1. HIGH PERFORMANCE(PRESSURE) LIQUID CHROMATOGRAPHY

2. HPLC Presented By - Mr. Shaise Jacob Faculty Nirmala College of Pharmacy Muvattupuzha, Kerala India Email – jacobshaise@gmail.com

3. 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.

4. 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  Note:The modern form of liquid chromatography is now referred to as “flash chromatography”

5. Four types of high performance liquid chromatography (HPLC): partition adsorption (liquid-solid) ion exchange size exclusion or gel

6. ◊ improved performance ◊ high pressure HPLC- Separation is accomplished by partitioning b/w a M.P & Stationary column material. Packing material small, uniform particle gives high column efficiencies High pressure to achieved desired flow rates

7. Types of HPLC techniques Based on Modes of chromatography 1. Normal phase mode: S.P is polar M.P is non polar 2. Reverse phase mode: S.P is non polar M.P is polar Different columns used: ODS,C18,C8,C4…

8. Based on principle of separation 1. Adsorption chromatography 2. Ion exchange “ 3. Ion pair “ 4. Gel permeation / Size exclusion “ 5. Affinity “ 6. Chiral phase “ Based on elution technique Isocratic separation Gradient separation

9. Based on scale of operation Analytical HPLC Preparative HPLC Based on the type of Analysis Qualitative analysis Quantitative analysis HPLC offers numerous advantages ♠ Capable of handling “macromolecules” ♠ Suitable for pharmaceutical compounds ♠ Efficient analysis of liable natural products ♠ Reliable handling of inorganic & ionic species

10. ♠ Dependable analysis of biochemical's PRINCIPLE Adsorption Particle size of the S.P material plays a crucial role in HPLC Micro particulate column packing : Silica particles → uniform, porous, with spherical or irregular shape Diameter → 3.5 to 10 µm

11. HPLC instrumentation comprises: 1.M.P reservoirs 2.Eluent degas module 3.Solvent delivery pumps 4.Manual / Auto injector 5.Analytical column 6.Detector 7.Data processor

16. Mobile phase reservoir stores M.P (HPLC grade solvents) ♠ Resolution & Speed of analysis } Flow rate, polarity & pH of M.P Can't use metallic reservoir Eluent degas module Dissolved gases in M.P pose a number of problems ∆ flow ∆ excessive detector noise ∆ Rt fluctuation ♠ Bubbling the pump & detector,

17. Degas module with reservoir of inert gases He or N 2 1.Vacuum filtration 2. Helium purging 3.Ultrasonication (converts ultra high frequency to mechanical vibrations.) SOLVENT DELIVERY PUMPS Reciprocating pumps: » widely used » maintain accurate flow rate

20. Cross-sectional diagram of a simple single – piston reciprocating pump

21. Solvent delivery systems two types: 1. Isocratic system 2. Low pressure gradient 3. High pressure gradient Injection system a. Syringe system:- results best b. Injection valve :- [Rheodyne injector] » Loading through the sample loop (20-50µl)

22. u c. Automated injection device :- commercially available, automatically inject 100samples

23. Guard column Pre-filter :- useful for industry

24. Analytical column Heart of any chromatographic system » Actual separation of components takes place Several S.P available  depending upon tech. or mode of separation Column material S.S, glass, polyethylene, PEEK Column length Column diameter Particle size 5-30 cm 2mm-50mm 1µ-20µ

25. Particle nature: Spherical, uniform sized porous material Surface area 1g S.P provides surface area 100-860 sq.m Functional group Depends on the type of chromatographic separations. Normal phase mode: hydroxy group Reverse phase mode: C 18 (octa decyl silane) Bondapak ( waters) C 8 octyl column, C 4 butyl column, CN Nitrile column NH 2 Amino column

27. b

28. Column packing three forms 1.Microporous support 5-10 µm in d.m 2. Pellicular Porous & 40 µm in d.m 3.Bonded phase S.P bonded onto an inert support

29. DETECTORS 1. UV DETECTOR : Based on UV light ab. > fixed WL detector (254nm) > variable WL detector (190-600nm) 2. REFRACTIVE INDEX DETECTOR : Non specific / Universal detector ↓ sensitivity & specificity 3. PHOTODIODE ARRAY DETECTOR (PDA) similar to UV detector, non destructive 190-600 nm for quantization & identification Spectra is 3D, Response vs time vs WL

30. Photodiode Array Detector

31. Flourimetric detector Excitation & emission WL can be selected ↑ sensitive than UV Disadvantage: Some comp. are not fluorescent Conductivity detector based on electrical conductivity Amperometric detector Reduction / oxidation

32. RECORDERS & INTEGRATORS Recorders – to record the responses Integrators - data processing capabilities ◊ record individual peaks with Rt, height, width of peaks, peak area, % of area.. Selection of solvent systems Solvent compatibility with the detector e.g.. Hexane, chloroform, ACN, Methanol… Most widely used solvent in HPLC is water Millipore Milli-Q apparatus produce water

33. Selection of Column Non polar & moderately polar comp. → ADSORPTION CHRO. Highly polar molecules by → R.P Chro. Acids & Bases by → Ion exchange Chro.

34. APPICATIONS OF HPLC ♥ Pharmaceutical field ♥ Chemical & Petrochemical industry ♥ Forensic ♥ Biochemical separations ♥ Food analysis Qualitative analysis Checking the purity of a compound

35. Quantitative Analysis Direct comparison method injecting the sample & std. separately & comparing their peak areas. Area of the peak = peak height x width of peak at half height Calibration curve method Multi component analysis Isolation & identification of drugs Stability studies