High-performance liquid chromatography, or HPLC for short, is an analytical method used to separate the components of a mixture from one another and to make each separated component visible individually. For samples such as venom, which carry dozens of components, it is the most common way of obtaining a profile.
The method owes the high performance in its name to its fine-grained packing material. The smaller the particles, the sharper the separation, but the greater the resistance to flow; overcoming this resistance requires pumps that operate at high pressure. In modern systems, the pump, injection unit, column oven and detector are controlled together along a single flow path, and all conditions are recorded by software.
Operating principle
The heart of the system is a column tightly packed with packing material. The pump drives the solvent, called the mobile phase, through this column at high pressure. When the sample is injected into the flow, each component interacts with different strength with the packing material (the stationary phase) and the solvent. A component that is retained more strongly by the stationary phase stays longer in the column, while one that is retained less elutes first. The detector at the column outlet measures the substances passing through and plots a signal against time; this graph is called a chromatogram.
Keeping the column temperature constant is important for reproducibility; when the temperature changes, the retention behaviour of the components also changes and the peaks shift. For the same reason, the column is expected to be equilibrated at the starting conditions before each run. Instrument model, column type and program conditions are written into reports so that the result can be reproduced in another laboratory. In the reference batch analysis, the separation was carried out on a Shimadzu Prominence-i LC2030 3D system.
What does reversed phase mean?
In classical normal-phase chromatography, the stationary phase is polar and the mobile phase non-polar. In reversed phase (RP-HPLC), this relationship is inverted: the stationary phase is made non-polar with long hydrocarbon chains bonded to a silica surface, while the mobile phase is aqueous and polar. Separation is therefore based on the hydrophobicity of the molecules. Because peptides carry both water-loving and water-repelling surfaces, this approach has become the standard in peptide analysis.
In practice, the mobile phase is not kept at a single composition. At the start it is mainly water; over time, the proportion of organic solvent is increased gradually. In this method, called gradient elution, hydrophilic components elute early and hydrophobic ones late, and a wide polarity range is covered in a single run. In the reference batch analysis, a reversed-phase column with a shallow gradient was used, the proportion of organic solvent being raised from zero to sixty per cent.
Why detect at 280 nm?
Proteins and peptides absorb light at around 280 nanometres because of the tryptophan and tyrosine residues in their structure. Reading at this wavelength makes protein-containing peaks selectively visible while limiting the noise from buffer components. The physics of absorbance is covered in the article on measurement at 280 nm.
Steps of a run
- The sample is weighed and prepared at a defined concentration in a suitable solvent; in the reference batch analysis, the sample was dissolved at 1000 ppm.
- The solution is filtered to remove particles; this step is not skipped, so that the column does not become blocked.
- The column is equilibrated at the starting conditions.
- The sample is injected and the gradient program is started.
- The detector signal is recorded, and the peaks are assessed by retention time and area.
- If necessary, the peaks are collected individually and passed on for further analysis.
Sample preparation is the source of most problems. Undissolved particles block the column inlet, while an unsuitable solvent distorts peak shape. The sample is therefore prepared in a solvent close to the starting composition of the mobile phase and filtered before injection. Keeping the injection volume reasonable for the column also prevents peak broadening.
What does the chromatogram tell you?
| Parameter | Meaning |
|---|---|
| Retention time | The moment the component leaves the column; a reproducible identifier under the same conditions |
| Peak area | Related to the relative amount of the component |
| Number of peaks | Shows the number of components that can be resolved |
| Peak width | Gives information on separation efficiency and column condition |
| Baseline | Drift points to problems arising from the system or the gradient |
In the reference batch analysis, 37 proteins and peptides were resolved at 280 nm. It should be remembered that this number depends on the conditions: when the column type, gradient slope and detection wavelength change, a different number of peaks may be obtained from the same sample. Reading a chromatogram is explained in detail in the article on chromatograms.
Peak areas are usually assessed in relative terms. Giving the absolute amount of a component requires calibration with solutions of the same component prepared at known concentrations. Because pure standards are not available for most venom components, the profile is used as a comparative fingerprint rather than as a quantitative measurement.
The separated peaks are not only monitored; they can also be collected. When the flow from the column outlet is directed into separate containers over time, each peak is obtained as its own fraction. These fractions can be freeze-dried and stored, and studied as single components in later analyses. Isolating a peptide of interest from a complex mixture usually starts this way. If it is not recorded which run and which time window a collected fraction came from, the fraction's identity is lost too; this is why the collection plan is written before the analysis.
Limitation of the method
HPLC separates components but cannot, on its own, tell you what they are. Determining which peptide a peak corresponds to requires molecular weight and sequence information; chromatography is therefore mostly used together with methods such as SDS-PAGE and mass spectrometry. Our entire analytical workflow is summarized on the analysis page.
