Separating your way through Liquid Chromatography Instruments

Liquid chromatography (LC) is a technique used to separate a sample containing ions and molecules into its individual parts. A liquid sample is injected/introduced onto a column (a tube packed with tiny particles) where the individual components are moved through the column by a liquid via a pump. These components are separated from one another based on the physical or chemical interaction between the molecules and the packed particles. The separated components exit the column and enter a detector tat measures their amount. The measurement is recorded as a chromatogram.

There are 2 main types of liquid chromatography instruments: 1) Fast Protein Liquid Chromatography (FPLC), 2) High Performance Liquid Chromatography formerly known as High Pressure Liquid Chromatography (HPLC).

HPLCs and FPLCs are made up of several components that can be customized to suit your application. These typically include:

Component	Description
Pump	A pump is used to move liquid (mobile phase) through the column and system.
Injector	An injector introduces the liquid sample into the flow stream of the mobile phase. This may be manual or automated. HPLC sample injectors are exclusively 6 port valves that are overfilled via syringe. Typical sample volumes are 10 to 50 uL but can be larger.
Column	This is the “stationary phase” of the system that carries out the separation of the sample components.
Detector	Measures the amount of the individual molecules that come out from the column.The most common are UV-Vis absorbance detectors which include single wavelength (filter), variable wavelength (monochromator), or multiple wavelengths (PDA).Other detectors include Fluorescence, Electrochemical, and Mass Spectrometric.
Computer	Controls the modules of the LC and stores the signal from the detector to make qualitative and quantitative analysis of each sample.
Fraction Collector (optional)	A device that allows for regular or specified samples to be taken from the column elution and stored in a retrievable form.
Column Heater /Cooler (optional)	Controls temperature of a column to help with improved reproducibility.

HPLC or FPLC?

HPLCs are typically used for quantitative and qualitative analysis of peptides, other molecules, and proteins. The system can be modified for use in a small preparative scale by changing out components such as the pump to allow for a higher flow rate. But an FPLC can be more readily scaled from analysis quantities to production quantities with industrial sized FPLCs.

An FPLC is generally only applied to proteins as a separation tool rather than a precise qualitative tool. With some techniques you are able to quantify the protein(s) you’ve separated. HPLCs (usually come with a standard stainless steel tubing configuration) can certainly purify proteins. However, if you are at all concerned about the stability and activity of your protein due to increased temperature produced by the stainless steel tubing, you may want to use an FPLC or reconfigure your syste with polyetherketone (PEEK) tubing.

One of the biggest differences between an HPLC and an FPLC is the solvent pressure. An FPLC has a relatively low solvent pressure compared to an HPLC (typically less than 5 bar compared to 50-350 bar) while the flow rate is relatively high comparatively. FPLCs have a wider choice of resins and buffers providing for broader application for proteins. However, if the pressure of the HPLC can be limited, nearly every FPLC column may also be used in an HPLC system.

The most common type of chromatography used in HPLC is reversed-phase. This means that the mobile (or liquid) phase is highly polar, while the stationary phase (column) is non-polar. Other general types of chromatography include partition, adsorption, size-exclusion chromatography, and ion-exchange. Scientists are cautious about using the ion-exchange method on an HPLC as the corrosive nature of the high salt buffers used reduc

If don’t plan to use reversed phase columns, especially in analytical scale, buy an FPLC. However, if you plan or hope to use your system for semi-prep or analytical RP-HPLC, buy an HPLC.

	HPLC	FPLC
Pressure	50-600 bar	<5 bar
Flow Rate
Qualitative	Yes	No
Quantitative	Yes	Limited
Analytical ScaleInformation only (compound ID and quantification)
Qualitative	Yes	No
Quantitative	Yes	Limited
Semi-prepSame as above and < 0.5 gram of purified compound	Yes	Yes
Prep Scale>0.5 gram of purified compound	Limited	Yes
ProcessGrams to kilograms of purified compound	No	Yes
Proteins	Yes*	Yes
Peptides	Yes	No
Sugars	Yes	No