stirnation of
ars in
A Routine J.
E. JEFFERY,
E. V. BARTLOW, and W. d . POLGLASEI
Olympic Research Division, Rayonier Incorporated, Shelton, Wash.
A method is presented for the rapid routine determination of arabinose, xylose, galactose, glucose, and mannose in wood cellulose hydrolyzates without interference from uronic acids. It i s based on 6s procedure developed in this l a b o r d o r y several years ago employing a reflectance measurement on a colored chromatogram, and has been used extensively in analyzing the constituents of wood pulp hydrolyzates. A plot of Kwbelka and Munk K/S values vs. per cent sugar results in a line of very slight curvature over a wide range of c o n c e n ~ r ~ ~ ~ o n § ~
temperature of relatively large volumes of dilute sugar solutions. In the present procedure, the hydrolyzing acid is neutralized by the addition Qf barium carbonate which forms a barium sulfate-barium carbonate slurry. This mixed precipitate is filtered (without washing) and an aliquot of the filtrate is taken for evaporation. This use of an aliquot is justified, since control experiments showed no evidence of sugar adsorption by the insoluble barium sulfatebarium carbonate residue. A problem involved in determining monomeric sugars in the hydroljxates of woody plant residues is caused by the HE FIRST -4SALYGES Of wood Celpresence of 4-O-methy1-~-glucuronoxlulose hydrolyzates by paper chroylans in these materials. During the matography were reported by Gustafacid hydrolysis of 4-0-methyl-n-glucurosson, Sundman, and Lindh (5). Elunoxylans, an aldobiouronic acid [2-0tion of the chromatographically sep(4 - 0 - methyl - LY - D - glucopyranosyl arated sugars, folloLved by colorimetric uronic acid) - D - xylose] accuinulates estimation by various procedures, has in the acid hydrolyzates and is rebeen widely used in nearly all fields sistant to further acid hydrolysis (?'). where partition chromatography is This accumulation of aldobiouronic acid applicable (2, 8, 9, 1.2). The use of i s nob only rePponsible for some apdirect reflectance of the colored areas parent chromatographic loss of xylose, on the sprayed chromatogram has not but also may lead to erroneously high been as widely employed ( 1 1 ) . More estimations of mannose, since both recently, Saeman et al. reported the mannose and this aldobiouronic acid various chromatographic techniques inhave nearly identical mobilities on volved in the determihation of pulp paper chromatograms when using many constituents by either colorimetry or (or possibly all) acid irrigating solvents. reflectance (16). Analysis of pulp I n view of the latter coneideration, a n hydrolyzates by partition chroniatogirrigating solvent containing pyridine raphy and direct densitometry has (butyl acetate-pyridine-95% ethyl albeen reported recently (3) and the use cohol-water, 8 : 2 :2 : 1) was chosen for of paper chromatography in the ~ o o d the present method, since alkaline ircellulose field has also been reviewed (1). rigating solvents cause uronic acids to I n most methods of chromatographic remain on or near the starting line of analysis of hydrolyzates, the sugars paper chromatograms during irrigaare separated from the hydrolyzing tion, and thus prevent their interference acid either b y the use of a cation exwith the determination of neutral monochange resin, or b y precipitation of the meric sugars (8). Another favorable acid as an insoluble fialt. The ion exfeature of the above irrigating solvent change resin (or the precipitate) is is that it allows rapid and satisfactory then washed with copious quantities chroniatographic separation of the five of Iyater so as to recover the last traces sugars (galactose, glucose, mannose. of sugars. These procedures entail the arabinose, and xylose) most commonly time-consuming evaporation a t low found in wood cellulose and holocellulose hydrolyzates. Durso and Paulson (3) reported that 1 Present address, Department of Biocolor development may be improved chemistry, University of British Columbia, by suspending chromatograms, preVancouver, British Columbia, Canada.
1774
e
ANALYTICAL CHEMlSTRV
viously irrigated and sprayed n-ith color developing reagents, in an atmosphere saturated ~ i t hwater vapor. They speculated that humidification aided in the distribution of the spray reagent on the paper. This effect was also investigated. I n the present method the reflectance of the colored sugar spots is measured directly on the chroniatogram and the sugar concentrations are obtained from a plot of Kubelka-Nunk K / S values us. per cent sugar concentratior, of standard solutions. The K/S function is one of the many relationships derivable from the Kubelka and Munk theory (IO) and can be readily calculated from reflectance values using the forinula suggested by Foote (4):
Reflectance ( E ) can be described in terms of two coefficients which represent the fractions of incident light lost b y absorption ( K ) and by scattering (8) per unit thickness of material. Theoretically, the reflectance should he from a pile of sufficient thicknesfi that no change in reflectance is observed when the backing is changed frorn black t o white. I n the present method, only a single thickness of chromatographic paper is used so that some reflectance takes place from the backing. However, the K / S function is still useful. REAGENTS AND APPARATUS
Standard Sugar Solution. Appropriate sugars are dissolved in 0.2570 benzoic acid (for preservation) and standards prepared t o cover the range from 0.05 to 0.4y0 sugars. For analysis of pulp hydrolyzates, equal quantities of D-xylose and Dmannose are used in each standard with sufficient D-glucose to g:lve a total sugar content of 10%. Evaporation Equipment. Rotary evaporators or flasks fitted with capillary air inlet tubes are used for concentration at reduced pressure. Humidity Cabinet. A stainless steel box large enough to hold 23l/? X 23l/2 inch paper sheets, with a n inlet a t the bottom for air saturated with
water vapor and an outlet a t the top. Water-saturated air can be supplied conveniently by passing compressed air through a fritted-glass disk inserted in the bottom of a 2-foot column of mater thermostatically controlled a t about 40" C Spectrophotometer. Beckman Model B with reflectance attachment. The sample holder is modified by replacing the three spring-loaded platforms by a single platforni with a single open cover plate and by adding a gear train which allows transverse movenient in addition to the normal backward and forn-ard movements.
draft oven (110' C.) for 5 or 6 minutes I / to develop the color of the sugar spots. 1
2.01 I
m
I
\
Y
METHOD
Hydrolysis. For simplicity in subsequent calculations, the weight of sample employed for analysis is such t h a t 1 gram of monomeric sugars would result from acid hydrolysis if no losses occurred. For routine rvork, i t is assumed t h a t dry pulp in equilibrium with the moisture in the air a t room temperature is 95% dry (oven-dry basis). The initial weight of air-dry sample employed, therefore, is 0.95 gram, However, sample size may be varied if desired. The pulp is fluffed in a blender-type disintegrator -e.g., Osterizer Mode 110-placed in a 200-ml. round-bottomed flask f 2 4 , ! ~ , an! $ mi. of 77% (by weight) sulfuric acid introduced a t room temperature. The contents are alternately stirred (glass rod) and the flask evacuated several times to promote rapid solution of the pulp in the acid. After 30 minutes, 145 nil. of distilled water is introduced and the flask placed in a boiling water bath for 4 hours. h stoppered water condenser attached to the flask throughout this period prevents changes in volume. Neutralization and Evaporation. After cooling to room temperature in a cold water bath, the hydrolyzate is transferred (without dilution) to a 400-ml. beaker and anhydrous barium carbonate added slovr~lywith stirring until the solution is neutral to litmus paper and carbon dioxide evolution has ceased. The resulting slurry is filtered in a Buchner funnel through a triple layer of dry 3-inch filter paper (E and D No. 613) into a dry receiver. An aliquot (75 ml.) of the filtrate is evaporated a t low temperature (35" to 45" C.) and a t reduced pressure to about 2 ml. This solution is then transferred (with intermediate filtration through a plug of purified cellulose in a piece of 7-mm. tubing drawn to a tip) into a 5-ml. volumetric flask. The evaporating flask is rinsed 2 or 3 times with 0.25% aqueous benzoic acid solution (1-nil. portions) t o complete the quantitative transfer of the sugar solution from the original flask into the 5 m l . volumetric flask, and also to bring the solution level up t o the mark. Chromatographic Separation. Duplicate aliquots (20 ~ 1 . )of both the appropriate standard sugar solutions and hydrolyzates which are to be determined are applied with transfer pipets to
K/S= I t /
I
1
I
I
_ I
2R I
I
.2 .3 .4 .5 .6 .7
% SUGAR Figure 1. Typical calibration curves for standard solutions of xylose and mannose obtained from per cent reflectance (R) measurements at 375
w 231/2-inch-square S &- S YD 90. 598 chromatographic paper which is serrated at one end. All paper for quantitative estimations is dipped into distilled mater and dried (without tension) prior to use. The sugar solutions are applied 1 inch apart on a line 5 inches from the nonserrated end of the paper, and after drying in air (about 15 minutes), the chromatogram is placed in a chromatographic cabinet (Research Eqbipment Corp., hlodel B 300 or equivalent) where it is irrigated by descending flow of a solvent composed of butyl acetate (practical grade)pyridine (practical grade)-95% ethyl alcohol-water (8:2:2: 1). The duration of the irrigation is about 16 hours if only glucose, mannose, and xylose are present in the hydrolyzates, but if arabinose and/or galactose are also present, the time should be extended to about 24 hours. Since, in either case, the irrigating solvent will drip from the end of the paper, the lower edge should be serrated to allow the solvent t o drip off the sheet evenly. Humidification and Color Development. After drying a t room temperature in a forced air hood (15 t o 20 minutes) to evaporate the major portion of t h e irrigating solvent, the chromatogram is hung for 2 hours in the humidity cabinet. Following removal from this cabinet, the chromatogram is air-dried (room temperature) for 5 or 10 minutes and then dipped in the color developing solution which consists of 0.7% aniline and 1.4% monochloroacetic acid dissolved in diethyl ether. After air drying (3 t o 4 minutes), the chromatogram is again hung for 2 hours in the humidity cabinet. After removal from this cabinet, the chromatogram is placed in a forced
Estimation of Sugars. The chromatogram iscut into stripsabout 1inch wide (parallel to the direction of solvent flow during the irrigation) containing t h e sugar spots to be measured. Reflectance measurements are then made a t 375 mp using a Model B Beckman spectrophotometer. The instrument is set at 100% reflectance using a strip of unused chromatographic paper in the sample holder. The minimum percentage reflectance of each sugar spot is then read and converted to Kubelka-hIunk K / S values, using a table (4). Similarly, several background readings are made adjacent to the sugar spots which also are converted to K/S values. These background readings correspond to zero sugar concentration and are an integral part of the plot of the K/X values US. percentage sugar (solution concentration) of the standards. The unknowns are determined from a separate plot for each sugar, such as shown in Figure 1. Calculation. Xylose is converted to xylan as follows: Per cent xylose from graph X 0.88 X 10 Oven-dry weight of sample = per cent xylan in pulp
likewise : Per cent,mannose from graph X 0.90 X 10 Oven-dry weight of sample = per cent mannan in pulp
For most routine samples, the calculation is simplified by using 0.95 gram of air-dried pulp and assuming that this is equivalent t o 0.90 gram of Ovendried pulp. DISCUSSION
The initial dissolution of the pulp i j similar to the procedure described by Saernan (14, except that 77% rather than 72% WzS04 is used. K e have found no quantitative differences in results at these two acid concentrations, but have preferred the 77% H,S04, since difficultly soluble samples dissolve more readily. Evacuation of the flask during treatment of the pulp sample with 77% H2SO4 also facilitatee the dissolving of certain samples (6). Because the duration of the heating period following dilution is not critical (in the range of 3 to 5 hours), 4 hours was chosen as the standard time. Dipping of chromatographic papers prior to use improves subsequent spot uniformity and decreases variations in the distances traveled by a given sugar at the various positions across the width of the sheet during chromatographic irrigation Dipping is believed to relieve strains formed in the chromatographic paper during the manufacturing process. For each lot of VOL. 32, NO. 13, DECEMBER 1960
0
1776
chromatographic paper there is usually
a preferred direction for chromatographic irrigation (in respect to spot uniformity) either v,ith or across the machine direction; however, this preferred direction must be established by trial for each new lot of paper. Work in this laboratory with known mixtures or" sugar standards has established that the presence of a relatively high concentration of c-glucose in a mixture of D-glUCOhe, D-mannose, and ~ x y l o s e such , as is found in wood pulp hydrolyzates, causes a small but definite apparent losg of mannose but not of xylose, following chromatographic irrigation and color development. 'To correct for this small apparent chromatographic loss of niannose in the presence of relatively high concentrations of glucose, the latter is added to the standard sugar solutions in sufficient quantities to approximate the ratio of glucose to mannose (or xylose) actually encountered in wood pulp hydrolyzates-Le., t o mixtures containing equal amounts of xylose and mannose (0.1, 0.2, 0.3, or 0.4 gram per 100 ml. of both xylose and mannose), sufficient glucose is added to bring the total sugar concentration to 10.0 gram per 100 ml. For maximum accuracy in the determination of any sugars, it is good practice t o have the concentrations of all of the sugars in the standards of the same order of magnitude as the sugars in the unknowns. During routine chromatographic work, it is convenient either to introduce or remove chromatograms from the chromatographic cabinet without causing undue changes in the composition of the irrigating solvent due to losm of highly volatile solvent vapors, Routine use of the 6:2:2:1 irrigating solvent for many months has clearly dexonstrated that i t is much jess sensitive in this respect than are similar solvent systems featuring ethyl acetate as the main component. Another soivent system which has been used successfully is butyl alcohol saturated with water and containing 2.5% phthaiic acid (5). After elution with this solvent, the chromatographic paper is dried and dipped in a 4% aniline in ether solution and the color developed by heating a t 110" C. for 10 minutes (6). The colors of the sugar spots are similar to those obtained with the method described in this paper-that is, brownish for the hexoses and pink for pentoses. The uronic acids do not stay on the starting line with this latter solvent and if the aldobionronic acid is present, i t will interfere with the mannose determination. This solvent also does not separate galactose from glucose or mannose from arabinose as well as the 8 :2 :2 :I solvent. However, for roufine
determinations of mannose and xylose only in cellulose hydrolysates, the saturated butyl alcohol solvent has some advantages, since it appears to be slightly more stable than the 8:2:2:1 solvent and less sensitive to minor variations in technique. For most routine determinations of xylan and mannan, these two solvent systems give essentially the same results, provided appreciable amounts of aldobiouronic acid are not present. Humidification was found to improve the chromatograms in several ways: Background colors of humidified chromatograms were (following color development) considerably lighter in color than were s h i l a r nonhumidified chromatograms. This improvement in background color is believed to be related to the removal of reeidual pyridine and butyl acetate (as azeotropes) from the chromatograms during humidification, which prevents the subsequent reaction of these compounds with the color developing reagents. Pronounced odors of both pyridine and butyl acetate during humidification of chromatograms. even after drying overnight, indicate that these reagents are strongly absorbed by chromatographic paper. Sugar spot densities on the finished chromatograms were also intensified markedly by the use of a humidification step. It is believed that this intensification of spot density may be explained on the basis of the relative accessibility of cellulosic chromatographic paper to nonpolar and polar liquids-i .e., the irrigating solution contains polar solvents and upon evaporation of this solution from the chwmatographic paper following irrigation, the sugars are partially deposited in regions of the three-dimensional structure of the chromatographic paper which are not accessible to nonpolar liquids. Since, in the present procedure, the color developing reagents are applied to the paper dissolved in a nonpolar solvent, it appears reasonable t o believe that the water absorbed during subsequent humidification transports the color developing reagents deeper into the structure of the paper where they may react with sugars not previously available to these reagents in nonhumidified chromatograms. For routine work, it is desirable to use a single wave length for measuring the reflectance of the colored sugar spots. The wave length of 375 mM was, therefore, selected as a compromise between the minimum reffectanee of pentose spots a t about 365 mp and the minimum reflectance of hexose spots at about 400
mM. The sugar concentrations may be estimated from the reflectance readings in several ways. A plot of per cent reflectance against per cent sugar concentrations of the standards gave a,
line with considerable curvature A plot of absorbance us. per cent sugar was linear a t low concentrations (below Q.2Yo),but deviated appreciably from linearity at bigher concentrations. A plot of logarithm of per cent refieetanee us. per cent sugar gave a fairly satisfactory linear relationship; honever, zero sugar concentration (background reading) cannot be used as one of the points on the standard curve. The preferred method is t o convert the reflectance readings of the standard sugars to K / S values and plot K / S against per cent sugar. This plot gives acceptable linearity orer a relatively wide concentration range, For accurate work, standards must be included with the unknowns on each chromatogram, since a number of factors can cause small variations in sugar spot densities from chromatogram to chromatogram and consequently change the slope of the plot of K/#V8. per cent sugar. PRECISION AND ACCURACY
Reproducibility tests were run on four samples reprerenting dissolving and paper grades of sulfite and kraft pulps. Eight xylan and mannan tests were carried out on each sample over a period of several weeks. The results are shown in Table I. The use of the terms xylan and mannan in Table I does not imply that these carbohydrates were present in the original sample as such, but rather are used as a convenient way of reporting composition on an anhydro sugar basis. As mentioned previously, some xylose is not determined, since it remains as aldobiouronic acid. Some losses of carbohydrate material are to be expected during the acid hydrolysis step prior t o chromatographic analysis. To correct for these losses, it viould be desirable to carry out similar hydrolyses with other pure carbohydrate polymers representative of those which occur in the various soft and hardwood pulps prepared by both acidic and alkaline proeesses-eg., gIucan (cellulose), gluconiannans, galactoglucomannans, arabogalactans, araboxylans, xylans, 4 - 0 - methylglucurono-araboxylans, 4 - 0 - methylglucurono-xyians, etc. A thorough study of this kind R-oulc! be difficult An alternative and time-consuming. approach is to subject the pure sugars such as glucose, mannose, xylose, ete., to hydrolysis and check for recovery. Justification for this alternative approach has been presented by Saeman and coworkers in studies of this nature on monomeric sugars and they have shown (14), using their method, that the recovery varies from about 97% for galactose to 91% for xylose. Re-
sults obtained in this laboratory using pure monomeric sugars have shown reasonable agreement with those of Saeman. Work on this problem is still continuing. For absolute determinations, corrections for losses which occur during hydrolysis and subsequent steps must be applied. I n routine work, however, where the change in hemicellulose content is being studied with respect to various process variables, correction €or these minor losses can be disregarded, since a relative comparison is equally useful. Wood hydrolysates have been analyzed chromatographically in this laboratory for about 9 years. About 4 gears ago, xylan and mannan tests were riln on three pulps submitted by the joint ASTM-ACS-TAPPI Committee on Chromatographic Methods. The agreement with results obtained at the Forest Products Laboratory b y Saeman (13) was very good with the exception of the mannan of pulp A (Table 11). The method described in this paper using the 8:2:2:1 solvent has been in use for approximately 2 years without change. The method requires reasonable care, but can be handled easily by laboratory technicians with limited training. On an 8-hour-day basis, one laboratory technician can conveniently average 6 samples per day. Working on a three-shift basis, results could be obtained in 25 to 30 hours of elapeed time. ACKNOWLEDGMENT
The authors express their thanks to
J. K. Hamilton for his interest and advice throughout this work, and to
Table 1. Precision of Method Sulfite Kraft Sulfite Kraft (Dissolving) (Dissolving) (Paper) (Paper)-Xylan Alannan Xylan Mannan Xylan Mannan Xylan Mannan
%
%
%
%
%
L‘io
%
%
1.1
1.0
8.7
0.9
1.2 1.3 1.2
2.2 2.2
8.7 8.7 8.9 8.9
7.0 6.9
1.0
1.8 2.0 1.8 1.9 1.8 2.0 2.0 2.0 1.9 0.10 0.04
1.2
1.0 1.0 1.0
0.9
1.1
1.1
1.1
1.0 0.10
1.2 Av. Std. dev. Std. error
1.1
0.09 0.03
1.0 1.0
1.2
0.04
Otto Goldschmid for assistance in preparing the manuscript.
1.3
1.0 1.2
1.1
1.2 1.2 0.10
0.04
8.9 8.6
2.2
2.3
8.7
2.2
0.06
0.02
Table II.
9.2
8.7 8.7
9.1
...
9.0
2.3 2.2 2.1 2.2
8.5
...
8.9 0.21 0.07
8.8
(19.58). , ~ ~ - - I
(9) Hough, L., Jones, J. K. N., Wadman, W. H., Nature 162, 448 (1948). (10) Kubelka, P., Munk, F., 2. tech. Physilc 12, 593 (1931). 1111 RlcCreadv. R. hl.. McComb. E. A.. ANAL.CHE
