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HPLC and UHPLC Separations at Mid to High pH: Part 2


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.


 • 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 lmax 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: the ACE® 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.

ACE Super C18

ACE SuperC18 Features and Benefits

• Unique bonding chemistry provides increased resistance to degradation under alkaline conditions (up to pH 11 with 35% aqueous with 0.1% NH4OH at 40°C) compared to conventionally-bonded silica based products (including ACE C18).  (See Note 1 below.)

• Can be used at pHs ranging from 1.5–11 with all analytes types.

• Designed for use with LC-MS-compatible mobile phase buffers, and performs well with ACN, MeOH, and their blends.

• Does not show hysteresis effects when changing between low and high pH mobile phases, and vice-versa.

• Slightly more retentive than ACE C18, C18-AR, and C18-PFP phases

Note 1:  Ammonium hydroxide at 0.1% (v/v) in water is equivalent to approximately 15 mM (0.015 M), and has a calculated pH of ~10.7 (ChemBuddy Bate pH Calculator).

Performance evaluations of the ACE SuperC18 in the laboratory by the author have revealed the following practical information:

• The ACE SuperC18 column produces highly symmetrical peaks (see Figure below) for bases, acids, and neutral analytes using both ACN and MeOH with pH 10.5 aqueous mobile phases (10 mM NH4OH or 10 mM NH4OAc adjusted to pH with NH4OH).  ACE SuperC18 also performed very well with bases, acids and neutral compounds at low and mid pH.

• Baselines were flatter (with UV/Vis detector) when NH4OH or NH4OAc/NH4OH were premixed with the organic modifier in the B solvent (see Note 2). 

Note 2:  For example, use 10 mM NH4OH in A solvent, and 95:5 organic modifier/200 mM NH4OH as the B solvent.  This approach keeps the NH4OH (or NH4OAc) concentration constant in the gradient at 10 mM.)  This control of buffer or additive concentration is frequently used and necessary for HILIC method development and analyses to keep additive concentration and ionic strength constant. 

Tables are available on request from MAC-MOD Analytical to help prepare premixed mobile phases for RPLC and HILIC separations so that additive concentrations are held constant during gradient runs.  Email us at and ask for premixed mobile phase tables.

ACE Excel SuperC18 is available now from MAC-MOD Analytical in 2, 3 and 5 micron particle sizes in ACE Excel hardware, which can handle pressures up to 1000 bar (14,500 psi).  Once formally introduced, ACE SuperC18 will also be available in semi-preparative and preparative geometries.  

Please contact MAC-MOD via phone at 800-441-7508 or email us at for more information or to place an order prior to official product introduction at PittCon 2013.


1. “Mass spectrometry-compatible ICH (International Conference on Harmonization) impurity analysis with a high-pH mobile phase:  Advantages and pitfalls.”  M.W. Dong, G.D. Miller, and R.K. Paul.  Journal of Chromatography A, 987 (2003) 283–290.

2. “Mobile-Phase Buffers, Part I—The Interpretation of pH in Partially Aqueous Mobile Phases”, G. W. Tindall, LCGC North America, Volume 20, Number 11, November 2002.

3. HPLC for Pharmaceutical Scientists, 2007, John Wiley & Sons, Inc., Yuri Kazakevich and Rosario LoBrutto, eds., chapter 4, p158 ff.

4. LabNote for January 2013, 1/17/2013, “HPLC and UHPLC Separations at Mid to High pH:  Part 1”, MAC-MOD Analytical, 2013.

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