Carbohydrate analysis by gas-liquid chromatography

Authors：

Category

Isolation & structural analysis of glycans

Protocol Name

Authors

Sato, Chihiro *
Bioscience and Biotechnology Center, Nagoya University

Kitajima, Ken
Bioscience and Biotechnology Center, Nagoya University

*To whom correspondence should be addressed.

KeyWords

Trimethylsilylation Methanolysis GLC Acetylation Gas-liquid chromatography

Reagents

●	Myo-inositol as an internal standard (Wako Pure Chemical Industries, Ltd. Osaka, Japan)
●	0.5N HCl-MeOH (Kokusan Kagaku Co., Ltd. Tokyo, Japan)
●	Absolute MeOH (Sigma-Aldrich, St. Louis, MO)
●	Acetic anhydride (Sigma-Aldrich)
●	Absolute pyridine (Sigma-Aldrich)
●	Hexamethyldisilazane（HMDS） (Wako Pure Chemical Industries, Ltd.)
●	Trimethylsilyl chloride (TMSC) (Wako Pure Chemical Industries, Ltd.)

Instruments

●	GLC (GC-14A: Shimadzu Corp., Kyoto, Japan)

Methods

Methanolysis

1)	Sample (1–10 µg) with an internal standard (1–10 µg).	Comment 0

2)	Dry up with rotary evaporator and then in a desiccator in vacuo (over P₂O₅).	Comment 0

3)	Add 0.5 mL of 0.5 N HCl-methanol, and heated at 65°C for 16 h.	Comment 1

4)	Dry up with evaporator and in the desiccator in vacuo (over P₂O₅).	Comment 0

Methylglycosides (see Note 1)

Comment 0

Re-N-acetylation

1)	Add 0.5 mL of absolute MeOH, 10 µL of absolute pyridine, and 50 µL of acetic anhydride to the sample, and leave at room temperature for 15min.	Comment 0

2)	Dry up with N_2-gas and then in a desiccator (over NaOH).	Comment 0

Methylglycosides with or without re-N-acetylation

Comment 0

TMS-derivatization

1)	Add 50 μL of absolute pyridine, 10 μL of HMDS and 5 μL of TMSC to the sample and leave at room temperature for 30 min.	Comment 0

2)	Centrifuge at 3,000 rpm for 10 min.	Comment 1

3)	Transfer the supernatant into a small glass tube.	Comment 0

4)	Dry up with N₂-gas.	Comment 0

5)	Solubilize with 50 μL of hexane.	Comment 0

TMS-derivatives

Comment 0

GLC

Inject 2–10 μL of the hexane solution into GLC.

Condition: for example: 140°C (10 min)・140°C–260°C (5°C/min)・260°C (10 min)

Detection FID, Column, CBJ-5 (30 m × 0.32 mm)

Comment 0

Notes

1. If samples contain a large amount of lipids, you had better to perform hexane extraction procedure as described below before re-N-acetylation step.

1) methanolyzed sample

↓ add 0.5 mL of hexane and mix.

↓ centrifuge at 3000 rpm for 5 min.

2) methanol layer

↓ dry up with rotary evaporator and then in the desiccator (over P₂O₅)

Initial amount

1–10 μg/sugar

Discussion

Carbohydrates have to be derivatized for volatilization before application to GC, because they are involatile. Trimethylsilylation is often used for volatilization due to the facileness. Trimethylsilylated monosacharides can be separated on GC, and detected by flame ionization detector or mass spectrometry (MS) like electron impact MS. Methylation is also used for volatilization of carbohydrates; however, it is more often applied to the linkage analysis of glycosides.

Figure & Legends

Fig. 1. Gas liquid chromatogram of Medaka hyosophorin.

Carbohydrate composition of N-linked glycan chain derived from hyosophorin sample was analyzed by GLC (Column: DB-5, Initial Temp: 140°C, Initial Time: 10 min, Final time: 260°C, Programmed rate 5°C/min).

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Date of registration：2015-03-24 13:52:16