J A N K A R R a n d L L O Y D W . N O R M A N Received for publication October 30, 1973
Introduction
T h e polarimetric quantitation of sucrose in the presence of other optically active compounds has been recognized as a significant prob- lem in the sugar industry (1,3, 12, 13)
1. As the ratio of sucrose to other optically active components of the system decreases the accuracy of the polarimetric analysis may be severely impaired.
Henglein et al. (7) described a procedure for the preparation of the octa trimethylsilyl ether of sucrose and other carbohydrate silyl ethers. These ethers were thermally stable and had a higher vapor pressure than the parent compounds. Using this procedure, Sweeley et al. (16) developed a qualitative gas chromatographic method for the detection of mono and disaccharides. A modification of this method was used by Walker (17) for qualitative investigation of molasses. Using sorbitol as an internal standard and the silylation procedure of Sweeley, Dowling and Libert (6) reported a gas chro- matographic method for mono and disaccharides that had an accuracy comparable to that of the standard polarimetric method for low purity samples. Brobst and Lott (2) developed a similar technique for the analysis of mono, di, tri, and tetra saccharide components of corn syrups. Using the disaccharide trehalose as an internal standard, Rumpf (15) published a technique for the analysis of carboxyllic acids and carbohydrates found in potato extracts.
The silylating reagent commonly used is a mixture of hexamethyl- disilazane and trimethylchlorosilane. In addition to silylating hydroxyl groups, amino and carboxyl moieties are partially silylated (16). N- trimethylsilylimidazole was shown by Horning (8) to be selective for hydroxyl groups. T h e use of such a derivatizing reagent eliminates possible interference from amino acids and polypeptides.
The primary objective of this work was to develop a method for the determination of sucrose in C.S.F. that was accurate, precise and specific for sucrose. A secondary objective was to determine whether there was any correlation between the polarimetric result and the gas chromatographic result and develop an equation for the correction of the polarimetric result.
1Numbers in parentheses refer to literature cited.
54 JOURNAL OF THE A. S. S. B. T.
E x p e r i m e n t a l
Reagents and standards
T r e h a l o s e , t h e i n t e r n a l s t a n d a r d u s e d , was o b t a i n e d f r o m N u t r i - t i o n a l B i o c h e m i c a l C o m p a n y , C l e v e l a n d , O h i o a n d r e c r y s t a l l i z e d twice f r o m a n a l c o h o l - w a t e r system. T h e silylating r e a g e n t , N - t r i - m e t h y l s i l y l i m i d a z o l e , was p u r c h a s e d f r o m O h i o Valley S p e c i a l t y C h e m i c a l C o m p a n y , M a r i e t t a , O h i o . Analytical r e a g e n t g r a d e N N d i m e t h y l f o r m a m i d e was u s e d a s o b t a i n e d f r o m J . T . B a k e r . Equipment
A H e w l e t t P a c k a r d R e s e a r c h Gas C h r o m o t o g r a p h , M o d e l 7 6 2 0 A , e q u i p p e d with a d u a l flame i o n i z a t i o n d e t e c t o r , a digital i n t e g r a t o r , M o d e l 3 3 7 0 A , a n d a n a u t o m a t i c injector, M o d e l 7 6 7 0 A , was u s e d i n t h e c o u r s e o f this s t u d y . D u a l stainless steel c o l u m n s , o n e - f o u r t h i n c h d i a m e t e r , six feet i n l e n g t h w e r e p a c k e d with t e n p e r c e n t O V - 1 7 o n C h r o m s o r b W , 8 0 / 1 0 0 m e s h , ( A p p l i e d Science L a b o r a t o r i e s , I n c . , State C o l l e g e , Pa.) a n d coiled. P r e c o n d i t i o n i n g o f t h e c o l u m n s a t 290°C o v e r n i g h t with a h e l i u m f l o w r a t e o f 2 0 m l p e r m i n u t e was n e c e s s a r y for base line stability. T h e c o l u m n s w e r e o p e r a t e d i s o t h e r m a l l y a t 265°C with a h e l i u m f l o w r a t e o f 6 0 m l p e r m i n u t e . T e m p e r a t u r e s o f t h e injection p o r t a n d d u a l f l a m e i o n i z a t i o n d e t e c t o r w e r e m a i n t a i n e d a t 280°C a n d 325°C respectively. Flow r a t e s o f h y d r o g e n a n d c o m - p r e s s e d a i r w e r e 4 0 a n d 6 0 0 m l p e r m i n u t e . T h e s a m p l e s w e r e injected i n t o t h e gas c h r o m o t o g r a p h u s i n g a 2 5 u l H a m i l t o n S y r i n g e , M o d e l 7 0 2 . All w e i g h i n g s w e r e p e r f o r m e d o n a M e t t l e r M - 5 m i c r o b a l a n c e . Infrared spectroscopy
A s t r e a m s p l i t t e r was i n t r o d u c e d i n t o t h e gas c h r o m a t o g r a p h j u s t p r i o r t o t h e d e t e c t o r . T h e p e a k s o f t h e s u c r o s e a n d t r e h a l o s e d e r i v a - tives w e r e collected in d r y glass t u b i n g . S e v e r a l injections w e r e re- q u i r e d t o o b t a i n a sufficient a m o u n t o f m a t e r i a l for i n f r a r e d analysis.
A f t e r t h e s a m p l e s w e r e c o l l e c t e d , t h e t u b e s w e r e s e a l e d p r i o r t o analysis. T h e i n f r a r e d analysis was p e r f o r m e d u s i n g a P e r k i n - E l m e r M o d e l 6 2 1 s p e c t r o p h o t o m e t e r with a s o d i u m c h l o r i d e m i c r o cell.
T h e s a m p l e was dissolved i n 3 0 m l o f t e t r a c h l o r o m e t h a n e a n d injected i n t o t h e m i c r o cell. E a c h s a m p l e was r u n a g a i n s t t e t r a c h l o r o m e t h a n e b l a n k .
Mass spectroscopy
A s t r e a m s p l i t t e r was i n t r o d u c e d i n t h e gas c h r o m a t o g r a p h j u s t p r i o r t o t h e d e t e c t o r . T h e T M S s u c r o s e a n d t r e h a l o s e p e a k s w e r e col- lected i n d r y glass t u b i n g a n d s e a l e d . Mass s p e c t r a o f s u c r o s e a n d t r e h a l o s e d e r i v a t i v e s w e r e r u n o n a C E C 2 1 - 1 1 0 m a s s s p e c t r o m e t e r u s i n g e l e c t r o n m u l t i p l i e r d e t e c t i o n a n d 7 0 e v i o n i z i n g voltage. T h e s a m p l e s w e r e i n t r o d u c e d directly i n t o t h e ion s o u r c e . T h e t e m p e r a t u r e of volatization was 130°C.
VOL. 18, No. 1, APRIL 1974 55
Methods Gas chromatographic procedure
Approximately 15 mg of trehalose and 100 mg of a well mixed C.S.F. sample were accurately weighed into a tared serum bottle. The container was capped, 3 ml of N-trimethylsilylimidazole and 3 ml of N N dimethylformamide added. The bottle was heated at 60°C for a period of 15 minutes and cooled to room temperature. A seven microliter aliquot was injected into the gas chromatograph. T h e areas under the sucrose and trehalose peaks were integrated. The following equation was used to calculate the percent sucrose.
Wt. Trehalose X Area Sucrose x K
% Sucrose (W/W) =
Wt. Sample x Area Trehalose K= Response Per Unit Mass of Trehalose x 100
Response Per Unit Mass of Sucrose
Polarimetric procedureA 13.00 g aliquot of a well mixed C.S.F. sample was quantitatively transferred to a 200 ml volumetric flask. T h e sample was neutralized to a phenolphthalein end point with 20% acetic acid. After the addi- tion of 20 ml of 55 brix basic lead acetate solution and thorough mix- ing, the volume was adjusted to 200 ml with distilled water. T h e resultant solution was thoroughly mixed and filtered. The filtrate was polarized in a 200 mm tube. The resultant reading was multiplied by four to determine the percent sucrose in the sample.
Results and Discussion
The kinetics of the silylation of the disaccharides has previously been reported (9). Derivatization was shown to be complete in fifteen minutes at room temperature. The pure octa trimethylsilyl ethers of sucrose and trehalose were isolated gas chromatographically and subjected to infrared and mass spectrographic analysis, Figures 1 and 2. Similarly the sucrose and trehalose peaks from C.S.F. samples were isolated and subjected to infrared and mass spectrographic analysis.
Infrared analysis of the TMS ethers of sucrose and trehalose
showed the absence of a hydroxyl band at 33-3600 cm
- 1indicating
that both carbohydrates were completely silylated. Methyl and
methylene bands (14) were noted at 2950, 2900, 1600-1500 and 1400-
1420 cm"
1in both cases. T h e bands at 1250, 900-700 cm
- 1coincide
with the SiCH
3absorption bands reported by Liu and Yahg (11). Also
the Si-O-C absorption bands at 1080 and 1060 cm
- 1reported by Liu
and Yahg (11) were noted in both spectras. The infrared spectras of
5 6 JOURNAL OF THE A. S. S. B. T.
V O L . 18, No. I, APRIL 1974 57
Table 1.—Mg sucrose per 100 Mg sample.
A d d e d 0.00 0.90 1.84 2.64 3.58
Total 10.21 11.15 11.95 12.89
Found ± Std. Dev.
9.31 ± 0.04 10.18 ± 0.01 11.15 ± 0.03 11.96 ± 0.06 12.94 ± 0.06
sucrose and trehalose were significantly different in the 900-1100 c m
- 1range so that they are readily differentiated from each other.
Infrared analysis of similar fractions prepared from C.S.F. were in agreement with the respective standards.
Partial mass spectra recorded for pure samples of sucrose and trehalose trimethylsilyl ethers above m/e 150 are shown in Figure 2.
Additional intense peaks occurred at m/e 73,89, 117, 129, 133 and 147
in both compounds. Molecular ions are absent from the spectra, but
the M-CH
3ion {mle 903) provides indirect verification of molecular
weight, as reported for similar compounds (15, 16). The sucrose TMS
ether produces an intense rearrangement ion at m/e 437 which is of
low intensity in the trehalose spectrum, so that the compounds are
easily differentiated. Significant differences in the spectra also occur
in the 500-600 m/e region, a region reported (5, 10) to be of value in
the assessment of glycosidic linkages. Mass spectra of the chromato-
graphic effluents of TMS sucrose and trehalose from C.S.F. were
comparable to that of the pure standards. No impurities were detected
in those samples, which indicates that the sucrose and trehalose TMS
peaks contain single components.
58 JOURNAL OF THE A. S. S. B. T.
A s a m p l e of C.S.F. was t a k e n a n d split i n t o five r e p r e s e n t a t i v e s a m p l e s . T o f o u r o f t h e a l i q u o t s s u c r o s e was a d d e d . E a c h o f t h e five r e s u l t a n t s a m p l e s w e r e a n a l y z e d i n triplicate b y t h e G . C . m e t h o d . T h e r e s u l t s a r e s h o w n in T a b l e 1.
O v e r t h e c o u r s e o f t h e 1970-71 b e e t c a m p a i g n a t Moses L a k e , C.S.F. s a m p l e s w e r e t a k e n a n d a n a l y z e d p o l a r i m e t r i c a l l y a n d b y gas liquid c h r o m a t o g r a p h y . A plot o f t h e p o l a r i m e t r i c r e s u l t v e r s u s t h e gas c h r o m a t o g r a p h i c r e s u l t i s s h o w n i n F i g u r e 3 . T h e d a t a was f i t t e d t o a c u r v e b y t h e m e t h o d o f least s q u a r e s (4). T h e l i n e a r r e l a t i o n s h i p , y = mx + b, g a v e t h e best fit for forty p a i r s of d a t a , a n d h a d a coefficient o f d e t e r m i n a t i o n o f 0 . 9 3 1 . T h e e q u a t i o n g e n e r a t e d was: % S u c r o s e = 0.877 ( P o l a r i m e t r i c Result) + 5 . 2 3 . W h i l e t h e fit is n o t p e r f e c t for t h e c o r r e c t i o n of t h e p o l a r i m e t r i c r e s u l t , it is w o r t h y of c o n s i d e r a t i o n for a m o r e realistic e v a l u a t i o n o f t h e Steffen o p e r a t i o n losses w h e r e t h e gas c h r o m a t o g r a p h i c m e t h o d i s n o t b e i n g e m p l o y e d .
S u m m a r y
A gas liquid c h r o m a t o g r a p h i c m e t h o d for t h e d e t e r m i n a t i o n o f s u c r o s e in c o n c e n t r a t e d Steffen filtrate is d e s c r i b e d . T h e m e t h o d is a c c u r a t e , p r e c i s e a n d selective for s u c r o s e . C o m p a r a t i v e a n a l y s e s , p o l a r i m e t r i c vs. gas c h r o m a t o g r a p h i c , d e m o n s t r a t e d a significant e r r o r i n t h e p o l a r i m e t r i c p r o c e d u r e . T h e e r r o r was c o n s i s t e n t . U s i n g t h e m e t h o d o f least s q u a r e s , a n e q u a t i o n was g e n e r a t e d f o r t h e c o r r e c - t i o n o f t h e p o l a r i m e t r i c r e s u l t s a t t h e C o l u m b i a Basin r e f i n e r y . Utiliza- tion o f t h e e q u a t i o n h a s p e r m i t t e d a m o r e realistic e v a l u a t i o n o f Steffen o p e r a t i o n a t t h e C o l u m b i a Basin r e f i n e r y .
A c k n o w l e d g m e n t s
G r a t e f u l a c k n o w l e d g m e n t i s given t o M r s . P a m e l a M c B r o o m a n d M r . F o u a d S h a k e r for t h e i r t e c h n i c a l assistance. T h e m a s s s p e c t r o - g r a p h i c analytical s u p p o r t b y D r . William H a d d o n o f t h e W e s t e r n R e g i o n a l U . S . D . A . L a b o r a t o r y i s d e e p l y a p p r e c i a t e d . I n f r a r e d s p e c t r a l s u p p o r t w o r k b y D r . B r u c e E t t l i n g , W a s h i n g t o n State U n i v e r s i t y i s d u l y a c k n o w l e d g e d .
Literature Cited
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D e p a r t m e n t of C o m m e r c e , United States G o v e r n m e n t Printing Office, Washington, D.C.
(2) BROBST, K.M., AND C.E. L O T T , J R . , 1966, Determination Of Some Com- ponents In Corn Syrup By Gas-Liquid Chromatography Of T h e Trimethylsilyl Derivatives, Cereal Chem., 43, 35.
(3) BROWNE, C.A. AND F.W. ZERBAN, 1941, Physical and Chemical Methods of Sugar Analysis, 3rd Ed., Wiley & Sons Inc., New York, 372-373.
V O L . 18, No. 1, APRIL 1974 5 9 (4) DANIELS, F., J. MATHEWS, J. WILLIAMS, P. BENDER AND R. ALBERTY,
1956, Experimental Physical Chemistry, McGraw-Hill Book Co., Inc., New York, p. 339.
(5) D E J O U G H , D C , T. RADFORD, J.D. HRIBAR, S. HAUESSIAN, M. BIEBER, G. DAWSON, AND C.C. SWEELEY, 1969, Analysis of Trimethylsilyl De- rivatives of Carbohydrates by Gas Chromatography and Mass Spec- trometry, J. Am. Chem. S o c , 91, 1728.
(6) DOWLING, J.F. AND J.F. LIBERT, 1966, Applications of Gas-Liquid Chromatography in the Sugar Industry, Proc. 1966 Tech. Session Cane Sugar Refinery Research, 113-125.
(7) HENGLEIN, F.A., G. ABELSNES, H. HENEKA, K. LIEHARD, O. NAKHRE AND K. SCHEINOST, 1957, Organosilyl Derivatives of Dicarboxyllic Acids, Hydroxy Carboxyllic Acids and Sugars, Makromol. Chem., 24, 1.
(8) H O R N I N G , M.G., A.M. Moss, E.A. BOUCHER AND E.C. HORNING, 1968, T h e GLC Separation of Hydroxyl-Substituted Amines of Biological I m p o r t a n c e Including T h e Catecholamines. Preparation of De- rivatives For Electron Capture Detection. Anal. Letters, 1, (5), 311.
(9) KARR, J. AND L.W. NORMAN, 1970, A Gas Liquid Chromatographic Method for the Determination of Sucrose in Molasses, presented at the Sixteenth General Meeting of the Am. Soc. Sugar Beet Tech- nologists, Denver, Colorado.
(10) KocHETKov, N.N., O. CHIZHOV, AND N. MOLODTSOV, 1968, Mass Spect- rometry of Oligosaccharides, Tetrahedron, 24, 5587.
(11) Liv, S. AND M. Y A H G , 1964, Some Bis (trimethylsiloxy) Compounds, J.
of Chinese Chem. Soc. (Taiwan), 11, 202-204
(12) M C G I N N I S , R.A., En., 1971, Beet Sugar Technology, 2nd Ed., Beet Sugar Development Foundation, Fort Collins, Colorado, Chapter 2.
(13) MEADE, G.P., E D . , 1963, Cane Sugar Handbook, 9th Ed., Wiley & Sons, Inc., New York, 434-40.
(14) NAKANISHI, K., 1964, Infrared Absorption Spectroscopy, Holder-Day, San Francisco.
(15) RUMPF, G., 1969, T h e Silylation of Substances Occurring in Natural Products and Detectable by Gas Chromatography, J. Chromato- graphy, 43, 247.
(16) SVVELLEY, C . C , R. BENTLEY, M. MAKITA AND W.W. WELLS, 1963, Gas- Liquid Chromatography of Trimethylsilyl Derivatives of Sugars and Related Substances, J. of Am. Chem. S o c , 85, 2497.
(17) WALKER, H . G . , J R . , 1965, GLC Examination of Molasses Carbohydrates, Internal. Sugar J . , 6 7 , 237.