(9) (10) (1 1) (12) (13) (14) (15) (16) (17) (18)
E. Grushka, Anal. Chem., 45, 1626 (1973). J. J. Kirkland and J. J. DeStefano, J. Chromatogr. Sci., 9, 206 (1971). I. Halasz and I. Sebestian, Angew. Chem. lnt. Ed. Engl., 8, 453 (1969). H. N. M. Stewart and S. G. Perry, J. Chromatogr.,37, 97 (1968). E. Grushka and E. J. Kikta, Anal. Chem., 46, 1370 (1974). W. A. Aue, C. R. Hastings, and J. M. Augl, J. Chromatogr., 5 3 , 487 (1970). J. Wartrnan and H. Deuel, Helv. Chim. Acta, 42, 1166 (1959). W. A. Aue and C. R. Hastings, J. Chromatogr., 42, 319 (1969). E. W. Abel, F. H. Pollard, P. C. Uden, and G. Nickless, J. Chromatogr.,22, 6 (1969). I. Halasz and I. Sebestian, J. Chromatogr. Sci., 12, 161 (1974).
(19) (20) (21) (22) (23) (24) (25) (26)
J. J. Kirkland and J. J. Destefano, J. Chromatogr. Sci., 12, 337 (1974). J. J. Kirkland and J. J. Destefano, J. Chromatogr. Sci., 8 , 309 (1970). J. J. Pesek and J. H. Frost, Anal. Chem., 45, 740 (1973). R. K. Gilpin and M. F. Burke, Anal. Chem., 45, 1383 (1973). E. J. Kikta and E. Grushka, Anal. Chem., 48, 1098 (1976). D. E. Martire and H. L. Liao, Anal. Chem., 44, 498 (1972). I. Shapiro and I. M. Kolthoff, J. Am. Chem. SOC.,72, 776 (1950). D. E. Martire and H. L. Liao, J. Am. Chem. SOC.,96, 2058 (1974).
RECEIVEDfor review July 26,1976. Accepted September 30, 1976.
Separation of Trioses and Tetroses as Trimethylsilyl Oximes by Gas Chromatography Dugan Anderle, * Jozef Kbnigstein, and Vladimk KovaEik Institute of Chemistry, Slovak Academy of Sciences, 809 33 Bratislava, Czechoslovakia
Separation of tetroses has been achieved by gas chromatography of their per-O-Me3Si oximes on a capillary column coated with OV-17. Trioses could be satisfactorily separated on a packed column with SP-2340 as a stationary phase. The achieved resolution R 3 1 fulfills criteria required for quantitative analysis. Mass spectra of the title derivatives are also presented.
The existing spectrophotometric, gravimetric, and titration methods of determining trioses and tetroses based on general reactions of carbonyl groups lack specificity and selectivity. As such they are suitable only for the determination of one member of the series but not for the determination of individual components in mixtures (1). The determination of these sugars in admixture with their dehydration products can be conveniently done by means of their derivatives, mainly imines and quinoxalines. Taking advantage of the different reactivity of aldoses and ketoses with primary aliphatic amines in the presence of o-phenylenediamine, an electrochemical method has been developed making it possible to determine aldoses indirectly as aldimines while ketoses, unreactive under similar conditions, can be determined directly by polarography as free carbonyl compounds. Dehydration products containing a-dicarbonyl arrangement when allowed to react with o-phenylenediamine may be determined in the form of quinoxalines ( 2 ) . Gas chromatography of glyceraldehyde and erythrose in the form of their per-0-MesSi derivatives was first described by Sweeley et al. ( 3 ) ;erythrose gave three peaks, threose, glycero- tetrulose, and dihydroxyacetone were not chromatographed. These authors also described the separation of trimethylsilylated oximes of hexoses. Using gas chromatography of MeaSi oximes, the products of Smith degradation have been analyzed by Yamaguchi et al. (4).The same technique has been more extensively used by Mason and Slover ( 5 )in the analysis of fructose and several aldoses in foods. The most detailed work on gas chromatography of T M S derivatives of oximes derived from aldoses, ketoses, and some deoxy sugars was presented by Petersson (6). Using three columns of different polarity, and mass spectrometry for peak-substance assignments, he was able to identify a number of substances of the class. But he has not, however, been able to separate trioses and tetroses satisfactorily enough to make it possible to determine this class of compounds in mixture quantitatively in the form of their MesSi oximes. Laine and
Sweeley (7) have also described separation and mass spectra of methyloxime MesSi derivatives of some carbohydrates (number of carbon atoms 3-7).
EXPERIMENTAL Instruments. Polarography was carried out using an OH-102 (Radelkis, Budapest) polarograph. During analysis and oxime formation the polarographical and reaction vessels were kept a t 20 and 70 f. 0.02 "C, respectively, using an U-10 (Prufgerate Medingen, Dresden) thermostat. Gas chromatography was done using a Hewlett-Packard Chromatograph, Model 5750, equipped with FID's. The injector port and the detector temperature were higher by 50 "C than that of the column. A stainless steel column 200 cm X 2-mm i.d. was packed with 3% SP-2340 on Supelcoport 100/120 mesh (Supelco Inc.). The column temperature was maintained isothermally at 70 "C with nitrogen as a carrier gas at 14 ml/min. Column efficiency for the last peak of trioses was 2300 theoretical plates. The second column was a stainless steel capillary 45 m X 0.2-mm i.d. coated with Silicone OV-17 (Supelco Inc.). The temperature of this column was maintained isothermally at 140 "C. Nitrogen was used as a carrier gas at inlet pressure 83 kPa. Column efficiency for the last significant peak of tetroses was 40 000 theoretical plates. The electronic integrator Hewlett-Packard 3370A was used for the area measurements. Mass spectra (23eV, emission 30 PA) were recorded with a JMS-100 (Jeol, Japan) instrument equipped with a JGC-2OK gas chromatograph. For MS measurements the SP-2340column was used and the separation was carried out under identical conditions except that helium, at inlet pressure 98.1 kPa, was used as a carrier gas. The temperature of the ionizing chamber was 180 "C. Chemicals. DbGlyceraldehyde (purum), dihydroxyacetone (crystalline dimer, puriss.), isobutylamine (purum),o-phenylenediamine (puriss.), and methylglyoxal dimethyl acetal were products of Fluka, A.G., Switzerland. Methylglyoxal was prepared by hydrolysis of its dimethylacetal with 1%sulfuric acid (8). D-Erythrose and D-threose were prepared by oxidation of D-glUCOSe and D-galactose, respectively, with lead tetraacetate (9).Purification of the crude product was performed by chromatography on a cellulose column using ethylacetate-glacial acetic acid-4% aqueous boric acid (9:l:l). Pure substances were dried over phosphorus pentoxide and sodium hydroxide pellets. L-glycero-Tetrulose was prepared by biochemical oxidation of erythritol with Acetobacter suboxidans ( I O , 11). The crude product was purified by chromatography on a cellulose column using acetone-butanol-water (8:l:l) as the solvent system. The pure product was dried as above. Powdered cellulose used for chromatography was a product of Whatman (England). For oxime preparations, reagent grade pyridine was refluxed over solid potassium hydroxide and distilled. Hydroxylamine hydrochloride was purchased from Lachema (Brno, Czechoslovakia). TRI-SIL Concentrate (Pierce Chemical Co., Rockford, Ill.) was used for silylation.
ANALYTICAL CHEMISTRY, VOL. 49, NO. 1, JANUARY
1977
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Table I. Relative Retention Times of per- 0-Me3Si Oximes, Trioses a n d Tetrosesa Me3Si oxime
3% SP 2340, 70 "C
OV-17, 140 "C
D ,L-Glyceraldehyde
-2
__.. .+
0
8
0.88 1.12 0.78 1.00 Dihydroxyacetone 1.00 1.00 D-Erythrose 2.28 2.78 n-Threose 2.95 3.00 L-glycero -Tetrulose 2.36 2.47 a Retention time relative to per-0-MesSi oxime of dihydroxyacetone.
.
!
:
2b
1b
:
:
:
32
$ .in'
Separation of per-O-Me3Si oximes of dihydroxyacetone, D,L-glyceraldehyde,L-glycero-tetrulose, D-erythrose, and D-threose on a capillary column coated with OV-17 Flgure 1.
Procedures. A solution of hydroxylamine hydrochloride in pyridine (15 mg/ml, 0.7 ml) was added to the sample of sugars (10 mg) obtained by concentration of a water solution and drying at 40 "C under reduced pressure. To assure quantitative reaction, the mixture was allowed to react in a tightly closed vial at 70 "C for 1 h. After cooling to room temperature, the silylation reagent was added and the resulting solution (1pl) was injected after 10 min. For the quantitative determination of the ratio of glyceraldehyde and dihydroxyacetone, mixtures of these trioses containing the substances in the ratio of 05; 1:4;2:3; 3:2; 4:l;and 5:O were prepared. The derivatives were made as described above. The peak areas for glyceraldehyde (2 peaks) and dihydroxyacetone (1peak) were determined and the ratios were compared. The area ratios for glyceraldehyde and dihydroxyacetone corresponded to the expected values. Five consecutive measurements showed a relative error less than 2.3%.Results were compared with polarographic determination.
RESULTS AND DISCUSSION A satisfactory separation for quantitative analysis of all tetroses was achieved using a capillary column coated with OV-17 (Figure 1).The mixture of trioses, however, could not
.-
+----0
8
lb tR
min
Figure 2. Separation of per-0-Me3Si oximes of dihydroxyacetone and o,L-glyceraldehyde on a column packed with SP-2340
Figure 3.
138
be separated even though this highly efficient column was used. These substances could be separated using a packed column with SP-2340 as t h e stationary phase (Figure 2). Trimethylsilylated oximes of glyceraldehyde and tetroses each gave two peaks corresponding, obviously, to the syn and anti oximes. A single peak was obtained from t h e same derivative of dihydroxyacetone from which, owing t o its molecular symmetry, only one isomer was formed. The retention times of Me3Si oximes, relative to dihydroxyacetoxime MesSi
Mass spectra (23 e V ) of Me3Si oximes of dihydroxyacetone, o,L-glyceraldehyde, L-glycero-tetrulose, and D-erythrose
ANALYTICAL CHEMISTRY, VOL. 49, NO. 1, JANUARY 1977
In order to confirm the separation, triose and tetrose MesSi oximes in a GC-MS analysis mass spectrometric fragmentation of compounds of this class were studied. The spectra of all compounds under investigation gave molecular ion and [M - 15]+ peaks (Figure 3), and peaks at mle 73 and 147, characteristic of MesSi derivatives (6, 7). Characteristic of the fragmentation of per-0-MeaSi dihydroxyacetoxime is the formation of [M - MesSi-OH]+ ions, not present in the spectra of D,L-glyceraldehyde. The spectra of the two isomeric MesSi glyceraldoximes (Table I) contain peaks characteristic of the cleavage of the C(2)-C(3) bond (Figure 3). T h e two isomers show qualitatively identical spectra, except for the presence of the [M - 15]+ions present in the spectrum of the first eluted isomer. The mass spectra of the MesSi oximes of L-glycero-tetrulose differ from those of the corresponding aldoses by the presence of [M - MesSi-OH]+ ion peaks. The spectra of the two pairs of isomeric MeaSi oximes of D-threose and D-erythrose (Figure 3) are qualitatively identical. Trioses from tetroses and aldoses from ketoses can thus be distinguished by means of their mass spectra alone, although the spectra are not informative as far as the stereochemistry of the substances is concerned. Compared with existing methods, the selectivity and specificity of the determination of trioses and tetroses by the method involving gas chromatography described herein is noteworthy. The polarographic method is selective only with respect to individual groups of aldoses, ketoses, and their dehydration products. Two substances of a class in mixture cannot be determined in this way. I t may be used as an analytical tool when only one substance of each class is present in a mixture. T h e appearance of polarographic maxima or catalytic (observed, e.g., when molybdic acid is present in the
reaction medium in high concentration (12) interferes with the determination of aldoses and ketoses. In such instances, or when products of transformation of tetroses (erythrose and threose) are to be monitored a method is required capable of determining the substances in question, present either individually or in a mixture. The new method is specific with respect to trioses and tetroses and can be successtklly applied in cases when polarography or other methods fail because of various interfering effects. It fills efficiently the gap in the existing analytical methods of determining trioses and tetroses. When 2- to 10-mg samples were analyzed, the mean relative error of determination of trioses is less than f2.3%.
LITERATURE CITED (1)D. C. Gutsche, D. Radmore, R. S. Buriks, and K. Nowotny, J. Am. Chem. Soc.. 89. 1235 (1967). (2) J. Konigstein and M. Fedorohko, "Proceedings of the 3rd Analytical Con-
ference", Vol. 2,Budapest, 1970,p I 13. (3)C. C. Sweeley, R. Bentley, M. Makita, and W. W. Wells, J. Am. Chem. Soc., 85, 2497 (1963). (4)H. Yamaguchi, T. Ikenaka, and Y. Matsushima, J. Biochem. (Tokyo), 68, _753 _ _ ( i w n-,.\ (5) B. S. Mason and H. T. Slover, J. Agric. FoodChem.. 19, 551 (1971). (6) G. Petersson, Carbohydr. Res., 33, 47 (1974). (7)A. Laine and C. C. Sweeley, Carbohydr. Res., 27, 199 (1973). (8)J. KrupiEka and J. J. K. Novak, Collect. Czech. Chem. Commun., 25, 1275 (1960). (9)R. L. Whistler and M. L. Wolfrom, "Methods in Carbohydrate Chemistry", Vol. 1, Academic Press, New York, London, 1962,p 64. (IO)H. Muller, C. Montegai, and T. Reichstein, Helv. Chim. Acta, 20, 1468 (1937). (11) V. Moses and R. I. Ferrier, Biochem. J., 83, 8 (1962). (12)J. Konigstein, Collect. Czech. Chem. Commun., in press. 1
RECEIVEDfor review July 21, 1976. Accepted October 6, 1976.
In Situ Generation of Standards for Gas Chromatographic Analysis D. J.
Freed* and A. M. Mujsce
Bell Laboratories, Murray Hill, N.J. 07974
Techniques for the direct generation of acrolein, acrylonitrile, and vinyl chloride for gas chromatographic analysis are described. Precolumns containing conversion reagents generate the desired compound from suitable precursors via direct injection. At the 5-ng level, a precision of better than 5 % is achieved, with high and reproducible yields being obtained over a wide dynamic range. The method obviates the necessity for manipulationand storage of hazardous or toxic materials and facilitates the preparation of precise standards.
Because of growing awareness of the hazards of industrial pollutants, methods for the determination of trace amounts of these materials are being introduced a t an ever-increasing rate. There are, however, relatively few standards or standardization methods for toxic or hazardous compounds. Exponential dilution ( I ) , permeation tubes ( 2 ) ,diffusion ( 3 , 4 ) , and standard mixtures (5, 6) have all been used for standardization and calibration. T h e above techniques are not without attendant difficulties, perhaps the greatest one being the necessity for storage and handling of relatively large quantities of potentially dangerous materials. In addition,
many of the most toxic materials are gases or low boiling liquids, thus requiring elaborate flow and temperature control systems for successful standard preparation. Also, since many of these compounds are highly reactive vinyl compounds, such as acrolein, the reliability and stability of dilute standard mixtures may be suspect. For the above reasons we have been prompted to explore alternative methods for preparing standards for trace gas analysis. This report describes techniques for the generation of vinyl chloride, acrolein, and acrylonitrile directly in the injection port of a gas chromatograph. Precursor compounds are used, which are not nearly as volatile or hazardous as the desired compounds. The yields of the desired compounds are uniformly high and the decreased volatility of the precursor facilitates the preparation of reliable standard solutions. Finally, the introduction of the standard into the chromatograph as a plug, rather than as a continuous flow, more nearly mimics the conditions used in most analytical procedures.
EXPERIMENTAL Instrumentation.A V a r i a n MAT 112 Gas Chromatograph-Mass Spectrometer was used f o r a l l investigations. Chromatograms were ANALYTICAL CHEMISTRY, VOL. 49, NO. 1, JANUARY 1977
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