Waters High Performance Carbohydrate Column User Manual

[ Care and Use ManUal ]

HIgH performanCe CarboHydrate Column

I. IntroduCtIon

Waters High Performance Carbohydrate Column (P/N WAT044355)

is an application-specific column for separation of monosaccharides

and disaccharides with short analysis times. T he columns are tested

in the Waters Quality Control Laboratory to ensure separation of

fructose from dextrose and maltose from lactose in less than twelve

minutes. This separation is representative of the applications for this

column.

®

The column contains 4 μm Nova-Pak

trifunctional amino propylsilane. The 4.6 mm x 250 mm steel car-

tridge columns are optimum for the carbohydrate separation. Guard-

Pak™ inserts with the same packing material are also available for

use with these columns. A Sentry Universal Guard Holder is required

for the Guard-Pak inserts. See Section VI for Ordering Information.

spherical silica bonded with

II. InstallatIon and equIlIbratIon

Before installing the column make sure the detector (refractive index

detector) is equilibrated with the mobile phase being used.

Contents

I. IntroduCtIon

II. InstallatIon and equIlIbratIon

III. sample preparatIon

IV. operatIon

V. Care and maIntenanCe

VI. orderIng InformatIon

Follow the procedure in the Waters Steel Cartridge Column Care &

Use Instructions for installing the column on the HPLC system. An

End Connector Kit (P/N WAT037525) is required for installing the

steel cartridge on the HPLC system.

The columns are shipped in a mixture of acetonitrile and water

(75/25 by volume). Equilibrate the column with the desired mobile

phase until a steady baseline is obtained on the detector.

High Performance Carbohydrate Column 1

[ Care and Use ManUal ]

III. sample preparatIon

All lipids and ionic contaminants should be removed from the sample

®

matrix before application to the column. Waters Sep-Pak

are available with reversed-phase packings to remove lipids or fats

and with ion-exchange packings to remove charged species. A list

of recommended Sep-Pak cartridges is provided in Section VI; a

complete list of cartridges is included in the Waters Chromatography

Columns and Supplies Catalog. Visit www.waters.com/chemcat.

cartridges

IV. operatIon

The effects of temperature, flow rate, and percent organic in the

mobile phase are discussed in this section.

a. Temperature

An improvement in resolution is obtained when the column is oper-

ated above room temperature. Operation of the cartridge at 30 °C

or 35 °C will give better resolution of both the monosaccharide and

disaccharide pairs. Figure 1 shows that the optimum resolution is

obtained between 30 °C and 35 °C.

gets significantly longer at the higher organic levels. The lower

the organic concentration that is used, the more soluble are the

sugars and the closer are the peaks. T herefore, the lowest organic

concentration that separates the sugars in your sample should be

used. T he optimum range for the high performance carbohydrate

column is 70% to 75% acetonitrile in the mobile phase. The organic

concentration may need to be adjusted after extended use. T he reten-

tion times of the sugars decrease with extended column use. This can

be compensated for by increasing the percentage of acetonitrile in

mobile phase.

Figure 1: Effect of Temperature on Resolution

b. Percent Organic in Mobile Phase

Figure 2 highlights the effect of percent organic in the mobile phase

on the resolution and analysis time. As the percent organic increases

the resolution of the sugars improves; however, the analysis time

High Performance Carbohydrate Column 2

Figure 2: Effect of Percent of Organic in the Mobile Phase on the

Resolution and Analysis Time

c. Flow Rate

The High Performance Carbohydrate Column gives high resolution

of the sugars at 1.4 ml/min and the analysis time remains below 12

minutes. Higher flow rates can be used if your separation permits.

The column should not be operated above the backpressure limit of

40 Mpa (400 atm or 6000 psi).

V. Care and maIntenanCe

The continued high performance of your column can be monitored by

carrying out the flowing column efficiency test and/or the functional

test on a new column and periodically while the column is in use.