Hplc ppt

principle theory and instrumenttion of hplc

1. High Performance Liquid Chromatography Submitted by- SHWETA TYAGI M . PHARM 1 St YEAR PHARMACOLOGY COLLEGE OF PHARMACY MEERUT INSTITUTE OF ENGINEERING AND TECHNOLOGY

2. Overview: Chromatography and its principle Liquid chromatography High Performance Liquid Chromatography ( HPLC ) The components of the high performance liquid chromatograph (HPLC).

3. Latest instrument

4. The process involves the interaction of the compounds in the analyte (which travels along with a mobile phase) across an immobile surface (stationary phase). The compounds bind at specific regions of stationary phase based on certain physical and chemical properties. These bound molecules are then eluted with a suitable buffer and the same are collected with time. These are – Polarity Charge Molecular weight Present of functional group Principle

5. Introduction HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution. HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector. Compounds are separated by injecting a sample mixture onto the column. The different component in the mixture pass through the column at differentiates due to differences in their partition behavior between the mobile phase and the stationary phase. The mobile phase must be degassed to eliminate the formation of air bubbles.

6. Liquid chromatography is a separation technique that involves : • the placement (injection) of a small volume of liquid sample • into a tube packed with porous particles (stationary phase) • where individual components of the sample are transported along the packed tube (column) by a liquid moved by gravity . The components of the sample are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles. The separated components are collected at the exit of this column and identified by an external measurement technique , such as a spectrophotometer that measures the intensity of the color , or by another device that can measure their amount.

7. Principles of HPLC Principle: The table shows relation between various parameters of HPLC. Trendline : Stationary phase have small particulate size and high surface areas. Columns: 20 cm or less Mobile phase pumped at high pressures of 200Bar, 3000 psi. Flow rates: 1-3 cm 3 per min Column length No. of theoretical plates per unit area Resolving power Column length Particle size Surface area

8. What is HPLC? 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) •where 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. These 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. These separated components are detected at the exit of this tube ( column) by a flow-through device (detector) that measures their amount. An output from this detector is called a “liquid chromatogram”.  In principle, LC and HPLC work the same way except the speed , efficiency, sensitivity and ease of operation of HPLC is vastly superior. s

9. HPLC system Flow chart of HPLC mechanism

10. Varian 9010 Solvent Delivery System Rheodyne Injector %A %B %C Flow Rate Pressure {H 2 O} {MeOH} (mL/min) (atmos.) Ready Ternary Pump A C B from solvent reservoir Column to detector to column through pulse dampener to injector through pump load inject

11. Picture of HPLC instrument

12. COMPOSITION OF A LIQUID CHROMATOGRAPH SYSTEM Solvent Solvent Delivery System (Pump) Injector Sample Column Detectors Waste Collector Recorder (Data Collection)

13. Instrumentation of HPLC ( Describing the 5 major components and their functions….) 1 2 3 4 5 Solvent reservoirs and degassing Not shown here 1 – Pump 2 – Injector 3 – Column 4 – Detector 5 – Computer

14. Pump : •The role of the pump is to force a liquid (called the mobile phase) through the liquid chromatograph at a specific flow rate, expressed in milliliters per min ( mL /min). •Normal flow rates in HPLC are in the 1-to 2-mL/min range. •Typical pumps can reach pressures in the range of 6000-9000 psi (400-to 600-bar). •During the chromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition (gradient).

15. Pump Module–types : Isocratic pump - Delivers constant mobile phase composition; •solvent must be pre-mixed; •lowest cost pump Gradient pump - Delivers variable mobile phase composition; •can be used to mix and deliver an isocratic mobile phase or a gradient mobile phase –Binary gradient pump –delivers two solvents –Quaternary gradient pump –four solvents

16. Injecto r: •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. •An auto sampler is the automatic version for when the user has many samples to analyze or when manual injection is not practical .

17. Sample Injection ……how is a sample actually put into an LC system Manual Injector : 1.User manually loads sample into the injector using a syringe 2.and then turns the handle to inject sample into the flowing mobile phase… which transports the sample into the beginning (head) of the column, which is at high pressure Auto sampler : 1.User loads vials filled with sample solution into the auto sampler tray (100 samples) 2.and the auto sampler automatically a. measures the appropriate sample volume, b. injects the sample, c. then flushes the injector to be ready for the next sample, etc., until all sample vials are processed …

18. Manual Injectors 19 Front View Inject Rear View Load - Inject Sample Loop

19. Automatic Injectors 20 Step 1 Step 2 Step 3

20. 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 are what 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 chromatograph.

21. HPLC Columns Within the Column is where separation occurs. Key Point –Proper choice of column is critical for success in HPLC Materials of construction for the tubing Stainless steel (the most popular; gives high pressure capabilities) Glass (mostly for biomolecules) PEEK polymer (biocompatible and chemically inert to most solvents Packing material: The packing material is prepared from SILICA particle, ALUMINA particle and ion exchange RESIN. Porous plug of stainless steel or Teflon are used in the end of the columns to retain the packing material. According to the mode of HPLC , they are available in different size , diameters, pore size or they can have special materials attached ( such as antigen or antibody ) for immuno affinity chromatography.

22. Types of columns in HPLC : Guard Coalumn Fast Column Preparative( i.d . > 4.6 mm; lengths 50 –250 mm) Capillary( i.d . 0.1 -1.0 mm; various lengths) Nano ( i.d . < 0.1 mm, or sometimes stated as < 100 μm ) Analytical[internal diameter ( i.d .) 1.0 -4.6-mm; lengths 15 –250 mm]

23. Guard Column These are placed anterior to the separating column. This serves as protective factor. They are dependable columns designed to filter or remove : Particles that clog the separation column Compounds and ions that could ultimately cause “ Baseline drift ”, decreased resolution, decreased sensitivity and create false peaks. These columns must be changed on a regular basis in order to optimize their protective function .

24. Guard column 25

25. Capillary Column It is also known as micro columns It has a diameter much less than a millimeter and there 3 types: Open tubular Partially packed Tightly packed They allow the user to work with nanoliter sample volume , decreased flow rate and decreased solvent usage volume , led to cost effectiveness

26. Capillary column 27

27. Preparatory Column Used when objective is to prepare bulk ( milligrams) of sample for laboratory preparatory application. It has usually a large column diameter , which is designed to facilitate large volume injections into the HPLC system

28. Prepratory column 29

29. 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 the chemist can quantitatively analyze the sample components. •The detector provides an output to a recorder or computer that results in the liquid chromatogram(i.e., the graph of the detector response).

30. UV-Vis Detectors 31 b c Detector Flow Cell I I Log I = A = abc I Principles : The fraction of light transmitted through the detector cell is related to the solute concentration according to Beer’s Law. Characteristics : Specific, Concentration Sensitive, good stability, gradient capability. Special : UV-Vis Spectral capability (Diode Array Technology ).

31. Electrochemical Detectors Gold for carbohydrates. Platinum for chlorite, sulfate, hydrazine, etc. Carbon for phenols, amines. Silver for chloride, bromide, cyanide. 32

32. Variable UV/Vis Detector ABS AUFS l RunTime EndTime 0.001 2.000 238 0.00 min 10.0 min Ready

33. Computer : • Frequently 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: 1. determine the time of elution (retention time) of the sample components (qualitative analysis) and 2. the amount of sample ( quantitative analysis) .

34. chromatogram 35

35. Varian 9060 Polychrom Detector UV Spectrum Chromatogram Reset Ready UV Spectrum {shows full UV abs.} Chromatogram {shows peaks, R t } ABS. Time ABS. Wavelength UV max UV max R t R t

36. HPLC is optimum for the separation of chemical and biological compounds that are non-volatile . Typical non-volatile compounds are:  Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol)  Salts like sodium chloride and potassium phosphate  Proteins like egg white or blood protein  Organic chemicals like polymers (e.g. polystyrene, polyethylene)  Heavy hydrocarbons like asphalt or motor oil  Many natural products such as ginseng, herbal medicines, plant extracts  Thermally unstable compounds such as trinitrotoluene (TNT), enzymes HPLC used for

37. HPLC uses This technique is used for - chemistry and biochemistry research analyzing complex mixtures purifying chemical compounds developing processes for synthesizing chemical compounds isolating natural products, or predicting physical properties. It is also used in quality control to ensure the purity of raw materials, to control and improve process yields, to quantify assays of final products, or to evaluate product stability and monitor degradation.

38. HPLC Applications 39 Chemical Environmental Pharmaceuticals Consumer Products Clinical polystyrenes dyes phthalates tetracyclines corticosteroids antidepressants barbiturates amino acids vitamins homocysteine Bioscience proteins peptides nucleotides lipids antioxidants sugars polyaromatic hydrocarbons Inorganic ions herbicides

39. References HPLC instrumentation – Agilent Technologies Introduction to HPLC – Agilent Technologies WEB REFERENCES www.slideshare.net www.wikipedia.com

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