Application Packets
Product Bulletins
Technical Reports
Videos & Webinars
Posters & Presentations
Chromatographic Terms

Lab Notes

LabNotes Subscription
Subscribe to our LabNotes e-mail to receive helpful hints and practical adivce on HPLC delivered right to your inbox.


An Overview of Systematic RPLC Method Development Using Multiple Organic Modifiers and Different pH Buffers

An Overview of Systematic RPLC Method Development 
Using Multiple Organic Modifiers and Different pH Buffers

An efficient and effective strategy for reversed-phase method development for HPLC and UHPLC usually utilizes several stationary phases with different selectivities (Table 1). Those stationary phases, ideally, should have different mechanisms for retaining and separating various types of analytes, such as hydrophobic interactions (common for all alkyl bonded phases), p–p interactions, dipole-dipole interactions, hydrogen bonding interactions, and steric interactions (shape selectivity). For each of the phase types we’ve listed some pertinent ACE column phases that fall into those categories. It’s also wise to select columns from a manufacturer that has a proven history of producing and delivering high quality, reproducible (column-to-column and batch-to-batch) columns in a variety of geometries and particle sizes.

Table 1 Analyte-Phase Interactions for Common RPLC Stationary Phase Types

Phase Type/Description


Most Selective for

C18, C8

ACE SuperC18

UltraCore SuperC18

  • Hydrophobic
  • Diverse polar, moderately polar, and nonpolar acidic, basic, and neutral analytes



UltraCore SuperPhenylHexyl

  • Hydrophobic
  • p–p
  • Dipole-Dipole
  • Hydrogen-bonding
  • Analytes with electron-withdrawing groups 
    (e.g., halogens, nitro groups, ketones, esters, acids)
  • Analytes having aromatic rings, differing dipole moments, or protic groups


ACE C18-Amide

  • Hydrophobic
  • Hydrogen-bonding
  • Proton donors (e.g., alcohols, phenols, polar acids)
  • Proton acceptors (e.g., amines, anilines, ketones)



  • Hydrophobic
  • Dipole-Dipole
  • Analytes with double or triple bonds, or with polarizable functional groups (acids, amines, alcohols, esters, carbonyl compounds, ethers, organic halides, aromatics, alkenes, alkynes)
  • Works well for normal-phase separations



  • Hydrophobic
  • p–p
  • Dipole-Dipole
  • Hydrogen-bonding
  • Steric (Shape selective)
  • Analytes with electron-donating groups 
    (e.g., phenols, alcohols, aromatic ethers, amines)
  • Analytes having aromatic rings, different dipole moments, or protic groups
  • Structural isomers

Example Using 2 Organic Modifiers and Aqueous Buffers Having 3 pHs

In this example (Scenario A of Figure 1), we show how a single, versatile phase with a broad useable pH range, ACE SuperC18, can be used in screening experiments for RPLC method development. In these experiments, we compared the separations of a 15-analyte mixture of acids, bases, and neutrals under various conditions. Notably, the ACE SuperC18 phase is not subject to memory effects (hysteresis) from previous conditions at low or high pH or with different modifiers, when cycling between various conditions. This benefit makes it ideal for method scouting and development.

In this LabNote we present an example in which one of these phases, the ACE Excel SuperC18, is used in a one-factor-at-a-time (OFAT) screening approach (with two different organic modifiers and several pHs) to identify conditions to use for a sample of fifteen acids, bases, and neutral analytes.

Method Development Strategies

Systematic method development can vary from simple to quite complex (Figure 1), depending on the nature of the sample. Some methods can be developed quickly by carrying out several gradient separations with different gradient slopes at only a single temperature, with a preferred organic modifier at a single pH (Scenario A). More complex projects such as related substances methods or multi-analyte environmental methods may require a comprehensive approach (Scenario B and scenarios in Figure 1). Other advantages of using gradient mode for method development are: (1) it allows you to quickly assess the complexity of your sample; (2) it ensures that you won’t “miss” any analytes that may be present; and (3) it allows cleaning of the column with each run so that late-eluting components do not affect subsequent runs.

When evaluating different combinations of stationary phase, organic modifier, pH, and temperature, it is often important to be able to track peak identities among those various conditions from one run to another. This is especially important when you want to use those runs as inputs into a computer simulation and optimization program such as DryLab®. Peak tracking is most easily accomplished when you have access to LC-MS detection, but can also be accomplished using LC-UV spectra from diode-array detectors or using peak areas, if they’re sufficiently dissimilar. However, you must be careful when changing pH dramatically as the UV spectra and peak areas can vary dramatically for many analytes, especially for large pH changes.

Figure 1 Examples of Different Strategies of Varying Complexity for RPLC Method Development

A short efficient column works well for this type of screening gradient or method development approach. For this example, a 2.1 x 50 mm ACE Excel SuperC18 2 mm UHPLC column was used at 0.5 mL/min at 30°C with short 8-minute gradient times from 5 to 80% organic using both acetonitrile and methanol as organic modifiers, with pH 2.75, 4.75 and 10.5 aqueous components (Figure 2). 

Figure 2 Gradient Separations Using ACE SuperC18 with 2 Organic Modifiers and 3 pHs

Note: Unfortunately, chromatographic runs using methanol at pH 10.5 could not be carried out due to an instrument problem at the end of a limited method development window. It is expected that those results would also have been useful in selecting conditions for further development and optimization.

By comparing separations carried out using such an experimental design, one can quickly decide which combination(s) of organic modifier and pH are most promising for optimization and finalization of a method, based on the number of peaks detected and peak shapes of the various analytes. Based on the chromatograms shown in Figure 2, the separations obtained with methanol at both pH 2.75 and 4.75, with 14 out of 15 detected peaks, showed the most promise for additional work. The overall separation and peak shapes were also quite good for the pH 10.5 conditions with acetonitrile, although there were several co-elutions and little retention for one of the acidic analytes. You can get additional information by requesting a PDF copy of our recent PittCon 2014 poster, “Application of Unique Stationary Phases for Effective RPLC Method Development” by contacting us at

In summary, a systematic method development strategy can be a very productive approach for surveying various parameters that affect selectivity, resolution, and peak shape for RPLC. The ACE SuperC18 phase, with its superior inertness and stability over a broad pH range (1.5 to 11.5), is an excellent column choice, combined with the ability to study the effects of different organic modifiers, modifier blends, and pH.

For more information on ACE SuperC18, please visit theMac-Mod Analytical web site, and take advantage of the on-going offer to see what ACE SuperC18 can do in your laboratory.

Guidance for Selecting the Correct Pore Size of Your Analytical Columns for Better RPLC Separations of Biomolecules (part 1)
Using the correct pore size for peptides and proteins is important for narrow peaks, proper retention and excellent peak shape. more...
Improve Resolution and Recovery of Peptides by Increasing Column Temperature
How to Improve Resolution and Recovery of Peptides by Increasing Column Temperature more...
Reduce Baseline Noise For Peptide Gradient LC-UV Analysis
Reduce Baseline Noise For Peptide Gradient LC-UV Analysis more...
The Moderate Polarity Power of 3: ACE Excel SuperC18
This lab note describes the features and benefits of ACE SuperC18 and links to a video. more...
Advancing Selectivity With the Polar Power of 3
An Application of different stationary phases having different modes of retention more...
Specific Applications for HALO Peptide ES-C18 and ES-CN Columns
Some specific applications of HALO Peptide columns are shown, and advantages vs. other commercial columns given. more...
HALO BioClass Columns for Peptide and Protein Separations
The recent introductions of HALO Protein C4 and ES-C18 are highlighted with example applications. more...
Go Ballistic...with HALO-5 Fused-Core 5-mm HPLC Columns!
With some instruments and autosamplers you can use an injector program to delay the injection a predetermined amount of time. more...
HPLC and UHPLC Separations at Mid to High pH: Part 1
In this LabNote we will review the findings that were published by Kirkland, Claessens and co-workers in the 1990s. more...
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. more...