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What is HPLC/ High Performance Liquid Chromatography?

HPLC stands for High Performance Liquid Chromatography.

Its earlier name was High Pressure Liquid Chromatography because it involved use of liquid mobile phase requiring higher pressures than gases used in Gas Chromatography. The technique has found immense scope of applications in both academic and industrial laboratories requiring identification and quantification of mixtures of organic compounds.

Why not start with a short video?

Are you new to High Performance Liquid Chromatography? Simply spend about 2 to 3 min on the introductory video giving you an insight into what constitutes a HPLC system.

Glossary of HPLC terms

The glossary will help you to understand the terminology in case you aren’t already familiar with the technique.

HPLCseparation technique for components of organic mixtures involving retention of components on stationary phase packed inside column on the basis of physico – chemical interactions followed by sequential elution
Stationary phase solid bed inside column whose particles are coated with the retention phase
Mobile phase liquid carrier medium used for transporting the sample through the HPLC system
Normal phase separationseparation mode in which the retention material is polar and mobile phase is nonpolar. Retained sample components are eluted in ascending order of polarity
Reverse phase separation separation mode in which the stationary phase is nonpolar and mobile phase is polar. Elution order of components is in decreasing order of polarity.It is the most commonly used mode of HPLC separations.
Column efficiencyexpressed in terms of HETP which expresses resolution power of the HPLC column.
Columna steel tube packed with the stationary phase for separation of sample components
Autosamplera device for automated precise selection and introduction of programmed sample volume into the HPLC system
Injectormanual or automated device capable of precise sample volume injection of sample into the HPLC system
Filterfrit fitted with a screen membrane to remove solid suspensions from mobile phase of sample
Degassingprocedure for removal of dissolved air from mobile phase using vacuum filtration, helium purging or online degassing
Pumpa module of the system for metering controlled flow rate mobile phase through the system
Detectordevice for providing response of the component after separation by the HPLC column
Column Oven a housing for the HPLC column which can be maintained at a constant temperature. It permits constant flow rate of mobile phase
Ion-exchange chromatography branch of HPLC involving separation of charged sample components by attraction to the oppositely charged stationary phase
Gel permeation chromatography separation technique in which separation is based on molecular size of component molecules. Smaller molecules get trapped in pores of stationary phase and are eluted after the large molecules.
Chiral chromatography chromatographic separation technique for optically active sample components
Analytical scale separations separations in the mass range mg-ng involving use of columns packed with 3-5μm size particles
Semi-preparative HPLC HPLC separations involving mass range from mg-gm involving collection of separated fractions.
Preparative HPLC mass range gm-kg involving collection of separated fractions
UHPLCUltra-high-Pressure or Performance HPLC. Particle size is less than 2 µm. Such systems and columns are capable of handling back pressures in the range of 15,000 up to 18,000 psi
Isocratic Elution mobile phase composition does not change during the chromatographic run
Gradient Elutionmobile phase composition varies during the run and can be programmed before starting the chromatographic run
Retention timetime taken between injection and the maximum of the peak response of a component
Mobile phase reservoir a container that is filled with the mobile phase
Fraction collector a carousel packed with empty vials in which different components get collected after separation on the HPLC column
Syringehand held glass device capable of injecting selected volume of sample into the chromatograph
Bulk property detector detector based on detection of total property of mobile phase and sample component as a single system
Specific property detectordetector based on specific property of eluting molecules and is independent of changes in the mobile phase composition
HETPHeight equivalent to theoretical plate. It is a measure of column efficiency and is expressed as a numerical value without units
HETP = L/N
Where L is column length and N the number of theoretical plates.The larger the number of theoretical plates the lower is HETP and better is the column efficiency.

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Did you know there are only 18 key steps to learning how to run a HPLC? Read our 18 step simplified guide to High Performance Liquid Chromatography and get started!

Certificate Course on HPLC

Do you want to master the fundamentals and operation of HPLC? You can join our certificate course on HPLC and become an expert and get a certificate online!

HPLC certificate program is an advanced level program that is designed keeping the needs of the laboratory’s worker in mind. It covers the subject in detail and will instill in you the confidence to start work on HPLC the day you come face to face with it. The course includes self-evaluation quiz sessions in addition to several opportunities to interact with our experts in regular webinar sessions besides participation in discussion forums. It is not a time bound programme and you can complete it as per your convenience. Our subscribers found it to be even more beneficial than regular hands on training programs.

How about a more detailed introduction to HPLC operation? Watch out “What really goes into running a HPLC?” video!

To know more about the program and joining guidelines please go through the link to Join HPLC Certificate Programme.

Want to learn more about HPLC? Continue with our library of articles on HPLC below –

We regularly publish articles specially to help you upgrade your laboratory skills and to expose you to new concepts and developments in the field of HPLC.

You’ll find the list is ever growing with inclusion of newer published articles. We are confident that you’ll find the article content of immense use. Continue learning more about High Performance Liquid Chromatography by clicking any of the articles that interest you.