However, C and N analysis could be carried out in 90 seconds (see Figure 3). Table I1 is a comparison of this procedure with others reported in the literature. Certainly a standa-d deviation of 1.0.17 for N is not realistic based on the results of only a few samples, but it does indicate that the procedure works and is not out of line with previous work. The next step in the overall procedure is to add halogens to the system.
RECEIVEDfor review November 18, 1965. Resubmitted August 15, 1966. Accepted August 15, 1966. Work supported by the National Aeronautics and Space Administration through operating funds to carry out this project, and by the National Science Foundation through funds to purchase the chromatograph and the recorder. Presented at the 1st Midwest Regional ACS Meeting, Kansas City, Mo., November 1965. This study represents partial fulfillment (S.P.) of the requirements for the Ph.D. degree in chemistry.
Determination of the Carbohydrate Composition of Wood Pulps by Gas Chromatography of the Alditol Acetates Edwin P. Crowell and Bruce B. Burnett Research and Developntent Dicision, Union Camp Corp., P.O. Box 412, Princeton, N. J.
THERE IS A NEED in the ield of wood and wood pulp chemistry for a rapid and more accurate method for the determination of the five sugars normally obtained by the hydrolysis of wood products. The traditional paper chromatographic method is very time consuming, cumbersome and tedious to apply ( I ) . Gas chromatographic analysis of monosaccharides has received much attention of late as a result of the pioneer work of Sweeley and coworkers with trimethylsilyl derivatives (2). Other workers have followed the silyl ether derivative approach to the quanatitative analysis of sugar samples ( 3 - 3 , Three reports have appeared which utilize the silyl ether derivatization in the analysis of pulp hydrolyzates (6-8). The trimethylsilyl derivatization is simple and rapid, but it does have shortcomings. Because water reacts with the reagents, it is necessary to employ extensive drying procedures. The chromatography is difficult to interpret because each of the five sugars produces several anomers which result in chromatograms with 14 components. Complete resolution has not yet been achieved for this system. Consequently, the chromatographic conditions must be rigidly controlled to maintain analytical precision, and the data reduction is cumbersome. It has been our experience that silanization of simple sugars is not always quantitative and sometimes requires extended reaction times. In addition. the literature also indicates that there have been problems with lack of reproducibility in the conversion of steroids to trimet iylsilyl ethers for gas chromatography (9).
It appeared to us thai the successful development of a gas chromatographic method for pulp sugars capable of routine
(1) J. F. Saeman, W. E. Moore, R. L. Mitchell, and M. A. Millet,
Tuppi, 37, 336 (1954). (2) C. C. Sweeley, R. Bentley, M. Makita, and W. W. Wells, J. Am. Chem. Soc., 85, 2497 (15163). (3) R. J. Alexander and J. T. Garbutt, ANAL.CHEM., 37, 303 (1965). (4) J. Kagan and T. J. Malxy, ANAL.CHEM., 37, 288 (1965). (5) J. S. Sawardeker and J . H. Sloneker, ANAL.CHEM.,37, 945 (1965). (6) 0. Bethge, C. Holmstrom, and S. Juhlin, Scensk Pupperstidn 68, 60 (1965). (7) H. E. Brower, J. E. Jeffery, and M. W. Folsom, ANAL.CHEM., 38, 362 (1966). (8) D. W. Clayton and M. E. MacMillan, American Chemical Society Meeting, Phoenix, Ariz., January 1966. (9) H. L. Lou, J. Gus Chromatog., 4, 136 (1966).
application depended on a derivatization scheme that gave one volatile product for each sugar. Gunner, Jones, and Perry (IO)showed that single peaks are obtained for the alditol acetates of the sugars of interest in our work. Sawardeker, Sloneker, and Jeanes (11) extended this observation and obtained preliminary quantitative data which appeared encouraging. Their column consisted of a 37, liquid phase of an organosilicone polyester of ethylene glycol succinate chemically combined with a silicone of the cyanoethyl type. The purpose of this work was to investigate the alditol acetate approach to the gas chromatographic analysis of sugar mixtures resulting from the hydrolysis of southern pine wood pulps. Derivatization and chromatographic conditions were investigated and optimized. In the final procedure the sugar mixture in aqueous solution is reduced with sodium borohydride to form the alditols, which in turn are acetylated with acetic anhydride-pyridine. The acetates are isolated, dissolved in methylene chloride, and chromatographed. With the chromatographic conditions employed, excellent resolution of the five pulp components and the internal standard is obtained in 40 minutes. EXPERIMENTAL
Apparatus. The gas chromatograph used in this work was a dual column F & M Model 700 instrument equipped with a flame ionization detector. Chromatographic peak area measurements were made with an Infotronics Model l l H S electronic integrator connected directly to the electrometer. A 6' X 1/4''-o.d.column packed with 3z ECNSS-M on Gas Chrom Q (Applied Science Laboratories) was employed at a helium flow rate of 90 cc/min. The iiijection port of the instrument was modified to reduce the dead volume of the vaporizer assembly and to allow on-column injection. The column was operated isothermally at 180" C with an injection port temperature of 250" C and a detector temperature of 290" C. Standards. The monosaccharides employed as standards (D-glucose, D-galactose, D-mannose, D-xylose, D-arabinose, L-rhamnose) were obtained from Mann Research Labora-
(10) S. W. Gunner, J. K. N. Jones, and M. B. Perry, Can. J. Ckem., 39, 1892 (1961). (11) J. S. Sawardeker, J. H. Sloneker, and A. Jeanes, ANAL.CHEM., 37, 1602 (1965). VOL. 39,
NO. 1, JANUARY 1967
121
Table I. Empirical Calibration Component Factor, k Glucose 0.864 Xylose 0.798 Mannose 0.829 Arabinose 0.927 Galactose 1.75
Std dev 0.008 0.015 0.018 0.088
0.11
tories, N. Y. The standard monosaccharides were chemically assayed by the periodate oxidation method (12). Gas chromatographic purity checks of the alditol acetates of the standard sugar samples indicated that xylose and mannose contained small but significant amounts of one or more of the other sugars. Results of these determinations were used to arrive at corrected values for the components in the mixtures used for calibration. Procedure. The neutralized aqueous hydrolyzate from 0.35 gram of wood pulp was obtained using the procedure published by Saeman et al. ( I ) . To this solution 200 mg of rhamnose were added as an internal standard. The solution was concentrated to about 10 ml in a Rinco thin film evaporator, water bath temperature 60" C. The sugars were reduced with 0.4 gram of sodium borohydride contained in 10 ml of water. After 1 hour at room temperature the excess reagent was destroyed by treatment with acetic acid until gas evolution ceased. The solution was evaporated to dryness and dehydrated by adding 20 ml of methanol and again evaporating to dryness. The dehydrated residue was acetylated by refluxing with 10 ml of 1 : 1 acetic anhydridepyridine overnight. The acetylation mixture was evaporated to a syrupy residue, treated with 2 ml of water and evaporated to dryness to remove the acetylation reagents. The resulting acetates were dissolved in 10 ml of methylene chloride and one microliter of this solution was injected into the gas chromatograph for analysis. Calculation. The following equation is used to determine the concentration of the individual components.
x Polysaccharide
=
A X W , X C X 100 A, X W, X k
where: A = chromatographic area of component peak; A , = chromatographic area of rhamnose peak; WT= weight of rhamnose; W , = oven-dry weight of sample; C = conversion factor for monosaccharides to polysaccharides; 0.88 for pentoses, 0.90 for hexoses; and k = calibration factor for the individual component (See Table I). The results calculated as above are absolute values. It has been the practice in the field of wood chemistry to calculate the carbohydrate composition as sugar units, in which case it is not necessary to use an internal standard. The following calculations are then employed. A X C u, = -
k
u, x
100
xu,= zu,
where: U, is the relative sugar units of each component. RESULTS AND DISCUSSION
Reduction. A study of the sodium borohydride reduction of sugars by Abdel-Akher et al. (13) showed that the reduction rate at room temperature is a function of the amount of borohydride used. Accordingly, it was felt that the 3-hour reduction time used by Sawardeker (11)could be shortened by using (12) N. A. Cheronis and T. S. Ma, "Organic Functional Group Analysis," Interscience, New York, 1964. (13) M. Abdel-Akher, J. K. Hamilton, and F. Smith, J. Am. Chern. SOC.,73, 4691 (1951). 122
ANALYTICAL CHEMISTRY
87t85
a higher proportion of borohydride. Samples containing glucose and arabinose with mannitol as an internal standard were reduced with 0.4 gram of NaBH4 for reaction times varying from 30 minutes to 3 hours. After the reactions were stopped with acetic acid, all solutions were found to be nonreducing to boiling Fehling's solution. Subsequent acetylation and chromatography also demonstrated that the shorter reduction time was feasible. Acetylation. The acetylation reaction was studied to establish the optimum conditions for reproducible conversion of the reduced sugars. The degree of acetylation was determined by gas chromatography using mannitol hexaacetate as an internal standard. Initial studies using alditols corresponding to the sugars of interest showed that 1 gram of the alditols was completely acetylated with 20 ml of reagent in less than one hour at room temperature. However, when the sugars were carried through the reduction reaction, it was found that the resulting alditols were not completely acetylated at room temperature. It was suspected that the boric acid resulting from the reduction inhibited the acetylation rate. Therefore, the rate of acetylation was investigated starting with glucose. A sample was prepared containing glucose reduced with 0.4 gram of NaBH4and mannitol hexaacetate. Acetylation was carried out under reflux temperature in a flask equipped with a septum port for periodic removal of sample aliquots. The aliquots were chromatographed directly. Figure 1 presents the results of this study for acetylation times up to 27 hours. An independent experiment showed that the relative response for glucitol and mannitol hexaacetates is 1.00. Therefore, the acetylation is an absolute reaction efficiencyvalue, This study demonstrated that in the presence of the borate it is necessary to acetylate the alditols under reflux conditions for a minimum of 10 hours and that no further acetylation occurs up to 27 hours, the maximum time investigated. In other experiments with mixtures of the five pulp sugars and rhamnose, the internal standard, it was found that xylose and arabinose acetylated faster than rhamnose while glucose, galactose, and mannose acetylated slower. , Glucose exhibited the slowest acetylation rate of the six sugars. Two approaches were investigated for removing the borate prior to acetylation. First, hydrochloric acid was added to the methanol used in the dehydration step to convert the boric acid to the volatile methyl borate. Two treatments with 20 ml of 0.24N methanolic HC1 effectively removed the boric acid resulting from 0.4 gram of NaBH4. However, extremely small peak areas were found for the chromatograms of the samples
x
! r,
x
xs
IO L _ -
I ,--
x5
x 3 2-
0
I2
24 - u c
,.\
36
4 8
6G
48
Figure 2. Chromatograms of pulp sugar sample Top chromatogram: acc:tylation mixture injected directly Bottom chromatogram: methylene chloride solution of acetates Components: (1) rhamnose; (2) arabinose; (3) xylose; (4) mannose; (5) galactose; (6) glucose
treated in this manner. This result was attributed to either degradation of the sugar alcohols or interference with the acetylation reaction by .the high concentration of hydrochloric acid produced by the evaporation. In the second approach, the reduction mixture was treated with 50 ml of 0.24N methanolic HC1 in a distillation flask and the methyl borate was distilled over with methanol. During the distillation, methanol was added at a rate sufficient to keep the liquid level constant. The distillate was continuously monitored for methyl blxate by the characteristic green flame test. At the completion of the distillation, the hydrochloric acid was neutralized with ammonium hydroxide prior to evaporation and acetylation. The approach was somewhat successful but was of limited utility because the distillation took from 2-4 hour.j, required considerable attention, and was not convenient for handling large numbers of samples. After it was found that a series of samples could be hydrolyzed and reduced in one working day, overnight acetylation (14-16 hours) became the most efficient solution to the problem and was adopted. Gas Chromatography, Our experience has confirmed the conclusions of Sawardeker et af. (11) that the 3 ECNSS-M column is currently the most efficient chromatographic system for resolving the alditol acetates of interest. Sawardeker was interested in the resolution of 10 monosaccharides and as a result employed conditions resulting in a chromatographic time of 80 minutes. Because resolution of our six component system was somewhat simpler, it was possible to decrease the chromatographic time to 40 minutes by reducing the column length from 10 feet to 6 feet. In Sawardeker's procedure it was stated that the acetylation mixture was chromatographed directly. However, the chromatograms presented in his report were apparently produced with alditol acetates isolated from the acetylation reagent mixture prior to chromatography. In practice, direct injection of the acetylation mixture results in a chromatogram with considerable solvent and reagent tailing which interferes with the quantitative epaluation of the rhamnose and arabinose peaks. This is illustrated by the chromatogram in Figure 2. Programmed column temperature operation was investigated in an effort to reduce the solvent tailing effect and it was found that a programmed rate of 0.5" C per minute from 165" to 195" resulted in a suitable chromatogram as shown in Figure 3. In practice, however, it was very difficult to reproduce the
Figure 3. Temperature programmed chromatogram Components: (1) rhamnose; (2) arabinose; (3) xylose; (4) mannose; (5) galactose; (6) glucose
stable base line shown. Considerable difficulty was encountered in duplicating these results with different batches of the column packing, a factor also reported by other workers (6). Because isothermal operation is inherently easier to reproduce, we returned to it and employed derivative isolation methods to reduce solvent tailing. Under isothermal conditions we have had no difficulty with chromatographic reproducibility throughout an operating period of six months. Isolation of Derivatives. Efforts were made to separate the alditol acetates from the acetylation mixture prior to chromatography. Initially, we attempted to precipitate the acetates by pouring the acetylation mixture over ice, filtering and dissolving the residue in chloroform or acetone for chromatography. The pentoses were selectively precipitated, so this approach did not offer promise for quantification. Removal of the acetylation reagents was investigated, and it was found that most of the pyridine could be evaporated with a thin film evaporator at reasonable temperatures. To remove the acetic acid and anhydride, the residue from the pyridine evaporation was treated with 2 ml of water and again evaporated to dryness, Solubility studies indicated that methylene chloride was the best solvent for the alditol acetates and offered no problems to the subsequent chromatography. The chromatograms in Figure 2 demonstrate the effectiveness of this procedure. Internal Standards. It is most desirable to select an internal standard that undergoes the derivative-forming reactions (reduction and acetylation) and possesses solubility characteristics similar to the pulp sugars so that it can be added immediately after the hydrolysis step. Obviously, a monosaccharide not normally found in wood pulp hydrolyzates would best fit this requirement. From the chromatographic data reported by Gunner et ul. (10) ideal candidates would be allose or idose because they would be expected to elute between the pentoses and hexoses commonly found in wood pulp. However, allose and idose are not readily available in reasonable quantities. Inositol was explored for use as an internal standard and was found to elute just after glucose. However, selective losses, presumably due to the lower solubility of inositol in water, were found in the neutralization and concentration steps. In addition, since it is an alditol, inositol could not function as an internal standard for the reduction reaction. Rhamnose was found to be a suitable internal standard because it was readily available in high purity, it was not selectively lost in the sample workup procedure, and it participated in the derivatization reactions. The one experimental difficulty encountered with rhamnose was that it was the first component eluted and was most subject to interference by solvent tailing. By using isothermal chromatographic conditions and removing the acetylation reagents to minimize VOL. 39, NO. 1, JANUARY 1967
123
Table 11. Precision Data
Arabinan 0.6 0.8 0.8 0.8 0.8 0.7 Av 0 . 8 Std dev
-
Xylan
Mannan Galactan Glucan
8.1 8.1 8.2 8.2 8.3 8.2 8.2
6.2 6.2 6.2 6.1 6.3 6.6 6.3
0.5 0.6 0.6 0.7 0.5 0.7 0.6
72.8 70.6 73.2 71.6 73.3 73.1 __ 72.6 1.0
Table 111. Standard Pulp ICCA-4
Method GLC-Alditol acetates Paper chromatography GLC-Silyl ethersa Paper chromatography
2.5 2.5 1.9 2.2
88.1 86.3 89.0 87.4 89.2 89.3 88.2 1.2
Sugar Units
Glucose Mannose Xylose 95.4 95.3 96.0 95.7
Total carbohydrates
2.0 2.2 2.1 2.1
Ref. This work
(14 (6)
(6)
Not corrected for hydrolysis survival.
solvent tailing as discussed above, no interference problem was encountered. All wood samples and pulps from hardwoods probably will contain trace quantities of rhamnose. For such samples it is advisable to analyze samples both with and without internal standard addition and to make appropriate corrections. Calibration. Twelve sample mixtures were prepared by weighing the individual components with a microanalytical balance. These calibration mixtures were prepared to simulate the compositions of southern pine pulp samples. The following concentration ranges were used for the individual components: glucose 180-275 mg, xylose, 18-65 mg, mannose 9-46 mg, arabinose 2-20 mg, and galactose 1-6 mg. Each 10 mg. mixture contained a total sugar content of 309
*
The mixtures were treated as pulp samples and were put through the entire procedure including hydrolysis. Therefore, the resulting calibration factors reflect hydrolysis survival, reaction efficiencies,and chromatographic detection response factors. The calibration factors, k , obtained are given in Table I along with the standard deviations found. The factors and their precision did not show any concentration dependence throughout the concentration ranges examined. The peak areas resulting from the low concentrations of galactose were not measured reproducibly with the electronic integrator. Therefore, the area of this component was measured by triangulation. Since the internal standard area and the galactose area are in different units, the galactose factor given cannot be compared with the other factors. Analysis of Pulp Samples. To determine the precision that can be expected from this method, a typical southern pine kraft pulp was hydrolyzed and analyzed repeatedly. The results of these analyses are shown in Table 11. The consistency of the results for the minor components and the standard deviation for glucan and for the total carbohydrates demonstrates the excellent precision attainable by this method. Although it is difficult to establish the accuracy of a determination of this type, triplicate analyses of Whatman cellulose powder gave an absolute carbohydrate content of 97.5x. This is consistent with the assumption that this material is essentially a high molecular weight glucose polymer (a-cellulose). This indicates that the method is capable of good accuracy. To see if the results obtained with this method agree with results obtained by other procedures, a standard pulp sample, ICCA-4, was analyzed. Table I11 compares our results with those reported by other workers. All the data are in Sugar Units. The results compare favorably with those obtained by the traditional paper chromatographic method and the silyl ether-gas chromatographic method. ACKNOWLEDGMENT
The authors thank Dr. James H. Sloneker of the Northern Regional Research Laboratory, Department of Agriculture, Peoria, Ill., for his advice in the initial stages of this work, and Drs. S. Aronovic and R. G. Barker of this laboratory for their helpful discussions. We are particularly indebted to R . A. Schmidt and C. M. Schindewolf for their diligent efforts on the experimental work connected with this investigation. RECEIVED for review July 25, 1966. Accepted November 10,
(14) R. W. Detrick, Tappi, 43, 552 (1960).
124
0
ANALYTICAL CHEMISTRY
1966.
