Topic 1: Peak tailing in RP HPLC

Problems of asymmetry of peaks, mainly, in reversed phase HPLC methods.
Main definitions are refreshed for the participants, as well as major consequences of this phenomenon effecting quality and reliability of analytical results.

The reasons for causing peak tailing are reviewed, as well as the general attitude to solving this problem:

• Effect of strength of sample solvent (diluent) – aqueous/organic ratio
• Sample mass overload
• Void in the column
• Residual silanols interaction with amino groups of the analyte
• Residual silanols interaction with carboxylic groups of the analyte

Aside from the presentation, a story of development “problems-free” stationary phases is presented. The focus is made on the importance of silanol activity of RP stationary phases.

As a specific case of peak asymmetry, the case of fronting is discussed separately.

The reasons for causing peak fronting and the general attitude to solving this problem are reviewed:

• Column ageing and / or fouling
• Influence of injection volume
• Effect of strength of sample solvent (diluent) – aqueous/organic ratio and its pH
• Effect of matrix components and / or presence of other analytes
• Effect of temperature

Topic 2: Retention time changes

Problem of consistency / variability of retention times in liquid chromatography.

On one hand, it is a very common problem, on the another – can be a useful diagnostic tool to identify problems with a separation following this symptom.

A systematic “drill-down” attitude to root cause of retention time changes issue is suggested and the key reasons of this phenomenon are discussed:

• Longer retention as a result of flow rate reduction due to:
  • Air bubbles
  • Check valves malfunction
  • Leaks (pump seals, tubings and fittings)
• Retention time drift as a result of:
  • Weak on-line blending
  • Temperature changes
  • Mobile phase composition and pH changes
  • Column ageing
• Retention time variations within a run which is a specific problem of:
  • Gradient methods

Topic 3: Gradient Elution Troubleshooting

Specific problems related to gradient RP HPLC methods and the ways of solving them.

1. The most common problem: the influence of dwell volume mismatch between different instruments, which results in difficulties in transfer of gradient methods.

This problem is illustrated with typical examples of why some brilliant methods are hard to transfer and how to solve such issues in intra- and interlaboratory studies.

Various types of gradient equipment are presented and the simple method of dwell volume determination is explained.

2. The gradient shape: how accurate is the mixer and what is the reason that in the gradient methods retention time drift may be different in the different segments of the chromatogram.

Simple but efficient methods of testing of functioning of the mixers in the gradient chromatographic systems to ensure that the proper gradient shape is explained.

3. Gradient artifact peaks. The most complicated part of the problems associated with baseline issues.

The “components” of gradient baseline shape are analyzed, including their influence on analytical result.

The main aim of this part is to understand how to diagnose gradient artifact peaks and how to get rid of them. The questions of quality of solvents and reagents, as well as the ways of HPLC systems cleanup are in the focus of this topic.

The seminar is intended for the analytical chemists having experience in HPLC or related techniques, working in Quality Control or Research and Development laboratories and for university students and post-graduates in the field of modern instrumental methods of analysis.