Waters High Performance Carbohydrate Column - Manual (Page 3)

[ Care and Use ManUal ]
High Performance Carbohydrate Column 3
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.
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.
Rs = 2(RT
2
-RT
1
)
(W
1
+W
2
)
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.
Table 1: Functional Test Conditions
Table 2: Functional Test Sample Preparation
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
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
Sugars Amount /10 ml
Fructose 60 mg
Dextrose 120 mg
Sucrose 100 mg
Maltose 100 mg
Lactose 100 mg
t
r
Time min
min