Liquid chromatography of carbohydrate monomers and oligomers

[15]

LIQUID CHROMATOGRAPHY

145

[15] L i q u i d C h r o m a t o g r a p h y of C a r b o h y d r a t e M o n o m e r s and Oligomers

By J. K.

LIN,

B. J.

JACOBSON,

A. N.

PEREIRA,

and M. R. LADISCH

Introduction Liquid chromatography is in wide use for separating monosaccharides and oligosaccharides. There are four basic modes of separation involved: (1) gel-permeation chromatography (GPC), (2) ion-exchange chromatography (IEC), (3) adsorption chromatography, and (4) partition chromatography. Numerous papers have indicated use of these four modes in carbohydrate analysis by classical liquid chromatography as well as highperformance liquid chromatography (HPLC).1-3 GPC is based on size exclusion, and has been extensively employed to fractionate homologous series of oligosaccharides, including cellodextrins, cyclodextrins, xylodextrins, and maltodextrins. Packings capable of separating various carbohydrate oligomers include BioGel P-2, 4, and 6 ( p o l y a c r y l a m i d e g e l ) , 4-6 Bio-Glas (granular porous glass), 7 Sephadex ( d e x t r a n g e l s ) , 8,9 and /zBondagel and ~Porasil GPC 60 -A columns, l° Maltodextrins and xylodextrins with a degree of polymerization (DP) of 13-15 are fractionated in 11-20 hr. 4,5 Water or aqueous alcohols are generally used as the eluent in GPC. GPC is capable of fractionating water-soluble oligosaccharides but not monosaccharides which have similar sizes. The main problem associated with GPC gels are their poor mechanical strength and inability to withstand high pressures associated with higher flow rates. Thus analysis time in GPC is also long requiring up to 24 hr per analysis. In comparison, adsorption chromatography, based on the affinity of a solute for the adsorbents, has been shown to give good separation of monosaccharides i A. Heyraud and M. Rinaudo, J. Liq. Chromatogr. 4 (Suppl. 2), 175 (1981). 2 M. R. Ladisch and G. T. Tsao, J. Chromatogr. 166, 85 (1978). 3 M. R. Ladisch, A. L. Huebner, and G. T. Tsao, J. Chromatogr. 147, 185 (1978). 4 N. K. Sabbagh and I. S. Fagerson, J. Chromatogr. 120, 55 (1976). 5 M. John, J. Schmidt, C. Wandrey, and H. Sahm, J. Chromatogr. 247, 281 (1982). 6 K. Hamacher, G. Schmid, H. Sahm, and C. Wandrey, J. Chromatogr. 319, 311 (1985). 7 G. Belue and G, D. McGinnis, J. Chromatogr. 97, 25 (1974). 8 W. Brown, J. Chromatogr. 52, 273 (1970). 9 W. Brown and 0. Anderson, J. Chromatogr. 57, 255 (1971). i0 p. j. Kundsen, P. B. Eriksen, M. Fenger, and K. J. Florentz, J. Chromatogr. 187, 373

(1980). METHODS IN ENZYMOLOGY, VOL. 160

Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

146

CELLULOSE

[15]

and oligosaccharides using carbon, 11,12 silica, 13 and polysaccharide ~4 based supports in classical liquid chromatography using nonaqueous or partially aqueous solvents. Separation of carbohydrate monomers and oligomers reported for cation-exchange and anion-exchange resins include glucose, ribose, xylose, sucrose, raffinose, galactose, fructose, lyxose, mannose, arabinose, uronic acids, aldonic acids, aldobionic acids, xylooligosaccharides, and maltodextrins using water as eluent (refer to Table 2-9 in Ref. 1). Partition chromatography is based on the relative solubilities of the solute in the mobile phase versus the support. Partition chromatography falls into two categories, normal-phase and reversed-phase chromatography. Normal-phase chromatography can be done on underivatized silica packing or bonded phases such as cyanopropyl, aminopropyl, and polyfunctional amine to silica.15 Separation of carbohydrate monomers and oligomers by chemical bonded phases have been achieved by using aminopropyl-bonded phase 16,17(for starch oligomers up to DP 7), polyfunctional amine-bonded 18,19(for starch oligomers and maltodextrins up to DP 20) in 1 hr with acetonitrile/water as the eluent. Furthermore, the carbohydrate columns such as /~Bondapak and Partisil-10 PAC with amino- and cyano-bonded phases are also commercially available for separating mono- and oligosaccharides.2°-24 This system appears to be a rapid method for sugar analysis. However, an aqueous organic eluent is not suitable for oligosaccharides such as cellodextrin~ which have low solubility or samples which have noncarbohydrate components which precipitate in a semiaqueous environment. Thus, this form of partition chromatography has limitations. In reversed-phase chromatography, the silica with nonpolar functional groups such as C~8, phenyl, C8, cyano, and amine is less polar than the mobile phase. Thus, the compounds having a low polarity are eluted later 11 G. L. Miller, J. Dean, and R. Blum, Arch. Biochem. Biophys. 91, 21 (1960). n D. French, J. F. Robyt, M. Weintraub, and P. Knock, J. Chromatogr. 24, 68 (1966). la E. E. Dickey and M. L. Wolfrom, J. Am. Chem. Soc. 71, 825 (1949). 14 S. Gardell, Acta Chem. Scand. 7, 201 (1953). 15 Millipore Corporation, "Waters Sourcebook for Chromatography." Waters Chromatography Division, Millipore Corporation, Massachusetts, 1985. 16 R. Schwarzenbach, J. Chromatogr. 117, 206 (1976). 17 B, B. Wheals and P. C. White, J. Chromatogr. 176, 421 (1979). 18 K. Aitzetmiiller, J. Chromatogr. 156, 354 (1978). 19 C, A. White, P. H. Corran, and J. F. Kennedy, Carbohydr. Res. 87, 165 (1980). 2o j. C. Linden and C. L. Lawhead, J. Chromatogr. 105, 125 (1975). 2J B. Zsadon, K. H. Otta, F, Tud6s, and J. Szejtli, J. Chromatogr. 172, 490 (1979). 22 F. M. Rabel, A. G. Caputo, and E. T. Butts, J. Chromatogr. 126, 731 (1976). 23 G. J. L. Lee and H. Tieckelmann, Anal. Biochem. 94, 231 (1979). 24 G. J. L. Lee and H. Tieckelmann, J. Chromatogr. 195, 402 (1980).

[15]

LIQUID CHROMATOGRAPHY

147

than polar compounds. In comparison with normal phase, these packings are faster to equilibrate, and use less organic solvent. Carbohydrate separations on reversed-phase HPLC have been achieved by using aminobonded p h a s e 25,26 ( f o r starch oligomer up to DP 8, cyclosophoraose up to DP 35, curdlan up to DP 15, dextrans up to DP 25, inulin up to DP 20, and cellodextrins up to DP 5) and C18-bonded p h a s e 27-3° ( f o r starch oligomers up to DP 14, maltodextrins up to DP 13, cellodextrins up to DP 6, amylose up to DP 30). In most cases, water or acetonitrile-water mixture is used as the eluent and analyses take less than 60 min. For rapid analysis of oligosaccharides, the Dextro-Pak and Sugar-Pak columns are also suitable. 15 These columns are available in cartridge form for use in radial compression devices. Ion-exchange resins form a major class of chromatographic supports in sugar separations. Two types of ion-exchange resins, cation- and anionexchange resins, have been shown to be capable of separating sacc h a r i d e s . 2,3,31,32 Cation-exchange resins separate mono- and oligosaccharides in 30 min using water as the sole eluent. 2,3Anion-exchange resins also give good separation of monosaccharides using aqueous ethanol 31 or acetonitrile/water. 32 However, anion-exchange resins tend to promote sugar conversion reactions. Thus, application of this type of resin to carbohydrate separation is limited. 33 Cation-exchange chromatography has been successfully applied to rapid separation of mono- and oligosaccharides. Ladisch e t al. 2,3 developed a low pressure LC system designed for rapid analysis of carbohydrate. Cation-exchange resins such as Aminex Q15S and Aminex 50W-X4 in the calcium form give excellent separation of monosaccharides in 30 min and oligosaccharide, e.g., cellodextrins (up to DP 7) in 30 min using water as the sole eluent. Several counterions on cation exchange resins are applicable for different separations. These counterions include silver, calcium, lead, hydrogen, and cadmium. 2,3,34-38 Of these counterions, the 25 B. Porsch, J. Chromatogr. 320, 408 (1985). 26 M. D'Amboise, D. No~l, and T. Hanai, Carbohydr. Res, 79, 1 (1980). 27 N. W. H. Cheetham and P. Sirimanne, J. Chromatogr. 2117, 439 (1981). 28 N. W. H. Cheetham and G. Teng, J. Chromatogr. 336, 161 (1984). 29 L. A. T. Verhaar and B. F. M. Kuster, J. Chrornatogr. 284, 1 (1984). 30 p. Vratny, J. Coupek, S. Vozka, and Z. Hostomska, J. Chrornatogr. 254, 143 (1983). 31 R. Oshima, N. Takai, and J. Kumanotani, J. Chromatogr. 192, 452 (1980). 32 D. No~l, T. Hanai, and M. D'Amboise, J. Liq. Chromatogr. 2, 1325 (1979). 33 p. Jandera and J. Chur~tc6k, J. Chromatogr. 98, 55 (1974). 34 H. D. Scobell and K. M. Brobst, J. Chrornatogr. 212, 51(1981). 35 L. E. Fitt, W. Hassler, and D. E. Just, J. Chromatogr. 187, 381 (1980). 36 Bio-Rad Laboratories, "Bio-Rad HPLC Column for Carbohydrate Analysis." Bio-Rad Laboratories, Richmond, California, 1986. 37 B. J. Jacobson, M.S. thesis. Purdue University, West Lafayette, Indiana, 1982. 38 j. Schmidt, M. John, and C. Wandrey, J. Chrornatogr. 213, 151 (1981).

148

CELLULOSE

[15]

calcium form with water as eluent is most suitable for the separations of most monosaccharides, oligosaccharides, cellodextrins (up to DP 7), and starch oligomers (up to DP 8). 2,3,35-37 The lead form with water as eluent gives separation of monosaccharides derived from hemicellulose hydrolysis (i.e., xylose, arabinose, mannose, glucose, and galactose). 36,37 The silver forms of Aminex 50W-X4, Aminex Q15S, and Aminex A-7 separate oligosaccharides, found in corn syrup, to a greater extent than the calcium form of the same resins .34 However, the columns packed with resin in the silver form are less stable than those in the Ca 2+ form. 37 The principles of separation using cation-exchange resins have been reviewed by Jacobson. 37The separation of carbohydrates may result from size exclusion, complexing with counterions, and/or adsorption effects. Of these effects, a key factor for saccharide separation in an aqueous system is the choice of the appropriate counterion since the cations adsorbed on a resin significantly influence the separation. The advantages of using cation-exchange resins for carbohydrate monomers and oligomers are readily apparent. The system is capable of speedy analysis at low pressure. Furthermore, water is used as the sole eluent. This enhances the stability of the column used. The column may be continuously used for over 2600 hr and some columns in our laboratory have lasted for over 18 months of intermittent use. 2 Perhaps most important is the stability of properly packed cation-exchange resins when used with chemically "dirty" samples containing noncarbohydrate components, which otherwise foul normal- and reversed-phase supports. Liquid chromatography of carbohydrates, cellulose and biomass hydrolyzates, cellodextrins, maltodextrins, sugar alcohols, volatile fatty acids, fermentation broths, and plant sugars is almost exclusively carried out using cation-exchange resin columns packed in our laboratory. Methods which have evolved in our laboratory over a period of 10 years are described below. Thus, in this chapter, emphasis is placed upon appropriate techniques for using cation-exchange resins for separation and quantification of carbohydrate monomers and oligomers. Methods Instrumentation

Basic instrumentation for liquid chromatography is supplied by many manufacturers and consists of an eluent reservoir, pump, injector, column, detector, and connecting tubing. An example is given in Fig. la. All components between the injector, where the sample is introduced, and the detector cause dispersion which can interfere with the column's performance. This is referred to as band spreading. A typical instrument

[15]

LIQUID CHROMATOGRAPHY

149

can be checked for band spreading by removing the column and inserting a " z e r o " dead volume union in its place. A sample containing a solute detectable by the detector is then injected. For a differential refractometer, glucose at 1 mg/ml concentration will be convenient. For a normal instrument, solute contained in a 10/xl sample injection should elute in a volume of 150/xl or less (as measured from the peak width at the baseline). If the volume (peak width or instrument band width) is significantly greater, the instrument components should be checked for an improperly functioning injector, poor tubing connections between various instrument components, a detector cell which has a dead volume greater than 5-10 /xl, or tubing connections having improperly seated ferrules. A variety of detectors are available for detection of sugars and oligosaccharides. Our experience has shown the differential refractometer type of detector to be the best suited for separations involving carbohydrates. The component in the liquid chromatograph responsible for fractionating the solubles is, of course, the column. The remainder of this chapter describes preparation of packed columns for separating carbohydrate monomers and oligomers using aqueous eluents at isocratic conditions.

Packing Materials The column packing support is usually either Aminex 50W-X4 (BioRad, Richmond, CA; 4% cross-linking, 20-30/xm particle size) or Aminex Q15S (Bio-Rad, 8% cross-linking 22 tzm particle size) in either the calcium or hydrogen forms. The higher degree of cross-linking provides the mechanical stability to the resin matrix, although this resin excludes oligomers of DP 3 or higher. A column packed with Aminex QI5S may be operated at higher flow rates, making it suitable for separation of monosaccharides and oligosaccharides (corn syrup up to DP 3). 39,4°For a resin with a low degree of cross-linking, the resin matrix is more open, and therefore, more sensitive to compaction and plugging. The advantage of the Aminex 50W-X4 is that it will separate oligosaccharides (cellodextrins up to DP 7 and corn syrup up to DP 8). 2,40

Resin Preparation The cation-exchange resins in the sodium form were purchased from Bio-Rad (Rockville Center, NY) (Aminex Q15S, 22 -+ 3 ~m in diameter and Aminex 50W-X4, 20-30/zm in diameter), or from Calbiochem (San Diego, CA) and then converted to calcium or other counterion form be39 J. K. Palmer and W. B. Brandes, J. Agric. Food Chem. 22, 709 (1974). 4o H. D. Scobell, K. M. Brobst, and E. M. Steele, Cereal Chem. 54, 905 (1977).

150

CELLULOSE

[15]

fore use. The procedure used in our laboratory for preparing cation-exchange resin prior to packing is given below. Calcium chloride is used for converting to the calcium form. In the case that the resin is to be prepared in another counterion form, the chloride salt of that ion will typically be used in place of CaCI2. Otherwise the procedure is the same for CaC12. 1. Place 25-50 g (wet) resin into a l-liter graduated cylinder, and fill with 1000 ml of deionized water in 200 ml increments while swirling. Let Sample

~n9

Pressure Goucje

Water Out

[_~.1T= 80

Relief~

Injector I

I

1

Jacketed Column

1

l Water In

Pump

i

Detector

RI

to 85 °

Recorder

Zero Dead Volume Fitti ncj s Required

"

Solvent Reservoir Pump (piston or pneumatic type)

Pressure ~ "~ Gau qe

I Relief Packer Bulb

Valve

Water Out

o,,=oT=2o,toi1 I

at t= 2h; T=80 85°

Jacketed Column Assembly

Water

FIG. 1. Schematicdiagramof key steps in packingappropriatelytreated resin. (a) Instrument configuration;(b) packingconfiguration;(c) fillingprocedure.

[15]

LIQUID CHROMATOGRAPHY

151

Important: Immediately(!) Connect to Pump in Packing Configuration after Filling

Swirl to keep

~1

Resin Suspended

Step (i)

Step (ii) FIG. 1.

Step (iii)

Step (iv)

(continued)

the resin settle overnight and decant the water. This procedure is repeated 10 times or more until the supernatant above the settled resin is devoid of fines, with subsequent resin settling times of about 1-2 hr. Several days are usually needed to complete step 1. 2. Remove the excess water and rinse the resin with 1 liter of the acidic solutions, respectively, in the following order: 0.5 N HC1, 2.0 N HCI, and 6.0 N HC1. The slurrying/settling procedure is the same as step 1. 3. If the H ÷ form is desired, leave the resin in 6 N HC1 solution and go to step 5. Otherwise, rinse with deionized water using the procedure in step 1, until the pH is between 5 and 7 and then go to step 4. 4, Rinse the resin with 1.5-2 liters of CaClz solutions, respectively, in the following order: 0.5, 2.5, 5.0, and 10% CaClz. A gradual increase in salt concentration is used here to minimize fracturing of the ion-exchange resins due to osmotic pressure shock otherwise induced by a large change in ion concentration. Leave the resin in the 10% CaC12 solution. 5. The resin is heated to boiling under reflux for 30 min and then allowed to cool to room temperature. 6. Rinse with 10 volumes of deionized water three to five times. The resin slurry is then degassed at room temperature before packing.

Column Preparation Column diameters may vary from 2, 3.2, and 4 mm i.d. (0.25 in. o.d.) to 6 and 8 mm i.d. (0.375 in. o.d.). Column lengths may vary from 5 to greater than 60 cm. Column diameters of 3.2 to 4 mm i.d. (0.25 in. o.d.) or

152

CELLULOSE

[15]

6 mm i.d. (0.375 in. o.d.) are recommended, based on the experience in this laboratory. The 2-mm-i.d. columns give both excellent resolution and sensitivity by minimizing solute dispersion. However, these columns are difficult to reproducibly pack unless one has had significant experience in slurry packing of LC columns. The 3.2- and 4-mm-i.d. (0.25 in o.d.) columns in a 30 to 60 cm length give good results, particularly for resolution of oligomers of DP -< 3, monosaccharides, alcohols, selected ketones, and furfurals when packed with Aminex 50W-X4 in the Ca 2+ or H + form. In some cases a larger diameter column (6 mm i.d., 0.375 in. o.d.) of the same length packed with the same resin will give resolution of oligomers of DP up to 8. A thorough study on the impact of column diameter and length, for the same chromatographic support, is given by J a c o b s o n 37 who packed over 50 columns on an internally consistent basis and then compared their performance. The 8-mm-i.d. column can also give excellent results particularly when packed with Q15S or other resins of relatively high cross-linking. However, when Aminex 50W-X4 (or equivalent resin) is used, the packing characteristics of a 6-mm-i.d. column are dramatically different from an 8-mm-i.d. column. 2,3 The 8-mm-i.d. column gives high pressure drops (up to 3000 psig) 3 while a 6-mm-i.d. column gives lower pressures (100200 psig) with little loss in resolving capabilities. 2,3 Consequently, the 6mm-i.d, column is recommended for Aminex 50W-X4. The column constructed of 316 stainless-steel tubing must be cut evenly, preferably by an experienced machinist. The ends are then smoothed and deburred. Before packing, the interior surface of the column should be treated as followsnl: I. rinse with deionized water at 1 ml/min for 10 min and connect standard liquid chromatography end fittings with either 2-, 5-, or 10-~m cut-off sintered disks; 2. pump 50% nitric through the column acid for 10 min at room temperature; 3. pump deionized water through the column until a pH of 5 is obtained; 4. pump 10-100 ml (i.e., 3 to 4 column volumes) of acetone through the column; and 5. then disconnect column from pump, remove end fittings, and dry thoroughly with air.

41 j. K. Lin, S. J. Karn, and M. R. Ladisch, Biotechnol. Bioeng., in press (1986).

[15]

LIQUID CHROMATOGRAPHY

153

This procedure is particularly important for resin in the H + form, where contact of the resin with the wall can cause corrosion and pitting, resulting in the formation of gas. The gas may only partially dissolve in the eluent, thus passing as bubbles into the detector cell. 4~This causes spikes in the resulting chromatogram which render the chromatogram useless.

Column Packing and Operation The column can be packed by either a pneumatic amplifier pump 2,37 (constant pressure) or by a constant-flow pump. 3,41 However for a 2-mmdiameter column, the constant-flow pump is more suitable for column packing. 37,4j Details of the column packing are given elsewhere, ~,3,37,41 with the key steps summarized in Fig. lb and c. The resin, prepared as described above, was finally slurried in 150 ml of degassed water in a beaker. The slurry of resin was loaded, by pipetting, into an empty column assembly (capped with a 10-/~m end fitting) at ambient temperature (Fig. lc). The column (3/8 in. o.d., 6 mm i.d., by 50 cm long, with 10-/zm outlet end-fitting) was then connected to the prepacker assembly which was subsequently also filled with resin slurry. It is important to keep the resin slurry evenly suspended in the beaker during the loading procedure. Intermittent swirling of the beaker during pipetting is usually sufficient. Water from a feed reservoir attached to the pump inlet was then pumped into the prepacker column assembly (Fig. lb). During packing, the column was slowly heated to 85° over a 2-hr period with a circulating water bath. The pressure was gradually increased to 120 psi, and the column was allowed to pack for 16 hr. The pressure was kept constant, with flow rate decreasing as the resin packed. The type of pump used in our laboratory is either a Milton Roy positive displacement pump or equivalent, or a pneumatic amplifier pump. For laboratories where only a few columns are to be packed, a positive displacement pump is recommended. In this case the pump must be given constant attention, and the flow rate controlled by manual adjustment to keep the pressure constant. A pressure gauge and relief valve as indicated in Fig. lb is thus required for packing as well as for instrument operation (see Fig. la). When a large number of columns are to be packed a pneumatic amplifier pump is recommended. 2,3 This pump automatically adjusts volumetric flow rate to maintain a constant pressure at changing flow resistance such as occurs when a column is packed. This type of pump may require a greater degree of mechanical maintenance than a constant displacement pump. Microprocessor controllable constant displacement pumps which maintain a steady pressure can also be purchased and are suitable for both packing

154

CELLULOSE

[15]

(constant pressure mode) as well as for normal instrument operation (constant volume mode). After column packing, the column is connected to the liquid chromatograph and maintained at 80 °. The liquid chromatograph includes a degassed, distilled water reservoir, an injector system, a pump, and a differential refractometer connected to an integrator/chart recorder. All analyses are carried out at a constant flow rate using water as the eluent. The typical flow rates for the 4-mm and 6-mm-i.d. column × 60 cm long are 0.25 and 0.5 ml/min at pressure drops of 200 psig or less, respectively. 37 The chromatograms in Figs. 2 to 7 illustrate a variety of separations.

Troubleshooting of LC System Troubleshooting a liquid chromatograph can be made easier, if one knows the symptoms produced by an instrument malfunction, leaks, or

15cm x 3 . 2 m m i.d.

2 5 c r u x 5.2 mm i.d.

or¢ u l.m.l ll.aJ o~

k 24

24 :5 T I M E (minutes)

6

36

FIG. 2. Separation of sucrose (S), glucose (G), fructose (F); cellobiose (C), glucose (G), fructose (F) at 2 mg/ml, each. Disproportionate glucose peak probably caused by sample overlap. Packing: Calbiochem resin (Lot No. 002156), Ca 2÷ form; 10 ~1 sample; 8X attn; chart speed of 20 cm/hr; column temperature of 85°.

[15]

LIQUID CHROMATOGRAPHY

155

column problems. The most common problems and possible remedies in the LC laboratory are summarized below. The manufacturer's instructions should, of course, always be consulted and followed with respect to details on correctional procedures. 1. Bubbles in detector cells cause spikes on the chart paper output. The bubbles can be flushed out by disconnecting the detector, connecting a 10-ml hypodermic syringe with tubing or a swagelock fitting, and pushing the degassed eluent through. Then confirm that the eluent in the solvent reservoir is also properly degassed. 2. Contamination in a detector cell appears as a noisy baseline. The cells should be flushed with appropriate acid, e.g., 6 N HNO3 and/or methanol/water as given in manufacturer's specification. 3. Baseline drift may occur due to room temperature changes (baseline drifts upward by 2-5 cm/hr). Check working environment for constant temperature and cooling, where appropriate. Note that as little as 0.1 ° fluctuation can cause full scale deflection of the recorder pen. 4. An increase in column pressure can occur when the column is plugged. This is normally caused by a "dirty" sample. The sample should be filtered before injection. The column may be regenerated by disconnecting from instrument, connecting column outlet to pump, and slowly flushing overnight, with the inlet fitting draining directly into a small

dose

o0

z o

uJ n.

~lts and Proteins

oI--

Meso ,2 3-Butonediol

bJ ic

II

TM

~ar~ Acet0in

~No.-Meso

15

J ~ 2,3--l~utonediol 30 45 60 ELUTION TIME, MINUTES

FIG. 3. Liquid chromatograph of fermentation broth after 8-hr fermentation of xylose by

Klebsiella pneurnoniae. Column dimensions: 6 mm i.d. × 60 cm long. Temperature at 85 °, water as eluent at 0.5 ml/min. Column packed with Aminex 50W-X4, Ca 2+ form, 20-30/zm (Bio-Rad). [Reprinted with permission from M. Voloch, M. R. Ladisch, M. Cantarella, and G. T. Tsao, Biotechnol. Bioeng. 26, 557 (1984). Copyright © 1984 by John Wiley & Sons.]

156

CELLULOSE

[15]

beaker. The column is then turned around again and reconnected. If the pressure remains high, a void volume will likely form at the column inlet, and cause band broadening and loss of resolution. The column should then be repacked. 5. Poor peak response can occur due to leaks in the system, especially the injector or in a dirty cell (see item 2 above). In case of injector problems, the rotor seal in the injector may be worn and needs replacing. In this case, repeated injections of the same volume of sample will give peaks of widely different heights and baseline widths and/or shoulders. 6. Loss of resolution may occur after the column has been used for a long period of time. This may be due to loss of the counterion or fouling. The resin needs to be unloaded, and if a cation-exchange resin, boiled in 6 N HCI under reflux. Then, repeat resin preparation and column packing procedures as described previously.

G6G5 GT G4 G3

~ G2

I

nol

I

I

I

15

50

45

TIME (rain)

FIG. 4. Chromatogram of cellodextrins with water adjusted to pH 2 (using H2SO4) as eluent. Column 6 mm i.d. x 60 cm long packed with Aminex 50W-X4; H ÷ form, 20-30 tzm particle size (Bio-Rad). [Reprinted with permission from M. Voloch, M. R. Ladisch, V. W. Rodwell, and G. T. Tsao, Biotechnol. Bioeng. 23, 1289 (1981). Copyright © 1981 by John Wiley & Sons.]

[15]

LIQUID CHROMATOGRAPHY

157

7. When a differential refractometer is used as the detector, pre- or postpeak negative dips can occur due to improper mask adjustment. The mask needs to be properly adjusted. 4J 8. No response on chart recorder or a slow drifting of the base-line can also occur if the detector cell is broken due to back pressure changes exceeding the tolerance limit (usually at 50 psig or less). A new detector cell would then be required. A more likely cause is a dirty cell surface due to microbial growth or protein fouling. In this case the cell needs to be cleaned based on the manufacturer's instructions. Column performance should be routinely checked by injecting standards on a daily basis. Major shifts in retention time and increase in peak width may indicate void volume formation in the column. The column would then need to be repacked. Decreases in peak height indicate a dirty detector cell.

Commercial

k

s

GT

70 %

{~ 70% ;6 Ethanol Supernote

>G7 G5 G3

\

64

j I

$00

I

I000

I 1500

I 500

I 1500

I

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\

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I

1500

ELUTION TIME, SEC FIG. 5. Chromatogram of maltodextrins. Column size, type of packing, and conditions the same as given in Fig. 4.

158

CELLULOSE GA'M

[15] CQ 2+ G M

I.=J O3

n,' n-"

1

I

I

29 31

I

I

32 33

31

33

T I M E (minutes)

FIG. 6. Separation of glucose (G), galactose (GA), and mannose (M); xylose (X) and arabinose (A); and glucose (G) and mannose (M) for 50-cm × 3.2-ram-i.d. column using Aminex 50W-X4 resin (control No. 20654, Lot No. 15970) in the Ca 2÷ form; 10/~1 sample; 8X attn.; chart speed of 20 cm/hr; 85° column temperature.

p b2-+ w z o Q. 03 nn~

G

M GA

i

i

i

29 33 37

~

31

I

i

32

:>9

37

T I M E (minutes)

FIG. 7. Separation of glucose (G), galactose (GA), and mannose; xylose (X) and arabinose (A); and glucose (G) and mannose (M) for 50-cm × 3.2-mm-i.d. column using Aminex 50W-X4 resin (control No. 2654, Lot No. 15970) in the Pb 2÷ form; 10/xl sample; 8X attn. ; chart speed of 20 cm/hr; 85° column temperature.

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QUANTITATIVETLC OF SUGARS

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Conclusions Liquid chromatography, as described in this chapter, is a useful tool for quantitating carbohydrate monomers and oligomers in a short analysis time using aqueous based eluents. The key is proper choice of sorbent and eluent, together with appropriate packing techniques. An attempt was made in this chapter to describe the salient techniques. Acknowledgments The procedures in this chapter were independently confirmed by Mandy Mobedshahi, K. Kohlmann, B. Woodruff, R. Hendrickson, and Jill Porter who repeated procedures written in this chapter in order to confirm clarity of presentation. Their helpful suggestions in preparing this manuscript are greatly appreciated. The material in this work was supported by NSF Grant CPE8351916 and USDA 83-CRSR-2-2250.

[16] Q u a n t i t a t i v e T h i n - L a y e r C h r o m a t o g r a p h y of Sugars, S u g a r Acids, a n d P o l y a l c o h o l s

By R. KLAUS and W. FISCHER Introduction The main procedures used for the quantitative determination of sugars (and sugar substitutes ~) are polarimetry, chromatography, and enzymatic methods. Though offering extremely high selectivity, enzymatic analysis is based on a specific reaction mechanism for each sugar. 2,3 The other two procedures adopt a fundamentally different approach and permit the simultaneous analysis of multiple component mixtures. For the analysis of pure substances polarimetry is ideal both in terms of effort involved and accuracy. With multiple component mixtures, however, polarimetry very quickly reaches the limit of its capabilities. For instance, in a twocomponent mixture (presupposing a difference in rotatory dispersions) H. F6rster, Med. Monatsschr. Pharm. 2, 42 (1978). 2 H. O. Beutler and J. Becker, Dtsch. Lebensm. Rundsch. 73, 182 (1977), s A. Lomard and M. L. Tourn, J. Chromatgr. 134, 242 (1977).

METHODS IN ENZYMOLOGY, VOL. 160

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