Quantitative Determination of Monosaccharides as Their Alditol Acetates by Gas Liquid Chromatography
SIR: Rapid separations and precise quantitative data obtained make the technique of gas liquid chromatography (GLC) ideally suited for identification and quantitation of sugars in biological materials. Reviews have been published that describe the use of GLC in carbohydrate chemistry (3, 10). However, GLC has not been applied extensively to the quantitative analysis of mixtures of monsaccharides (8, 9, 11). To be successful, the method requires that a volatile derivative be preparable in quantitative yield from each monosaccharide and that a mixture of the derivatives being analyzed be resolved completely. Though a number of possible derivatives fulfill these requirements, the main difficulty in working with monosaccharides is the formation of as many as four glycosides per monosaccharide resulting from anomeric and ring isomerization, each of which produces a peak on the chromatogram. Thus, in a complex mixture containing a number of monosaccharides, the multiplicity of peaks produced prevents complete separation of all the peaks from one another, and accurate quantitation cannot be achieved. I n view of this difficulty, reduction of monosaccharides to their alditols and then separation of the alditol derivatives would offer better possibilities for their quantitation because this procedure would eliminate the problem of multiple peaks since the alditols cannot anomerise. This approach is possible because reduction of monosaccharides is simple and quantitative with sodium borohydride ( 1 , 2 ) . We now have established the conditions necessary for complete separation of 10 alditol acetates derivable from 10 commonly occurring monosaccharides on a single liquid phase and we report the general applicability of this procedure to the quantitative analysis of monosaccharide mixtures. EXPERIMENTAL
Apparatus and Materials. The gas chromatograph used was Model 810R from F & M Scientific Corp., Avondale, Pa., equipped with a dual flame detector system, a Y-153 MinneapolisHoneywell recorder, and a 201-B Disc integrator. The injection port of the F & M model was modified as follows: The insert liners supplied in the injection ports were replaced by high-pressure 1/4-inch 0.d. stainless-steel tubing with a 3/32-in~hi.d. The inner bore of the 1602
ANALYTICAL CHEMISTRY
tube was flared a t the ends to a inch opening to facilitate the entrance of the syringe needle on one end and to fit against the column a t the other. These inserts served to minimize the loss of efficiency resulting from large dead volumes of the original insert liners. Chromosorb W 80-100 mesh (HMDS treated), XE-60, and Carbowax 20J1 terminated with terephthalic acid were obtained from Wilkens Instrument and Research Inc., Walnut Creek, Calif. Gas Chrom Q 100-120 mesh precoated with 3% ECNSS-M was purchased from Applied Science Laboratories, State College, Pa. Three columns of 0.25-inch 0.d. copper tubing were used, each containing different packing: 8 feet with 5% x E - 6 0 , 4 feet with 10% Carbowax 20.W terminated with terephthalic acid, and 10 feet with 3% ECNSS-M. Alditol acetates used as standards in this investigation were: glycerol acetate, b.p. 258-260' C.; erythritol acetate, m.p. 83" C.; rhamnitol acetate, sirup; fucitol acetate, m.p. 127' C.; arabinitol acetate, m.p. 76' C.; ribitol acetate, sirup; xylitol acetate, m.p. 61'-62' C.; mannitol acetate, m.p. 126' C.; galaclitol acetate, m.p. 171' C.; and glucitol acetate, m.p. 99' C. The acetates were prepared in the laboratory as described (1) and recrystallized until they were chromatographically pure. Alditols were used as starting materials, except for rhamnitol and fucitol where the parent monosaccharides were used. Reduction and Acetylation Procedure for Glycose Mixtures. Glycose mixtures were reduced with sodium borohydride for 3 hours ( I ) . Excess borohydride was neutralized with acetic acid and the solution evaporated to dryness. The dry residue was refluxed for 4 hours with a mixture containing equal amounts of acetic anhydride and pyridine (ca. 1 cc./lOO mg. of sugar). The solution was cooled and directly injected in the gas chromatograph for analysis. Method. The column packings were prepared by the solution coating technique described earlier (9) except for the 3% ECNSS-M. The phasecoated support was packed into columns under 40 lb. of nitrogen pressure by using a Column Pac, Model G-3 from Illinois Instrument Group, Des Plaines, Ill. The columns were conditioned a t their maximum temperatures for 16 hours. Detector Calibration and Calculations. Xylitol acetate was chosen as an internal standard. Linear calibration curves were obtained for each alditol acetate by chromatographing varying amounts of each alditol acetate with constant amounts of xylitol acetate and by plotting the
ratio of the area of each alditol acetate to t h a t of the standard against the ratio of the weight of the alditol acetate to that of the standard. The detector response ( K ) values were then determined by the slopes of these curves. Peak area of the alditol acetate/ peak area of the internal standard K = Wt. of the alditol acetate/ wt. of the internal standard For all the acetates K was 1.00. For analysis of a glycose mixture, a known amount of xylitol was added to the mixture which was reduced and acetylated, and then a small fraction of this was injected directly into the gas chromatograph. Peak areas of unknowns were then related to their amounts by the expression: Wt. of the unknown acetate = Peak area of the unknown X wt. of the internal standard Peak area of the internal standard X appropriate K value RESULTS AND DISCUSSION
Preliminary consideration suggested that separation of alditols as their trimethylsilyl (TMS) ether derivatives would be suitable because of the rapid and quantitative formation of this derivative and its successful application to monosaccharides and oligosaccharides (10). However, we found that the TMS ether derivatives of all 10 alditols were not adequately separated on any liquid phase tried, apparently because TMS ether groups are nonpolar in character and, a t temperatures required for elution, are not retained
Table 1.
Retention Times for Aldito Acetates
Retention time (minutes) Carbo-
wax ECNSS-M XE-60 20M" Component 190" C. 160" C. 190" C. 3.6 1.2 3.2 Glycerol 13.2 5.8 12.0 Erythritol 30.0 30.5 GRhamnitol 12.4 30.0 34.0 GFucitol 13.8 40.0 19.4 42.8 Rabitol 42.8 44.4 L-Arabinitol 21.8 Lyxitol 21.8 ... 44.4 61.0 52.8 Xylitol 30.2 57.0 122 127.2 D-Mannitol 144.4 66.0 125 Galactitol 132 144.4 76.4 Glucitol a
Terminated with terephthalic acid.
Acetate 1 Glycerol 2 Erythritol 3 t4hamnitol 4 t+ucitol 5 Ribitol 6 t4rabitol 7 Xylitol 8 o4annitol 9 Galactitol 1 0 o.Glucitol
1
Y YI e
t z
e % G
3
4
A A
5 A
6
7
9.10
a
120
130
140
150
Time, min.
Figure 1 . Gas chromatogram of alditol acetates separated on a &foot column containing 10% Carbowax 20M terminated with terephthalic acid Sensitivity. Range 100, attn. 8x, column temp. 190' C., detector temp. 2 7 0 ' C., injection port temp. 3 0 0 " C., helium flow 85 ml./minute, sample size 4 pl. of 1 % solution
eluted without degradation (4). However, when polarity of the Carbowax 20M column was reduced by esterifying the terminal hydroxyl groups of the polymer chain with terephthalic acid, this phase produced an excellent column which separated all the alditol acetates with the exception of galactitol from glucitol acetate. A chromatogram obtained on this phase shows nine symmetrical peaks (Figure 1). The XE-60 column of relatively less polarity is capable of resolving the three isomeric hexitol acetates from one another but does not resolve ribitol from arabinitol acetate and rhamnitol from fucitol acetate. Aliquid phase with its polarity intermediate between the Carbowax 20M terminated with terephthalic acid and the XE-60 phase was so tried. ECNSS-M, an organosilicone poly-
long enough by the liquid phase to be resolved. I n contrast, alditol acetates are relatively more polar and showed possibilities for better resolution. Attempts by earlier investigators (5-7, 1 2 ) to resolve alditols as their acetates were partially successful. Also a rather successful attempt was made to extend the separation to the analysis of monosaccharides ( 5 ) . However, separation of glucitol from galactitol, rhamnitol from fucitol, and ribitol from arabinitol, was too poor for quantitative analysis ( 5 ) . The degree of polarity of the liquid phase used was a very critical parameter for complete separation of alditol acetates (Table I). On the polar Carbowax 20M phase, a highly selective phase for the TATS ether derivatives of monosaccharides (9), alditol acetates rvere severely retarded and could not be
ester phase consisting of ethylene glycol succinate chemically combined with a silicone of cyanoethyl type, had the ideal polarity for this separation. Figure 2 illustrates separation of all 10 alditol acetates obtained on this phase. Alditol acetates show single and symmetrical peaks with no evidence of decomposition or any thermal change occurring. The analysis is complete within 80 minutes. When the sample was analyzed with the original injection ports, the column temperature had to be lowered to 170' C. to obtain complete resolution. Under these conditions resolution took almost twice as long. Several synthetic glycose mixtures were quantitatively analyzed by reduction to the corresponding glycitols followed by conversion to their fully
Acetate 1 Glycerol 2 Erythritol 3 tTIhamnito1 4 t.Fucitol 5 Ribitol 6 t.Arabinitol 7 Xylitol 8 o-Mannitol 9 Galactitol 10 o.Glucitol
6
7 0
Figure 2.
I
I
I
I
I
I
I
I
10
20
30
40 Time, min.
50
60
70
80
Gas chromatogram of alditol acetates separated on a 10-foot column containing 3% ECNSS-M
Sensitivity: Range 100, attn. 8x, column temp. 190' C., detector temp. 2 7 0 " C., injection port temp. 300" C., helium flow 60 ml./minute, sample size 4 pt. of 1 % solution
VOL. 37, NO. 12, NOVEMBER 1 9 6 5
1603
Table II. Analyses of Two Monosaccharide Mixtures
Recovery, yoComponents A" Bb D-Fucose 98.2 98.0 &Arabinose 100.8 99.2 n-Ribose 100.8 99.0 n-Mannose 98.9 98.2 D-Galactose 98.9 98.0 D-Glucose 102.4 97.8 0 5.0 mg. of each component. * 2.5 mg. of each component.
acetylated derivatives. The resulting mixtures of acetylated derivatives were separated and quantitated with xylitol as the internal standard (Table 11). Data obtained indicate that both reduction and acetylation of monosac-
charides are quant,itative and that the method is excellent for analysis of glycose mixtures. LITERATURE CITED
(1) Abdel-Akher, M.,Hamilton, J. K., Smith, F., J . Am. Chem. SOC.73, 4691
(1951). (2) Bishop, C. T., Can. J . Chem. 38, 1636 (1960). (3) Bishop, C. T., Advun. Carbohydrate Chem., 19, 95 (1964). (4)Bishop, C. T., Cooper, F. P., Murray, R. K., Can. J . Chem. 41,2245 (1963). (5) Gunner, S. W., Jones, J. K. N., Perrv. M. B.. Chem. Ind. (London) 196l"255. (6) Gunner, S. W., Jones, J. K. N., Perry, M. B., Can. J . Chem. 39, 1892 (1961). (7) Hause, J. A,, Hubicki, J. A., Hazen, G. G., ANAL.CHEM.34, 1567 (1962). (8) Richey, J. hl., Richey, M. G., Jr., Schraer. R.. Anal. Biochem. 9.272 (1964). (9) Sawardeker, J. S., Sloneker, J. H:, ANAL. CHEM.37, 945 (1965).
(10) Sweeley, C. C., Bentley, R., Makita, M., Wells, D. D., J . Am. Chem. SOC. 85, 2497 (1963). (11) Sweeley, C. C., Walker, B., ANAL. CHEM.36, 1461 (1964). (12) VandenHeuvel, W. J. A., Homing, E. C., Biochem. Biophys. Res. Commun. 4,399 (1961). JAWAHAR S. SAWARDEKER JAMES H. SLONEKER ALLENEJEANES
Northern Regional Research Laboratory Peoria, Ill. Division of Carbohydrate Chemistry, 150th Meeting ACS, Atlantic City, N. J., September 1965. The Northern Regional Research Laboratory is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U. s. Department of Agriculture. hlention of firm names or trade products does not imply that they are endorsed or recommended by the Department of Agriculture over others not mentioned.
Determination of Magnesium in Uranium by Atomic Absorption S pectro metry SIR: Magnesium may be determined in luranium by atomic absorption spectrometry (AAS), using the method of additions, without prior separation. Previously this method has been used only after separation of most of the uranium by solvent extraction (1). The procedure consists simply of bringing the sample into a hydrochloric acid solution, dividing into three or more aliquots to which are added different quantities of a magnesium standard solution, and measuring the atomic absorbances. If hydrogen peroxide is required to dissolve the sample, it is subsequently destroyed by evaporating to dryness and redissolving in acid. The standard solution is magnesium metal dissolved in a minimum volume of hydrochloric acid and diluted as required. Atomic absorption measurements were made by the author at
Table I.
Results with Several Samples
Range of Mg
By AAS, Sample p.p.m. 1-A 6.5 1-B
2-A
2-B
3 4 5
1604
6.S
8.4 8.3 7.6 3.6 0.6
std. additions, pg.
By.
emismon spectrography, p.p.m.
0-30
... ...
0-60
10 f ca. 3
0-30 C-30 e30 0-60 0-75
