LC Technical Note

Highly Sensitive and Simple Method for Sugar Analysis
Using HPLC-ECD

LT101
GL Sciences Inc.

In this note, sugar was detected with electrochemical

There are several methods to analyze sugar by HPLC. Our
previous notes showed an HPLC method using fluorescence

detector (ECD). Sensitivity of this method wasdetector coupled with post-column derivatization (No. 6) andcomparable to that of fluorescence detection althoughthat using RI detector (No. 91).

derivatization is not necessary.

A chromatogram obtained from standard solutionConditionsSystemColumn

210 mg/L
10 mg/L
10 mg/L10 mg/L
10 mg/L

2.8

3.0

3.2

1

1. Fucose
2. Glucose
3. Fructose
4. Lactose
5. Sucrose

Eluent Flowrate Col.
Temp.
Detection: LC800 system with ECD: InertSphere Sugar-1(5 μm, 150×4.6 mm I.D.): 100 mM NaOH*: 0.5 mL/min: 25 ℃: ECD Pulse Mode (ED723, Gold)E1: 150 mVE2: -1500 mVE3: 600 mV
E4: -200 mVuA
2.6

Inj. Volume : 10 μL
352.0
1.801020
Time [min]30

Calibration Curves

How are sugars separated in this method?
Sugars are ionized under strong alkaline condition. Therefore,sugars can be retained and separated on anion-exchange column
using alkaline aqueous solution as mobile phase.

Alkaline condition

t1: 600 ms
t2: 50 ms
t3: 50 ms
t4: 100 ms
ts: 50 ms* Eluent was stored in a polypropylene bottle with CO2
trap cartridge.

2.2

2.44（C. Aoyama）

GL Sciences LC Technical Note

Detection of sugar in HPLC
Sugars cannot be detected using UV or fluorescence detector without derivatization
because sugars do not possess chromophore, such as double bond or benzene ring,in their structure. On the other hand, sugar analysis using electrochemical detector(ECD) does not require any derivatization because sugars are detected by oxidationof carbohydrates on a working electrode surface and monitoring the resulting
current. The sensitivity is approximately 1000-fold higher than that of refractiveindex (RI) detector, which can also detect sugars without derivatization. ECD hasmajor advantages in sugar analysis.
Detector

Electrochemical detector(ECD)
Fluorescence detector(FL, coupled with post-column derivatization)
Refractive index detector (RI)

Sensitivity (approx.)
Cautions
Standard
HPLC
pumpand autosampler can be used
10 ng
if materials for their wetted parts are alkaliresistant.

10 ng

10 μg

Additional pump and reaction unit are required forderivatization.
Gradient elution cannot be carried out.

Evaporative light scattering detector (ELSD)

1 μg

Non-volatile agents cannot be performed, andcalibration curve is not linear.

Cleaning of electrode surface and pulsed-amperometric detection mode
In sugar analysis using ECD, oxidized analyte is adsorbed to the working electrode surface. It is necessary to
remove analyte bound to the surface because it may diminish the sensitivity.
ED723 can be used with not only dynamic current mode (DC), in which constant potential is applied throughout
an analysis, but also pulsed-amperometric detection mode (PAD). For example, potential program shown below
is applied periodically. Analytes bound to working electrode surface is removed by applying strong reduction andoxidation potential after each time current is measured. Surface of working electrode can be maintained in goodcondition by applying PAD mode.
E3E1

Applied potential in a sugar analysis

tsEnlarged view

E4

E2
Time (msec)

Time (min)E1:

Potential for detection

E2:

Potential for reduction

E3:

Potential for oxidation

E4:

Potential for reductionts:

Sampling time

Cleaning of working
electrode surface

GL Sciences LC Technical Note
Clean-up of serum sample using MonoSpin C18
MonoSpin is a series of spin columns for solid phase extraction (SPE). Owing to the highpermeability of monolithic silica disk packed into the spin column, the procedures, such as
conditioning, sample loading, washing, and elution can be carried out only by centrifuging thecolumn. It is also the advantage that the elution volume is only 200 µL.
MonoSpin C18, which is used in this note, has octadecyl group on the surface of silica as afunctional group. Sugar is not retained on MonoSpin C18 at all, whereas hydrophobic
compounds are retained. Therefore, hydrophobic interfering substances can be easily
removed only by passing sample solution through MonoSpin C18.

Example of procedures

Centrifuge

Centrifuge

Conditioning Adsorption

Centrifuge

Rinsing

Centrifuge

ElutionProcedures for clean-up
Monolithic silica disk

Attach the spin column to tube for waste fluid↓ ＋ Methanol

200 μLCentrifuge at 10,000 g for 1 min↓ ＋ Water

MonoSpin

200 μLCentrifuge at 10,000 g for 1 min
Put the spin column into collection tube

Enlarged view ofmonolithic silica↓ ＋ Serum sample
10,000 g for 1 min

Preparation of sample
Serum sample：

200 μL Centrifuge at

Collected sample was injected into HPLC system after 5fold dilution with water

Three times volume of acetonitrile was added to thesample, and the solution was centrifuged. Obtained
supernatant was loaded onto MonoSpin C18.

A chromatogram obtained from serum sample after clean-up using MonoSpin C182uA

1.6

1.8

2. Glucose
3. Fructose

Not only glucose, concentration of which is relatively highin serum (70-110 mg/dL during fasting state), but also
fructose were successfully detected with ECD because of
its high sensitivity although it is known that concentrationof fructose in serum is 100-fold lower than that ofglucose!

1.0

1.2

1.4

301020GL Sciences LC Technical Note

HPLC column for sugar analysis, InertSphere Sugar-1
Cat.No. 5020-11001■InertSphere Sugar-1 is an anion-exchange column packed withpolymer particles. Quaternary ammonium group is chemically bonded.

■Oligosaccharides and sugar phosphates also can be retained andseparated with use of gradient elution.
■InertSphere Sugar-1 can be washed with methanol .
■Cartridge guard column is also available.

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

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