Chapter 5 HPLC of carbohydrates with cation- and anion-exchange silica and resin-based stationary phases (original) (raw)
Related papers
Analytical Chemistry, 1998
Barium ion is shown here as an effective component of alkaline mobile phases for improving the separation of carbohydrates in anion-exchange chromatography with pulsed amperometric detection. We demonstrate that the introduction of barium ensures a complete removal of carbonate, making it possible to carry out a large number of separations without column reequilibration after each run. This is particularly helpful when diluted alkaline eluents (i.e., <20 mM NaOH) are employed. Under such experimental conditions, the analysis time of carbohydrates is drastically reduced and the reproducibility of the chromatographic data greatly improved. For example, the normalized capacity factor (k′/k′ 0 ) of lactulose, which represents the most retained compound investigated using 16 mM NaOH as the eluent, exhibits only a slight decrease from 1.00 to 0.94 after 7.5 h of chromatographic run. The net improvement in the peak shape of D-ribose, D-allose, and D-talose has been interpreted by considering the complex formation between barium and sugar molecules having an axial-equatorial-axial sequence of three OH groups on the six-membered ring. The presence of Ba(II) (0.5-0.7 mM) in alkaline eluents has been demonstrated by ion chromatography with conductivity detection.
Column selection strategies for high performance liquid chromatographic analysis of carbohydrates
High performance liquid chromatographic analysis of carbohydrates is a real challenge; because these compounds exhibit similar chemical and physical properties, they are more difficult to analyze than most other classes of compounds, to date no single chromatographic column or method being capable of separating all carbohydrates. Choosing the best column for carbohydrates analysis requires consideration of stationary phase chemistry, retention capacity, particle size and column dimensions. This research focuses on mono-and di-saccharides analysis, represented here by arabinose, fructose, glucose, saccharose and maltose, different commercially available columns being tested; isocratic separations were achieved using as mobile phase different acetonitrile / water mixtures at 1.2 mL min-1. A test carbohydrate mixture was used and the resulted separations were compared in order to select the best suited column.
Journal of Chromatography A, 1992
The influence of experimental variables such as the wavelength, composition of the fluorogenic reagent, reaction time, reaction and measurement temperatures, and concentration on the post-column derivatization of carbohydrates by reaction with an ethanolamineboric acid mixture was studied. In order to enhance the signals given by di-and trisaccharides, they were hydrolysed, after elution, with p-toluenesulphonic acid, which also provided lower detection limits for monosaccharides. The procedure was applied to the determination of eleven carbohydrates in wines.
Fresenius Journal of Analytical Chemistry, 2000
This article reviews recent advances of carbohydrate analysis by high-performance anion-exchange chromatography with pulsed amperometric detection. Starting from the paper of Dennis C. Johnson [1] in which the great analytical promise of such a technique was anticipated, a multitude of exciting new research possibilities have recently emerged. The great attractiveness of high-performance anion-exchange chromatography is largely due to its compatibility with such a sensitive, selective and reliable detection method as pulsed amperometry. This very good match between liquid chromatography and electrochemical detection has allowed the determination of carbohydrates in a variety of complex matrices, for instance, foods, beverages, diary and biotechnological products, vegetal tissues, and also in the area of clinical diagnostics. For this reason, the introduction of HPAEC-PAD into regulated methods is becoming increasingly accepted. A comprehensive collection of applications to carbohydrates and samples of interest is given, with special focus on the separation of closely related sugar compounds using dilute alkaline eluents. Advances in pulsed potential waveforms are also discussed, and a comparison with other liquid chromatographic methods addressed.
Analysis of carbohydrates by anion exchange chromatography and mass spectrometry
Journal of Chromatography A, 2005
A versatile liquid chromatographic platform has been developed for analysing underivatized carbohydrates using high performance anion exchange chromatography (HPAEC) followed by an inert PEEK splitter that splits the effluent to the integrated pulsed amperometric detector (IPAD) and to an on-line single quadrupole mass spectrometer (MS). Common eluents for HPAEC such as sodium hydroxide and sodium acetate are beneficial for the amperometric detection but not compatible with electrospray ionisation (ESI). Therefore a membrane-desalting device was installed after the splitter and prior to the ESI interface converting sodium hydroxide into water and sodium acetate into acetic acid. To enhance the sensitivity for the MS detection, 0.5 mmol/l lithium chloride was added after the membrane desalter to form lithium adducts of the carbohydrates. To compare sensitivity of IPAD and MS detection glucose, fructose, and sucrose were used as analytes. A calibration with external standards from 2.5 to 1000 pmole was performed showing a linear range over three orders of magnitude. Minimum detection limits (MDL) with IPAD were determined at 5 pmole levels for glucose to be 0.12 pmole, fructose 0.22 pmole and sucrose 0.11 pmole. With MS detection in the selected ion mode (SIM) the lithium adducts of the carbohydrates were detected obtaining MDL's for glucose of 1.49 pmole, fructose 1.19 pmole, and sucrose 0.36 pmole showing that under these conditions IPAD is 3-10 times more sensitive for those carbohydrates. The applicability of the method was demonstrated analysing carbohydrates in real world samples such as chicory inulin where polyfructans up to a molecular mass of 7000 g/mol were detected as quadrupoly charged lithium adducts. Furthermore mono-, di-, tri-, and oligosaccharides were detected in chicory coffee, honey and beer samples.
Hydrophilic-interaction chromatography of complex carbohydrates
Journal of Chromatography A, 1994
Complex carbohydrates can frequently be separated using hydrophilic-interaction chromatography (HILIC). The mechanism was investigated using small oligosaccharides and a new column, PolyGLYCOPLEX. Some carbohydrates exhibited anomer separation, which made it possible to determine the orientation of the reducing end relative to the stationary phase. Amide sugars were consistently good contact regions. Relative to amide sugars, sialic acids and neutral hexoses were better contact regions at lower levels of organic solvents than at higher levels. HILIC readily resolved carbohydrates differing in residue composition and position of linkage. Complex carbohydrate mixtures could be resolved using volatile mobile phases. This was evaluated with native glycans and with glycans derivatized with 2-aminopyridine or a nitrobenzene derivative. Both asialo-and sialylated glycans could be resolved using the same set of conditions. With derivatized carbohydrates, detection was possible at the picomole level by UV detection or on-line electrospray mass spectrometry. Selectivity compared favorably with that of other modes of HPLC. HILIC is promising for a variety of analytical and preparative applications.
Journal of Chromatography A, 1994
Sp~~r~ca~ ~~g~J~ ~ross-~~~k~d s~yrene-div~ny~be~~ene ~o~oly~e~ particles were ~hem~~al~y m~di~~d by direct nitration, followed by reduction with tin metaI and quatern~2at~on with iodomethane to produce a superficial layer of quaternized amino functions. Resides being simple and economical, the proposed method of synthesis leads to an anion exchanger that allows the use of shorter columns (75 x 4.6 mm I.D.) than those currently employed for carbohydrate separation by high-performance anion-exchange chromatography with pulsed amperometric detection under alkaline conditions. The new sorbent has proved to be highly selective for isomeric disaccharides and individual oligomers of an homologous series of maltooligosaccharides of up to 21 glucose residues. Extended exposure of columns packed with this sorbent to high pressure and strong alkaline solutions did not have any untoward effect on mechanical stability and chromatographic performance.
Improved thin-layer chromatographic method for sugar separations
Journal of Chromatography A, 1979
A method is described for the utilization of pre-coated, non-impregnated silica gel thin layers in one-and two-dimensional separations of carbohydrates and related compounds. Boric and phenylboronic acids were added to the organic elution systems in different concentrations and their interactions with the sugar molecules during the chromatographic process were studied. A comparison was made between solvent systems containing boric or phenylboronic acid and systems devoid of both acids as eluents. With boric acid-containing solvents the migration of some sugars was considerably inhibited, whereas phenylboronic acid produced an increase in the RF values of certain sugars. The combination of these two types of solvent in two-dimensional development resulted in the clear separation of a group of mono-and disaccharides of bioclinical interest_ 2 M. GHEBREGZABHER, S. RUFINI, G. M. SAPIA, M. LAT.0 Although the separation of more complex sugar mixtures by such a chromatographic system is, in our opinion, difficult to achieve, nevertheless other more appropriate boric acid-containing mobile phases would enable one to use pre-coated thin layers directly without any loading pre-treatment, giving considerable advantages in comparison with the TLC of sugars on home-made or on pre-coated homeloaded layers, riz., (a) better reproducibility of the results; (b) higher sensitivity of the examined compounds to the detecting reagents; and (c) better tractability of the chromatograms, i.e., after single or multiple elution and even after several applications of the spray reagents the chromatographic layer does not become friable and retains a firmness similar to that of the paper. Selectivity based on a particular type of solute-solvent interaction also occurs when phenylboronic acid-containing mobile phases are used for the separation of polyhydroxy compounds, as Boume et al." have demonstrated in their work on the paper chromatography of sugars and poly-alcohols_ Substances with polyhydroxy groups in appropriate steric arrangements react with phenylboronic acid to give esters whose solubility in organic solvents is probably increased by the presence of the phenyl group. Phenylboronic esters of several carbohydrates have been obtained in crystalline form, and the molecular structures of some of them have been elucidated5-'. One of the properties of these esters is their instability in water-containing organic solvents'. The formation of sugar phenylboronates and their aqueous hydrolysis also seem to occur during the chromatographic process'. In this instance, not only the solubility of the compounds under examination in the mobile phase but also their hydrolysis equilibrium constants will affect the chromatographic separation of the sugars. Therefore, the study of new solvent systems with different amounts of phenyldihydroxyboric acid, suitabIe for the separation of carbohydrates and related compounds on silica or cellulose thin layers, is of interest. In addition, such mobile phases should permit the direct use of pre-coated thin layers with the above-mentioned advantages_ We have therefore examined various concentrations of boric acid and phenyldihydroxyboronic acid in different eluent systems which, in a chromatographic study of sugars of bio-clinical interest, should give good separations by a simple technique on pre-coated silica gel thin layers. In this paper we present the results of studies on (a) quantitative aspects Of sugar-boric acid interactions that can occur during the chromatographic process when this acid is present in the solvent system, (b) the effect of phenylboronic acid on the chromatographic behaviour of sugars when it is present in the mobile phase in various concentrations, and (c) the capabilities of eluents containing boric or phenylboronic acid for the one-dimensional TLC separation of sugars. .Also,-we have examined the combined use of boric acid-and phenylboronic acid-containing solvent systems in the two-diemensional TLC of carbohydrates and related compounds. EXPERIMENTAL Materials and apparatus All chemicals were of analytical-reagent grade and were used without further purification.
HPAE-PAD - a sensitive method for the determination of carbohydrates
Fresenius' Journal of Analytical Chemistry, 1998
High performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) was used for the determination of eleven monosaccharides. Three analytical columns with different selectivities were tested, and the resulting separations were compared calculating precision and detection limits. The monosaccharides could be separated on CarboPAC PA10 in one analysis run with the lowest detection limits and a high precision. For the determination of polysaccharides and humic bound carbohydrates in natural organic matter, an hydrolysis step had to be carried out. With the exception of fructose, the recoveries varied between 56% and 83%. The described methods were applied for the determination of bound carbohydrates in a bog lake water and a soil extract without preconcentrating the samples.
Journal of Separation Science, 2014
After 20 years of development, evaporative light-scattering detection (ELSD) has become the mainstream choice for the detection of various classes of natural products. ELSD continues to grow in popularity as a "quasi-universal" technique because of the specificity of the detection method, which is based on the scattering of laser light from nonvolatile analyte particles. It represents an attractive alternative compared to other types of detection, such as refractive index detection and/or ultraviolet detection. This review presents issues concerned with the separation of carbohydrates in plant materials by HPLC and ELSD, as well as the advantages and limitations relating to the ELSD method. Additionally, an overview of possible ELSD applications in the analysis of carbohydrates in natural products is presented.