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HPLC and UHPLC Separations at Mid to High pH: Part 2 Introduction This month’s LabNote continues our discussion from January of the use of higher pH for method development and analysis. There are a number of advantages for some analytes, particularly basic compounds, for using higher pH mobile phases. Some of these advantages are given below. In addition, some items to be aware of when working at higher pH are also summarized below. Advantages • Orthogonal selectivity (different elution order) can be obtained for many analytes, which

can allow compounds, which are inseparable at lower pH, to be separated. • Basic analytes are retained longer, and have better loading capacity, linearity, and peak

shape at pHs above their pKas • Signal and signal-to-noise ratios are usually improved for basic analytes in LC-MS

separations at higher pH. • Chromatographic separations at higher pH allow compounds that are unstable at lower

pH to be analyzed without on-column degradation, and higher pH also permits analysis of analytes that have low solubility at lower pH. What to Watch Out For at High pH • Analytes that are unstable at higher pH could degrade on column. • With LC-UV detection the UV spectra and max values of ionizable analytes often change

dramatically, which can make peak tracking more challenging when comparing separations carried out at different pHs during column and mobile phase screening in method development. • Most commercial, non-hybrid silica-based bonded-phase columns can be used with

high-pH mobile phases that contain phosphate, carbonate, or borate buffer salts. However, column lifetimes for all (non-hybrid) silica-based columns will be shorter using these buffers, because such mobile phases have a higher apparent pH than those prepared with additives such as ammonium hydroxide (see References 2, 3, and 4 for more information). • In some very rare instances, on-column reaction (dimerization) or decomposition can

occur for metal-sensitive analytes, due to accumulation of insoluble metal impurities

on the column at higher pH. This phenomenon can often be remedied by periodic column flushing with acidic mobile phases (see Reference 1). Method Development Effective, systematic RPLC method development should include an evaluation of the important parameters that affect selectivity. These parameters include, in approximate order of effectiveness: 1. Column bonded phase 2. Organic modifier choice (ACN, MeOH, ACN/MeOH blend, etc.) 3. Mobile phase pH (for ionizable analytes and excipients) 4. % Organic modifier (isocratic) or gradient steepness 5. Column temperature 6. Buffer identity and concentration (e.g., TFA, phosphate, ammonium formate, ammonium acetate, citrate, NH4OH, etc.) The ability to use mid- and high-pH conditions with LC-MS-compatible mobile phases can make it easier and more effective to evaluate changes in retention, selectivity, and peak shape with changes in mobile phase pH—especially for basic analytes, which often have poorer peak shapes at low pHs. At PittCon 2013 in Philadelphia, PA in March, Advanced Chromatography Technologies, (ACT) and MAC-MOD Analytical will introduce officially the newest addition to the ACE column family: theACE® SuperC18 phase. ACE SuperC18 is a new ultra-inert, stationary phase, based upon porous silica particles that provides ultra-robust and ultra-stable HPLC, UHPLC, and preparative columns. It produces excellent peak shape for acidic, basic and neutral analytes across an extended eluent pH range (pH 1.5 – pH 11). In comparison tests, the ACE SuperC18 has one of the most inert and deactivated silica surfaces, leading to extremely high performance. The unique encapsulated bonding chemistry created for the ACE SuperC18 provides additional protection to the silica surface across this extended eluent pH range, ensuring excellent column robustness and lifetime. For more details, visit us at

Hplc and uhplc separations at mid to high ph  

This document breaks down how to do a proper chromatography from a pH of 1 to a pH of 7. This breaks down the do’s and don’ts of doing this...

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