Waters Sugar-Pak I Column - Manual (Page 3)

[ Care and Use ManUal ]
Sugar-Pak I Columns 3
foil to minimize evaporation. Bring the mobile phase to
its boiling point for a few minutes just before use, but
maintain the temperature between 70-90 °C during use.
This practice ensures that gases (especially C0
2
) do not
redissolve in the mobile phase. It also prevents the growth of
microorganisms.
 Keep the mobile phase reservoir clean and covered and supply
freshly prepared mobile phase every 24 hours.
c. Equilibration
To ensure that a new column is fully equilibrated with calcium prior to
analysis, the following procedure is recommended.
1. Install the column with the normal direction of flow reversed
(arrow on label pointing to column inlet tubing). A backflush
valve may also be used.
2. Back flush the column with at least 100 mL of 0.001 M calcium
EDTA solution at 90 °C using a flow rate of 0.5 mL/min. A
concentration of 500 mg/L of calcium EDTA approximates
0.001 M.
3. Reverse the flow direction again (back to the normal direction)
and flush the column with the stable baseline indicating that the
column is ready for use.
III. CARe AnD Use
Liquid chromatography columns have a finite life influenced by their
care and use, number of injections, sample and solvent cleanliness,
frequency of solvent changeover, and handling and storage procedures
(among other factors). If a change is observed in the:
 retention of a particular compound,
 resolution between two compounds, or
 peak shape
take immediate steps to determine the reason for the changes. Until you
have made this determination, you must not rely upon the results of any
separation using the column. Follow generally accepted procedures for
quality control and methods development when using these columns.
Note: Before running the first analysis on your new column, perform
the test sample separation given in the test conditions section.
Before using your new Sugar-Pak I column for the first time, it is
recommended that the following procedure be observed:
1. Connect the column (refer to Section II a)
2. Set the flow rate at 0.2 mL/min until the column temperature
reaches 70 °C.
3. Once the column temperature reaches 70 °C, increase the
flow rate to 0.6 mL/min in steps of 0.1 mL/min. Wait for the
backpressure to stabilize between each 0.1 mL/min increase.
a. Precautions
Normal recommended pressure should not exceed: 2000 psi.
Maximum flow rate: 0.6 mL/min (at or above 70 °C).
 DO NOT use calcium chloride, calcium nitrate, or any other acidic
calcium salt in the mobile phase, as this can corrode the column
and damage the packing.
 Maximum mobile-phase organic content is 5% V/V. Small amounts
of acetonitrile, ethanol, methanol, and isopropanol in the sample
will not effect column performance.
 Column temperatures should not exceed 95 °C. Generally, high
temperatures provide greater resolution, but little improvement
occurs between 90 °C and 95 °C. Temperatures below 70 °C do not
provide adequate resolution of many sugars due to the separation
of anomers. For quantification of ethanol, a column temperature of
75 °C should be used.
 Column temperature changes can be rapid without adverse
effects. If the column temperature is below 60 °C, a maximum
flow rate of 0.2 mL/min should be used. Higher flow rates may
produce excessive backpressure due to the higher viscosity of
water at lower temperatures.
 Reverse flow direction through the column (after every 5 liters
of mobile phase). This procedure will prolong column life
significantly. Flow rate changes should be made slowly and not
exceed 0.5 mL/min
2 (0.5 mL/min per minute). It is advisable to
change the flow rate in steps of 0.1 mL/min and wait until the
backpressure stabilizes before the next step change.