Study of retention mechanisms in high performance liquid chromatography

Full project title: Study of retention mechanisms in high performance liquid chromatography and their application in biomedical and pharmaceutical science

Duration: Ongoing

Project lead for CBR: Professor David McCalley

Research partners/collaborators: 

These scientists have each worked within my research group and have gone on to fund joint research now that they are working in industry.

• Dr James Heaton, GlaxoSmithKline
• Dr Stephan Buckenmaier (Agilent Technologies)

Historically, my group has worked on the analysis of small molecules (MW<500) that account for about 70% of all analyses performed by HPLC.  While this technique (reversed phase) is often the method of choice, a substantial proportion of these separations can be problematic. Basic compounds can give strong interactions with acidic column groups, leading to misshapen peaks and poor separation in reversed-phase mode.  My group has looked at overloading by basic drugs which can also give drastic deterioration in peak shape of these basic compounds. Other alternatives include working at high pH to prevent ionisation of the basic solutes. We have made much progress in this subject area, culminating in a major invited review published in Analytical Chemistry in 2023.

More recently, we have carried out mechanistic studies involving hydrophilic interaction chromatography. This technique involves using highly organic mobile phases together with a small quantity of water. It is thought that the water forms a layer on the surface into which polar solutes can selectively partition. This method can produce good retention times and separation of polar or ionised molecules. These molecules are often insufficiently retained by reversed phase techniques. Another advantage is that the mobile phase used is favourable for mass spectrometry work due to the ease of its removal prior to detection.

A disadvantage of this technique can be the relatively high concentrations of acetonitrile in the mobile phase, giving rise to problems of disposal of this toxic solvent. A promising way around this disadvantage is to use miniaturisation techniques where short narrow bore columns containing very small particles can give equivalent performance to standard HPLC columns- but using as little as 10th of the volume of mobile phase. We have used this technique very successfully for the analysis of antibiotics. There is increasing recognition in microbiology that using the correct amount of antibiotic is crucial in the performance of these compounds. Too little may mean the dose is ineffectual and can encourage resistance to the microorganisms which it is designed to treat. Too much may produce effects of toxicity to the patient.

Our work has moved on to the analysis of larger molecules such as proteins and oligonucleotides. Recently we have worked on methods which require little or no organic solvent; in addition, these procedures do not use fluorinated compounds which are another source of toxicity in the environment. These compounds are sometimes called “forever chemicals” because they are extremely difficult to eliminate from body fat.  We have had considerable success in initial work involving oligonucleotide separations and ion exchange.

• McCalley, D. V., Buckenmaier, S. M. C., & Heaton, J. C. (2026). Per-aqueous liquid chromatography and/or weak ion exchange chromatography as a green analytical technique for the separation of neutral, acidic and basic solutes. Journal of Chromatography A, 1777, 466904.
• McCalley, D. V. (2024). Practical examination of flow rate effects and influence of the stationary phase water layer on peak shape and retention in hydrophilic interaction liquid chromatography. Journal of Chromatography A, 1715, 464608. 
• McCalley, D. V. (2023). Understanding and managing peak shape for basic solutes in reversed-phase high performance liquid chromatography. Chemical Communications, 59(51), 7887-7899.
• Taylor, M. R., Kawakami, J., & McCalley, D. V. (2023). Managing sample introduction problems in hydrophilic interaction liquid chromatography. Journal of Chromatography A, 1700, 464006.
• McCalley, D. V. (2022). Influence of metals in the column or instrument on performance in hydrophilic interaction liquid chromatography. Journal of Chromatography A, 1663, 462751.
• McCalley, D., & Stoll, D. R. (2021). But my peaks are not Gaussian! Part 3: Physicochemical causes of peak tailing. LCGC North America, 39(11), 526–533, 539.
• Lardeux, H., Duivelshof, B. L., Colas, O., Beck, A., McCalley, D. V., Guillarme, D., & D’Atri, V. (2021). Alternative mobile phase additives for the characterization of protein biopharmaceuticals in liquid chromatography – Mass spectrometry. Analytica Chimica Acta, 1156, 338347.
• McCalley, D. V. (2021). Evaluation of a linear free energy relationship for the determination of the column void volume in hydrophilic interaction chromatography. Journal of Chromatography A, 1638, 461849.
• McCalley, D. V. (2020). Managing the column equilibration time in hydrophilic interaction chromatography. Journal of Chromatography A, 1612, 460655.
• McCalley, D. V., & Guillarme, D. (2020). Evaluation of additives on reversed-phase chromatography of monoclonal antibodies using a 1000 Å stationary phase. Journal of Chromatography A, 1610, 460562. 
  
  

For further information about the project, please contact Professor David McCalley (david.mccalley@uwe.ac.uk).