Waters Sugar-Pak I Column - Manual (Page 5)

[ Care and Use ManUal ]
Sugar-Pak I Columns 5
2. Wash the methanol from the cartridge with two 5 mL portions of
pure water.
3. Blow excess water from the cartridge with the syringe.
4. Slowly pass the sample through the Sep-Pak C
18
Cartridge.
a. Reject the first 2 mL.
b. Collect the next few milliliters for subsequent analysis.
e. In-Line Cleanup of Proteins and Lipids
Protect your column from small amounts of lipophilic material by
using the Waters Guard-Pak Holder (P/N WAT080040) and Guard-
Pak C
18
Inserts (P/N WAT085824). As the Guard-Pak Insert becomes
contaminated with lipophilic sample components, it can be simply
replaced and the old one discarded.
f. Cleanup of Polysacchacrides
Note: Since C
18
-guard columns will adsorb some neutral polysaccharides,
they should not be used for the analysis of such samples.
Neutral polysaccharides will pass through the Sugar-Pak I Column.
For some samples, such as starch hydrolyzates, the analysis of such
polysaccharides is desirable and the use of guard columns containing
resins (as described under “Removal of Salts and Acids”) is
recommended. These resins remove most residual proteins and lipids,
but allow neutral polysaccharides to pass through.
g. Removal of Salts and Acids
Salts and organic acids present in crude samples can co-elute with
sugars and interfere with the analysis. Typically, salts of strong acids
such as sodium chloride, elute near the void volume. Salts of weak
acids, such as lactate and acetate, elute later.
For some samples of low MW sugars, Waters Sep-Pak Alumina
Type A Cartridges (P/N WAT051800) are suitable for removing
salts and acids.
The recommended procedure is as follows:
1. Pass 5 mL of pure water through the Sep-Pak Alumina Type A
Cartridge.
2. Place 10 mL (maximum) of sample in the syringe and slowly
pass the sample through the cartridge.
a. Reject the first 5 mL.
b. Collect the next few milliliters of sample for injection.
Make sure this procedure is suitable for quantitative recovery for your
particular samples.
h. In-Line Cleanup for Samples Not Prone to Hydrolysis
Use a Waters Guard Column (P/N WAT084550) between the column
and the injector to remove salts and acids from the sample. Pack the
guard column with a mixture of strong cation resins (in the H
+ form)
and strong anion resins (in the OH - form) with a maximum particle
size of 200/400 mesh. These resins are available from a number of
sources (e.g., CG120 and CG400 resins made by Rohm and Haas), but
have to be converted into the appropriate ionic forms just before use.
In particular, the anion-exchange resins are unstable in the OH
- form
during prolonged storage. Repack the guard column with fresh resin
when necessary.
Note: Do not use a guard column containing the mixed bed resin for
sucrose analysis, as this will cause severe inversion of the sucrose (or
other sugars prone to inversion). Only use the mixed bed resin in the
guard column when 100% pure water is used as the mobile phase.
Any calcium EDTA in the mobile phase will saturate the column. Any
other type of guard column that exceeds the bed volume of the Waters
guard column (about 0.2 mL) is not recommended for use with Sugar-
Pak I Columns. The increased bed volume will cause excessive band
spreading and loss of resolution.
V. CoLUMn ReGeneRAtIon PRoCeDURe
The need to regenerate your Sugar-Pak I Column will vary according
to the relative purity of your samples. The cruder the samples
(particularly those containing heavy or transition metals or organic
acids), the more frequent regeneration will be required due to
inversion of the column.
You can test for inversion by injecting sucrose. If a tail is produced on
the sucrose peak, the column should be retreated as follows:
 Make up 500 mL of regeneration solution (500 mg calcium EDTA/L)
and allow it to run through the column overnight at 0.5 mL/min at
90 °C. Make sure the direction of flow is reversed before starting
regeneration and returned to normal following regeneration.