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
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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.