Hplc

In HPLC, the samples are dissolved in a solvent and injected into the system. The analytes are then separated from other sample constituents by the differential rates of dissolution into the mobile phase and the stationary phase. The rate of this process is a characteristic of the analyte, mobile, and stationary phases used in the system. Increased or decreased separation can be obtained by altering the composition of the mobile phase solvent (i.e., changing the solvent polarity). Analytes are detected upon exiting the column by several types of detectors (i.e., UV-VIS, fluorescence, electrochemical, mass spectrometers, Fourier Transformed Infrared (FTIR) detectors). The main limitation with HPLC is the ability to dissolve the

FIGURE 1 Chromatographic Separation. In GC, compounds are acted on by two forces: the carrier gas (mobile phase) which sweeps the molecules along the column (but does nothing to separate molecules), and dissolution of the compounds into the stationary phase. Separation is accomplished by the differences in the rate of dissolution of the molecules into and out of the stationary phase. The circles represent molecules with lower vapor pressures, which spend more time dissolved in the stationary phase. The circles are held up by the stationary phase, whereas the molecules represented by the squares have a higher vapor pressure (lower boiling point), and spend more time in the mobile phase, which sweeps these molecules out of the column faster than the circles. Therefore, the squares are swept through the column to the detector faster than the circles. (The squares have a shorter retention time.) In HPLC, these interactions are similar. The difference is that a solvent is used in the mobile phase, and it contributes to the forces that separate the molecules.

FIGURE 1 Chromatographic Separation. In GC, compounds are acted on by two forces: the carrier gas (mobile phase) which sweeps the molecules along the column (but does nothing to separate molecules), and dissolution of the compounds into the stationary phase. Separation is accomplished by the differences in the rate of dissolution of the molecules into and out of the stationary phase. The circles represent molecules with lower vapor pressures, which spend more time dissolved in the stationary phase. The circles are held up by the stationary phase, whereas the molecules represented by the squares have a higher vapor pressure (lower boiling point), and spend more time in the mobile phase, which sweeps these molecules out of the column faster than the circles. Therefore, the squares are swept through the column to the detector faster than the circles. (The squares have a shorter retention time.) In HPLC, these interactions are similar. The difference is that a solvent is used in the mobile phase, and it contributes to the forces that separate the molecules.

sample in a solvent. This difficulty, however, is much less of a problem in HPLC than sample vaporization is in GC. The limit of detectability is usually lower with GC than HPLC (10 to 100 times), depending on the type of detector used. Generally, UV-VIS and fluorescence detectors in HPLC provide less sensitivity than GC detectors, but electrochemical and mass spectrometric detectors could provide equivalent sensitivity to GC systems.

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