Waters High Performance Carbohydrate Column - Manual (Page 2)

[ Care and Use ManUal ]
High Performance Carbohydrate Column 2
III. sample preparatIon
All lipids and ionic contaminants should be removed from the sample
matrix before application to the column. Waters Sep-Pak
® cartridges
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.
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.
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
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. Therefore, the lowest organic
concentration that separates the sugars in your sample should be
used. The 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. The 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 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.