High-performance liquid chromatography (revision 34)

High-performance liquid chromatography (HPLC) separates the components of a mixture by passing a solution through a column packed with fine particles. Components that interact more strongly with the packing move more slowly and emerge later; the detector records what emerges against time, producing a chromatogram.^[1]

For peptides the dominant mode is reverse phase, in which the stationary phase is hydrophobic and the mobile phase is a water–acetonitrile mixture whose organic content is increased during the run. Detection is usually by ultraviolet absorbance at 214 nm, where the amide bond absorbs, so response is broadly proportional to the number of peptide bonds rather than to any particular side chain.^[2]

Almost every number on a peptide certificate of analysis originates here. Understanding what the technique measures — and what it cannot measure — is therefore the single most useful piece of analytical background for reading such a document.^[2]

A sample is injected into a stream of mobile phase and carried onto the column. Each component partitions continuously between the mobile phase, in which it moves, and the stationary phase, in which it does not. The fraction of time spent in each determines how long it takes to traverse the column — its retention time.^[1]

In reverse-phase separation the stationary phase is a hydrocarbon chain, commonly C18, bonded to silica particles. Hydrophobic molecules are retained more strongly. Increasing the proportion of organic solvent in the mobile phase weakens that retention, so a gradient of increasing organic content elutes components in approximate order of hydrophobicity.

Resolution between two peaks depends on their retention difference, on peak width, and on the efficiency of the column. Efficiency improves with smaller particles and with longer columns, at the cost of higher back-pressure — the constraint that drove the development of sub-2-micron particles and the instruments capable of running them.^[3]

The chromatogram is detector response against time. Peak area is proportional to the quantity of the eluting species multiplied by its response factor at the detection wavelength; peak position identifies it only by comparison with a known.^[1]

The last caveat is the one most often overlooked. Species that do not absorb at the detection wavelength — inorganic salts, many sugars, water — are invisible, and species that do not elute within the run remain on the column and are simply absent from the chromatogram. A clean trace is evidence about what was detected, not about what was present.^[2]

Two determinations of the same material can differ substantially if the methods differ, and the difference is not error: the methods are answering different questions.^[1]

A short gradient completes quickly and resolves poorly, merging close-eluting impurities into the main peak and returning a higher purity figure. A longer, shallower gradient resolves them and returns a lower one. Both figures are correct for their method. Similarly, detection at 280 nm rather than 214 nm under-detects impurities lacking aromatic residues, and column chemistry alters selectivity as well as retention.

This is why a certificate that does not name its column, gradient, flow rate and detection wavelength cannot be compared with any other document or reproduced by any other laboratory. Method transparency is what converts a number into a checkable claim, and it is a more robust criterion for reading a certificate than the value of the number itself. It is also the precondition for the robustness assessment that validation requires. See Purity claim inflation and Analytical method validation.^[2][4]

Coupling the column outlet to a mass spectrometer gives LC-MS, which adds mass information to retention information and converts a weak identification into a strong one. This is the ordinary way a peptide identity is confirmed.^[2]

Scaling the same separation up, with wider columns and higher loadings, gives preparative HPLC, which is a purification step rather than an analytical one. The chemistry is the same; the objective is collecting a fraction rather than measuring one.

The relationship between the two matters for interpreting a certificate. Material purified by preparative chromatography and then analysed by an analytical method derived from the same separation will look clean, because impurities that co-eluted during purification will co-elute during analysis. An orthogonal method — different column chemistry or different mode — is what tests that assumption.^[1]

• Reverse-phase HPLC
• Area percent purity
• Retention time
• Mass spectrometry
• Analytical method validation
• Preparative HPLC purification

• Mant CT, Hodges RS. "Analysis of peptides by high-performance liquid chromatography." Methods in Enzymology 271:3–50 (1996).