Page 10 - Application Notebook - Solution for Food Development

LAAN-A-LC-E251

Application High Performance Liquid Chromatography

News Analysis of Oligosaccharides in Japanese Sake Using

an Evaporative Light Scattering Detector
No.L474

An evaporative light scattering detector (ELSD) is an addition, an ELSD detector has an advantage over an
HPLC detector often called a "universal" detector RID with its capability for analysis using gradient elution
because it can detect almost all non-volatile sample conditions.
components, including those that do not absorb light. Here we introduce an example of analysis of
Although a differential refractive index detector (RID) oligosaccharides in Japanese sake using the Nexera-i
can also be used for analysis of compounds with no integrated high-performance liquid chromatograph,
chromophore, the ELSD removes any potential which includes a built-in UV detector. The ELSD-LTⅡ
interference from the solvent peak that is eluted at the evaporative light scattering detector was connected
column void volume because detection occurs after directly to the Nexera-i through an A/D acquisition
volatilization and evaporation of the mobile phase. In board.

n Analysis of a Standard Mixture of Isomaltooligosaccharides
A differential refractive index detector is typically used mV
in the analysis of sugars, but it is limited because 110
gradient elution cannot be used, therefore resulting in 100 n Peaks
longer run times. Fig. 1 shows the chromatogram 90 1. Glucose
2. Isomaltose
obtained from the analysis of a standard mixture of 80 3. Panose
isomaltooligosaccharides using isocratic conditions, and 70
Table 1 shows the analytical conditions that were used.
60
50
Table 1 Analytical Conditions: Isocratic Elution
40
Column : Asahipak NH2P-50 4E (250 mm L. × 4.6 mm I.D.) 30
Mobile Phase : A : 10 mM Ammonium Acetate Buffer
B : Acetonitrile 20 3
Isocratic 10 2
B 70 % 1
Flowrate : 1.0 mL/min 0
Column Temp. : 40 °C 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min
Detection : ELSD-LT Ⅱ
Temperature : 40 °C
Gain : 7
Nebulizer Gas : N2 Fig. 1 Standard Solution of Isomaltooligosaccharides: Isocratic Elution
Gas Pressure : 350 kPa

Under isocratic conditions, components that have long
retention times tend to have broader peak shapes and mV
diminished sensitivity because of the less intense 110 3
response. Fig. 2 shows an example where a gradient 100 n Peaks
was used for the same sample. The analytical 90 1. Glucose
conditions are described in Table 2. Using gradient 80 2. Maltose
3. Isomaltose
elution with the ELSD permits separation of many 4. Panose
components with high sensitivity because the narrower 70 5. Isomaltotriose
6. Isomaltotetraose
peaks produce a much higher signal. 60
50
2
40
Table 2 Analytical Conditions: Gradient Elution 4 5
30 6
Column : Asahipak NH2P-50 4E (250 mm L. × 4.6 mm I.D.)
Mobile Phase : A : 10 mM Ammonium Acetate Buffer 20
B : Acetonitrile 10 1
Linear Gradient
B 70 % → 40 %, 25 min 0
Flowrate : 1.0 mL/min 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min
Column Temp. : 40 °C
Detection : ELSD-LT Ⅱ
Temperature : 40 °C
Gain : 7 Fig. 2 Standard Solution of Isomaltooligosaccharides: Gradient Elution
Nebulizer Gas : N2
Gas Pressure : 350 kPa