Bio-Rad Aminex Organic Acid and Alcohol Analysis Columns - Manual (Page 8)

5.2 Sample Preparation
Some of the sample components may not be soluble in some solvents. To prevent
any problems of this sort, always dissolve the sample in the mobile phase. Filter
the sample solution through a 0.45 µm filter to remove particulates.
5.3 Pressure Checks
The HPLC pump pressure limit device should be adjusted so that a pressure
increase (10–20% above the standard operating pressure) will cause the pumps to
turn off. This will protect the system from accidental over-pressure. On some
HPLC pumps, the pressure sensor cannot be adjusted to this close a tolerance at
the low pressure required. In this event, extra care must be taken to insure that the
pressure limit is not exceeded.
Always pressure-up slowly to normal flow rates (ca. 1–2 min). This method is
gentle on the column, and provides maximum longevity and retention of resolution
and efficiency. Starting the column at full flow rate may compress the packing and
create an inlet void, which will reduce performance.
When first connecting the column to an HPLC system, check the pressure while
operating at normal flow rates, after the slow start-up. The total back pressure of
the system will be approximately 700–1,100 psi. (Note: some HPLC pumps do
not give accurate pressure readings at low pressures; use of a 0–1,000 psi gauge
placed upstream of the injector may be necessary.) To determine the back
pressure of the column, read the total system operating pressure, disconnect the
tubing between the guard column and the analytical column, and note the
pressure drop. The decrease should be approximately 100–200 psi. (Note:
remember to reduce the flow rate of the pump before reconnecting the column,
then slowly return to the full flow rate over a 1–2 min period.)
If the total back pressure of the system increases during use (other than the normal
increase caused by the higher concentration solvents which may be used during
elution), repeat the above procedure to determine the cause. If the back pressure
increase is small, and is due to the guard column, the system can be operated
normally. If the increase in guard column pressure is greater than 150% of the
pressure when new, the guard column should be replaced.
If the system pressure increase is caused by the column, the column may be
partially clogged and flow rates should be reduced to stay within the operating
limits. Sometimes column back pressure increases are caused by non-polar
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