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 3โ€“10 ยตm. 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.

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

  1. suggest Me about research topic in cancer plzz

    • 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.

    • Clear cut information. Tq

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

    thank u dada.. it will help us

  3. anant sherkhane // July 30, 2015 at 3:30 pm // Reply

    Nice HPLC information

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

    thank you for this information…

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

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

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

    Thank you…………

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

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

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

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

  9. Good infrmation

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

  11. Very helpful sir

  12. Thank-you for the given good information

  13. Santosh v lokhande // July 13, 2016 at 10:15 am // Reply

    Thanks sir

  14. PRATIK NAYI // July 15, 2016 at 9:30 am // Reply

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


  15. Anjalichaurasiya // July 31, 2016 at 4:16 pm // Reply

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

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

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

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

  19. thanks for good information

  20. K M Ravi Kumar // September 4, 2016 at 12:52 pm // Reply

    nice brief description

  21. Thank u sir

  22. miraben nayak // September 21, 2016 at 1:31 pm // Reply

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

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

  24. Very helpful… Nicely done.?

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

  26. Odinga Tamuno-boma // October 12, 2016 at 9:50 pm // Reply

    Nice write up in clear words , articulate and helpful

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

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

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

  30. Simple and precise. Very helpful

  31. Jaswant Bhardwaj // December 4, 2016 at 4:35 pm // Reply

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

  32. Thank you dear its really helpd me lot to Understand

  33. Nice information

  34. Very nice information…easily understandable

  35. Divya bharathi // March 15, 2017 at 4:25 pm // Reply

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

  36. Shiv Kalia // March 23, 2017 at 3:45 pm // Reply

    It’s very helpful to me sir and thanks

  37. 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

  38. shailesh Rathod // April 5, 2017 at 8:22 am // Reply

    Sir can you add some more details about detector

  39. priyankara // April 10, 2017 at 5:13 am // Reply

    thanks bro

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

  41. thank you so much!! This helps me ๐Ÿ˜€

  42. Interesting information. Thanks.

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

  44. thank you so much

  45. Gopinath Mal // January 9, 2018 at 4:01 am // Reply

    Very nice.

  46. Gopinath Mal // January 9, 2018 at 4:04 am // Reply

    Sir thank you very much..

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