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.
The glossary will help you to understand the terminology in case you aren’t already familiar with the technique.
|HPLC||separation 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 separation||separation 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 efficiency||expressed in terms of HETP which expresses resolution power of the HPLC column.|
|Column||a steel tube packed with the stationary phase for separation of sample components|
|Autosampler||a device for automated precise selection and introduction of programmed sample volume into the HPLC system|
|Injector||manual or automated device capable of precise sample volume injection of sample into the HPLC system|
|Filter||frit fitted with a screen membrane to remove solid suspensions from mobile phase of sample|
|Degassing||procedure for removal of dissolved air from mobile phase using vacuum filtration, helium purging or online degassing|
|Pump||a module of the system for metering controlled flow rate mobile phase through the system|
|Detector||device 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|
|UHPLC||Ultra-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 Elution||mobile phase composition varies during the run and can be programmed before starting the chromatographic run|
|Retention time||time 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|
|Syringe||hand 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 detector||detector based on specific property of eluting molecules and is independent of changes in the mobile phase composition|
|HETP||Height 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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Refresh your concepts by registering for the free course which will provide you an introduction to the technique and even prepare you for an interview if you are applying for a job in the laboratory equipped with HPLC systems.Want to read all the High Performance Liquid Chromatography free course modules right now? Here are all links to all the modules for you!
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.
- Benefits of Automated injections in chromatographic analysis
- Suggestions for trouble free operation of your HPLC system
- Importance of HPLC grade solvents in HPLC analysis
- Guidelines on use of buffers in HPLC separations
- HPLC – The new age analytical technique!
- Critical considerations in HPLC solvent selection
- Important role of preventive maintenance in smooth functioning of HPLC systems
- How are analytical scale and preparative scale HPLC different?
- Which technique to use – GC or HPLC?
- Role of HPLC in Cleaning Validation of Laboratory Glassware
- Restoration of HPLC Column Performance
- Important suggestions for generating reliable HPLC results
- Time saving tips for the HPLC user
- How to minimize cost on HPLC column replacements?
- Advantages of Filtering HPLC mobile phase solvents
- Potential applications of HPLC in Clinical laboratories
- Contribution of HPLC to Nutritional Labeling of Foods
- 10 Similarities between High Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC)
- How are Gas Chromatography (GC) and High Performance Liquid Chromatography(HPLC) different?
- Advantages of HPLC over Thin-layer Chromatography (TLC)
- Introduction to High Performance Liquid Chromatography and it’s parts
- What are guard columns and what benefits they offer in HPLC analysis?
- Solutions to First HPLC Program Quiz
- Why is HPLC the most popular technique in analytical laboratories today?
- HPLC – which modules require regular calibration?
- Essential precautions in handling and use of HPLC mobile phase
- What are the Desirable Properties of HPLC Mobile Phase?
- HPLC Quiz : Test Your Eligibility for Discount on Certificate Program on HPLC
- How to Mix HPLC Mobile Phase Solvents for consistency of results?
- What are the Benefits of UHPLC over normal Analytical HPLC?
- The role of Stationary Phase particle size on HPLC separations
- On-line Certificate Program on High Performance Liquid Chromatography – Join Now
- USP Classification of HPLC Columns
- What are the Emerging Trends in HPLC Analysis?
- How to ensure trouble-free HPLC System Operation
- HPLC Free e-course : Introduction to High Performance Liquid Chromatography
- Cost Saving Tips for the HPLC User
- How to Deprotenize Biological Fluids prior to HPLC analysis?
- Common Peak Shape Distortions in HPLC and their Prevention
- General applications of HPLC based on solute – stationary phase interaction classification
- Prevention of Retention Time Drifts in HPLC Analysis
- Advantages and Disadvantages of different HPLC Pump Configurations
- Advances in HPLC Stationary phases: Zirconia HPLC columns
- Advances in HPLC Stationary Phases: Monolithic HPLC columns
- Ion Pair Chromatography – a viable alternative for Separation of High Polarity molecules by Reverse Phase HPLC
- Role of Buffers in Reverse Phase HPLC Separations
- Join us on FREE Webinar on 18 Steps for learning HPLC operation
- Classification of HPLC Separations by Column Dimensions
- Core Shell Column Packings- A Challenge to UHPLC
- Care of HPLC Columns
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!
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.