LC Technical Note

Purification of 2-AB Derivatized Glycans Using MonoSpin

LT172

GL Sciences Inc.

When analyzing glycans, Fluorescent derivatization method is often used before measurement byHPLC. Although large amounts of unreacted derivatizing reagents remain in the solution immediately
after the derivatization reaction, only unreacted reagents can be removed by performing solid-phaseextractions prior to injecting them into the HPLC.
Using MonoSpin, a spin-type solid-state column, separate sugar chains derivatized with 2aminobenzamide (2-Aminobe​nzamide,2-​AB)​ from unreacted reagents. As a result of evaluating thetwo types of MonoSpin Amide and MonoSpin NH2, both of them recover sugar chains of more thantrisaccharide.
(Y. Yui, C. Aoyama, S. Ota)

Sample Solution Measurement Without Purification
Unreacted Reagent

Sample Solution Measurement After Purification using MonoSpin Amide
Significantly remove unreacted reagents
Little GU12-AB can be recovered
GU22-AB yields recoveries as high as 30 %Glycans over trisaccharides yielded recoveries approaching 100%

HPLC Condition
System : GL7700 HPLC SystemColumn : InertSustain Amide (5 µm, 250 X4.6 mm I.D.)
Eluent : A) CH3CNB) 50nM HCOONH4 in H2O (pH 4.4, HCOOH)
Time [min]A (vol%)B (vol%)080

2020653580

5050905050

Flow Rate: 1.0 mL/minColumn Temp. : 50 ℃
Eluent: FL Ex 330 nm Em 420 nm(FL7753 FL Detector)
Injection Volume: 25 µLSample: 2-AB Labeled Glucose
Homopolymer LadderGL Sciences LC Technical Note
◆MonoSpin
The MonoSpin series is a spin-column using silica
monoliths with uniform continuum pores. Silica
monoliths with high porosity are used as carriers,
so they can be passed through by centrifugal
manipulation alone. Therefore, it is possible topurify and concentrate samples by a simple
operation in a short time. It is also suitable when
the sample volume is small because the bed
volume is small and the liquid can be cut off.

Enlarged view ofthe silica monolith

The procedure is centrifugation.

Silica monolith
disk◆Purification using MonoSpin
MonoSpin Amide

MonoSpin NH2

A column modified amid group. Mainly used as
HILIC mode and is optimal for extracting wide
range of hydrophilic compounds.

A column modified aminopropyl group on its
surface of silica monolith. It is used as not only
HILIC mode but also as weak anion-exchange
columns

Purification Procedure
Centrifuge: 9,000 x g 1 min

Centrifuge

1. Conditioning
Solution A 200 µL↓
Solution B 200 µL

Centrifuge

Centrifuge

2. Adsorption
Sample Solution
200 µL

3. Rinsing
Solution B 200 µL

Centrifuge

4. Eluent
Solution A 200 µL

Purified Sample

Solution A
Water : Acetonitril : Formic acid = 50:50:0.1 (v/v/v)
Solution B
Water : Acetonitril : Formic acid = 10:90:0.2 (v/v/v)
Sample Solution
In this study, 10μL of water and 180μL of acetonitrile were mixed with 10μL of a solution
containing 2-AB sugar chains dissolved in DMSO:acetic acid=70:30 (v/v).

GL Sciences LC Technical Note

If MpnoSpin NH2 is Used Instead of MonoSpin Amide
MonoSpin NH2 works not only in the HILIC mode but also in the negative ion-exchange mode,
but the effects of this study were almost equivalent to those of MonoSpin Amide.

Purification with MonoSpin Amide

Purification with MonoSpin NH2

Unreacted reagents can be removed
as well as MonoSpin Amide.

The recoveries over trisaccharides were also comparable to MonoSpin Amide

GL Sciences disclaims any and all responsibility for any injury or damage which may be caused by this data directly or indirectly.
We reserve the right to amend this information or data at any time and without any prior announcement.

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