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High-performance liquid chromatographic analysis of oligosaccharides
Journal qf Chromatograph)J, 440 (1988) 281-286 Elsevier Science Publishers B.V., Amsterdam -
CHROM.
in The Netherlands
20 212
HIGH-PERFORMANCE OLIGOSACCHARIDES I. SEPARATION CROSS-LINKING*
H. DERLER, Institute
Printed
ON
LIQUID
AN ION-EXCHANGE
H. F. HiiRMEYER
qf Radiochemistry,
CHROMATOGRAPHIC
STATIONARY
ANALYSIS
OF
PHASE OF LOW
and G. BONN*
University
of Innsbruck,
Innrain 52a. A-6020 Innsbruck
(Austria)
SUMMARY
The separation of oligomeric carbohydrates was investigated for the first time on a 2% cross-linked ion-exchange material with H+ as the counter ion. In spite of the low resistance to pressure, good oligosaccharide separations were obtained, which are comparable to those described previously on 4-6% cross-linked and silver-loaded ion exchangers. The mechanism responsible for the separations is, predominantly, size exclusion. By investigating the dependences on the temperature and flow-rate, the chromatographic conditions could be optimized.
INTRODUCTION
The separation of oligomeric carbohydrates, such as, e.g., gluco-oligomers, is required in many fields of the applied sciences, such as medicine, food chemistry and biomass analysisip3. Apart from gel chromatographic separations4v5, several highperformance liquid chromatographic (HPLC) methods have been described, most of which make use of aminopropyl, C is or ion-exchange stationary phases+‘O. With regard to the ion-exchange materials, cation exchangers loaded with Ag+ and Ca2 + and having different degrees of cross-linking have been employed most frequently. The different extents of cross-linking are obtained by copolymerization of polystyrene with different concentrations of divinylbenzene. Conventionally, the amount of crosslinkage is expressed as the percentage of divinylbenzene. Polymers with low percentages of cross-linking are characterized by a high degree of permeability, high equilibrium rates and reduced physical stability. Furthermore, particle size must be considered l l. Silver as the counter ion leads to a particularly high resolution of gluco-oligomers, with an optimum at 70% loading and 4% divinylbenzene cross-linkings. In addition to size exclusion, ligand exchange and matrix adsorption contribute to this
l
September
Presented in part at the 24th International 8-10. 1987.
0021-9673/88/$03.50
0
Symposium
1988 Elsevier Science Publishers
on Advances in Chromatography,
B.V
Berlin,
282
H. DERLER,
H. F. HORMEYER,
G. BONN
separation. Since, however, many “optimizations” have to be applied to acidic samples, considering, e.g., the large number of cases where oligomers only form upon acid hydrolysis, an exchanger loaded with 70% Ag+ would prove rather problematic with regard to the reproducibility both of the retention time and of the extent of cation loading; furthermore, in-flow regeneration would not be possible, thus adding to the difficulties in application. The possibility of obtaining analogous separation of identical resolution with other materials was investigated in the present work by the use of a weakly crosslinked support and substitution of Ag+ by H+ in order to eliminate ligand exchange in favour of the size-exclusion mechanism, whereas the separation capacity was maintained. An additional comparison was drawn with regard to the influence of silver loading of a 2% cross-linked material. EXPERIMENTAL
Apparatus The HPLC system consisted of a pump (Bio-Rad Labs., Richmond, CA, U.S.A.), an injection valve with a volume of 20 ~1 (Altex, Berkeley, CA, U.S.A.), a column oven (Bio-Rad), a refractive index detector (Bio-Rad RI Monitor) and a C-R3A integration system (Shimadzu, Kyoto, Japan). Columns The stationary phases used were (a) a sulphonated styrenedivinylbenzene copolymer, loaded with H+, 2% cross-linked, 25 pm, 300 mm x 7.8 mm I.D. (probably soon to be commercialized as HPX22H by Bio-Rad), and (b) a sulphonated styreneedivinylbenzene copolymer, loaded with Ag’, 2% cross-linked, 25 ym, 300 mm x 7.8 mm I.D. (HPX22A, Bio-Rad). As precolumns, ion-exclusion micro-guard cartridges (Cation-H, Bio-Rad) were employed. The eluents were 0.01 N sulphuric acid (p.a.; E. Merck, Darmstadt, F.R.G.) and deionized water, respectively. All eluents were degassed in an ultrasonic water bath before use. Samples For standard solutions of oligomers, an enzymatic hydrolysate of starch as well as hydrothermally degraded gluco-oligomers from cellulose were investigated12-r4. All other reference solutions were obtained from analytical grade products commercially available (Fluka, Buchs, Switzerland; E. Merck; Sigma, St. Louis, MO, U.S.A.) and dissolved in deionized water. RESULTS AND DISCUSSION
The low degree of cross-linking (2%) of the sulphonated poly(styrene-divinylbenzene) copolymer (hydrogen form) causes the exchanger material to be less stable towards high pressure, which may lead to changes in the polymeric support. Hence, lower flow-rates must be applied than with the silver-loaded ion-exchange phases presently available commercially, which achieve higher pressure stability by higher cross-linking (4-6%; HPX42A). A major advantage of hydrogen-loaded ion exchangers lies in the fact that the use of 0.01 N sulphuric acid as the eluent brings about
HPLC
OF OLIGOSACCHARIDES.
283
I.
Fig. 1. Separation of oligosaccharides derived from an enzymatic hydrolysate of-starch. Chromatographic conditions: column, 2% cross-linked hydrogen-loaded sulphonated poly(styrene-divinylbenzene) ion exchanger; mobile phase, 0.01 N sulphuric acid; flow-rate, see chromatograms; temperature, 76°C. The numbers on the peaks indicate DP values.
continuous regeneration, in contrast to the 70% silver-loaded columns, which can be operated only with deionized water as the mobile phase. Through the use of sulphuric acid and water, respectively, as the mobile phase and refractive index detection, the sensitivity in the determination of mono-, di- and oligosaccharides reaches the nanogram range. Thus, the applicable range was examined for flow-rates which did not result in an high pressure build-up, i.e., < 200 p.s.i., ea. 14 bar. Because of the low degree of cross-linking of the polymeric ion-exchange matrix, the pressure build-up, depending on the flow-rate and temperature, becomes very important. At room temperature, e.g., the system cannnot be operated at flow-rates higher than 0.154.25 ml/min if irreversible changes in the matrix, which would occur at pressures above 300 psi., are to be avoided. At 60°C the flow-rate can be raised to ca. 0.4 ml/min, at 90°C to 0.5 ml/min, without a drastic increase in pressure. Figs. l-3 show the separation of oligomeric carbohydrates on a 2% crosslinked hydrogen-loaded ion-exchange resin as functions of the flow-rate and the column temperature. With a constant flow in the range of 0.2-0.4 ml/min, resolution is lost with increasing temperature. At constant temperature, increased flow-rates tend
0 Zml/min
3’0 %
Fig. 2. Separation of oligosaccharides derived from an enzymatic hydrolysate of starch. Chromatographic conditions as in Fig. 1, except temperature is 88°C. The numbers on the peaks indicate DP values.
min
H. DERLER,
284
H. F. HORMEYER,
G. BONN
1
2 3
1 kJ32
4
7F5 _;-i
0.3ml/min
0.4ml/min
i
---I-. I
2’0
30
40
I i: 10 20
30
min
Fig. 3. Separation of oligosaccharides derived from an enzymatic hydrolysate of starch. Chromatographic conditions as in Fig. 1, except temperature is 98°C. The numbers on the peaks indicate DP values.
to produce marked changes in the chromatograms. Thus it becomes possible at 88°C and 0.4 ml/min to separate oligomeric carbohydrates with degrees of polymerization (DPs) up to 12 within only 27 min. At a column temperature of 98°C there is no resolution at 0.2 ml/min, and the resolution is improved at 0.4 ml/min (Fig. 3). The chromatographic retention-time behaviour observed with the weakly cross-linked ion exchangers investigated, as a function of the temperature and flowrate, can obviously be explained by a combination of mechanisms. With H+ as the counter ion, ligand exchange plays a minor role in the separation. Whereas in the case of Agi, strong monodentate complexes are formed with monosaccharides’ 5, this is not the case when H’ is used as the counter ion so that, apart from electrostatic interactions between the protons and lone-pair electrons of the sugar oxygens, the prevalent mechanism is size exclusion. The fact that hydrogen-bonding interactions of carbohydrates with counter ions such as those of Tris, i.e., (HOCH&CNH3+, had been shown to be weak15 also shifted attention away from the investigation of the rather similar but stronger electrostatic interactions provided by hydrogen loadings. The relative importance of mere size-exclusion effects and the above-mentioned attractive forces thus remains largely unknown, so that the contribution of the latter must not be neglected a priori, even though, for the stationary phase investigated, it may not reach the significance of the size-exclusion mechanism. Fig. 4 shows the linear semilogarithmic plot of the molecular weights of the oligomeric carbohydrates vs. their retention times, which is analogous to that observed in molecular exclusion chromatography. In the same figure, a similar linear dependence is shown for a 4% cross-linked silver-loaded ion-exchange material available commercially (HPX42A)’ 6. An increase in the flow-rate does result in shorter retention times, but it can be applied only in a restricted sense due to the instability of the matrix at higher pressures. However, the resolution is partly lost at lower flow-rates due to the smaller number of theoretical plates. The fact that no separation occurs at 98°C and low flow-rates can be explained as a consequence of diffusion effects when assuming a size-exclusion mechanism. This effect is particularly pronounced at low linear velocities. The loss in resolution caused by the thermal mobility of the compounds becomes
HPLC
OF OLIGOSACCHARIDES.
285
I. DP
IogMG
T
I ,
I
I
I
600
1200
1000
800
1
1400
I
1600
I
I
1800
I
2000
Fig. 4. Semilogarithmic plot of molecular weight (MG) vs. elution time (s) of the oligo-carbohydrates. (0) Chromatographic conditions: column, 2% cross-linked hydrogen-loaded sulphonated poly(styrenedivinylbenzene) ion exchanger; mobile phase, 0.01 N sulphuric acid; flow-rate, 0.4 ml/min; temperature, 4% cross-linked silver-loaded sulphonated 88°C. (0) Chromatographic conditions: column, polystyrene-divinylbenzene ion exchanger; mobile phase, deionized water; flow-rate, 0.5 ml/min; temperature. 85°C.
prominent as soon as their movement through the column is too slow to compensate for diffusive band broadening within the pores. A further decisive advantage of the column material investigated is the possibility of analyzing, with 0.01 N sulphuric acid as the eluent, samples resulting from acidic degradations as well as from enzymatic and fermentative conversion without time-consuming sample work-up procedures. In addition to the oligomeric carbohydrates, other compounds such as xylose, arabinose, glucuronic acid, hydroxymethylfurfural and furfural (see Table I) can be detected in the same analysis. TABLE
I
RETENTION RANGEMENT
BEHAVIOURS PRODUCTS
OF
CARBOHYDRATES,
THEIR
CONVERSION
Chromatographic conditions: column, hydrogen-loaded poly(styrene-divinylbenzene) cross-linking); mobile phase, 0.01 N sulphuric acid; flow-rate, 0.4 ml/min; column Compound Oligosaccharides, D.P. Glucuronic acid Glucose Xylose Arabinose Hydroxymethylfurfural Furfural
Retention time (min)
1l-2
12.624.2 26.6 27.0 27.9 28.9 37.1 50.1
AND
REAR-
ion exchanger (2% temperature 88°C.
286
H. DERLER,
H. F. HijRMEYER,
G. BONN
In regard to the possible coupling of this column with other ion-exchange systems, the use of deionized water as the mobile phase instead of 0.01 N sulphuric acid was also investigated. No decrease in resolution occurred, so that this column packing material can be regarded as applicable in combination with ion-exchange columns loaded with Na+, K+, Ca*+ and Pb*+. Thus the way is open to the determination in a single analysis of gluco-oligomers, monomeric carbohydrates and their degradation products with water as the mobile phase, obtaining similar retention times to these already describedlO. When using a 2% cross-linked silver-loaded ion-exchange material, ligand-exchange effects are expected in addition to size exclusion of the carbohydrates. Our first experiments in this direction show resolution of the gluco-oligosaccharides up to DP 15, which suggests a combination of mechanisms. ACKNOWLEDGEMENTS
We thank Messrs. Gray (Bio-Rad, Vienna, Austria) for their valuable help.
CA, U.S.A.) and Leinstein (Bio-Rad
REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
N. K. Sabbagh and I. S. Fagerson, J. Chromatogr., 86 (1973) 184. G. Bonn, J. Chromatogr., 387 (1987) 393. H. D. Scobell and K. M. Brobst, J. Chromatogr., 212 (1981) 51. M. John, G. Trenel and H. W. Dellweg, J. Chromatogr., 42 (1969) 476. W. Brown, J. Chromatogr., 52 (1970) 213. K. Aitzetmtiller, J. Chromatogr., 156 (1978) 354. L. A. Th. Verhaar, B. F. M. Kuster and H. A. Claessens, J. Chromatogr., 284 (1984) 1. E. Rajakyll, J. Chromatogr., 353 (1986) 1. G. Bonn, R. Pecina, E. Burtscher and 0. Bobleter, J. Chromatogr., 287 (1984) 215. G. Bonn, J. Chromatogr., 322 (1985) 411. E. Lederer and M. Lederer, in Chromatography - A Review of Principles and Applications, Elsevier, Amsterdam, 1957. 0. Bobleter and G. Pape, Aust. Put., 263,661 (1968). G. Bonn, R. Concin and 0. Bobleter, Wood Sci. Technol., 17 (1983) 195. G. Bonn, H. F. Hormeyer and 0. Bobleter, Wood Sci Technol., 21 (1987) 179. R. W. Goulding, J. Chromatogr., 103 (1975) 229. R. Pecina, Ph.D. Thesis, University of Innsbruck, Innsbruck, 1985.
CHROM.
in The Netherlands
20 212
HIGH-PERFORMANCE OLIGOSACCHARIDES I. SEPARATION CROSS-LINKING*
H. DERLER, Institute
Printed
ON
LIQUID
AN ION-EXCHANGE
H. F. HiiRMEYER
qf Radiochemistry,
CHROMATOGRAPHIC
STATIONARY
ANALYSIS
OF
PHASE OF LOW
and G. BONN*
University
of Innsbruck,
Innrain 52a. A-6020 Innsbruck
(Austria)
SUMMARY
The separation of oligomeric carbohydrates was investigated for the first time on a 2% cross-linked ion-exchange material with H+ as the counter ion. In spite of the low resistance to pressure, good oligosaccharide separations were obtained, which are comparable to those described previously on 4-6% cross-linked and silver-loaded ion exchangers. The mechanism responsible for the separations is, predominantly, size exclusion. By investigating the dependences on the temperature and flow-rate, the chromatographic conditions could be optimized.
INTRODUCTION
The separation of oligomeric carbohydrates, such as, e.g., gluco-oligomers, is required in many fields of the applied sciences, such as medicine, food chemistry and biomass analysisip3. Apart from gel chromatographic separations4v5, several highperformance liquid chromatographic (HPLC) methods have been described, most of which make use of aminopropyl, C is or ion-exchange stationary phases+‘O. With regard to the ion-exchange materials, cation exchangers loaded with Ag+ and Ca2 + and having different degrees of cross-linking have been employed most frequently. The different extents of cross-linking are obtained by copolymerization of polystyrene with different concentrations of divinylbenzene. Conventionally, the amount of crosslinkage is expressed as the percentage of divinylbenzene. Polymers with low percentages of cross-linking are characterized by a high degree of permeability, high equilibrium rates and reduced physical stability. Furthermore, particle size must be considered l l. Silver as the counter ion leads to a particularly high resolution of gluco-oligomers, with an optimum at 70% loading and 4% divinylbenzene cross-linkings. In addition to size exclusion, ligand exchange and matrix adsorption contribute to this
l
September
Presented in part at the 24th International 8-10. 1987.
0021-9673/88/$03.50
0
Symposium
1988 Elsevier Science Publishers
on Advances in Chromatography,
B.V
Berlin,
282
H. DERLER,
H. F. HORMEYER,
G. BONN
separation. Since, however, many “optimizations” have to be applied to acidic samples, considering, e.g., the large number of cases where oligomers only form upon acid hydrolysis, an exchanger loaded with 70% Ag+ would prove rather problematic with regard to the reproducibility both of the retention time and of the extent of cation loading; furthermore, in-flow regeneration would not be possible, thus adding to the difficulties in application. The possibility of obtaining analogous separation of identical resolution with other materials was investigated in the present work by the use of a weakly crosslinked support and substitution of Ag+ by H+ in order to eliminate ligand exchange in favour of the size-exclusion mechanism, whereas the separation capacity was maintained. An additional comparison was drawn with regard to the influence of silver loading of a 2% cross-linked material. EXPERIMENTAL
Apparatus The HPLC system consisted of a pump (Bio-Rad Labs., Richmond, CA, U.S.A.), an injection valve with a volume of 20 ~1 (Altex, Berkeley, CA, U.S.A.), a column oven (Bio-Rad), a refractive index detector (Bio-Rad RI Monitor) and a C-R3A integration system (Shimadzu, Kyoto, Japan). Columns The stationary phases used were (a) a sulphonated styrenedivinylbenzene copolymer, loaded with H+, 2% cross-linked, 25 pm, 300 mm x 7.8 mm I.D. (probably soon to be commercialized as HPX22H by Bio-Rad), and (b) a sulphonated styreneedivinylbenzene copolymer, loaded with Ag’, 2% cross-linked, 25 ym, 300 mm x 7.8 mm I.D. (HPX22A, Bio-Rad). As precolumns, ion-exclusion micro-guard cartridges (Cation-H, Bio-Rad) were employed. The eluents were 0.01 N sulphuric acid (p.a.; E. Merck, Darmstadt, F.R.G.) and deionized water, respectively. All eluents were degassed in an ultrasonic water bath before use. Samples For standard solutions of oligomers, an enzymatic hydrolysate of starch as well as hydrothermally degraded gluco-oligomers from cellulose were investigated12-r4. All other reference solutions were obtained from analytical grade products commercially available (Fluka, Buchs, Switzerland; E. Merck; Sigma, St. Louis, MO, U.S.A.) and dissolved in deionized water. RESULTS AND DISCUSSION
The low degree of cross-linking (2%) of the sulphonated poly(styrene-divinylbenzene) copolymer (hydrogen form) causes the exchanger material to be less stable towards high pressure, which may lead to changes in the polymeric support. Hence, lower flow-rates must be applied than with the silver-loaded ion-exchange phases presently available commercially, which achieve higher pressure stability by higher cross-linking (4-6%; HPX42A). A major advantage of hydrogen-loaded ion exchangers lies in the fact that the use of 0.01 N sulphuric acid as the eluent brings about
HPLC
OF OLIGOSACCHARIDES.
283
I.
Fig. 1. Separation of oligosaccharides derived from an enzymatic hydrolysate of-starch. Chromatographic conditions: column, 2% cross-linked hydrogen-loaded sulphonated poly(styrene-divinylbenzene) ion exchanger; mobile phase, 0.01 N sulphuric acid; flow-rate, see chromatograms; temperature, 76°C. The numbers on the peaks indicate DP values.
continuous regeneration, in contrast to the 70% silver-loaded columns, which can be operated only with deionized water as the mobile phase. Through the use of sulphuric acid and water, respectively, as the mobile phase and refractive index detection, the sensitivity in the determination of mono-, di- and oligosaccharides reaches the nanogram range. Thus, the applicable range was examined for flow-rates which did not result in an high pressure build-up, i.e., < 200 p.s.i., ea. 14 bar. Because of the low degree of cross-linking of the polymeric ion-exchange matrix, the pressure build-up, depending on the flow-rate and temperature, becomes very important. At room temperature, e.g., the system cannnot be operated at flow-rates higher than 0.154.25 ml/min if irreversible changes in the matrix, which would occur at pressures above 300 psi., are to be avoided. At 60°C the flow-rate can be raised to ca. 0.4 ml/min, at 90°C to 0.5 ml/min, without a drastic increase in pressure. Figs. l-3 show the separation of oligomeric carbohydrates on a 2% crosslinked hydrogen-loaded ion-exchange resin as functions of the flow-rate and the column temperature. With a constant flow in the range of 0.2-0.4 ml/min, resolution is lost with increasing temperature. At constant temperature, increased flow-rates tend
0 Zml/min
3’0 %
Fig. 2. Separation of oligosaccharides derived from an enzymatic hydrolysate of starch. Chromatographic conditions as in Fig. 1, except temperature is 88°C. The numbers on the peaks indicate DP values.
min
H. DERLER,
284
H. F. HORMEYER,
G. BONN
1
2 3
1 kJ32
4
7F5 _;-i
0.3ml/min
0.4ml/min
i
---I-. I
2’0
30
40
I i: 10 20
30
min
Fig. 3. Separation of oligosaccharides derived from an enzymatic hydrolysate of starch. Chromatographic conditions as in Fig. 1, except temperature is 98°C. The numbers on the peaks indicate DP values.
to produce marked changes in the chromatograms. Thus it becomes possible at 88°C and 0.4 ml/min to separate oligomeric carbohydrates with degrees of polymerization (DPs) up to 12 within only 27 min. At a column temperature of 98°C there is no resolution at 0.2 ml/min, and the resolution is improved at 0.4 ml/min (Fig. 3). The chromatographic retention-time behaviour observed with the weakly cross-linked ion exchangers investigated, as a function of the temperature and flowrate, can obviously be explained by a combination of mechanisms. With H+ as the counter ion, ligand exchange plays a minor role in the separation. Whereas in the case of Agi, strong monodentate complexes are formed with monosaccharides’ 5, this is not the case when H’ is used as the counter ion so that, apart from electrostatic interactions between the protons and lone-pair electrons of the sugar oxygens, the prevalent mechanism is size exclusion. The fact that hydrogen-bonding interactions of carbohydrates with counter ions such as those of Tris, i.e., (HOCH&CNH3+, had been shown to be weak15 also shifted attention away from the investigation of the rather similar but stronger electrostatic interactions provided by hydrogen loadings. The relative importance of mere size-exclusion effects and the above-mentioned attractive forces thus remains largely unknown, so that the contribution of the latter must not be neglected a priori, even though, for the stationary phase investigated, it may not reach the significance of the size-exclusion mechanism. Fig. 4 shows the linear semilogarithmic plot of the molecular weights of the oligomeric carbohydrates vs. their retention times, which is analogous to that observed in molecular exclusion chromatography. In the same figure, a similar linear dependence is shown for a 4% cross-linked silver-loaded ion-exchange material available commercially (HPX42A)’ 6. An increase in the flow-rate does result in shorter retention times, but it can be applied only in a restricted sense due to the instability of the matrix at higher pressures. However, the resolution is partly lost at lower flow-rates due to the smaller number of theoretical plates. The fact that no separation occurs at 98°C and low flow-rates can be explained as a consequence of diffusion effects when assuming a size-exclusion mechanism. This effect is particularly pronounced at low linear velocities. The loss in resolution caused by the thermal mobility of the compounds becomes
HPLC
OF OLIGOSACCHARIDES.
285
I. DP
IogMG
T
I ,
I
I
I
600
1200
1000
800
1
1400
I
1600
I
I
1800
I
2000
Fig. 4. Semilogarithmic plot of molecular weight (MG) vs. elution time (s) of the oligo-carbohydrates. (0) Chromatographic conditions: column, 2% cross-linked hydrogen-loaded sulphonated poly(styrenedivinylbenzene) ion exchanger; mobile phase, 0.01 N sulphuric acid; flow-rate, 0.4 ml/min; temperature, 4% cross-linked silver-loaded sulphonated 88°C. (0) Chromatographic conditions: column, polystyrene-divinylbenzene ion exchanger; mobile phase, deionized water; flow-rate, 0.5 ml/min; temperature. 85°C.
prominent as soon as their movement through the column is too slow to compensate for diffusive band broadening within the pores. A further decisive advantage of the column material investigated is the possibility of analyzing, with 0.01 N sulphuric acid as the eluent, samples resulting from acidic degradations as well as from enzymatic and fermentative conversion without time-consuming sample work-up procedures. In addition to the oligomeric carbohydrates, other compounds such as xylose, arabinose, glucuronic acid, hydroxymethylfurfural and furfural (see Table I) can be detected in the same analysis. TABLE
I
RETENTION RANGEMENT
BEHAVIOURS PRODUCTS
OF
CARBOHYDRATES,
THEIR
CONVERSION
Chromatographic conditions: column, hydrogen-loaded poly(styrene-divinylbenzene) cross-linking); mobile phase, 0.01 N sulphuric acid; flow-rate, 0.4 ml/min; column Compound Oligosaccharides, D.P. Glucuronic acid Glucose Xylose Arabinose Hydroxymethylfurfural Furfural
Retention time (min)
1l-2
12.624.2 26.6 27.0 27.9 28.9 37.1 50.1
AND
REAR-
ion exchanger (2% temperature 88°C.
286
H. DERLER,
H. F. HijRMEYER,
G. BONN
In regard to the possible coupling of this column with other ion-exchange systems, the use of deionized water as the mobile phase instead of 0.01 N sulphuric acid was also investigated. No decrease in resolution occurred, so that this column packing material can be regarded as applicable in combination with ion-exchange columns loaded with Na+, K+, Ca*+ and Pb*+. Thus the way is open to the determination in a single analysis of gluco-oligomers, monomeric carbohydrates and their degradation products with water as the mobile phase, obtaining similar retention times to these already describedlO. When using a 2% cross-linked silver-loaded ion-exchange material, ligand-exchange effects are expected in addition to size exclusion of the carbohydrates. Our first experiments in this direction show resolution of the gluco-oligosaccharides up to DP 15, which suggests a combination of mechanisms. ACKNOWLEDGEMENTS
We thank Messrs. Gray (Bio-Rad, Vienna, Austria) for their valuable help.
CA, U.S.A.) and Leinstein (Bio-Rad
REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
N. K. Sabbagh and I. S. Fagerson, J. Chromatogr., 86 (1973) 184. G. Bonn, J. Chromatogr., 387 (1987) 393. H. D. Scobell and K. M. Brobst, J. Chromatogr., 212 (1981) 51. M. John, G. Trenel and H. W. Dellweg, J. Chromatogr., 42 (1969) 476. W. Brown, J. Chromatogr., 52 (1970) 213. K. Aitzetmtiller, J. Chromatogr., 156 (1978) 354. L. A. Th. Verhaar, B. F. M. Kuster and H. A. Claessens, J. Chromatogr., 284 (1984) 1. E. Rajakyll, J. Chromatogr., 353 (1986) 1. G. Bonn, R. Pecina, E. Burtscher and 0. Bobleter, J. Chromatogr., 287 (1984) 215. G. Bonn, J. Chromatogr., 322 (1985) 411. E. Lederer and M. Lederer, in Chromatography - A Review of Principles and Applications, Elsevier, Amsterdam, 1957. 0. Bobleter and G. Pape, Aust. Put., 263,661 (1968). G. Bonn, R. Concin and 0. Bobleter, Wood Sci. Technol., 17 (1983) 195. G. Bonn, H. F. Hormeyer and 0. Bobleter, Wood Sci Technol., 21 (1987) 179. R. W. Goulding, J. Chromatogr., 103 (1975) 229. R. Pecina, Ph.D. Thesis, University of Innsbruck, Innsbruck, 1985.