (1mL), and the solution was allowed to stand for 24 h. The reaction mixture was deionized by passing it through a column of Amberlite CG-120 (H' form,
Ri
R X
OCH C h
3
H R +-
