Mobile phase miscibility

Considerations when choosing solvents for mobile phases Miscibility... 

In reversed-pViase chromatography (RPC), the mobile phase modulator is typically a water-miscible organic solvent, and the stationary phase is a hydrophobic adsorbent. In this case, the logarithm of solute retention factor is commonly found to be linearly related to the volume fraction of the organic solvent. 

Of course, LC is not often carried out with neat mobile-phase fluids. As we blend solvents we must pay attention to the phase behavior of the mixtures we produce. This adds complexity to the picture, but the same basic concepts still hold we need to define the region in the phase diagram where we have continuous behavior and only one fluid state. For a two-component mixture, the complete phase diagram requires three dimensions, as shown in Figure 7.2. This figure represents a Type I mixture, meaning the two components are miscible as liquids. There are numerous other mixture types (21), many with miscibility gaps between the components, but for our purposes the Type I mixture is Sufficient. 

Multidimensional HPLC offers very high separation power when compared to monodimensional LC analysis. Thus, it can be applied to the analysis of very complex mixtures. Applications of on-line MD-HPLC have been developed, using various techniques such as heart-cut, on-column concentration or trace enrichment applications in which liquid phases on both columns are miscible and compatible are frequently reported, but the on-line coupling of columns with incompatible mobile phases have also been studied. 

Why are methanol or acetonitrile preferred to other water miscible solvents for the preparation of mobile phases for reverse phase chromatography ... 

If the performance of a column is no longer satisfactory it can sometimes be reconditioned by washing with a suitable solvent, or series of solvents. Some bonded phase columns, C-18 for instance, tend to collect non-polar impurities, which can sometimes be removed by washing the column with a non-polar solvent, eg heptane. Assuming the mobile phase normally used with the column is CH3OH/H2O 50 50, we cannot wash directly with heptane because of miscibility problems, we have to get to heptane via a miscible solvent or series of solvents. 

You could first wash the column with methanol, then trichloromethane, then heptane (or methanol, ethyl ethanoate, heptane). You cannot go directly from methanol to heptane because the two are only partly miscible. The column needs to be washed with about 20 dead volumes of each solvent (about 50 cm3 of each solvent for a 25 cm x 4.6 mm column). To get back to CH3OH/H2O 50 50 you would have to go through the sequence of solvents in reverse. If buffer solutions or ion-pairing reagents have been used in the mobile phase, very much longer equilibration times may be needed. 

The use of extraction cartridges in the separation of azines, discussed in the last Section, is an example of on-column concentration using off-line column switching. A chromatogram can be cut off-line by collecting the zones of interest at the detector outlet followed by reinjection of the collected fraction onto a secondary column. The mobile phases used with the two columns should be compatible, eg they should be miscible and the mobile phase used with the first column should not have too high an eluting power in the second column. If the mobile phases are incompatible it may be possible to evaporate the primary mobile phase and redissolve the sample in a suitable solvent. 

In achiral-chiral LC-LC, the mobile phases used with the achiral and chiral columns must be miscible with one another. Since the enantiomeric separation is usually the most difficult to optimize, it is usually the separation that dictates the mode of operation of the total analysis. Thus, it makes sense that a chiral column that operates in the normal phase mode would require an achiral column that also works in the normal phase mode. Polar organic mode chiral separations are universal in that they can be paired with an achiral column that operates in either the reverse phase or normal phase mode. The choice of the achiral column is always determined after selecting the chiral column and the mode of operation. As with traditional liquid chromatography, different achiral columns will give different selectivity. 

Additionally, the inj ected matrix must also be miscible with the solvents used in the separations. For normal phase mode separations, all water must be removed from the injected matrix. Since many of the complex matrixes, such as plasma, urine, and other biological fluids contain a large amount of water, this requires more time consuming sample preparation. However, water can be injected into a polar organic or reverse phase mode separation. Even within the same mode, mobile phases that are very different can cause large disturbances in the baseline. Oda et al., (1991) solved this problem by inserting a dilution tube followed by a trap column in order to dilute the mobile phase used on the achiral column. Following the dilution tube, a trap column was used to reconcentrate the analyte of interest before the enantiomeric separation. 

Liquid-liquid chromatography in its simplest form involves two solvents that are immiscible. However, many recently developed media consist of a liquid (the stationary phase) that is firmly bound to a solid supporting medium. As a result, it is possible to use a second solvent (the mobile phase) which under normal conditions would be miscible with the first solvent. The second solvent is permitted to move in one direction across the stationary phase to facilitate the separation process. The presence of a supporting medium introduces some problems in the system and, in theory, it should be completely inert and stable, showing no interaction with the solutes in the sample. However, this is not always the case and sometimes it affects the partitioning process, resulting in impaired separation. 

The combination of normal (silica) and reversed (C18) phase HPLC in a comprehensive 2D LC system was used for the first time for the analysis of alcohol ethoxylates [64] the NP separation was run using aqueous solvents, so the mobile phases used in the two dimensions were miscible, resulting in the easy injection of the entire first-dimension effluent onto the second-dimension column. 

Elution in reversed-phase chromatography is often carried out using a gradient, produced from water and some water-miscible organic solvent. The solute components are thus distributed between the stationary and mobile phases mainly on the basis of their polarities. In reversed-phase chromatography hydrophilic compounds elute before hydrophobic ones. 

Different solvents and the composition of the mobile phase used in HPLC may effect lmax. Generally speaking, the lmax in hexane, ethanol, and petroleum ether will show little if any change, but chloroform, for example, will show a shift to a longer wavelength. Other factors which may effect the spectral characteristics are water in water-miscible solvents, protein in carotenoproteins, and low temperatures. 

Inject 10 to 50 pi of sample (see Support Protocol 2) and any optional internal standard dissolved in a solvent miscible with the mobile phase (e.g., ethanol, methanol, acetonitrile). 

Samples extracted into strong organic solvents (hexane, ether, pet ether, ethyl acetate, etc.) must be transferred into a solvent miscible with the mobile phase. A small volume (e.g., 1 ml) of the organic extract should be evaporated under N2 gas and dissolved in reagent alcohol. Further dilutions with alcohol may be necessary to obtain 5 to 10 mg/liter concentration before HPLC injection. 

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