High pH Mobile Phase

Analysis of bases has been carried out with low, intermediate, or high pH mobile phases, which are discussed in the following sections. 

If the HPLC mobile phase is operated close to the pA of any solute or if an acidic or basic buffer is used in the mobile phase, the effects of temperature on retention can be dramatic and unpredicted. This can often be exploited to achieve dramatic changes in the separation factor for specific solutes. Likewise, the most predictable behavior with temperature occurs when one operates with mobile phase pH values far from the pA s of the analytes [10], Retention of bases sometimes increase as temperature is increased, presumable due to a shift from the protonated to the unprotonated form as the temperature increases. As noted by Tran et al. [26], temperature had the greatest effect on the separation of acidic compounds in low-pH mobile phases and on basic compounds in high-pH mobile phases. McCalley [27] noted anomalous changes in retention for bases due to variations in their pA s with temperature and also noted that lower flow rates were needed for optimal efficiency. 

Berg T, Lundanes E, Christophersen AS, Strand DH (2009) Determination of opiates and cocaine in urine by high pH mobile phase reversed phase UPLC-MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci 877(4) 421—432. doi S 1570-0232(08)00951-3 [pii] 10.1016/j.jchromb.2008.12.052... 

Formic or acetic acid concentrations of 0.1—1% (v/v) are recommended when preparing low pH mobile phases to enhance ionization in electrospray. Trifluoroacetic acid is preferred for protein and peptide separations but should be avoided when negative ion mode is utilized. Ammonium hydroxide, or, in rare cases, triethylamine, are recommended for high pH mobile phases. 

An impurity method for a drug product with two APIs illustrating the use of high-pH mobile phases and the optimization of pH, organic modifier and column... 

Figure 8.15. A chromatogram of a cocktail of two API and some potential impurities for an impurity method of a multi-component drug product. This method uses high-pH mobile phase.

Figure 10.9. Comparative chromatograms of a good and a failed silica-base C18 column due to exposure to high pH mobile phase. Severe peak fronting indicated channeling of the packed bed while peak tailing of the basic analyte (amitriptyline) indicated increase of silanophilic activity due to loss of endcapping or bonded groups. Figure courtesy of Waters Corporation.

Recently, graphitized-carbon stationary phases have become available for reversed-phase carbohydrate analysis. They exhibit a stronger hydrophobic interaction than C,g bonded phases. They are also compatible with a high-pH mobile phase, which speeds up mutarotation and prevents peak splitting due to anomer separation. 

Phenomenex. 2009. Analysis of basic drugs byLC/MS/MS with high pH mobile phases (www.phenomenex.com). 

In recent years, charged aerosol detection is increasingly used for the detection of carbohydrates if the high sensitivity of IPAD in combination with anion-exchange chromatography using high pH mobile phases is not required (see Section 3.10). With... 

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