High performance liquid chromatography

High performance liquid chromatography

1. High Performance liquid Chromatography Presented by : Deepa Kumari Karn First Semester, M Pharm School of Health and Allied Sciences Pokhara University, Dhungepatan , Pokhara Lekhnath-30, Kaski , Nepal

2. Contents Introduction General History Classification Instrumentation Operation Advantages and Disadvantages Applications Difference between TLC and HPLC References May 29, 2017 2

3. Introduction Chromatographic technique used to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying or purifying the individual components of the mixture HPLC is a separation technique that involves: The injection of a small volume of liquid sample into a tube packed with tiny particles (3 to 5 micron (µm) in diameter called the stationary phase) May 29, 2017 3

4. Individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump In principle, LC and HPLC work the same way except the speed, efficiency, sensitivity and ease of operation of HPLC is vastly superior Components are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles May 29, 2017 4

5. Separated components are detected at the exit of this tube (column) by a flow-through device (detector) that measures their amount May 29, 2017 5

6. General History The acronym HPLC , coined by the late Prof. Csaba Horváth for his 1970 Pittcon paper, originally indicated the fact that high pressure was used to generate the flow required for liquid chromatography in packed columns In the beginning, pumps only had a pressure capability of 500 psi. This was called high pressure liquid chromatography, or HPLC May 29, 2017 6

7. These new HPLC instruments could develop up to 6,000 psi [400 bars] of pressure, and incorporated improved injectors, detectors, and columns. HPLC really began to take hold in the mid-to late-1970s May 29, 2017 7 Figure: Csaba Horváth working on pittcon paper

8. May 29, 2017 INS 591: Presentation 8 Figure: Traditional HPLC Instrunment

9. Classification Depending upon the phase system (stationary and mobile phase) HPLC is classified into following types: 1) Normal Phase HPLC : It has polar stationary phase and non-polar mobile phase 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 May 29, 2017 9

10. 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 May 29, 2017 10

11. 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 May 29, 2017 11

12. 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 May 29, 2017 12

13. 5) Based on the mobile phase gradient: Isocratic: Mobile phase solvent composition remains constant with time Often used in quality control applications that support and are in close proximity to a manufacturing process b) Gradient : Mobile phase solvent (“B”) composition increases with time Linear gradients are most popular May 29, 2017 INS 591: Presentation 13

14. May 29, 2017 INS 591: Presentation 14 Fig: mobile phase gradient representation

15. The HPLC system consists of following components: Solvent reservoir Pump Sample injector HPLC column Detector Chromatogram Instrumentation May 29, 2017 15

16. Figure: - Instrumentation of HPLC system May 29, 2017 16

17. May 29, 2017 INS 591: Presentation 17 Figure : Overall view of the HPLC

18. Solvent reservoir : - It holds the solvent called mobile phase which moves through the various parts of the system   Pump : - The role of the pump is to force a liquid (called the mobile phase) through the liquid chromatography at a specific flow rate, expressed in milliliters per min (ml/min) Normal flow rates in HPLC are in the 1to 2mL/min range Typical pumps can reach pressures in the range of 6000-9000 psi (400to 600bar) May 29, 2017 18

19. During the chromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition (gradient) Sample injector: - The injector serves to introduce the liquid sample into the flow stream of the mobile phase Typical sample volumes are 5-to 20-microliters (μL) The injector must also be able to withstand the high pressures of the liquid system May 29, 2017 19

20. HPLC column: - Considered the “heart of the chromatograph” the column’s stationary phase separates the sample components of interest using various physical and chemical parameters The small particles inside the column cause the high back pressure at normal flow rates The pump must push hard to move the mobile phase through the column and this resistance causes a high pressure within the chromatography May 29, 2017 20

21. Detector: - The detector can see (detect) the individual molecules that come out (elute) from the column A detector serves to measure the amount of those molecules so that we can quantitatively analyze the sample components The detector provides an output to a recorder or computer that result in the liquid chromatogram (i.e., the graph of the detector response) May 29, 2017 21

22. Computer and Chromatogram: - Also called the data system, the computer not only controls all the modules of the HPLC instrument but it takes the signal from the detector and uses it to determine the time of elution (retention time) of the sample components (qualitative analysis) and the amount of sample (quantitative analysis) Acquisition software is installed in computer. This software collects data from Detector and will draw a graph (Called Chromatogram)   May 29, 2017 22

23. Operations Overall process of HPLC can be described by the following flow diagram that involves following process:- Injection of sample Measurement of retention time Detection Interpreting the output from the detector May 29, 2017 23

24. May 29, 2017 INS 591: Presentation 24 Figure: Various components of the HPLC

25. Injection of sample: - It may be manual sample injector or the auto sampler for the injection of the sample in the HPLC Manual injector manually load the sample into the injector using the syringe and then turns the handle to inject sample into the flowing mobile phase May 29, 2017 25 Manual sample injector

26. In the auto sampler user loads vials filled with sample solution into the auto sampler tray (100 samples) and the auto sampler automatically measures the appropriate sample volume and injects the sample May 29, 2017 INS 591: Presentation 26 Auto sampler

27. The loop is connected with the high pressure mobile phase which carries the sample onto the column May 29, 2017 INS 591: Presentation 27

28. 2) Measurement of retention time:- The time taken for a particular compound to travel through the column to the detector is known as its retention time This time is measured from the time at which the sample is injected to the point at which the display shows a maximum peak height for that compound Different compounds have different retention times. For a particular compound, the retention time will vary depending on: The pressure used (because that affects the flow rate of the solvent) May 29, 2017 28

29. The nature of the stationary phase (not only what material it is made of, but also particle size) The exact composition of the solvent The temperature of the column That means that conditions have to be carefully controlled if we are using retention times as a way of identifying compounds. May 29, 2017 29

30. 3) Detection: - There are many detection principles used to detect the compounds eluting from an HPLC column The most common are: Spectroscopic detection ( UV absorption and Mass spectroscopy) Refractive index detection Fluorescence detection   May 29, 2017 30

31. 4) Interpreting the output from the detector: - The output will be recorded as a series of peaks - each one representing a compound in the mixture passing through the detector The output is shown as a graph of various peak in a chromatogram It looks like: May 29, 2017 31

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34. In above chromatogram, there are three peaks detected. As per Peak table; Peak RT (Retention Time), Area, Area Percentage, Height and Height Percentage detail is given. From chromatogram and peak table HPLC we can determine the substances detail, substance is present or not and if present, then how much is the concentration of that substance May 29, 2017 34

35. May 29, 2017 INS 591: Presentation 35 Figure: HPLC chromatogram of mixture of compound

36. Problems with the Chromatogram Peak Tailing May 29, 2017 INS 591: Presentation 36 Possible Causes Solution Blocked frit Reverse flush column (if it is allowed) or replace frit (if it is allowed) or replace column Column void Fill void Interfering peak Use longer column or change mobile phase and/or column selectivity Wrong mobile phase pH Adjust pH Sample reacting with active sites Add ion pair reagent or volatile basic modifier or change column

37. 2. Peak Fronting: May 29, 2017 INS 591: Presentation 37 Possible causes Solution Low temperature Increase column temperature Wrong sample solvent Use mobile phase for injection solvent Sample overload Decrease sample concentration Bad column Reverse flush column (if it is allowed) or replace inlet frit (if it is aalowed ) or replace the column

38. 3. Split Peaks May 29, 2017 INS 591: Presentation 38 Possible causes Solution Contamination on guard or analytical column inlet Remove guard column and attempt analysis. replace guard column if necessary. If analytical column is obstructed, reverse and flush. If problem persists, column may be fouled with strongly retained contaminants. Use appropriate restoration procedure. If problem persists, inlet is probably plugged. Change frit or replace column Sample solvent incompatible with mobile phase Change solvent. whenever possible, inject samples in mobile phase.

39. Advantages and Disadvantages Advantages:  Both qualitative as well as quantative analysis can be performed Separate and identify a multitude of compounds in low concentration in a complex mixture with very little assay optimization Gives result in a few minutes Gives high resolution, speed and accuracy Results are reproducible May 29, 2017 39

40. Gives result in a few minutes Gives high resolution, speed and accuracy Results are reproducible May 29, 2017 40

41.  Disadvantages: -   Expensive and not portable Costlier technique as compared to conventional chromatographic technique Operation is complex so trained person is required to operate HPLC   May 29, 2017 41

42. Applications May 29, 2017 INS 591: Presentation 42 Fig: Various fields for the applications of HPLC

43. Pharmaceutical Applications:- May 29, 2017 43 S. N. Applications 1 Pharmaceutical quality control, drug stability, 2 Shelf life determination of the pharmaceutical products 3 Identification of the counterfeit drug products 4 Separation of complex molecules 5 Determination of concentration of drugs in the blood after certain intervals of administration 6 Detection of alkyl sulfuric acid (used as catalyst, solvent and blocking agents in the synthesis of organic compounds and various pharmaceutical drugs) 7 Detection and separations of sugars, amino acids, polymers and others organic acids 8 Separation of antibiotics, analgesic, sedatives, steroids etc

44. 2) Environmental Applications: - Detection of phenolic compounds in drinking water Bio-monitoring of pollutants 3) Food and Flavour:- Measurement of Quality of soft drinks and water Sugar analysis in fruit juices Analysis of polycyclic compounds in vegetables Preservative analysis May 29, 2017 44

45. 4) Applications in Forensics: - Quantification of drugs in biological samples Identification of steroids in blood, urine etc Forensic analysis of textile dyes Determination of cocaine and other drugs of abuse in blood, urine  May 29, 2017 45

46. 5) Applications in Clinical Tests: - May 29, 2017 46 S.N. Applications 1 Analysis of Urine , antibiotics in blood, bilirubin , biliverdin in hepatic disorders 2 Detection of endogenous neuropeptides in extracellular fluid of brain 3 Diagnosis of many disorders related to body metabolism, those related to endocrine and exocrine gland secretion, alteration in body fluids 4 Separation of bile acids, drugs metabolites, estrogen, urine extracts etc

47. 6) Application in the Scientific Research : It is a mandatory tool in most of the labs involved in research which include medical, biological, chemical, biochemical, phytochemical (plant chemical research) Components with similar chemistry and properties can be easily distinguished May 29, 2017 INS 591: Presentation 47

48. Difference between TLC and HPLC May 29, 2017 INS 591: Presentation 48 TLC HPLC Type of Analysis Qualitative Both qualitative and quantitative Instrumentation minimal Much with many adjustable parameter Stationary Phase 2- dimensional column 3-dimensional column Sample Application spotting injection Mobile Phase Movement Capillary action High pressure Visualization of Result UV lightbox On-line detection Form of Results Spots, retention factor Peaks, retention times

49. Additional Things Theoretical Plate  In many separation process there is a hypothetical zone or stage in which two phases, such as the liquid and vapor phases of a substance, establish an equilibrium with each other The theory assume that in the column chromatography column is divided into the a number of zone called theoretical plates Such equilibrium stages may also be referred to as an equilibrium stage, ideal stage, or a theoretical tray May 29, 2017 INS 591: Presentation 49

50. The number of the theoretical plates in the column is used as measure of the efficiency of the column to separate the compounds The number of the theoretical plates can be calculated by using the formula: Where, N= no of theoretical plate, tr =retention time W= base width of the peak May 29, 2017 INS 591: Presentation 50

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52. References 1) Skoog, Holler and Crouch (2007) Principle of Instrumental Analysis (6 th edition), Thomos Brooks/Cole (762-782)   2) Giri D (2012) High Performance Liquid Chromatography (HPLC): Principle, Types, Instrumentation and Applications. Laboratoryinfo.com   3) Dr NR and Pacakova V (2009) Chromatography in biochemistry . Institute of Medical Biochemistry,Charles University in Parague.   4) Gupta A (2017) Pharma Loksewa , Goodwill Publication Pvt.Ltd, Bagbazar, 64-70.   5) Theory of HPLC ( www.chromacademy.com ) (Accessed on Feb 10, 2017)     6) http://chemguide.co.uk/analysis/chromatography/hplc.html (Accessed on Feb 12,2017)   May 29, 2017 52

53. Thank You May 29, 2017 53