Analytical Potential. With some modification according to particular need, the method described in this paper was found to be very useful when applied to the study of biological fluids containing carboxylic acids. With proper modification, many Other carboxylic acids could be and determined in the same manner.
The method can be modified to include other detecting reagents in case dichromate oxidation should prove to be unsuitable for other acids. LITERATURE CITED
(1) Mfre&son, B,, Bergdahl, s,, Samuelson, O., Anal. Chim. Acta 28, 371 (1963).
(2) Alfredsson, B., Gedda, L., Samuelson, O*, zbid*, 27, 63 (l962). (3) Goudie, A. J., Rieman 111, W., Zbid., 26, 419 (1962). (4) Hamilton, P. B., ANAL. CHEM.30, 914 (1958). (5) Shimomura, K., Walton, H. F., Zbid., 37, 1012 (1965).
RECEIVEDfor review April 21, 1966. Accepted June 27, 1966.
Thin Layer and Column Chromatography of Carbohydrates as Trimethylsilyl Ethers with Applications to Mucopolysaccharide Analysis JORMA
E. ~RKKA'INEN,EERO 0.HAAHTI,
and AAPO A. LEHTONEN
Department of Medical Chemistry, University o f Turku, Turku, Finland
b Thin layer and column chromatography of carbohydrates as their trimethylsilyl ethers on silica gel are described. The method is suitable for separation of anomers of sugars and for fractionation of sugar mixtures into neutral sugars, amino sugars, and uronic acids. Adsorption chromatography on silica affords a rapid way of prefractionating a complex mixture of sugars before final analysis by gas-liquid chromatography. Hydrolyzates of mucopolysaccharide fractions of bovine aorta and skin are determined using silicic acid column chromatography and gas-liquid chromatography in succession. Mannose, glucose, and galactose were present in the neutral sugar fractions. lduronic acid, glucuronic acid, galactosamine, and glucosamine were found in the fractions containing uronic acids and amino sugars.
I
determination of sugars by gas chromatography (GLC), trimethylsilyl ethers (TMS) have gained wide popularity due to their easy preparation and good thermal stability (7'). Difficulties are, however, often encountered in the analysis of sugar mixtures due to the great number of peaks arising from the different isomers. To a certain extent resolution can be improved by appropriate choice of stationary phases or by using sugar derivatives-e.g., methyl glycosides-for the analyses (8). I n complicated natural mixtures like mucopolysaccharide hydrolyzates a group fractionation previous to GLC is necessary. Ion exchange procedures have been successfully employed for the fractionation of mucopolysaccharides into sugar classes ( I , 8,4, a,but because of the use of aqueous solvents, which have to be removed before trimethylN THE
1316
ANALYTICAL CHEMISTRY
silylation, analysis times tend to b e come long. This paper describes the use of liquid adsorption chromatography of the trimethylsilyl ethers on silica gel for fractionation of sugar mixtures. Contrary to the common opinion, trimethylsilyl ethers appear to be stable enough for column or thin layer chromatography when dry conditions are employed. Because of the nonpolarity of the TMS ethers, development of the chromatograms is fast and separation clear. EXPERIMENTAL
Reagents
and
Standards.
D-
Glucosamine hydrochloride, Dgalactosamine hydrochloride (Mann Research Laboratories, Inc., New York, N. Y.), glucose, galactose, mannose, lactose, sucrose, rathose, xylose, fucose (E. Merck, A.G., Damstadt), glucuronic acid lactone (Fluka, A.G., Buchs SG), N-acetyl-D-glucosamine, and N-acetyPD-galactosamine (Sigma Chemical Co., St. Louis, Mo.) were used as reference compounds. All sugar standards were mutarotated in water for a minimum of 24 hours at room temperature before analysis. Hexamethyldisilazane, pumm 98%, trimethylchlorosilane, puriss. 99% (Fluka, A.G.) were used without further purification. Pyridine, reagent grade (May & Baker, Ltd., Dagenham, England), was redistilled and stored over KOH pellets. Iduronic acid standard for GLC was prepared with acid hydrolysis of chondroitin sulfate B obtained as a aift from K. von Berlepsch, F. HoffmahLa Roche, Ltd., Basel, Switzerland. Preparation of Trimethylsilyl Derivatives. The T M S derivatives were prepared by mixing the dry sample (0.01 to 1 mg.) with pyridine (0.14 ml.), hexamethyldisilazane (0.04 rnl-), and trimethylchlorosilane (0.02 ml.) in a conical centrifuge tube. The
solution was gently shaken and left to stand a t room temperature for 30 minutes. The excess silylation reagents were evaporated in a stream of nitrogen, and the TMS ethers were extracted from the precipitate into hexane using an ultrasonic vibrator. The extract was concentrated in a dry nitrogen stream to the desired volume. Thin Layer Chromatography. The thin layer plates were coated with a 0.25-mm. layer of Kieselgel G or aluminum oxide G (E. Merck A.G.) and dried at 120' C. for 2 hours. Before use the plates were activated a t 120' C. for 15 minutes. Sulfuric acid (70%, v./v.), saturated with potassium dichromate, was used for spraying. For preparative purposes the TLC fractions were made visible with Rhodamine 6 G (British Drug Houses, Poole, England); the areas of the support containing the fractions were scraped off the plates into centrifuge tubes and the TMS ethers were extracted into diethyl ether. Column Chromatography. Silicic acid (Unisil 200-325 mesh, Clarkson Chemical Co., Inc., Williamsport, Pa.) was dried at 150' C. for 30 minutes and stored under dry hexane. Columns of 90 x 4 mm. and 90 X 2 mm. were employed for 20- to 200-pg. samples. Elution of fractions was detected with a circulating chain detector for liquid chromatography (3) and the peaks were identified with GLC. Gas Chromatography. A BarberColman M-10 chromatograph equipped with a flame ionization detector was used. The columns were standard &foot x 4-mm. glass U-tubes treated with hexamethyldidazane before packing. The stationary phase SE30 (1% w./w.) on 100- to 140-mesh silicsnized acid-washed Gas-Chrom P (Applied Science Laboratories, State College, Pa.) was held at 150' C. Nitrogen flow was 40 to 60 cc. per minute. Samples were injected into the chromapgraph with a 10-pl. Hamilton syringe.
Preparation of Mucopolysaccharide Samples. Acetone-defatted samples of bovine skin and aorta were hydrolyzed with papain as described by Schiller, Slover, and Dorfman (6). Trichloroacetic acid (10% w./v.) was added to precipitate proteins and nucleic acids and the excess TCA was removed from the supernatant with ether. The mucopolysaccharides were precipitated at 4' C. with 4 volumes of ethanol which contained 0.5% sodium acetate and the precipitate was dissolved in water. The mucopolysaccharides were hydrolyzed in 1N hydrochloric acid in sealed tubes at 100' C. for 3 hours, after which the solvent was removed with a stream of nitrogen.
-START
m 3
I
Figure 1 . Separation of glucosamine ( l ) , glucuronic acid (2), and glucose (3) as trimethylsilyl derivatives on silica gel Amounts ea. 100 @(I.
RESULTS
Thin Layer Chromatography of Neutral Sugars. SEPARATION OF ANOM E R ~ . Table I shows the mobilities of some common T M S sugars on silica gel. All of the compounds moved with the front with diethyl ether and remained at the start with hexane. Benzene separated mutarotated fucose, glucose, galactose, mannose, and maltose into subfractions (Table I), which were found to be due to the resolution of different anomers when analyzed by GLC after their preparative isolation from the plate. Practically no separation of anomers was observed with mutarotated xylose, ribose, and lactose. Traces of polar material could be detected close to the start with benzene and chloroform, while with ethyl acetate all neutral sugars were eluted as single spots. Some differences of color were observed after charring with bichromate sulfuric acidT M S xylose, for example, was stained yellow. Thin Layer Chromatography of Amino Sugars and Uronic Acids. The T M S derivatives of amino sugars were not eluted from the start with
Table 1.
amino sugars on silica gel thin layer plates using benzene as solvent; also the two latter groups were almost totally resolved. Neutral sugars, uronic acids, and amino sugars could be completely separated using a mixture of 12% ethyl acetate and 1% acetic acid in hexane (Figure 1). Pure ethyl acetate as solvent separates amino sugars from other TMS sugars. After preparative TLC, recoveries as determined by GLC were subject to a relatively great variation (50 to 80%), and sometimes small additional peaks could be detected in the gas chromatograms. No selective loss of any particular sugar derivative occurred. Separations obtained with aluminum oxide as an adsorbent were similar to those on silica gel. Class Separation of Sugars by Column Chromatography and Combination with GLC. I n preliminary experiments silica gel which contained 10% removable moisture caused decomposition of T M S ethers, as indicated by low recoveries and by the appearance of extra peaks in the gas chromatograms. When silicic acid powder was dried (Ohaus moisture balance) a t 120' C. for 30 minutes before use, the recoveries were over 90% as estimated with GLC and no signs of decomposition were present. Neutral sugars, amino sugars, and uronic acids were separated using stepwise elution with benzene, which elutes neutral sugars, and 1% ethyl acetate in benzene, which separates uronic acids from amino sugars. Neutral sugars can be separated from amino sugars and uronic acids conveniently by eluting with benzene and ethyl acetate, respectively (Figure 2). The above procedure was applied to the fractionation of a mucopolysaccharide hydrolyzate of bovine aorta. Figure 3 illustrates the GLC records of the total and fractionated sample. I n addition to galactose, glucose, and mannose the neutral
benzene and that of glucuronic acid (in lactone form) was eluted only slightly (Table I). Ethyl acetate separated TMS glucosamine and galactosamine into two fractions, which in the case of glucosamine corresponded to the two main peaks obtained in GLC on SE30. The two hexosamines could not be completely separated. The polarity of the main TMS glucuronic acid derivative was intermediate between neutral sugars and amino sugars. The mobility of the smaller and more polar fraction of TMS glucuronic acid could be increased by adding 1% acetic acid to the solvent. The smaller portion probably represents the free acid form. Trimethylsilyl derivatives of N-acetylated forms of amino sugars, as well as that of N-acetyl neuraminic acid, were found to have slightly greater Rf values than the nonacetylated amino sugar derivatives. Class Separation of Sugars by Thin Layer Chromatography and Combination with GLC. Neutral sugars as a class could easily be separated from uronic acids and
Rf Values ( X 100) of Main Trimethylsilyl Derivatives of Some Common Carbohydrates on Silica Gel ( u and fI forms identified by GLC)
Xylose Fucose Ribose Glucose Galactose Mannose Maltose Lactose Sucrose Raffinose Glucuronic acid Glucosamine Galactosamine N-Acetyl glucosamine N-Acetyl galactosamine
Benzene 57 69;61 57 @(a); W 8 )
W u ) ; WfI)
56;44 65;12;6 38 53 51 8 0 0
... ...
Chloroform (1% methanol) 78 82 78 79 78, 69 72, 62
... ... ... ... ... ... ... ...
...
12% ethyl acetate and 1% acetic acid in hexane
Ethyl acetate 80 80 80 80 80 80 80 80 80 80 80 7467 67,61 75 78
... 80
... ... ... *.. ... 37,'23 0 0 6 13,4
50% ethyl acetate m benzene
... ... ... 82 ... ... ... ...
... ..
80 72,62 62,57 70 66
~~
VOL 38, NO. 10, SEPTEMBER 1966
0
1317
AMINO SUGARS URONIC ACIDS
GLUCURONIC ACID GALACTOSAMINE
1I
1
’
’
N-ACETYL GALACTOSAMINE
GLUCOSWINE
I
10
AND
I
I
I
LO
30
MIN$ES Figure 2. Gas chromatographic analysis of synthetic mixture of TMS sugars using silicic acid column chromatography for prefractionation Neutral sugars eluted with benzene, amino sugars and uronic acids with ethyl acetate. Stationary phase in GLC; 1% SE-30 at 150’C.
sugar fraction contained an unknown compound eluted after &glucose in gas chromatography. Glucosamine, galactosamine, glucuronic acid, and iduronic acid were present in the ethyl acetate eluate. A corresponding analysis of the mucopolysaccharides of bovine skin by the same method showed the presence of the same monosaccharide components except the unknown peak mentioned above. Neutral sugars, amino sugars, and uronic acids could also be separated by eluting with rising concentrations of ethyl acetate in hexane. Neutral sugars are totally eluted with 1% ethyl acetate in hexane, while a gradient from 4 to 10% ethyl acetate in hexane separates glucuronic acid from glucosamine.
are increased. It was found that for silylation of amino sugars a reaction time of at least 30 minutes should be
used. Hydrolysis during prolonged exposure of the sample to air before development of the chromatogram also resulted in an increase of the above polar fractions. Separation of uronic acids and hexosamines from neutral sugars is necessary in the analysis of mucopolysaccharide hydrolyzates by GLC, since some preparations of natural mucopolysaccharides contain pentoses and hexoses. Ion exchange procedures applied for this purpose usually involve conversion of the uronic acid into free acid form, but for GLC uronic acids should be brought back to the lactone form. The group fractionation method employing silica gel column chromatography seems to be a rapid and useful method in the study of natural polysaccharides, which often are composed of numerous subunits overlapping each other in GLC. Comparison of the chromatograms of natural MPS hydrolyzates with that obtained by hydrolyzing reference chondroitin sulfate B, showed the presence of a compound, tentatively identified as iduronic acid in both aorta and skin preparations. This peak of iduronic acid overlaps a small peak occasionally arising from partial silylation of glucosamine. Therefore a complete class separation to neutral sugars, uronic acids, and amino sugars is needed for
TOTAL HYDROLYZATE
DISCUSSION
Thin layer chromatography of trimethylsilyl ethers of sugars affords a rapid way of characterizing sugar mixtures. Gas-liquid Chromatography can be used for qualitative analysis of the fractions separated by TLC. Because the TMS ethers are slowly hydrolyzed upon exposure to atmosphere moisture, thin layer chromatography seems less suitable for quantitative work unless absolutely dry conditions are employed. For the same reason one solvent mixture is preferable to two developing solvents used successively. The polar fractions found as impurities in the thin layer chromatograms of TMS sugars probably represent incomplete silylation products, because their relative amount diminishes as silylation times 1318
ANALYTICAL CHEMISTRY
0-OLUCOSE
I
\
1
n
IWRONIC IWRONIC ACID ACID
AMINO SUGARS AND URONIC ACIDS
I
zo a@ (0 MINUTES Figure 3. Analysis of mucopolysaccharide hydrolyzate of aorta using combination of silicic acid column chromatography with gas chromatography 10
See legend for Figure 2
reliable quantitative determination of iduronic acid. Fklier methods such as that of Boas ( 1 ) for purification of amino sugars could be used in combination with silicic acid chromatography. Our studies indicate, however, that by refinement of the techniques presented this separation can be conveniently accomplished using adsorption chromatography of trimethylsilyl ethers.
LITERATURE CITED
(1) Boas, N. F., J. BWZ. Chem. 204, 553 (1953). (2) Dziewiatkowski, D.,Biochim. Biophys. Acta 56, 167 (1958). (3) Haahti, E.,Nikkari, T., Karkkainen, J., J. Gas Chranatog. 4, 12 (1966). (4) Karkkainen, J., Lehtonen, A., Nikkari, T.,J. Chromatog. 20, 457 (1965). (5)Lehtonen, A.,Karkkainen, J., Haahti, E., Ibid., in press.
(6) Schiller, S., Slaver, G., Dorfman, A,, J . BWl. Chem. 236, 983 (1961). (7)Sweeley, C. C., Bentley, R., Makta, M., Wells, W. W., J. Am. Chem. Soc. 85, 2497 (1963). (8) Sweeley, C. C.,Walker, B., ANAL. CHEM.36, 1461 (1964). RECEIVED for review March 28, 1966.
Accepted May 26, 1966. Work supported by PHS research grant HE0681805 from the National Heart Institute, Bethesda, Md., and by the Emil Aaltonen Foundation, Helsinki, Finland.
Qualitative Analysis of Petroleum and Related Materials Using Linear Elution Adsorption Chromatography L. R. SNYDER Union Oil Company of California, Union Research Center, Brea, Calif.
b By means of linear elution adsorption chromatography (LEAC) narrow petroleum fractions can be assigned an adsorptivity range, which in turn defines the various compound types which can or cannot be present in the fraction. This can simplify the subsequent qualitative analysis of the fraction by other techniques, since the number of possible component types which must be considered is greatly reduced. Application of the principle of the method to several past literature studies suggests some erroneous conclusions regarding the identificationof certain compound types in petroleum related samples.
A
CHROMATOGRAPHY has been used frequently in the separation of petroleum and related materials prior to analysis by other techniques. Its great value in this connection is based upon the fact that different compound types exhibit sharp differences in relative adsorptivity and are hence often separated cleanly by adsorption chromatography. By the same token the relative adsorptivity of a narrow petroleum fraction defines its composition to some extent. Until the present time, however, there has been no systematic attempt to use relative adsorptivity per se for the qualitative analysis of petroleum fractions. Two major problems have discouraged efforts in this direction: variability of adsorptivity with sample composition and separation conditions, and the complexity of petroleum. I n certain simple cases, however, it has been found that the techniques of linear elution adsorption chromatography (LEAC) can overcome these limitations on qualitative analysis of petroleum via
RP
-
1010
E,
1
0.305
\ I
1.0
El& I
I
0.1
I
I
I
I
0.2 0.3
0.4
l
0.5
l
1
06
t=
Figure 1. LEAC analysis of a narrow fraction from a heavy gas oil; fraction R ” values vs. eluent t o values
VSORPTION
adsorption. Thus the presence of nonvicinal aromatic and/or polysulfide types in petroleum was first demonstrated by qualitative LEAC analysis (see Figure 2 of ref. 8). With the recent publication (16)of an extended set of LEAC adsorptivity data for possible petroleum compound types, it is now possible t o apply LEAC routinely to the qualitative analysis of petroleum. The present paper describes this technique in detail, verifies its accuracy by several examples, and illustrates its usefulness by application to some previous literature studies. EXPERIMENTAL
Reagents. Philip’s 99% n-pentane is purified by passage over activated silica gel (20 ml. per gram). Other solvents used are reagent grade. Chromatographically standardized 3.8% H,O-AltOa (Alcoa F-20, equivalent linear retention volume 1.60 ml. per gram for elution of naphthalene by
n-pentane) is prepared as reported previously (9). Preparation of Narrow Fractions. The following procedure for qualitative analysis by LEAC is restricted to narrow fractions prepared by adsorption chromatography. Other samples must first be separated by nonlinear adsorption chromatography, the individual fractions analyzed by LEAC, and the resulting data composited as described in a following section. For nonlinear separation, 25 mg. of the sample are charged to a 10gram dry column as reported previously (7) and elution is begun with the series of eluents listed in Table I under “nonlinear separation.” Fifty-milliliter portions of each eluent are applied to the column in sequence (after wetting the column with the first eluent), beginning with n-pentane, and 50-ml. fractions are collected. When it is known that the original sample contains no very weakly or very strongly adsorbing components, one or more of the initial or terminal eluents of Table I may be omitted. The various fractions collected are evaporated under nitrogen and weighed, or redissolved in pentane or CCL, and their ultraviolet absorbance is determined (assumed proportional to weight, if necessary). Only those fractions which contain a p preciable sample are retained for LEAC analysis. LEAC Oualitative Analvsis. Elution curve‘s for the abovk fractions are obtained by LEAC in the usual manner (9, 10) (ultraviolet absorbance of small fractions), using 3.8% HeOAlrOa and solvents selected from the list of Table I under “LEAC Analysis.” Two or more elution curves (different eluents) must be obtained, using solvents whose eo values are similar to those for the solvent used to elute the original fraction in the nonlinear separation. One hundred micrograms of sample per gram of adsorbent (or smaller sample) is charged. values VOL 38,
NO. 10, SEPTEMBER 1966
1319
