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Practical HPLC Method Development, Second Edition

by Lloyd R. Snyder, Joseph J. Kirkland and Joseph L. Glajch


Copyright 1997 John Wiley & Sons, Inc.

APPENDIX VI
ADJUSTING MOBILE-PHASE
WATER CONTENT FOR
NORMAL-PHASE HPLC

As discussed in Section 6.7.4.2, columns packed with silica or other polar


inorganic solids (e.g., alumina) are affected by water. If the mobile phase
contains water, the column packing will tend to extract some of this water
from the mobile phase and become less retentive. In the case of less-polar
mobile phases for NPC (e.g., mthylne chloride/hexane), the solubility of
water in the mobile phase is often quite low (e.g., < 0.01%). The column, on
the other hand, can adsorb a considerable quantity of water (several percent
w/w). As a result, any change in mobile-phase water content will require a
large volume of the new mobile phase (as much as several hundred column
volumes) to achieve column equilibration and constant retention times. When
carrying out NPC separations, changes in mobile-phase water content are
common, because room humidity can vary and water can be adsorbed onto
the inside of glass containers used to hold the mobile phase. As a result, it is
common to see changes in retention from run to run in NPC.
In some cases, the effect of varying mobile-phase water concentrations on
sample retention can be minimized by adding 0.1 to 0.5% of methanol or
propanol to the mobile phase. However, this can sometimes result in peak
distortion and a drastic loss in column efficiency. A more reliable procedure
for eliminating the effect of water on NPC separations is to add a certain
quantity of water to the mobile phase, so that further (accidental) changes in
water content are relatively minor. This can be regarded as similar to the
action of a buffer in maintaining constant pH. The problem of varying water
is most severe for water-immiscible mobile phases which can dissolve no more
than 0.1% water. In these cases, the addition of half as much water to the
mobile phase as can be dissolved at saturation ("50% water saturation") has
been found to be effective.
744

APPENDIX VI

745

Since the solubility of water in typical binary-solvent NPC mobile phases


will usually not be known and is not easily measured, it is not feasible simply
to add a certain quantity of water to the mobile phase. Furthermore, the
dissolution of liquid water by adding and shaking can be quite slow and
impractical. An alternative procedure is therefore required. One approach is
to saturate a portion of mobile phase with water, then blend this portion with
an equal volume of ("dry") mobile phase that has not been treated with water.
In some cases the mobile phase can be saturated with water by adding excess
water and shaking for an extended period. However, this is less effective for
less-polar mobile phases that contain solvents such as hexane and mthylne
chloride. Similarly, if the mobile phase contains water-miscible solvents (methanol, THF, etc.), some of the latter solvent may be extracted into the excess
water used to saturate the mobile phase.
A more convenient and reliable means for saturating the mobile phase
with water is as follows. To 25 g of 100- to 200-mesh laboratory silica, add
5-mL portions of water, followed by shaking in a closed container after each
addition. Continue the addition of water until the resulting power is not free
flowing (lumps are formed that do not break up with continued shaking). At
this point, add back 5 g of silica, and shake until free flowing. The resulting
water-saturated silica is next used to prepare water-saturated mobile phase.
To 100 mL of mobile phase, add 2 g of water-saturated silica and stir vigorously
for 30 min. Allow the silica to settle, and decant the mobile phase into a
storage container. Add an equal volume of mobile phase that has not been
water saturated, mix, and use.
The procedure above assumes that the starting mobile phase is free of
water, which will be close enough to the actual situation when solvents from
the bottle are mixed and stored in closed containers. The nominally waterfree mobile phase can be further dried by the addition of activated silica,
stirring, and decanting (similar to the procedure for saturating mobile phase
with water). Activated silica can be prepared from silica by heating in air at
150C for 4 h, followed by cooling in a closed container. For further details
concerning the preparation of 50% water-saturated NPC mobile phases, see
Ref. 1.
REFERENCE
1. L. R. Snyder and J. J. Kirkland, Introduction to Modern Liquid Chromatography,
2nd ed., Wiley-Interscience, New York, 1979, pp. 374-383.

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