Table 111.
Source
Foreign ion
Cd +2 Sn +2 Sn +4 Pt+4 Fe +a Cr +3 Be +2 Zr + 4 Th +4
c5;4 v +5 U
Ti+4 Zn+2 Mn+:a
co
c o +a h-i +2 Ca + 2 Sr + 2 Ba +a w04-'
ASOs-3
m7oz4-6
Cr04-2 Se03-2 po4-3 CN SCN SZO~-~ EDTA-4 Tartrate-3 Oxalate-2 Citrate-3 Acetate+ hlalonate-2 Ascorbate-2
Effect of Diverse Ions
3CdSOa -8Hz0 SnC4 2H20 SnCl4 HzPt Cls .2H20 Fe(NO3)a CrC13 6Hz0 BeSO4 4Hz0 Zr(NOd4 Th(NO& 12H20 Ce(SO& 4H20 U02(N0a)z * 2H20 +
vosoc
Ti(S04)~ ZnSO4 *7Hz0 MnCll .6H20 Cos04 *7H20 C0(NOa)s*6H20 NiClz .6Hz0 CaS04.5H20 SrCl2'2H20 BaC12 '2H20 Na2W04 e2Ha0 NalAsOa (NH&M070r4 * 12H20 K2Cr04 NazSeOs (NHa)3P04 KCN KSCN NazSrOa * 5Hz0 EDTA (disodium salt) Tartaric acid Oxalic acid Citric acid Acetic acid Malonic acid Ascorbic acid
Tolerance limit, pg. Pd(I1) Pt(1V) 457 457 720 720 40 '78 80 39 120 950 760 ... 450 640 800 350 ... 0 ... 0 ... 500 464 ... 575 505 350 540 216 480 490 505 1000 450 450 ... 750 250 510 740 ... 640 ..* 640 640 ... 525 525 505 ... 385 384 580 870 530 605 0 540 0 200 0 0
550 0
... 0
500 500
440
500 480 500 500
...
300
1020 950 930 323
From 10 experiments the average recovery of palladium or platinum was 98.5 f 1.5%. Each determination took a total of 40 minutes. The standard deviation was *2%. The special feature of this method is its applicability to the selective extraction of palladium(I1) or platinum(1V) in the presence of a large number of other ions at microlevels.
LITERATURE CITED
(1) Ayres, G. H., Alsop, J. H., 111, ANAL. CHEM.31, 1135 (1959). (2) Ayres, G. H., Meyers, A. S., Zbid., 23, 299 (1951). (3) De, A. K., Rahaman, M. S., Analyst 89. 705 (1964).
(4) Dhara,'S. C:, Khopkar, S. M., Mikrochim. Acta 1965 5 . ( 5 ) Dhara, S. ( Khopkar, S. M., ANAL. CHEM. 37, 1158 (1965). ((5) KhoDkar. S. M., J . Sci. Znd. Res. (India), 24, :142 (1965). (7) Morrison, 3. H., Freiser, H., ANAL. CHEM.?'L, 73R (1962). (8) Morris()n, G. H., Freiser, H., Zbid., 36, l l l R (19-164). (9) Parshall, G . W., Wilkinson, G., Chem. Znd. (London) 1962, 261. (10) Sandell, E. B., "Colorimetriq, Determination of Tracclq nf Metals, 3rd ed., p. 726, Interscience, hlew York, ncn I
__
3
LYJY.
SHRIPAD M. KHOPKAR
1000
1000 660 660 680 800 680
Department of Chemistry Indian Institute of Technology Powai, Bombay-76 N.B., India Project sponsored by the Council of Scientific and Industrial Research (India).
Gas Chromatographic Sugar Analysis in Hydrolyzates of Wood Constituents Quantitative Determination of Mixtures of Arabinose, Xylose, Mannose, Galactose, and Glucose as Their Trimethylsilyl Ether Derivatives
SIR: A rapid and accurate quantitative method for the five sugars commonly found in wood product hydrolysates is needed. Paper chromatography requires about 16 hours to separate arabinose, xylose, mannose, and glucose and an additional 14 hours are needed to separate galactose from t,he high glucose concentrations normally encountered. If the concentration of a single sugar exceeds about 4 mg./ml., the original solution must be diluted and rerun before meaningful reflectance measurements (6) can be taken. I n this laboratory an average of 2.5 days is required, after hydrolyzing the sample, to report four or five sugars. Sweeley, et al. (8) gave a very detailed report of the gas-liquid chro362
ANALYTICAL CHEMISTRY
matography of trimethylsilyl ether (TMSE) derivatives of sugars and related compounds. They showed that arabinose] xylose, mannose, , galactose, and glucose could be identified on a column packed with EGS or SE-52 on Chromosorb-W. Quantitative determinations were not attempted. Richey et al. (6) reported a method using methyl-a-D-galactopyranoside as an internal standard for the analysis of sugars (fucose, mannose, galactose, and glucose). Results were somewhat erratic with an average error of 10%. They operated the gas chromatograph isothermally and used an argon ionization detector which requires a different calibration factor for many isomers and stereoisomers (3). Their standard is not available commercially. Other
methods ( I , 9) have been reported for the determination of glucose as its TMSE derivative. After this work was completed, the separation and determination of arabinose, mannose, galactose, and glucose using methyl-a-Dmannopyranoside as an internal standard was reported ( 7 ) . However, xylose could not be determined in the presence of glucose and an arabinose anomer overlaps a--mannose. Isothermal operation required 70 minutes to elute 8-glucose. I n the present method the problem of numerous calibration factors has been avoided by using a hydrogen flame ionization detector. This detector has been shown (3,4) to give uniform results on a weight basis for stereoisomers and alicyclic and aliphatic compounds of
widely varying molecular weight. Temperature programming eliminates any problem with the determination of arabinose resulting from tailing of the pyridine peak and also permits calculation by peak height, since all the peaks, including that of inositol which is used as an internal standard, have essentially equal half-peak widths. EXPERIMENTAL
Reagents. Standard sugar solutions were prepared using L-arabinose, D-xylose, D-mannose, D-galactose, and n-glucose. Benzoic acid was added as a preservative. Sugars and iinositol were obtained from Mann Research Laboratory and Pfanstiehl Laboratories, Inc., hexamethyldisilazane (HMDS) was obtained from Analabs, Inc., and trimethylchlorosilane (TMCS) from the General Electric Co., Silicone Products Division. Apparatus. The gas chromatograph was a Perkin-Elmer Corp. Model 800 with dual columns and hydrogen flame ionization detector. A Hamilton 10-pl. syringe with Chaney adaptor was used for sample injection. The columns evaluated consisted of 3% SE-52 (silicone gum rubber) on 60- to 80-mesh Chromosorb-W in 8foot, 5-mm. i.d. columns (Perkin-Elmer Corp.); 15% EGS (polyethylene glycol succinate) on 80-100-niesh, acid-washed Chromosorb-W in 1.5-mm. and 5-mm. i.d. columns (Applied Science Corp.) ; and 14% EGS on acid-washed Chromosorb-W (Applied Science Corp.). The latter material, which gave the best results, was packed in 8-foot, 5-mm. i.d. columns. Conditions. Helium flow was 140 ml./minute with 20% split (28 ml./ minute) to the detector; hydrogen, 35 ml./minute; and air, 310 ml./ minute. The injection block temperature was 300" C.; program rate, 2" C./minute from 110" to 190' C. Procedure. With a calibrated micropipet, a 0.500-1nl. aliquot of a neutralized wood cellulose hydrolyzate (6) (or any aqueous solution containing 100 mg./ml., or less, total sugars) and 0.250 ml. of an inositol solution containing 10.00 mg./ml. were added to a 1-dram (14.5 X 45 mm.) vial. The vial was then connected by means of an adaptor to a rotary evaporator. An apparatus which will evaporate water from four vials a t one time was constructed. The mixture of sample and standard was evaporated to dryness in a 40' C. water bath (10-15 minutes). Pyridine (1.4 ml.) was added and the sugars were dissolved. If galactose and glucose are to be determined together, there should be minimum time for the dissolution and no heating, otherwise the aqueous equilibrium concentrations of the anoniers may change. HMDS (0.4 ml., 2.0 mmoles) was added, the vial stoppered and shaken vigorously. TMCS (0.2 ml., 1.6 mmoles) was added and again the vial was shaken for a t least 1 minute. The vials should be fitted with polypropylene stoppers to prevent reaction with the
Time, m i n u t e s
Figure 1 .
Chromatogram of five sugars and inositol as TMSE derivatives
15% EGS on 80/100 Chromororb W, 5 mm. 1.d. 8-foot column, 2'C./minute
TMCS which is very corrosive. No significant difference was found in the equilibrium values when HMDS and TMCS were added immediately or 2-3 minutes after the pyridine addition. A fine suspension of NH&1 formed and eventually settled, but did not interfere with immediate analysis of the sample. Two microliters were injected into the gas chromatograph. Calculation. The following equation gives the concentration of an unknown sugar in mg./ml.: (peak height sugar) (10) (peak height inositol) (2)
mg./ml. sugar
where 10 is the concentration of the inositol standard in mg./ml. and 2 corrects for the difference between the aliquots of standard and sample. DISCUSSION OF RESULTS
Figure 1shows a chromatogram of the five sugars commonly found in wood hydrolyzates and inositol added as an internal standard. This sample was composed of 20% of each sugar and 5% inositol. I n a typical pulp hydrolyzate, the a-and @-glucosepeaks go off scale and no attempt is made to attenuate them. Peak heights are used in the calculation, since the half-peak widths of all the sugars and the standard are essentially equal. The peak heights of arabinose and xylose, respectively, are totaled. A minor xylose peak equal in height (area) t o the one labeled "trace" is present underneath the a-arabinose peak. Therefore, the height of the minor xylose peak is subtracted from arabinose and added to xylose. The a-and @-mannose peaks are totaled; if an inefficient packing is used and the @-mannose is not well separated from P-galactose, the a-mannose may be divided by 0.72, our value for the distri-
100-1 80°C.
bution of the a-anomer in an aqueous solution; Sweeley (7) also reported 0.72. T o determine glucose in the presence of galactose, the peak height of @glucose is divided by 0.60, which
Table 1. Response of the Hydrogen Flame. and Argonb Ionization Detectors to Pentose and Hexose TMSE on an Equal Weightc Basis
Name Mol. wt. Flame Argon Ribose 150 0.80 Arabinose 150 1.00 Xylose 150 1.00 Fucose 164 0.78 Mannose 180 1.00 0.79 Galactose 180 1.00 1.00 Glucose 180 0.99 1.00 This work; i-inositol = 1.00; average value (see text), relative standard deviatinn = Sal, ___._
J. MY %hey, et al. (6). carbohydrate, not TMSE Weight of carbohvdrate. derivative. derivatiGe. b
c
Table 11. Precision of TMSE Synthesis of Sugars Relative to Inositol"
Arabinose
Xylose
5.17 4.96 5.26 5.00 4.83 4.80 4.91 5.16 5.10 5.00
Average 5.02 Rel. std. dev.,
%
3.2
Mannose
Galactose
5.08 4.88 4.97 4.96 4.87 4.65 4.88 5.i6 5.09 5.13
4.83 4.80 4.95 4.86 4.79 4.90
5.15 4.75 4.70 5.05 5.04 4.95
4.92
A lQ
4.92 5.00 5.18
4.95 5.22 5.08
4.97
4.92
5.03
3.1
2.5
3.5
Concn. in mg./ml.; all samples contained 75 mg./ml. glucose.
VOL 38, NO. 2, FEBRUARY 1966
0
363
Table 111.
Sample No.
Gas Chromatography of Known Sugar Mixtures"
Arabinose
Added 1.13 Found 1.13 2 Added 10.0 Found 10.6 3 Added 0.47 Found 0.49 4 Added 0.22 Found 0.22 5 Added 5.00 Found 5.00 Av. of duplicate injections, mg./ml. 1
a
Table IV. Comparison of Gas and Paper Chromatography of Xylose and Mannose in Wood Cellulose Hydrolyzates"
Xylose, % Mannose, % SamG.C. P.C. ple G.C. P.C. 2.1 2.1 7.0 A 7.0 8.9 8.8 8.3 B 7.9 18.2 17.7 4.5 4.1 C 1.8 1.3 1.9 D 1.3 18.3 4.3 17.4 E 4.1 a Gas chromatography values are an average of three injections of the same TMSE solution; paper chromatographic values are an average of three chromatograms.
UI al
0 0
c
-
0 0 01 I
a
Xylose 5.08 4.98 4.00 4.20 0.98 1.02 0.17 0.16 5.00 5.13
Mannose Galactose Glucose 10.7 10.2 4.00 4.00 2.38 2.40 0.11
0.10 5.00 4.96
2.57 2.55 8.00 8.40 0.72 0.85 0.09 0.07 5.00 5.17
82.4
...
30.0 29.5 25.4 25.3 90.4
...
75.0
...
agrees with the value obtained by Sweeley. Figure 2 shows the distribution of the anomers of galactose from an aqueous solution after not more than 2 minutes in pyridine. Glucose is an impurity (6.5% total) as confirmed by paper chromatography. Galactose from two suppliers gave similar results. Our values of 26.4, 55.9, and 10.5% for CY-, p-, and y-galactose do not agree with previously reported (8) values of 31.9, 62.6, and 5.4%. A previously unreported (6, 7, 8) fourth galactose peak which appears as a shoulder immediately prior to b-galactose, when added to @galactose, changes our percentage to 63.2. With an SE-52 column, this separation is not observed and we obtained 61.0% for 8-galactose. To determine galactose in the presence of glucose in an unknown, the peak height of @-galactoseis divided by 0.56. Relative Response of Inositol and Sugar TMSE Derivatives. Inositol was selected as an internal standard, since it is readily available, gives a single peak, is not generally found in wood samples, and has the same molecular weight as the hexoses, although one more hydroxyl group. Table I shows the response of the five sugars relative to inositol. Duplicate TMSE derivatives of ten 5-sugar mixtures were prepared to contain 2.5 mg. of inositol and amounts of each
sugar varying randomly from 0.5 to 12.5 mg. Results in Table I1 show the precision of the method for ten TMSE syntheses of a mixture of four sugars with a large amount of glucose to simulate a pulp hydrolyzate. Table I11 lists the results obtained with known sugar mixtures ranging in individual concentrations from 0.01% to 3%. Base-line noise interferes at lower levels and the column overloads a t higher levels (3 to 4%). The 1.5-mm. i.d. columns began to overload when ca. 0.4% samples were used. This seems to agree with the calculations of De Wet and Pretorius (2) that, for small diameter columns, the volume of sample that can be handled a t any chosen efficiency is proportional to the square of the column diameter. Sample size can be increased or d e creased to determine smaller or larger amounts of sugar. Table I V shows good agreement between the gas and paper chromatographic (5) methods for xylose and mannose in wood cellulose hydrolysates over a wide range. SE-52 columns were initially tried and are well suited for programmed operation. In these columns, however, galactose cannot be determined in the presence of large amounts of glucose, since a minor ( ~ 1 . 3 % )glucose peak has the identical retention time of a-galactose, the only isolated galactose peak in the presence of glucose. ACKNOWLEDGMENT
The authors express their thanks to Otto Goldschmid for assistance in preparing the manuscript and to D. W. Teets for helpful discussions during the course of this work. LITERATURE CITED
( 1 ) Alexander, R. J., Garbutt, J. T., ANAL.CHEM.37, 303 (1965). (2) De Wet, W. J., Pretorius, V., Ibid., 32, 1396 (1960). (3) Ettre, L. S., "Gas Chromatogra hy," p. 231, Brenner, N., Callen, E.,
Weiss, M. D., Eds. Academic Press, New York, 1962. (4) Ettre, L. S., Kabot, F. J., J . Chromatog. 11, 114 (1963). (5) Jeffrey,J. E., Partlow, E. V., Polglase, W. J., ANAL.CHEM.32, 1774 (1960). (6) Richey, J. M., Richey, H. G., Jr., Schraer, R., Anal. Biochem. 9, 272 (1964). (7) Sawardeker, J. S., Sloneker, J. H., ANAL.CHEM.37, 945 (1965). (8) Sweeley, C. C., Bentley, R., Makita,
M., Wells, W. W., J . Am. Chem. SOC.
4 Figure 2. Chromatogram of galactose TMSE derivatives Galactose TMSE, aqueous equlllbrlum
a 364
ANALYTICAL CHEMISTRY
85, 2497 (1963).
(9) Wells, W. W., Chin, T., Weber, B., Clin. Chim. Acta 10, 352 (1964).
H. E. BROWER J. E. JEFFERY M. W. FOLSOM
Rayonier Inc. Olympic Research Division Shelton, Wash. Analytical Chemistry Section, Northwest Regional Meeting, ACS, Corvallis, Ore., June 14, 1965.
