taining compounds owing to the greater insolubility of silver sulfide than lead sulfide in 19N sulfuric acid. We now believe that it is more accurate to use blank solutions containing everything except protein because free tryptophan, in the absence of DAB, gives a very slight yellow coloration which contributes to the total absorption of test solutions in making tryptophan standard curves. The contribution of the proteins studied to total absorption in the absence of DAB in Procedure U was found to be nearly equivalent to that attributable to their tryptophan contents. Blank solutions previously used in Procedure N contained protein and no DAB although the tryptophan contents of six proteins averaged only 1 . 8 z higher without than with protein in the blank ( I ) . The bomb hydrolysis procedure is more convenient than the sealed-tube method because several samples can be sealed at once without glass-blowing skill. Aside from convenience, the bomb technique eliminated foaming with the proteins studied, because the heat capacity of the bomb and crucible holder prevented warming of the sample during closure and ensured slow warming up to the temperature of hydrolysis
and subsequent slow cooling to room temperature afterward. The calculated number of tryptophan residues per mole, Table V, is based on molecular weights of the proteins as given in the literature. ACKNOWLEDGMENT
The author is indebted to the following for suppling highly purified samples of proteins as listed in Tables 11, 111, and IV: J. J. Basch, W. G. Gordon, E. B. Kalan, M. P. Thompson, and J. H. Woychik, Eastern Utilization Research and Development Division, U. S . Department of Agriculture, Philadelphia, Pa., and J. C. Lewis and L. R. McDonnell, Western Utilization Research and Development Division, U. S. Department of Agriculture, Albany, Calif.
RECEIVED for review May 3, 1967. Accepted July 31, 1967. Reference to certain products or companies does not imply an endorsement by the U. S. Department of Agriculture over others not mentioned.
Rapid QuantitativeAnion-ExchangeChromatography of Carbohydrates Richard B. Kesler The Institute of Paper Chemistry, Appleton, Wis. 54911 While columns of anion-exchange resins i n the borate form have been used previously for separation of mixtures of carbohydrates as their borate complexes i n aqueous solutions, the potentialities of the technique as a rapid method of quantitative analysis seem to have been overlooked until very recently. Further development of this technique was undertaken and has resulted i n a procedure whereby multicomponent mixtures of mono-, di-, and trisaccharides can be separated and quantitatively measured i n four to six hours. The separation method has also been applied to mixtures of glucose polymer-homologs and to mixtures of soluble hemicelluloses. A pH-concentration gradient of borate buffers, formed i n a multichambered gradient device, was used to elute amounts of saccharides as small as 1 pg from a water-jacketed column of strong-base anion-exchange resin at 53' C. The column effluent was monitored continuously for carbohydrates by a Technicon AutoAnalyzer using the orcinol colorimetric method, resulting in a chromatogram consisting of a series of nearly symmetrical peaks presented on a strip-chart recorder. Between runs, re-equilibration of the resin required only 90 minutes. It was established that, for a given sugar, both its peak area and the net absorbance, measured from base line to peak-tip, were separate linear functions of the amount of sugar eluted from the column.
WHENwood, pulp, paper, and related carbohydrate-containing material is hydrolyzed in strong mineral acid, usually by the procedure of Saeman, et al. ( I ) , the carbohydrate polymers yield a mixture of their monosaccharide monomers: glucose, galactose, mannose, xylose, arabinose, and small (1) J. F.Saernan, W. C. Moore, R. L. Mitchell, and M. A. Millet, Tuppi,37, 336 (1954).
1416
ANALYTICAL CHEMISTRY
amounts of rhamnose. During hydrolysis small amounts of furfural and hydroxymethyl furfural are formed as degradation products of pentoses and hexoses, respectively. Uronic acids can also be present in the hydrolyzate because of their original presence in wood. After hydrolysis, it is usual to separate the carbohydrates in solution from the sulfuric acid and the lignin by neutralization with barium hydroxide and subsequent filtration or centrifugation. Additional steps of preparation are usually required to prepare the resultant clear solution of carbohydrates for analysis. In pulping experiments it is desirable to examine the initial wood and the product pulp for the various carbohydrate polymers present to determine the effect of the experimental conditions upon these polymers. The analytical techniques available do not yield results in a single day, and can, in the case of paper chromatography, require up to seven days. Obviously, such time lapses can greatly affect the scope and progress of research programs requiring many such analyses, particularly when the design of a subsequent experiment depends upon the results of the previous one. Khym and Zill(2) first introduced the technique of separating a mixture of sugar-borate complexes on a column of strong-base anion-exchange resin in the borate form. Later, Zill, Khym, and Chenial (3) extended their work to include the separation of related compounds. To obtain separation, requiring up to 60 hours, they made step-changes in the pH and concentration of the eluent. (2) J. X.Khyrn and L. P. Zill, J . Am. Chem. SOC.,74,2090(1952). (3) L. P. Zill, J. X. Khyrn, and G. M. Chenial, Zbid., 75, 1339 ( 1953).
MICRO PUMP
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Figure 1. Column-AutoAnalyzer system for separation and quantitation of carbohydrates About 10 years after publication of the work of Khym and Zill, Syamananda, Staples, and Block (4) reported an improvement in the method. Syamananda, et al., washed their column with a solution adjusted to pH 8.0 with sodium hydroxide and containing a constant borate ion concentration upon which was superimposed a smooth, positive chloride ion concentration gradient. This procedure reduced the time required by a factor of at least three over that of Khym and Zill. Neither group of workers, however, reported investigating the effect of various parameters such as column temperature variations, the function of chloride ions, and the nature of the resin. Recently, Green ( 5 ) reported the development of an automated carbohydrate analyzer in which 15 sugar-borate complexes were eluted from the ion-exchange column in about 12 hours. He found that the effect of increasing column temperature was to shift the eluted bands to longer elution times, while simultaneously enhancing resolution, and that resin particle size and size range was a function of resolution, smaller particles, and narrower size ranges enhancing resolution. Essentially the same procedure was applied by Samuelson and Simonson (6) to aldonic acid mixtures, and to hydroxy acids by Samuelson, Ljungquist, and Parck (7) and by Alfredsson, Gedda, and Samuelson (8,9). A review of recent methods for gas chromatographic analysis of carbohydrates shows that, since Sweeley, et al. (10) developed procedures for the volatile trimethylsilyl ether derivatives, many modifications have appeared, including those of Sawardeker and Sloneker (11) and Bethge, Holmstrom, and Juhlin (12). While these modifications have re(4) R. Syamananda, R. C. Staples, and R. J. Block, Contrib. Boyce Thompson Inst., 21, 363 (1962). (5) National Cancer Institute Monograph No. 21, “The Development of Zonal Centrifuges,” p. 447, U. S. Govt. Printing Office, Washington, D. C., June 1966. (6) 0. Samuelson and R. Simonson, Suensk Papperstid., 65, 363 (1962). (7) 0. Samuelson, K. J. Ljungquist, and C. Parck, Zbid., 61, 1043 (1958). (8) B. Alfredsson, L. Gedda, and 0. Samuelson, Zbid., 63, 758 (1960). (9) B. Alfredsson, L. Gedda, and 0. Samuelson, Anal. Chim. Acta, 27, 63 (1962). (10) C. C. Sweeley, R. Bentley, M. Makita, and W. W. Wells, J . Am. Chem. Soc., 85,2497 (1963). (11) J. S. Sawardeker and J. H. Sloneker, ANAL.CHEM.,37, 945 (1965). (12) P. 0.Bethge, C. Holmstrom, and S . Juhlin, Soensk Papperstid., 69, 60 (1966).
sulted in improved resolution and somewhat simplified sample preparation procedures, the appearance of several anomeric peaks for each sugar still persists as an inherent property of the trimethylsilyl ether method. Separation of monosaccharides as their alditol acetates, developed by Sawardeker, Sloneker, and Jeanes (13), and further advanced by Sjostrom, Haglund, and Janson (14) and Crowell and Burnett (15) yields only single peaks for each sugar. A system for partition chromatography of monosaccharides in ethanol-water solution, using columns of ion-exchange resins as supporting material, has been devised by Samuelson and coworkers (16-24), apparently with very good results. Recent work by Johnson and Samuelson (25) shows much improvement in speed of separation, with sugars present in wood hydrolyzates completely separated in a little over 2 hours. Despite these advances in systems and methods for analysis of carbohydrate mixtures, an improved method was sought. Preparation of the various derivatives for gas chromatography still requires many steps, much handling of the sample, and considerable time, although the final analysis of the product derivative is accomplished in an hour or two. Partition chromatography in ethanol-water usually requires at least 7 hours of column operation for multicomponent mixtures, involves the use of rather high temperatures for the volatile solvent system, and poses difficulties in quantitation because of rapid exhaustion of the AutoAnalyzer manifold tubes due to the high ethanol concentration. In addition, the solubility of polysaccharides in alcohol decreases as the degree of polymerization increases, thus limiting the ethanolwater chromatographic system to lower saccharides. EXPERIMENTAL
Equipment. Figure 1 is a schematic diagram of the system used for separation and detection of the carbohydrate mixtures. Most of the work was done with a borosilicate glass column 3 X 750 mm containing a resin bed of 700 mm. Other columns used were 6 X 750 mm, 254 X 1200 mm, and 6 x 1000 mm. All were glass-jacketed, with jacket water supplied by a Haake Type FJ constant temperature circulator. Column temperatures were measured in a thermometer well attached directly to the exit nipple of the water jacket. The columns, AutoAnalyzer, ninechamber gradient device (Autograd) and associated equipment (13) J. S. Sawardeker, J. H. Sloneker, and A. Jeanes, ANAL. CHEM.,37, 1602 (1965). (14) E. Sjostrom, P. Haglund, and J. Janson, Soensk Papperstid., 69, 381 (1966). (15) E. P. Crowell and B. B. Burnett, ANAL.CHEW,39, 121 (1967). (16) 0. Samuelson and B. Swenson, Acta Chem. Scand., 16, 2056 (1962). (17) J. Dahlberg and 0. Samuelson. Scensk Kem. Tidskr.. 75. i78 (1963). (18) 0. Samuelson and B. Swenson, Anal. Chim. Acta, 28, 426 (1963). (19) B. Arwidi and 0. Samuelson, Scensk Pappersfid., 68, 330 (1965). (20) B. Arwidi and 0. Samuelson, Anal. Chim. Acta, 31, 462 (1965). (21) J. Dahlberg and 0. Samuelson, Acta Chem. Scand., 17, 2136 (1963). (22) B. Arwidi and 0. Samuelson, Scensk Kem. Tidskr., 77, 84 (1965). (23) L.-I. Larsson and 0.Samuelson, Acta Chem. .jcand., 19, 1357 (1965). (24) 0. Samuelson, L.-I. Larsson, and 0. Ramnas, “Automation in Analytical Chemistry,” p. 169, Paper presented at Technicon Symposium, 1965, Mediad, New York, 1966. (25) P. Jonsson and 0. Samuelson, LKE Znstr. J . , 13, 1 (1966). I
VOL. 39, NO. 12, OCTOBER 1967
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Figure 2. AutoAnalyzer flow scheme for measuring boron concentration shown in Figure 1 were obtained from Technicon Chromatography Corp., Ardsley, N. Y . Peak areas were measured with a Technicon Model AAG integrator/calculator. A Beckman long, thin, probe-type combination electrode was used t o measure p H values of fractions. Resin. Three different resins were investigated : Dowex 1-X8 (200-400 mesh), A G 1-X8 (30-40 microns), and a n experimental resin obtained from Technicon. The A G 1-X8 is actually Dowex 1-X8 purified and sized by Bio-Rad Corp., Richmond, Calif. The Technicon resin, designated 3/28/VI, is a n 8 % cross-linked resin similar in chemical composition to the other two, but with a n average particle diameter of 21 microns and a range of 5-40 microns. As measured by a Coulter Counter, about 11 % of the Technicon resin particles exceeded 30 microns and about 5 % was smaller than 10 microns. All resins used were spherical in particle geometry. Chemicals and Reagents. Sugars and hydroxymethyl furfural were obtained mainly from K and K Laboratories, Inc., Plainview, N. Y . ,and all were of minimum 95% purity. Furfural was vacuum-distilled frequently during use, but after distillation it still retained a faint yellow color. Hemicelluloses, obtained from N. S. Thompson of the Cellulose Chemistry Group, The Institute of Paper Chemistry, were isolated by NaOH extraction from chlorite-delignified immature jack pine wood. Cellodextrins were prepared by partial hydrolysis of cellulose, and separated on carbon columns (26). Orcinol reagent for the AutoAnalyzer was a solution of 1.0 gram/Iiter of orcinol in 70% (v/v) suIfuric acid, stored in brown glass bottles. The eluent buffers were made by dilution of stock 0.80M H3B03, 5N NaOH being used for p H adjustment of the more dilute buffers, and 12N NaOH for the more concentrated ones. Carminic acid reagent for boron estimation (Figure 2) was made by mixing in a 1000-ml volumetric flask 25 ml of 0.1 % carminic acid in concentrated H2S04,250 ml of 9 5 % ethanol, 250 ml of concentrated H2S04, and sufficient distilled water t o bring the final volume t o 1000 ml of about 72' F. The solution was vacuum-filtered through a mediumporosity sintered-glass filter funnel just before use. Baker and Adamson potassium tetraborate tetrahydrate, analytical reagent grade, 0.20M, was used t o convert the resin from the chloride to the borate form, All solutions pumped through the columns were prefiltered through a Millipore filter having a pore size of 0.45 micron and in a n all-glass filter system.
(26) "Methods in Carbohydrate Chemistry," Vol. 111, Academic Press, New York, 1963, p. 134. 141 8
MM
TEMP: 53-C FLOW RATE: 0.30 ML/MIN.
ANALYTICAL CHEMISTRY
Figure 3. Chromatogram of a 17-component known solution Procedure. The columns were filled with resin by overnight settling from a specially-fitted glass bulb attached to the top of the column, which was partly filled with a resinwater suspension containing excess resin. Then, connecting the pump t o the column, the bed was packed by pumping water through the column a t a rate to produce a pressure of up to 600 psig. The packed bed of resin was next converted t o the borate form by washing with 0.2M K2B407solution, a t the same flow rates to be used in subsequent chromatographic runs, until the effluent showed a negative test for chloride ion with silver nitrate-nitric acid solution. After removing the liquid above the resin bed and replacing it with 0.10M H3BO3 adjusted to p H 7.0, and flushing the pumping system with the same buffer, the column was washed for 90 minutes with the p H 7.0 buffer. It was then ready for a chromatographic run. Packing, conversion, and washing all took place a t the same temperature, usually 53" C, as subsequent runs. Known mixtures of carbohydrates were dissolved in 0.10M H3B03 adjusted to p H 7.0, appropriate volumes of which were placed on the column with a micrometer pipet. The sample was forced into the resin bed with 50 psi nitrogen, and followed by an equal-volume wash of 0.10M H3B03 a t p H 7.0. The void space above the resin bed was then filled with the pH 7.0 buffer, the pump and Autograd were connected, the pump was started, and the sample line of the AutoAnalyzer was connected to the A2 fitting a t the bottom of the column. Hydrolyzates of pulp and paper were prepared by the procedure of Saeman, et al. (I). After neutralization with barium hydroxide solution and weight adjustment of the hydrolyzate, it was allowed t o stand for about 15 minutes and then filtered through a 0.45-micron-pore Millipore filter, decanting off the partly settled liquid first through the filter. With a pipet, 100 ml of the filtrate were placed in a 150-ml beaker containing 0.618 gram of boric acid and a magnetic stir-bar. After dissolution of the boric acid, the pH of the liquid was adjusted t o 7.0 with 5N NaOH, using a p H meter, and a capillary pipet for the NaOH. The volume of NaOH required was usually less than 0.2 ml, thus affecting the total volume of 100 ml very little. An aliquot (0.30 ml for the 6-mm diameter column) of this solution was then placed on the column. RESULTS
The order in which the carbohydrates come out of the column was established by adding one compound a t a time t o a previously established mixture, beginning with only two sugars-mannose and glucose-because, from the work of Syamananda, et ul. (4, these two are quite widely separated. The chromatogram in Figure 3, where the recorder range was electronically expanded by a factor of 2, shows the positions of 15 carbohydrates and two aldehydes. In addition t o these,
I
06
1
0 0
I
SUCROSE CELLOBIOSE
TiUE, H E .
Figure 4. Sample chromatogram of eight-component standard use for quantitative calibration
it has been established that under the same conditions sorbose is eluted with xylose, lyxose with ribose, d-2-desoxyribose with furfural, raffinose with cellobiose, and trehalose with sucrose. T o establish the relationship between peak area, peak height, and the amount of sugar represented by a given peak, a series of seven chromatograms of an eight-component standard solution was made under fixed conditions. The series was made using an increasingly larger aliquot of the standard solution for each run so as to cover an approximate ninefold range of amount spotted for each sugar. One of these chromatograms is shown in Figure 4. The relationship between peak area, as indicated by the number of counts made by the integrator, and the varying amounts of sugars is shown in Figure 5. For the same series, the net absorbance measured from base line to peak-tip has the relationship to sugar amount shown in Figure 6. Figure 7 shows the pH and boron concentration changes of the liquid entering and emerging from the column as a function of time, using the gradient and other conditions under which the chromatogram in Figure 3 was obtained. Three-milliliter fractions were collected in an automatic fraction collector to obtain these data. Figure 8 is a chromatogram of hemicellulose on a prep-size column filled with Dowex 1-X8, for lack of the required quantity of a more suitable resin. One hundred milligrams
MICROGRAMS
Figure 6. Quantitative calibration by peak height
of the solid were dissolved in 30 ml of 0.lOM H3B03adjusted to pH 8.0, placed on the column, and eluted with a solution at p H 8.0 containing 0.10 gram-mol/liter each of NaCl and H3B03. A Milton-Roy stainless steel adjustable-stroke piston pump was used to wash the column. The pump had a maximum capacity of 19.8 ml/minute at a discharge pressure of 1000 psi and a fixed rate of 29 strokes per minute. The column eRluent was simultaneously monitored for carbohydrates while the excess was collected in 25-1111 fractions. I1
I
I
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I
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1
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TIME, HRS. MI C ROGRAMS
Figure 5. Quantitative calibration by peak area
Figure 7. pH and total boron concentration in column influent and effluent produced by gradient shown in Figure 3 VOL. 39, NO. 12, OCTOBER 1967
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RESIN' DOWEX i-xa, 2 w - d ~ MESH ~ C h U M N 2 54 X I 2 0 CM
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-
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15.0 M L h I N .
1
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COLUMN 6 X 750 MM -RESIN TECHNICON TEMP 47'C FLOW-RATE I O ML/MIN -GRAD ENT l CHAMBER 2 CHAMBER I 90ML O F 0 6 ~ + 1 9 0 M L OFOISM+CCCUMN
I1 -I
TIME, HRS
Figure 8. Chromatogram of immature jack pine hemicellulose on prep column The original sample, upon hydrolysis, yielded galactose, glucose, mannose, and xylose. The fractions corresponding t o the four peaks shown in Figure 8 were pooled separately, dialyzed against distilled water to remove excess salts, concentrated to a thin sirup, deionized with Amberlite IR-120, distilled with methanol to remove boron, freeze-dried, then hydrolyzed and subjected to paper chromatography. The effluent between Peaks 2 and 3 and that collected during the first 75 minutes was treated similarly. The Peak 1 carbohydrates were composed of galactose, glucose, and xylose; Peak 2 the same as Peak 1 but less galactose; Peak 3 of galactose, glucose, xylose, and mannose; and Peak 4 was similar to Peak 2. The effluent collected during the first 75 minutes and that between Peaks 2 and 3 contained carbohydrate material consisting mainly of xylose. Figure 9 is a chromatogram of a known mixture of glucose polymer homologs. Figure 10 is a chromatogram of a hydrolyzate prepared from pulp made by chlorite delignification of aspenwood. DISCUSSION
The column chromatographic systems employed are suitable for use where pressures up to 1000 psi are encountered, with the exception of the prep column which is suitable only up to about 250 psi. In this work, the pumping pressures seldom exceeded 600 psi, and that of the prep column was below 200 a t the flow rate used. Of the three resins investigated, increasing resolution of eluted sugars was obtained in the order: Dowex 1-X8 (200-
COLUMN: 6 X 1000 MM RESIN: TECHNICON FLOW-RATE: 1.1 ML/MIN T.: 5 3 o c
0.4
3
5
4
TIME,
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Figure 10. Chromatogram of aspen pulp hydrolyzate
400 mesh), Bio-Rad AG 1-X8 (30-40 microns), and Technicon 3/28/VI, with the Technicon resin far superior to either of the other two. All three were packed into the column while still in the chloride form, and upon conversion to the borate form settled somewhat. When reconverted to the chloride form, as occurs when eluted with a salt-containing solution as in Figure 8, considerable additional settling took place. After several alternate conversions between the chloride and borate forms, a resin bed dimensionally stable was obtained, and one that contained about 2 0 z more resin than originally packed t o the same height. It was found that this more closely packed column produced in all cases better resolution of sugars than one packed once in the chloride form followed by a single conversion t o the borate form. It was never necessary to repack any of the columns, even after a year's use, but after about 25 runs, the top centimeter of resin became blackened and was replaced. It was found, in agreement with Green's results (5), that increasing column temperatures enhanced resolution and shifted the elution bands to longer elution times a t a given flow rate until little further advantage was gained between 55 O and 70 O C. Increasing or decreasing flow rates by 50 made little apparent difference in resolution of the sugars, but pressure limitations were soon encountered at flow rates much above 6.0 ml/minute cm2 of cross-sectional area. The properties of the borate buffers used to form the gradient for elution were initially derived from observations made in earlier experiments (27) with eluents containing sodium chloride, similar to that employed in Figure 8. It was then observed that the bands of mannose and glucose were nearly always asymmetric in a peculiar manner, and that the appearance of glucose always coincided with the abrupt presence of chloride ions in the column effluent. In a n attempt to explain these observations, and to explain the role of chloride ions, an experiment was made with a 6 X 750-mm column filled with Technicon resin at 47" C with simultaneous monitoring of the effluent for carbohydrates and for boron. Inasmuch as the flow rate was 1.00 ml/minute and the total requirement of the two AutoAnalyzer monitors was only about 0.6 ml/minute, the excess was collected in 10-minute fractions in a n automatic fraction collector for subsequent pH measurement and qualitative chloride ion analysis. The resin had been converted to the borate form with 0.8M K2B40i, followed by a five-hour wash with 0.10M BOs adjusted to pH 7.5. The eluent contained 0.10 gram-mol/liter each of H3B03 and NaCl, and was adjusted to p H 7.5. The results are illustrated in Figure l l .
TIME, H R S .
Figure 9. Chromatogram of known mixture of cellodextrins 1420
ANALYTICAL CHEMISTRY
(27) Unpublished work, The Institute of Paper Chemistry, 1966.
Additional experiments of this nature with the same column showed that: a sudden increase in boron content always occurred with the elution of mannose as a n asymmetric curve, elution of glucose coincided with the sudden appearance of chloride ions in the effluent and with the return of the p H and boron concentration of the effluent to that of the eluent, and during the run, p H and boron content of the effluent were inversely related. Although the conditions of these experiments are different from those of Everest and Popiel (28) in their ion-exchange studies of borate solutions, the results are in some respects comparable. These few experiments led to further studies of the nature of ionic species in aqueous borate solutions, the results of which will be reported later. However, the obvious conclusion was that the use of chloride ions in the column eluent can be dispensed with, and that a solution of borate buffers of changing p H and concentration is a more suitable eluent. The occurrence of unknown peaks in known solutions, such as shown in Figure 3, was observed only infrequently. Carubelli (29) apparently found that lactose underwent transformation t o lactulose during borate ion-exchange chromatography, along with suspected similar changes in other disaccharides. The keto sugar originally showed as a n unknown peak in the elution pattern. It is possible that Peak No. 12 in Figure 3 is a transformation product of one of the sugars originally present. As shown in Figure 5 , the area of the elution curve is a linear function of the amount of sugar it represents, except in the case of sucrose. The slight nonlinearity of the sucrose calibration curve is currently unexplained, but does not appear in Figure 6 where the net absorbance values reached by the same series of elution curves show this property to be also a linear function of the amount of sugar eluted. Because the peak height, expressed as absorbance, has this linear relationship to sugar amount, it appears that the band widths of the various sugars d o not change under constant conditions of elution. While this relationship has been firmly established for only the eight carbohydrates shown in Figure 6, it appears t o hold for quite a number of additional ones. Thus, the need for area measurement of elution curves can be eliminated. However, a n important factor affecting such a calibration as in Figure 6 is the constancy of conditions on the AuroAnalyzer, particularly the proportioning of sample t o reagent. This proportion is disturbed somewhat when the proportioning pump tubes are changed, which they must be because of wear. It has been found that a given set of manifold tubes will last for a t least 25 runs, and when it does become necessary t o change them, two standard runs are sufficient to reestablish the calibration. The compounds eluted from the column usually appear in groups, as shown in Figure 3, where the approximate halfhour time delay across the AutoAnalyzer is included in the time axis. In Figure 7 the p H and total boron concentration of both the influent and effluent of the column, produced by the gradient shown in Figure 3, are illustrated as a function of time. In Figure 7, the data points are “average” values for 10-minute intervals, as they were obtained from 10-minute (3 ml) collected fractions. However, no time delay is included in the time axis in Figure 7. Thus, to compare the two figures directly on a time basis, one-half hour must be
(28) D. A. Everest and W. J. Popie1,J. Chem. Soc., 1956, 3183. (29) R. Carubelli, Carbohydrare Res., 2, 480 (1966).
04
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10
I
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TIME, HRS.
Figure 11. pH and boron concentration changes during a run made with eluent containing chloride ions Peaks 1-9 = furfural, hydroxymethylfurfural, rhamnose, ribose, mannose, arabinose, galactose, xylose, and glucose, respectively
subtracted from the time values given in Figure 3. When this comparison is correctly made, it can be seen that, while the influent p H and boron concentration increase rather smoothly with time, the same two values in the effluent fluctuate and the three groups of elution bands 1-6, 7-10, and 11-18 are related t o these periods. During the first hour, bands 1-6 appear while the p H climbs steeply and the boron concentration increases only slowly. The second group of bands, 7-10, appear during the second hour while the p H is lowered, but the boron concentration increases sharply. Finally, as the p H and boron concentration cease to fluctuate and increase rather smoothly with time, the third group of sugar bands, 11-18, appear. There also seems to be a progressive broadening of individual band widths with longer elution times. The somewhat inverse relationship between pH and boron concentration during the first 2 hours would seem t o indicate a varying concentration of polyboron anions in the effluent, as lower pH values with attendant higher boron concentration favors condensation of monoboron anions t o polyboron anions (28). From paper chromatographic analysis of the pooled fractions of the run with hemicellulose, represented by Figure 8, it appears that the polymer consisting mainly of xylan was “smeared” through the whole run. Peaks 1, 2, and 4 contained the same sugars, but in different proportions. The notable result of this experiment is that the mannose-containing polymer was isolated in Peak 3, although it was not xylanfree. Applegarth and Dutton (30) encountered similar difficulty in obtaining xylan-free mannan by ion-exchange chromatography. Gradient elution with a more suitable resin is expected to produce better resolution, particularly of the xylose-containing portion of such mixtures. No molecularweight measurements have been made on any of the carbohydrates represented by the various peaks in Figure 8. The cellodextrins (Gi = celloheptaose, G6 = cellohexaose, etc.) chromatogram in Figure 9 was obtained by gradient elution after the column (converted to the borate form with 0.2M K2B407)was washed for 2 hours with 0.05M boric acid. In the Autograd, chambers 1, 2, 3, and 4 contained 100 ml each of 0.050M &Boar 0.100M H3B03 at p H 7.0, 0.125M
(30) D. A. Applegarth and G . G. S. Dutton, Tappi,48,204 (1965). VOL. 39, NO. 12, OCTOBER 1967
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H3BOaat pH 8.5, and 0.150M at pH 9.0, respectively. While all of the peaks are not completely resolved, an increase in column length would most likely improve resolution greatly. Initial chromatograms of the same mixture on a column 750 mm long showed celloheptaose and cellohexaose barely distinguishable as separate peaks. Using the same resin in a 1000-mm column gave the chromatogram in Figure 9. All of the cellodextrins except celloheptaose were easily soluble in 0.10M boric acid adjusted to pH 7.0 with sodium hydroxide. Celloheptaose entered solution completely after several hours stirring at about 40" C. Higher homologs of glucose have not been used in this system, but it is doubtful if any beyond the nonaose could be dissolved without increasing the alkalinity of the solvent. This would present the possibility of splitting, particularly at the column temperatures used. The peaks in the chromatogram of the pulp hydrolyzate in Figure 10 represent the following polymer contents of the pulp, expressed as per cent by weight of dry pulp: mannan 6.9, araban 0.7, galactan 0.9, xylan 5.6, and glucan 69.1. While the first 2 hours of the chromatogram are not shown in Figure 10, three very small, but sharp and narrow peaks occurred, two of which corresponded to the furfural and hydroxymethyl furfural positions, and the third to the position normally occupied by rhamnose.
CONCLUSIONS
Ion-exchange chromatography of borate-carbohydrate complex anions on columns of strong-base anion-exchange resins offers a relatively rapid method of quantitative analysis of sugar mixtures, particularly when column effluents are monitored for sugars by an automatic analyzer. There are no derivatives to prepare, and a properly sized column can be used simultaneously for analytical and preparative purposes. The same method is applicable to other areas of carbohydrate chemistry, having been shown to have the ability to rapidly resolve mixtures of oligosaccharides and hemicelluloses in solution. The amounts of individual saccharides measurable can vary in a wide range from less than a microgram, using a 3-mm diameter column, to several decigrams on a 1-inch column. ACKNOWLEDGMENT
The assistance of Donald Whitney, Lawrence University, and of Margaret Malueg in performance of much of the laboratory work is gratefully acknowledged.
RECEIVED for review June 7 , 1966. Resubmitted July 13, 1967. Accepted July 28, 1967. In part division of Cellulose, Wood and Fiber Chemistry, Winter Meeting, ACS, Phoenix, Ariz., January 17, 1966.
Fast Liquid Chromatography: An Investigation of Operating Parameters and the Separation of Nucleotides on Pellicular Ion Exchangers C. G . Horvath, B. A. Preiss, and S. R. Lipsky Section of Ph ysical Sciences, Yale University School of Medicine, New Hauen, Conn. A liquid chromatographic system featuring high inlet pressures and a sensitive UV detector was designed for fast analysis of nonvolatile organic compounds. To establish optimal operating conditions, band dispersion in the mobile phase was studied by using capillary tubes as well as small bore columns packed with glass beads. Although peak broadening in open tubes was less than predicted by theory, the use of packed columns was more promising for fast separations. Novel pellicular column materials were prepared by coating glass beads with ion exchange resin and other solid phases. Rapid separation of nanomole quantities of ribonucleoside mono-, di-, and triphosphates was achieved by using a pellicular basic ion exchanger and gradient elution with a phosphate buffer. As shown by cellular extract analyses, the stability and efficiency provided by such column materials makes it possible to achieve fast separation of complex mixtures by a liquid chromatographic technique similar in speed, resolution, and quantitative range to gas chromatography.
A TECHNIQUE for the separation of nonvolatile substances which encompasses the speed, efficiency, sensitivity, and versatility of gas chromatography has yet to be devised. The present study was undertaken in an effort to investigate the feasibility of developing a liquid chromatographic system which could approach this goal. The method which evolved 1422
ANALYTICAL CHEMISTRY
was then utilized for the analysis of the nucleoside phosphori:, acids. The time required for the separation of a solute pair is conveniently expressed by the retention time of the second peak, t , as t = "/ub
(1)
where N is the number of plates required for the particular separation, H i s the plate height, and U b is the band velocity. Although H is the basic measure of the peak broadening, H/ub, the time per plate, is more important if the speed of separation is of concern, as it is directly related to the separak), tion time as shown in Equation 1 . Since ub = u/(l Equation 1 can be written
+
H/ub = t/n
=
H(l
+ k)/u
(2)
where k is the column capacity ratio for the solute, and u the fluid velocity. The plate height depends on the flow velocity ( I , 2) and, as Giddings (3) pointed out, if the molecular diffusion is the (1) J. J. van Deemter. F. J. Zuiderweg. - and A. Klinkenberg, Chem. \-,
Eng. Sci., 5, 271 (1956). (2) M. J. E. Golay, ANAL.CHEM., 29,928 (1957). (3) J. C. Giddings, Zbid.,35, 2215 (1963).
