Reversed Phase Chromatography

1. Reversed Phase Chromatography

2. Reversed-Phase is the Most Common Type of HPLC - Columns are rugged, efficient, reproducible, and stable. - Aqueous mobile phases are less expensive then organic phases. - Less time is required for equilibration of the column between runs. - Detection is often easier.

3. Retention Times in Reversed Phase Polar (hydrophylic) molecules are eluted first. Non-polar molecules are are eluted last. Medium polarity molecules are eluted in the middle.

4. Stationary Phases Alkane chains of 8 (C-8) or 18 (C-18) carbon atoms bonded to silica are the most commonly used stationary phases For analyses conducted with a high pH mobile phase, alkanes bonded to a polymer support are used. Polymer supports are also preferred over silica for the analysis of basic compounds because the residual silanol groups on silica can cause severe band tailing. Polar stationary phases such as amino, cyano, diol, can also be used in reversed phase chromatography

5. Retention Time and pH in Reversed Phase When an acid or a base is ionized it becomes much less hydrophobic and will elute much earlier. Acids lose a proton and become ionized (negative charge) as pH increases. Bases on the other hand, gain a proton and acquire a positive charge as pH decreases.

6. Buffers and pH Control When the mobile phase pH is near the pKa of an analyte, the retention of the analyte varies considerably with small changes in pH (as small as 0.1 unit) and peak shapes can become skewed. It is therefore important that the mobile phase is well buffered, and the pH is accurately measured.

7. Buffers and pH Control It is also important to note that the analyte itself can change the pH of the mobile phase within its concentration band so concentrations of the analytes should not be too large and the sample pH should not be too different from the buffer pH. Buffers are limited in the pH range that they can control so it is best if the pKa of the analyte is similar to the pKa of the buffer.

8. Buffers and pH Control Citrate buffer has the advantage of being able to stabilize pH over a wide range (2.1-6.4). This is because the citrate molecule has 3 ionizable groups with different pKa’s. However, because of its UV absorbance, it can only be used with UV detectors at wavelengths above 230 nm. Citrate has also been claimed to be corrosive to stainless steel, so if it is used, extra care is needed to flush the system after a series of runs. Many other buffers are also available.

9. Amphoteric Molecules Some molecules, amino acids are good examples, have both acidic and basic groups on the same molecule. This property is called amphoteric. Amphoteric molecules have positive charges at low pH and negative charges at high pH. At intermediate pH values, both the acidic and basic groups are partially charged. Because both groups are charged, the molecule has its highest ionization and therefore its lowest retention at intermediate pH.

10. Water and C-18 Stationary Phases Mobile phases used should not contain less than 10% organic solvent when using stationary phases such as C-18. When the mobile phase contains more than 10% organic solvent the alkane chains are more or less expanded out straight from the surface. With less than 10% organic solvent, the C-18 chains collapse and it is difficult to bring them back to the straight conformation.

11. Organic Solvents Three water-miscible solvents, methanol, acetonitrile and tetrahydrofuran differ considerably in their selectivity properties and are also acceptable in terms of UV absorbance and viscosity and therefore are the most commonly used organic solvents for reversed-phase separations.

12. Reversed Phase and Basic Compounds Basic compounds interact with the silanols of silica-based columns resulting in increased retention, band tailing and column-to-column variability. - Use Type B columns which have a minimum of acidic silanols - Use a low pH mobile phase to minimize ionization of silanols - Use buffer cations that are strongly held by the silanols and will not readily exchange with the analyte ions. - Use high pH where basic analytes are not charged and will not be affected by the silanols. At higher pH, silica columns may degrade, so polymer based stationary phases should be used.

13. Non-Aqueous Reversed-Phase HPLC For very hydrophobic samples that are retained so strongly that they are not eluted with 100% acetonitrile, a non-aqueous mobile phase can be used. Typically methanol or acetonitrile is used as the more polar solvent, while chloroform, methylene chloride, methyl-t-butyl ether, or tetrahydrofuran are commonly used as the less polar solvent (stronger solvent). The separation of carotenoids such as a-carotene, b-carotene and lycopene is an example of a food science use of non-aqueous reversed-phase chromatography.

14. Effect of Temperature Increasing temperature has the effect of decreasing k so that compounds elute earlier. As temperature is increased the retention of planar compounds decreases faster than non-planar compounds resulting in a change in selectivity. Temperature can have a large effect on selectivity for ionizable compounds.