Highly efficient analysis of underivatized carbohydrates using monolithic-silica-based capillary hydrophilic interaction (HILIC) HPLC (original) (raw)
Related papers
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.
High-performance liquid chromatography of disaccharides on amine-bonded silica columns
Journal of Chromatography A, 1985
,/~,/~-trehalose, turanose, and xylobiose) were subjected to high-performance liquid chromatography on prepacked amine-bonded silica columns, using acetonitrile-water eluents. Glucopyranosyl-glucoses had increasing retention times in the order of linkage (1-,3), (1 ~4), (1 ~2) and (1-* 1), and (1 ~6). Replacement of one of the glucosyl residues by galactose led to longer retention times, while substitution by a fructosyl residue yielded shorter ones. Forced assumption of the furanose ring form by the fructosyl residue, as in sucrose and palatinose, gave greatly reduced retentions.
HPLC analysis of mono-and disaccharides in foods
A simple and reproducible high-performance liquid chromatography (HPLC) with refractive index (RI) method for the qualitative and quantitative analysis of five mono-and disaccharides (fructose, glucose, sucrose, maltose and lactose) in food products has been developed and validated. The best separation of sugars was achieved with mobile phase acetonitile-water (80:20) and flow rate 2 ml/min. HPLC method showed good linearity with determination coefficients exceeding 0.998. The limits of detection (DL) in these sugars were 0. 0.13, 0.21, 0.26 and 0.36 mg/ml, respectively; 0.47, 0.51, 0.69 and 0.70 mg/ml. Recoveries in all sugars were between 81 and 121%.
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.
HPLC Analysis of Mono-And Disaccharides in Food Products
A simple and reproducible high-performance liquid chromatography (HPLC) with refractive index (RI) method for the qualitative and quantitative analysis of five mono-and disaccharides (fructose, glucose, sucrose, maltose and lactose) in food products has been developed and validated. The best separation of sugars was achieved with mobile phase acetonitile-water (80:20) and flow rate 2 ml/min. HPLC method showed good linearity with determination coefficients exceeding 0.998. The limits of detection (DL) in these sugars were 0.13, 0.13, 0.21, 0.26 and 0.36 mg/ml, respectively; and the limits of quantification (QL) -0.44, 0.47, 0.51, 0.69 and 0.70 mg/ml. Recoveries in all sugars were between 81 and 121%.
2002
This chapter discusses different stationary phase matrices and functionalities available for the high-performance liquid chromatography (HPLC) separation of carbohydrates. Carbohydrates play an important role in many different research and industrial domains, such as biochemistry, clinical chemistry, biology, pharmacy, biotechnology, and food chemistry. Automated chromatographic analysis of carbohydrates is most frequently performed on ion-exchange stationary phases. The ion-exclusion phenomenon can be explained as the repulsion of ions with equidirectional charges. Spectrophotometric postcolumn labeling has played an important role in monitoring carbohydrates in borate complex anion-exchange chromatography because carbohydrates have no characteristic absorption in the ultraviolet (UV)-visible range. Measurement of the refractive index is insensitive and is prone to baseline drift because of temperature instability or changes in mobile-phase composition. The increase in retention times with increasing alkyl chain length as observed with linear acids, aldehydes, and alcohols is mainly caused by a reversed-phase mechanism. The elution of oligomeric analytes occurs in order of decreasing size. If there are no additional interactions between the analytes and the stationary phase, a linear correlation between the elution volume and the logarithm of molecular weight can be obtained.
Talanta, 2013
The qualitative and quantitative analysis of complex carbohydrate mixtures is a challenging problem. When tackled by GC/MS, close retention times and largely similar mass spectra with no specific features complicate unambiguous identification, especially of monosaccharides. An optimized pre-capillary ethoximation-silylation GC/MS method for determination of monosaccharides and disaccharides was applied to a wide range of analytes (46 compounds). The two-step derivatization resulted in a pair of syn and anti peaks with specific retention and intensity ratio. The resulting dataset of mass spectra was subjected to a PCA-based pattern recognition. An oxime peak identifier (OPI) of the carbohydrate analytes, based on the combination of an internal standard and the corresponding syn/anti peak ratios, increased the reliability of the identification of reducing carbohydrates. Finally, the introduced EtOx-TMS derivatization method was applied to four different carbohydrate matrices (agave sirup, maple sirup, palm sugar, and honey).
High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) has been widely used for separation and detection of carbohydrates ranging from simple monosaccharides to branched oligosaccharides, and large linear polysaccharides. 1,2 CarboPac ® columns are a family of high-resolution, strong anionexchange columns developed for carbohydrate separations with HPAE-PAD under alkaline conditions. They have been proven to be capable of separating monosaccharides; positional, linkage, and branch isomers of oligosaccharides; and homopolymer oligosaccharides that differ only in length. 3,4 Sodium hydroxide or potassium hydroxide is commonly used to elute carbohydrates on these columns. Sodium acetate is sometimes added to elute multiply-charged carbohydrate species that strongly bind to these columns. Here we demonstrate that the separation of carbohydrates on the CarboPac PA20 and CarboPac PA200 columns can be improved by adjusting the concentration of the eluting hydroxide solution.
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.