ABSTRACT
Reversed-phase (RP) high-performance liquid chromatography (HPLC) has been the predominant method for a vast array of separations over the last 30 years. Its principles have been applied to the analysis of compounds that include small organic and inorganic molecules as well as high molecular weight species such as proteins and peptides. However, RP HPLC is not a universal approach for all separation problems. While many molecules have suf“cient hydrophobicity to result in retention on typical RP materials such as octyl (C8) or octadecyl (C18), others are too hydrophilic so that under most circumstances they elute at or near the column void volume.1 This is especially the case when molecules are very polar or ionic. In order to enhance the retention of hydrophilic analytes, several alternative chromatographic me thods have been developed. The simplest is to make a derivative of the hydrophilic compound so that the product is more hydrophobic in nature.2,3 This can often be a time-consuming process that has the potential for error if the reaction is not quantitative. Another technique is to add ion-pairing reagents to the mobile phase in the case of ionic species so that the solutes being analyzed are neutral.4 Both of these approaches utilize RP as the chromatographic technique since the resultant analyte becomes more hydrophobic and thus can be retained on stationary phases like C8 or C18. Another strategy for retention of polar/ionic compounds is ion-exchange.5-7 In this case, the stationary phase retains a charge that can provide retention capabilities for hydrophilic species. Unfortunately, these methods often result in the use of mobile phases that are not compatible with mass spectrometry, a detection method that is becoming increasingly more prevalent.
