High-sensitive, High-selective Analysis of Saccharides

This is an example of a sensitive detection of cyclodextrin contained in plasma by pulse-amperometric electrochemical detector (PAD) using strong alkaline reagent mixed with eluent at post-column. Asahipak C8P-50 4E (a column for reversed phase chromatography) is used for the separation.

Sample : beta-Cyclodextrin
1. G-beta-CD
2. beta-CD

Columns      : Shodex Asahipak C8P-50 4E (4.6mmID*250mm)
Eluent       : 1mM Sodium hydroxide + 0.6% CH3CN
Reagent      : 1.5M Sodium hydroxide
Flow rate    : (Eluent); 0.6mL/min, (Reagent); 1.0mL/min 
Detector     : PAD
Column temp. : 50deg-C

Y.Kubota et al., Analytical Biochem., 201 (1992) 99.

For substances, which are not chemiluminescent themselves, if they can be converted into hydrogen peroxide through an enzyme reaction, it is possible to make a chemiluminescence detection of them. For example, glucose itself cannot be detected chemiluminescently. However, by reacting glucose with glucose oxydase, gluconic acid and hydrogen peroxide are generated. Then, it is possible to make a sensitive detection of the hydrogen peroxide generated here by both direct and indirect emission type. This is an example of glucose measurement by using AFpak AGO-494, a column for immobilizing enzyme, and a chemiluminescence detector (CL).

Sample : 1. Glucose

Column       : Shodex AFpak AGO-494 (4.6mmID*10mm)
Eluent       : H2O
Reagent      : 5mM Luminol + 5micro-M Hemoglobin in 0.1M Phosphate-Na
Flow rate    : (Eluent); 0.2mL/min, (Reagent); 0.4mL/min 
Detector     : CL
Column temp. : 37deg-C

The ligand of AFpak AWG-894 column is wheat germ agglutinin (WGA) which has affinity with acetylglucosamine. The column is suited to high-selective analysis and fractionation for saccharides or glycoproteins.

Sample :
1. N-Acetylglucosamine
2. N-Acetylchitobiose

Column       : Shodex AFpak AWG-894 (8.0mmID*50mm)
Eluent       : 1/15M Phosphate buffer(pH7.4) + 0.15M NaCl
Flow rate    : 0.5mL/min
Detector     : UV(280nm)
Column temp. : Room temp.

The ligand of AFpak ACA-894 column is concanavalin A which can distinguish sugars such as mannoisde and glucoside. The column is suited to high-selective analysis and fractionation of saccharides or glycoproteins.

Sample :
1. p-Nitrophenyl-alpha-D-galactoside
2. p-Nitrophenyl-alpha-D-glucoside
3. p-Nitrophenyl-alpha-D-mannoside

Column       : Shodex AFpak ACA-894 (8.0mmID*50mm)
Eluent       : 0.02M Tris-HCl buffer(pH7.4) + 0.25M NaCl
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : Room temp.

AFpak APB-894 is a column with aminophenylboronic acid bonded as ligand. As aminophenylboronic acid has affinity with vicinal hydroxyl groups, it can be used for the analysis and fractionation of saccharides, nucleic acids and catecholamines.

Sample :
1. Mannose
2. Mannitol
3. Sorbitol

Column       : Shodex AFpak APB-894 (8.0mmID*50mm)
Eluent       : 0.1M Sodium phosphate buffer(pH9.0)
Flow rate    : 1.0mL/min
Detector     : Shodex RI
Column temp. : 25deg-C

Steroid hormones like estradiol are excreted in animals as aggregates – a form presenting no hormonal activity. It has however been reported that upon removal of water, they disassemble to release the free active form, which has prompted efforts to characterize such aggregates. In this application, estradiol and its metabolic by-products, i.e. 11 compounds including estrone and a variety of estroriol glucuronides were analyzed with the polymer-based NH2P40-2D column in HILIC mode with LC/MS/MS detection.

Sample : 0.1ppm each, 5micro-L
1. Estrone-3-sulfate
2. β-Estradiol-3-sulfate
3. Estriol-3-sulfate
4. Dehydroepiandrosterone-3-sulfate
5. Estrone-3-glucuronide
6. β-Estradiol-17-glucuronide
7. β-Estradiol-3-glucuronide
8. Estriol-3-glucuronide
9. Estriol-16-glucuronide
10. Dehydroepiandrosterone-3-glucuronide
11. Androsterone-3-glucuronide

Column   : Shodex NH2P40-2D (2.0mmID*150mm)
Eluent  : A; 10mM NH4OH H2O
            B; 10mM NH4OH CH3CN
Gradient  : linear gradient: 
            B%, 22% (0-5min), 22-70% (5-10min), 70% (10-15min)
Flow rate : 0.2mL/min
Detector  : ESI-MS/MS (Polarity : Negative)
Column oven : 40deg-C

Data courtesy of Mr. Atsushi Yamamoto, Osaka City Institute of Public Health and Environmental Sciences

Pyridylaminated (PA) monosaccharides were analysed by Asahipak NH2P-50 4E. Carbohydrates are usually detected by refractive index detector (RI), but pyridylamination of saccharides enables high sensitivity analysis by fluorescence detector.

Sample : 5μL
1. 2-Aminopyridine, 0.1pmol/μL
2. PA-Rhamnose, 1pmol/μL
3. PA-Fucose, 1pmol/μL
4. PA-Ribose, 1pmol/μL
5. PA-Xylose, 1pmol/μL
6. PA-N-Acetylglucosamine, 1pmol/μL
7. PA-N-Acetylgalactosamine, 1pmol/μL
8. PA-Mannose, 1pmol/μL
9. PA-Glucose, 1pmol/μL
10. PA-Galactose, 1pmol/μL

Column       : Shodex Asahipak NH2P-50 4E (4.6mmID*250mm)
Eluent       : Phosphoric acid/H2O/CH3CN=1/14/85
Flow rate    : 0.5mL/min
Detector     : Fluorescence(Ex. 310nm, Em. 380nm)
Column temp. : 40℃
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