ABSTRACT:
In HPLC the mobile phase is pure or mixed solvents as well as solvents with solid modifiers. Chromatographers have a choice among hundreds of solvents for different application of HPLC. A particular selection is usually affected by solvent characteristics such as viscosity, refractive index, noncorrosiveness, toxicity, miscibility, transparency etc. Commercial availability in adequate purity at reasonable price is also important factor. The solvent strength or % organic solvent in mobile phase controls the retention time of the analyte. A useful rule of thumb in RPLC indicates that 10% decrease in the organic solvent in the mobile phase shows a 3-fold increase in k or tR. Whenever acidic or basic samples are separated, it is strongly advisable to control mobile phase pH by adding a buffer. Several considerations should be kept in mind in selecting a particular buffer e.g. buffer capacity, solubility, interaction with sample or column, corrosion of HPLC system etc. A buffer concentration in the range of 10 to 50 mM is adequate for most reversed phase applications. During mobile phase preparation, premixing is done by measuring the volume of each solvent separately and combining them in the solvent reservoir. Buffered mobile phase pH must be adjusted before adding organic solvent. This approach leads to some uncertainty in the actual pH value of the final mobile phase because the addition of organic solvent can change the pH. But this problem is much less important than poor reproducibility of the mobile phase pH if it is measured after addition of the organic solvent. Aqueous mobile phases containing buffers must be filtered through a 0.45 or 0.2 micrometer membrane filter and degassed by vacuum filtration or sonication.