[ 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.
[ Care and Use ManUal ]
t
r
Time
min
min
Note: Be sure to record results and instrument settings (and configura-
tions) to allow exact reproduction and camparison in the future.
a. Column Efficiency
The sucrose peak in the functional test chromatogram (Figure 4)
can be used to monitor the efficiency of the High Performance
Carbohydrate Column. Typical efficiencies of the sucrose peak can be
measured as outlined in Figure 3. Typical efficiencies of the sucrose
peak on a new column range from 7000 to 9000. The actual value
that you measure will be influenced by the bandspread of your HPLC
system.
Table 1: Functional Test Conditions
Parameter Conditions
Sample Sugars (Table 2) Fructose, Dextrose, Sucrose, Maltose, Lactose
Injection Volume 15 μl (containing a total of 0.72 mg sugar)
Mobile Phase Acetonitrile/water 75:25 (v/v)
Flow Rate 1.4 ml/min
Detector Waters Model 410 Refractive
Index Sensitivity of 8
Column 4.6 mm x 250 mm High Performance
Carbohydrate Column
Run Time 12 minutes
Table 2: Functional Test Sample Preparation
Sugars Amount /10 ml
Fructose 60 mg
Dextrose 120 mg
Sucrose 100 mg
Maltose 100 mg
Lactose 100 mg
Figure 3: 5 Sigma Method for Calculating Column Efficiency
b. Functional Test
A typical chromatogram for the functional test is shown in Figure 4;
the figure shows baseline resolution between fructose and dextrose
and between maltose and lactose.
(W
-RT1)
2
1+W2
)
Rs = 2(RT
The retention times (RT) and peak widths (W at tangent) are mea-
sured in cm.
A set of standards containing the sugars in your sample in the
desired concentration range should also be analyzed.
The sugars are dissolved in 4 ml water and brought to 10 ml volume
with acetonitrile. Mobile phase should be adjusted to maintain
baseline resolution and run time of approximately 12 minutes.
Figure 4: Functional Chromatography Test
High Performance Carbohydrate Column 3
[ Care and Use ManUal ]
VI. orderIng InformatIon
Description Part Number
High Performance Carbohydrate Column (4.6 x 250 mm)
(requires End Connector Kit for assembly
End Connector Kit (2 sets of end fittings with O-rings and
C-clips)
Carbohydrate Guard Column (2/pk) WAT046895
Sentry Universal Guard Holder WAT046910
Recommended Sep-Pak Plus Cartridges* (50/box) Part Number
C
18
Accell™ Plus CM WAT020550
Accell Plus QMA WAT020545
Additional Waters Sugar and Carbohydrate Columns Part Number
Ion Exchange/Size Exclusion Columns
Sugar-Pak I WAT085188
2+
SC-1011 (Ca
SP-0810 (Pb
) WAT034238
2+
) WAT036954
Partition Cartridges
Silica-Pak™ HP Cartridge (use with Sam Reagents) WAT010932
Silica-Pak Cartridge (use with Sam Reagents) WAT010935
Reversed-Phase Column
Dextro-Pak™ Cartridge Column WAT085650
Size-Exclusion Columns
KS-800, S-800 and S-800/S Series
WAT044355
WAT037525
WAT020515
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*For a complete list of cartridges and columns and their applications
refer to the Waters Chromatography Columns and Supplies Catalog.
Visit www.waters.com/chemcat
High Performance Carbohydrate Column 4
©2008 Waters Corporation. Waters, The Science of
What’s Possible, Nov-Pak, Guard-Pak, Sep-Pak, Accell,
Silica-Pak, and Dextro-Pak are trademarks of Waters
Corporation.
May 2008 WAT056972 Rev 2 KK-PDF
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