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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.


As discussed in Section, 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., méthylène 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.

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