Mobile phases strength

Figure 4.29 An example of the use of ternary solvents to control mobile phase strength and selectivity in reversed-phase liquid chromatography. A, methanol-water (50 50) B, tetrahydrofuran-water (32 68) C, methanol-tetrahydrofuran-water (35 10 55). Peak identification 1 - benzyl alcohol 2 phenol 3 3-phenylpropanol 4 2,4-dimethylphenol 5 benzene and 6 -diethylphthalate. (Reproduced with permission from ref. 522. Copyright Elsevier Scientific Publishing Co.)...

This chapter provides an overview of essential concepts in HPLC including retention, selectivity, efficiency, and resolution as well as their relationships with key column and mobile phase parameters such as particle size, column length and diameter, mobile phase strength, pH, and flow rate. The significance of several concepts important in pharmaceutical analysis such as peak capacity, gradient time, void volume, and limit of quantitation are discussed. 

Obtaining data on the effects of these parameters may allow one to judge whether a method needs to be revalidated when one or more parameters are changed. For example, if column performance changes over time, adjusting the mobile-phase strength to compensate for changes in the column may be allowed if such data are included in the validation. 

Polarity of Supercritical Fluids. In order to successfully perform any chromatographic separation, the analytes must be sufficiently soluble in the mobile phase. Efforts to ensure solubility have often been based on matching the polarity of the sample components and the mobile phase. For pure fluids, Giddings has reported a polarity classification based on the solubility parameter (18). In contrast to essentially constant values for incompressible fluids (liquids), the solubility parameter (mobile phase strength) of a supercritical fluid varies with its density. 

Initially the mobile phase was too strong and elutes all of the compounds at V0 The mobile phase strength is decreased stepwise until all four components are resolved. It is important to reemphasize that when using ion suppression, the additive used to adjust the pH must remain at a constant concentration during the method development process. Also, it should be mentioned that while acetic acid adjusts the pH to an approximate value of 3 and is often used in ion suppression, it is not a buffer. Therefore, use of a sodium acetate buffer (0.001 to 0.01 M) may be more appropriate for some bonded phases. 

Another injection-related effect that can diminish the separation performance is the diluent effect, also known as solvent mismatch. This occurs when the elution strength of the sample solvent is greater than the starting mobile-phase strength. The retention of the analyte on the stationary phase is less in the small plug of sample solvent than it is in the surrounding bulk... 

Adsorption or chemisorption on column packing or on different hardware components. Increase mobile phase strength add competing base (for basic compounds) or use base-deactivated packing ensure no reactive groups are present use inert tubing and flow-path components, e.g., PEEK. 

The influence of mobile phase strength on the separation is shown in Figure 9.3. Hexane is too weak a solvent for the analysis of this test mixture and the separation needs an unnecessarily long time. fcrf-Butyhnethyl ether is much too strong. A suitable eluent is the mixture hexane-fcrf-butylmethyl ether (9 1) (volume parts). 

Figure 2.12. Four RPLC chromatograms illustrating the effect of mobile phase strength and selectivity of acetonitrile (ACN) and methanol (MeOH). See Figure 2.10 for LC conditions.

R. G. Wolcott, J.W. Dolan, and L. R. Snyder, Computer Simulation for the Convenient Optimization of Isocratic Reversed-Phase Liquid Chromatography Separations by Varying Temperature and Mobile Phase Strength, J. Chro-matogr. A, 869 (2000) 3. 

Contamination can be the result of a buildup of retained sample material on the stationary phase. This can be a problem with gradient elution systems when the proportion of organic modifier in the mobile phase increases over the course of the chromatographic run. These unwanted materials can be washed off the column with the increase in mobile phase strength, which in turn can distort the baseline in the form of waves. In some circumstances, this can interfere with late eluting peaks of interest and can have a detrimental effect on the assay outcomes. 

The best mobile phase strength for a specific separation problem can be determined by thin-layer chromatography (TLC). It is represented by that solvent or solvent mixture which gives an value of about 0.3 in the thin-layer chromatogram. TLC results are readily transferable to HPLC and k values can be predicted if the stationary phase in both methods is the same product, albeit with a difference in particle size. A developing distance of 5 cm is sufficient for this test, so it can be carried out on small TLC plates in a very short time. Small sealable jam-jars make good developing tanks. 

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