High Performance Liquid Chromatography (HPLC) : Principle, Types, Instrumentation and Applications

Chromatography is a technique to separate mixtures of substances into their components on the basis of their molecular structure and molecular composition.

This involves a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase flows through the stationary phase and carries the components of the mixture with it. Sample components that display stronger interactions with the stationary phase will move more slowly through the column than components with weaker interactions.

This difference in rates cause the separation of variuos components. Chromatographic separations can be carried out using a variety of stationary phases, including immobilized silica on glass plates (thin-layer chromatography), volatile gases (gas chromatography), paper (paper chromatography) and liquids (liquid chromatography).

High perfomance Liquid Chromatography

High performance liquid chromatography (HPLC) is basically a highly improved form of column liquid chromatography.

Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much faster.

All chromatographic separations, including HPLC operate under the same basic principle; separation of a sample into its constituent parts because of the difference in the relative affinities of different molecules for the mobile phase and the stationary phase used in the separation.


Types of HPLC

There are following variants of HPLC, depending upon the phase system (stationary) in the process :

1. Normal Phase HPLC

This method separates analytes on the basis of polarity. NP-HPLC uses polar stationary phase and non-polar mobile phase. Therefore, the stationary phase is usually silica and typical mobile phases are hexane, methylene chloride, chloroform, diethyl ether, and mixtures of these.

Polar samples are thus retained on the polar surface of the column packing longer than less polar materials.


2. Reverse Phase HPLC

The stationary phase is nonpolar (hydrophobic) in nature, while the mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile. It works on the principle of hydrophobic interactions hence the more nonpolar the material is, the longer it will be retained.


3. Size-exclusion HPLC

The column is filled with material having precisely controlled pore sizes, and the particles are separated according to its their molecular size. Larger molecules are rapidly washed through the column; smaller molecules penetrate inside the porous of the packing particles and elute later.


4. Ion-Exchange HPLC

The stationary phase has an ionically charged surface of opposite charge to the sample ions. This technique is used almost exclusively with ionic or ionizable samples.

The stronger the charge on the sample, the stronger it will be attracted to the ionic surface and thus, the longer it will take to elute. The mobile phase is an aqueous buffer, where both pH and ionic strength are used to control elution time.


Instrumentation of HPLC

As shown in the schematic diagram in Figure above, HPLC instrumentation includes a pump, injector, column, detector and integrator or acquisition and display system. The heart of the system is the column where separation occurs.


1. Solvent Resorvoir

Mobile phase contents are contained in a glass resorvoir. The mobile phase, or solvent, in HPLC is usually a mixture of polar and non-polar liquid components whose respective concentrations are varied depending on the composition of the sample.

2. Pump

A pump aspirates the mobile phase from the solvent resorvoir and forces it through the system’s column and detecter. Depending on a number of factors including column dimensions, particle size of the stationary phase, the flow rate and composition of the mobile phase, operating pressures of up to 42000 kPa (about 6000 psi) can be generated.

3. Sample Injector

The injector can be a single injection or an automated injection system. An injector for an HPLC system should provide injection of the liquid sample within the range of 0.1-100 mL of volume with high reproducibility and under high pressure (up to 4000 psi).

4. Columns

Columns are usually made of polished stainless steel, are between 50 and 300 mm long and have an internal diameter of between 2 and 5 mm. They are commonly filled with a stationary phase with a particle size of 3–10 µm.

Columns with internal diameters of less than 2 mm are often referred to as microbore columns. Ideally the temperature of the mobile phase and the column should be kept constant during an analysis.

5. Detector

The HPLC detector, located at the end of the column detect the analytes as they elute from the chromatographic column. Commonly used detectors are UV-spectroscopy, fluorescence, mass-spectrometric and electrochemical detectors.

6. Data Collection Devices

Signals from the detector may be collected on chart recorders or electronic integrators that vary in complexity and in their ability to process, store and reprocess chromatographic data. The computer integrates the response of the detector to each component and places it into a chromatograph that is easy to read and interpret.


Applications of HPLC

The information that can be obtained by HPLC includes resolution, identification and quantification of a compound. It also aids in chemical separation and purification. The other applications of HPLC include :

    • Pharmaceutical Applications
      1. To control drug stability.
      2. Tablet dissolution study of pharmaceutical dosages form.
      3. Pharmaceutical quality control.


    • Environmental Applications
      1. Detection of phenolic compounds in drinking water.
      2. Bio-monitoring of pollutants.


    • Applications in Forensics
      1. Quantification of drugs in biological samples.
      2. Identification of steroids in blood, urine etc.
      3. Forensic analysis of textile dyes.
      4. Determination of cocaine and other drugs of abuse in blood, urine etc.


    • Food and Flavour
      1. Measurement of Quality of soft drinks and water.
      2. Sugar analysis in fruit juices.
      3. Analysis of polycyclic compounds in vegetables.
      4. Preservative analysis.


    • Applications in Clinical Tests
      1. Urine analysis, antibiotics analysis in blood.
      2. Analysis of bilirubin, biliverdin in hepatic disorders.
      3. Detection of endogenous Neuropeptides in extracellular fluid of brain etc.


Frequently Asked Questions


The principle of HPLC is based on analyte distribution between the mobile and stationary phases. It is important to keep in mind that the sample’s different constituents elute at different times before the sample ingredients’ separation is achieved. The intermolecular interactions between molecules of the sample and packaging materials determine their time on-column.


There are four primary types of HPLC –

  1. Normal phase HPLC (effective method for separating phospholipid classes)
  2. Reverse phase HPLC (the most common method used to separate compounds that have hydrophobic moieties)
  3. Size-exclusion HPLC/molecular sieve chromatography (Used in large molecules/macromolecular complexes such as industrial polymers and proteins)
  4. Ion-exchange HPLC (separates ions and polar molecules according to their ion exchanger.


The four types of chromatography are

  1. Liquid chromatography (test for pollution in water samples like lakes and rivers)
  2. Gas chromatography (detect bombs and useful in forensic investigations)
  3. Thin-layer chromatography (used to check the purity of organic compounds such as the presence of insecticide or pesticide in foods)
  4. Paper chromatography (uses a strip of paper in the stationary phase).


HPLC uses a moderate to high pressure to achieve the desired flow rate of the solvent through the chromatographic column as small particles have greater resistance to flow.


Your application can be run in different ways – isocratic and gradient. Isocratic is when the mixture of the mobile phase is consistent over the total testing time. with a gradient, the compounding of the eluent mixture is changed during measurement, which greatly affects analyte retention. It can accelerate or decelerate the separation process.


There are different solvents used in HPLC such as aqueous solvent (water) and organic solvent (methanol, acetonitrile, and propanol). To improve the chromatographic peak shape, acids can be used such as acetic acid, formic acid, trifluoroacetic acid.


The PDA and UV are both absorbance detectors, which provide sensitivity for light-absorbing compounds. The UV detector is most commonly used for HPLC analysis. The UV absorbance differs on the wavelength used, which is why it is important to choose the right wavelength based on the type of analyte. On the other hand, the PDA detector adds a third dimension wavelength, which is a more convenient way of finding out the wavelength without having the need to repeat the analysis.


The advantages of HPLC are as follows:

  • It can test both raw materials and finished products.
  • It can reverse engineer formulations.
  • It is helpful in solving product failure problems.
  • It can detect contaminants and other impurities.
  • It can perform competitor product analysis.
  • It can determine product stability and shelf life.
  • The testing can be done even with just a small sample size.
  • It enables you to modify the testing depending on the needed quantification level.
  • The results it produced are reliable.
  • It is helpful in developing better products.
  • It lets you gain a better understanding of the competitor’s products.


In chromatography, the RF value pertains to the distance a particular component traveled divided by the distance traveled by the solvent front. In other words, it is the characteristic of the component, which is helpful in the identification of the components.


There are different types of chromatography but the two primary types are liquid chromatography and gas chromatography.

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52 Responses to High Performance Liquid Chromatography (HPLC) : Principle, Types, Instrumentation and Applications

  1. Rakesh July 29, 2015 at 2:45 pm #

    suggest Me about research topic in cancer plzz

    • Dhurba Giri July 30, 2015 at 4:14 pm #

      Dear Rakesh, It’d be better if you first choose your priority area and specific type of cancer on which you want to do a research. Then searching related articles with specified keywords on internet search engines (like Google Scholar) or databases (like PUBMED) will help you out a lot for choosing the topic. I wish you a very good luck.

    • gangadhar September 4, 2017 at 10:18 am #

      Clear cut information. Tq

  2. sushmita poudel July 30, 2015 at 1:52 pm #

    thank u dada.. it will help us

    • Dhurba Giri July 30, 2015 at 4:05 pm #

      I’m glad that it helped you Sushmita. Keep visiting 🙂

    • jaydip khuman February 3, 2017 at 9:41 am #

      I think here tcd , ecd, flam ionization left ???

  3. anant sherkhane July 30, 2015 at 3:30 pm #

    Nice HPLC information

  4. Chandra Ghosh August 1, 2015 at 4:14 pm #

    thank you for this information…

  5. priyanka khade January 2, 2016 at 7:11 am #

    Thank you for information…. It helps me for my presentation

  6. sangani paresh January 5, 2016 at 6:27 am #

    Thank you…………

  7. priya pandita March 20, 2016 at 4:27 pm #

    thank youuuu sir … it helps me a lot in my exam

  8. Vaishali mehetre April 7, 2016 at 8:26 am #

    this s very very informative web site, Dhurba… excellent efforts… n pricision on ur knowledge.. congratulations!!! keep it up…

  9. Pallavi May 28, 2016 at 5:15 am #

    Good infrmation

  10. Rajni June 10, 2016 at 6:21 am #

    Written in simple way… easily understandable language… good work… thanks DRubaa

  11. senthil kumar June 18, 2016 at 1:50 am #

    super sir

  12. kumar June 25, 2016 at 10:02 am #

    Very helpful sir

  13. Naresh June 30, 2016 at 3:56 pm #

    Thank-you for the given good information

  14. Santosh v lokhande July 13, 2016 at 10:15 am #

    Thanks sir

  15. PRATIK NAYI July 15, 2016 at 9:30 am #

    Sir it is very useful for me and anybody can understand easily from this presentation.


  16. Anjalichaurasiya July 31, 2016 at 4:16 pm #

    The information that is available in this presentation is better for me.

  17. Parthee August 5, 2016 at 6:02 am #

    This is very useful to me… Thank you sir….

  18. sophie August 24, 2016 at 12:23 am #

    simple,short and straight to the point.very imformative and concise.well done

  19. sophie August 24, 2016 at 12:24 am #

    simple,short and straight to the point.very informative and concise.well done

  20. abhay tyagi August 24, 2016 at 2:24 pm #

    thanks for good information

  21. K M Ravi Kumar September 4, 2016 at 12:52 pm #

    nice brief description

  22. Vijaya September 9, 2016 at 7:30 am #

    Thank u sir

  23. miraben nayak September 21, 2016 at 1:31 pm #

    thnx a lot mr.dhurba…. your posts helped me a lot…thnx for making things a bit easier for me….:-)

  24. lylee September 26, 2016 at 2:41 pm #

    Explanation is easily understood and straight to the point. Very helpful! Thank you!

  25. seenu September 29, 2016 at 11:26 am #

    thanks boss

  26. Padma Ladon September 29, 2016 at 1:41 pm #

    Very helpful… Nicely done.?

  27. akumar October 2, 2016 at 2:34 pm #

    hplc analytical test about information is best given this manual and think is given perfect

  28. Odinga Tamuno-boma October 12, 2016 at 9:50 pm #

    Nice write up in clear words , articulate and helpful

  29. yuvraj arjun October 25, 2016 at 11:14 am #

    sir,can you give me more details about detectors used in HPLC?

  30. Trushna November 28, 2016 at 4:02 am #

    Want to know that, what pore size column we use for synthetic tripeptides in hplc

  31. Sai Shiva November 28, 2016 at 2:08 pm #

    Very Helpful..For Last Minute Preparation…Thank You!

  32. Neha Bisht December 4, 2016 at 3:14 pm #

    Simple and precise. Very helpful

  33. Jaswant Bhardwaj December 4, 2016 at 4:35 pm #

    Hplc or GC ka ki full knowledge Hindi me kese pata kru frnz anyone help me plz…

  34. vishal singh December 7, 2016 at 12:33 pm #

    Thank you dear its really helpd me lot to Understand

  35. Ashok December 9, 2016 at 6:35 am #

    Nice information

  36. nisha December 25, 2016 at 3:51 am #

    Very nice information…easily understandable

  37. Divya bharathi March 15, 2017 at 4:25 pm #

    It’s useful for me and am now little clear about HPLC

  38. Shiv Kalia March 23, 2017 at 3:45 pm #

    It’s very helpful to me sir and thanks

  39. Mamta March 28, 2017 at 10:15 am #

    Hello sir, I want to know about hplc.what is this. Ye test kaise hota hai. Actually ye test Dr. Ne mere bete ko prescribe kiya hai

  40. shailesh Rathod April 5, 2017 at 8:22 am #

    Sir can you add some more details about detector

  41. priyankara April 10, 2017 at 5:13 am #

    thanks bro

  42. Mich June 17, 2017 at 8:21 pm #

    ..volatile gas is not use,but inert gases like nitrogen,argon,hydrogen is use as mobile phase in GC…

  43. li wei August 14, 2017 at 3:57 pm #

    thank you so much!! This helps me 😀

  44. Richard September 6, 2017 at 10:30 am #

    Interesting information. Thanks.

  45. Tatfo September 6, 2017 at 2:39 pm #

    Hi Sir!!!
    Could we please establish a collaboration? In fact i am a PHD Student working on veterinary drugs in Cameroon

  46. anusha September 15, 2017 at 4:34 pm #

    thank you so much

  47. Gopinath Mal January 9, 2018 at 4:01 am #

    Very nice.

  48. Gopinath Mal January 9, 2018 at 4:04 am #

    Sir thank you very much..

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