The HPLC column is the consumable that does most to decide the quality of a separation. A badly chosen column weakens a well-written method; a well-chosen one shortens the run time and improves the repeatability of the results. This guide summarises the criteria to work through when choosing a column and illustrates each step with HALO and SunShell columns supplied by YGT Laboratory. When the column for a method is already known, the column defined in the method or monograph always takes precedence.
1. Is the column defined in the method or monograph?
Pharmacopoeial methods usually define the column by a description. In the USP, for example, the code L1 describes a packing with bonded octadecylsilane (C18). USP <621> Chromatography and Ph. Eur. 2.2.46 allow adjustments of method parameters within set limits; these limits and the current version of the column description should be checked in the pharmacopoeia concerned. When a new method is being developed, start with the criteria below.
2. Analyte size: pore size
The pore size decides whether a molecule can enter the pores inside the particle freely. For small molecules 90 Å or 120 Å is sufficient; peptides need wider pores and proteins much wider ones. According to the manufacturer, HALO 90 Å particles are designed for small molecules up to about 2000 Da and HALO 160 Å particles for peptides up to about 15,000 Da; for protein separations there are 400 Å (3.4 µm) particles, and 1000 Å for very large proteins and antibodies.
| Analyte | Pore size | Example column |
|---|---|---|
| Small molecules | 90 Å | HALO 90 Å C18 |
| Peptides | 160 Å | HALO 160 Å ES-C18 |
| Proteins | 400 Å | HALO 400 Å ES-C18 |
| Large proteins, mAbs | 1000 Å | HALO 1000 Å ES-C18 |
3. Particle structure and size
In core-shell (superficially porous) particles a solid core is surrounded by a porous layer, unlike fully porous particles. The shorter diffusion path is intended to give higher efficiency at lower back pressure. HALO Fused-Core columns use 2.7 µm particles; SunShell core-shell columns are offered in 2, 2.6, 3.5 and 5 µm. Smaller particles give higher efficiency but also higher back pressure, so the pressure limit of the instrument has to be considered. Larger particles suit conventional HPLC instruments and smaller particles suit UHPLC.
4. Phase chemistry
Phase chemistry determines selectivity. The summaries below are based on the manufacturers' product descriptions; detailed conditions should be checked in the manufacturer's documents.
- C18: the general-purpose and most common phase for a wide range of analytes. HALO 90 Å C18 is described as a universal C18 for acidic, basic and neutral solutes. SunShell C18 is supplied with Sunniest end-capping.
- C8: less hydrophobic than C18; used where shorter retention is wanted.
- Phenyl-hexyl and biphenyl: give different selectivity for aromatic compounds through pi-pi interaction. Example: HALO 90 Å Phenyl-Hexyl.
- PFP (pentafluorophenyl): offers alternative selectivity for aromatic and electron-rich compounds. Example: HALO 90 Å PFP.
- PCS (positively charged surface): designed to improve peak shape for basic compounds. Example: HALO 90 Å PCS C18.
- AQ-C18: compatible with 100 % aqueous mobile phases; used for polar analytes. Example: HALO 90 Å AQ-C18.
- HILIC: for polar analytes that are hard to retain in reversed-phase chromatography. Example: HALO 90 Å HILIC.
A common approach in method development is to try several phases (for example C18, phenyl-hexyl and PFP) with the same sample and similar conditions, and then choose the phase that gives the best selectivity and peak shape. This column screening wastes less time than sticking to a single phase and also documents why the chosen column was chosen.
5. pH range
The pH of the mobile phase must stay within the range the column can tolerate. Working outside it damages the packing and shortens column life. According to the manufacturers, SunShell C18 is rated for pH 1.5–10, ArmorShell C18 for pH 1–11 and the HALO ELEVATE series for pH 2–12. For methods that need an alkaline mobile phase, alkali-resistant phases such as HALO 120 Å ELEVATE C18 can be considered.
6. Column dimensions
Column length and inner diameter affect run time, solvent consumption and sensitivity. Shorter columns speed up the analysis; longer columns increase resolution for complex mixtures. Narrow-bore columns use less solvent and suit mass spectrometry, but they are more sensitive to the dead volume of the instrument and to the injection volume. When choosing the dimensions, check the dead volume of the instrument and the flow range of the pump.
7. Protecting the column
Column life depends not only on the column but also on the cleanliness of the flow that reaches it. Inlet filters on the solvent bottle, inline filters and guard cartridges keep particles away from the column. Dissolved gas in the mobile phase can upset pump and detector stability, so inline degassers can be used. Safety caps such as the SCAT Safety Cap are also used to reduce the release of solvent vapour into the laboratory.
Points to watch when changing to another column
Columns with the same bonded phase name may not give the same selectivity from different manufacturers or in different series. Bonding density, end-capping, silica purity and pore structure can change peak shape and elution order. When a new column is introduced, resolution, peak symmetry, retention time and pressure are therefore checked again; if the method is validated, the verification that the change requires is decided by the change procedure of the quality system.
Common mistakes in column selection
- Skipping the pore size: a 90 Å column for a peptide or protein separation can limit access of the molecule to the pores.
- Not checking the pH range: alkaline or strongly acidic mobile phases damage the packing of an unsuitable column.
- Ignoring the pressure limit of the instrument: small particles and long columns raise the back pressure.
- Looking only at the phase name: two different C18 columns can give different results in the same method.
- Neglecting protection: not using filters and guard cartridges shortens column life.
Short checklist
- Check the column defined in the method or monograph.
- Choose the pore size according to the molecular size of the analyte.
- Choose a particle size that suits the pressure limit of the instrument.
- Choose a phase chemistry that suits the polarity and structure of the analyte.
- Make sure that the pH of the mobile phase stays within the range of the column.
- Set the column dimensions according to the dead volume of the instrument and the resolution needed.
- Protect the column with an inlet filter, an inline filter and a degasser.
References
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