A glossary of chromatography terms

The words on a chromatogram are older than any one instrument, and they are easy to slide past each other. This glossary fixes the pairs people mix up, so a method file and an enquiry name the same object. The modes those words describe are surveyed in chromatography methods in life-science labs.

Chromatography, the chromatogram, and the peak

Chromatography, in the IUPAC sense, is a physical separation in which components distribute between a stationary phase and a mobile phase that moves in a definite direction. The chromatogram is the record of a detector's response against time or against volume. A peak is a rise and fall in that response. It has an apex, a width, and an area. It does not have a purity. Two molecules that share a retention draw one peak if the column does not resolve them. Calling the apex by a compound name is an interpretation you earn with a standard, a spectrum, or an assay.

HPLC is the instrument class that pushes liquid through fine particles at high pressure, usually with an injector, a column compartment, and a detector. It is not a stationary phase. Affinity chromatography, ion exchange, reversed phase, and size exclusion can all be run on HPLC hardware or on open columns. The word HPLC in a notebook does not tell the next person which distribution they are looking at.

Retention time and retention volume

Retention time is the clock time from injection to the apex under a stated flow. Retention volume is that time multiplied by the volumetric flow, or the volume delivered until the apex. Volume is the better number when you compare pumps. Neither number is portable to a different column chemistry or a different gradient. Extra-column volume, the tubing and the detector cell, adds a delay that is a larger share of a small column than of a large one. A method transfer that ignores it will call the column "less retentive" when the plumbing got shorter.

The retention factor compares how long the analyte spent in the stationary phase with how long an unretained marker spent in the mobile phase. It is a unitless way to talk about retention without the clock. It still belongs to one mobile phase. People who quote a retention factor from an isocratic run and then run a gradient are mixing two definitions. In a gradient, strength changes while the analyte is on the bed, and a single factor does not describe the path.

Void, included volume, and column volume

The void, or hold-up volume for a fully excluded solute, is the interstitial liquid that never enters the pores. A size-exclusion aggregate often travels there. The included volume adds the pore liquid. Small salts travel nearer that later volume when they are not adsorbed. Column volume is the cylinder of the packed bed. It is the unit of a wash: five column volumes of buffer means five bed volumes delivered, which you can only compute if diameter and bed height are known.

These three are not synonyms. A protocol that says "elute with two volumes" without saying which volume is how columns get stripped too early or washed all afternoon. Write millilitres or column volumes, and state the bed they refer to.

Selectivity, efficiency, and resolution

Selectivity is the chemical difference in retention between two analytes. Efficiency is how little the band spreads, often discussed as plate count. Resolution is whether the two peaks are actually separated, which takes selectivity, efficiency, and a load that has not flooded the sites. You can raise plates with a smaller particle and a longer bed and still fail if selectivity is near zero. You can have selectivity and lose it to a strong sample solvent that smears the inlet, or to a fraction so large it recombines the peaks in one tube.

Co-elution is unresolved overlap. It is the normal condition in a complex peptide map and in a lysate on a short resin. A symmetrical peak is compatible with co-elution. An orthogonal method, another chemistry or a mass measurement, is how you look inside the peak. Proteomics identifications that start from such a peak are reported under practices discussed by HUPO. The mass you hope to see can be checked in UniProt. The glossary term for the drawing remains peak.

Isocratic, gradient, and dwell

Isocratic means the mobile-phase composition is held constant. Gradient means composition is changed during the run so strongly retained analytes begin to move. Dwell volume is the volume from the mixer to the column inlet. It delays a gradient and barely changes the meaning of a long isocratic hold. Equilibration is the delivery of starting composition, usually for several column volumes, until the bed has forgotten the strong solvent. A second injection that comes earlier than the first is an equilibration failure described with these words, not a new analyte.

Backpressure, linear velocity, and fraction

Backpressure is the resistance the pump works against at a given flow. Watched in the same solvent and at the same temperature, it is a health signal for frits, precipitates, and a compressed bed. Linear velocity is volumetric flow divided by the cross-sectional area of the bed. It, not the millilitres per minute alone, is what a resin note usually limits. A fraction is the collected slice. Its volume should be smaller than the separation you are trying to keep. Injection volume is a fraction of another kind, applied at the inlet. ISO 8655-1 speaks to piston-operated devices when that volume is pipetted. The syringe in an autosampler needs its own check.

Words that should not be asked to do more

Baseline is the detector response between peaks. Drift is a baseline that walks. A ghost peak is a response in a blank, often from solvent or carryover. None of these is a component of the sample until a blank subtraction is justified and a repeat says so. Tailing and fronting name opposite asymmetries. Using "tailing" for every ugly peak sends the repair in the wrong direction.

A standard is a known substance run in the method. It is not the sample. A retention window is the allowed movement of that standard, and it moves again when temperature, column age, or mobile phase changes. System suitability is the decision that today's standard still meets the window and the shape you required. It is not a purity certificate for the unknowns that follow.

Using the glossary in an enquiry

Write chemistry, particle size, internal diameter, bed length, pressure rating, pH window, and analytical or preparative intent. If you mention retention, attach the mobile phase, the flow, and the standard's name. If you mention resolution, name the pair. Hardware classes live in the scientific instruments catalogue. The quote request is where that specification belongs. A protein method can be discussed from the custom protein expression and purification reference without treating the reference as a service in progress.

Safety of the things the terms describe

The terms are paper. The mobile phases are chemical hazards, and a lysate on an affinity resin is a biological material under your institutional rules. Pressure ratings are mechanical limits. A glossary does not authorise a diagnostic use of any peak it defines. ---