High Performance Liquid Chromatography (Mr.S)

Introduction To HPLC

1. H IGH P ERFORMANCE L IQUID C HROMATOGRAPHY BY @@@ Mr.S @@@ Pharmaceutical Analysis & Quality Assurance, Vikas College Of Pharmacy, Vissannapeta .

2. CONTENTS INTRODUCTION TYPES PRINCIPLE INSTRUMENTATION ADVANTAGES APPLICATIONS 26-Jun-16 VIKAS COLLEGE OF PHARMACY 2

3. INTRODUCTION High performance liquid chromatography (HPLC) formerly referred to as high pressure liquid chromatography. It is a technique in analytical chemistry used to separate the components in a mixture , to identify each component, and to quantify each component. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. 26-Jun-16 VIKAS COLLEGE OF PHARMACY 3

4. 26-Jun-16 VIKAS COLLEGE OF PHARMACY 4 Each component in the sample interacts slightly differently with the adsorbent material , causing different flow rates for the different components and leading to the separation of the components as they flow out the column . The development of HPLC from classical column chromatography can be attributed to the development of smaller particle is important . They offer more surface area over the conventional larger particle sizes .

5. 26-Jun-16 VIKAS COLLEGE OF PHARMACY 5 TYPES OF HPLC TECHNIQUES Based on modes of chromatography There are two modes of chromatography they are normal phase mode and reverse phase mode. These modes are based on polarity of SP and MP. The interactions which occur b/w solute, SP & MP. Polar –polar-affinity is more. Non-polar –Non-polar -affinity is more. Polar-Non-polar-affinity is less.

6. 26-Jun-16 VIKAS COLLEGE OF PHARMACY 6 Normal phase mode: In this stationary phase is polar and mobile phase is non polar Reverse phase mode: In this stationary phase is non polar and mobile phase is polar.

7. BASED ON ELUTION TECHNIQUE Isocratic elution technique : In this technique same mobile phase combination is used throughout the process of separation. The same polarity or elution strength is maintained throughout the process. Gradient elusion technique : In this technique, a mobile phase combinations of lower polarity or elution strength is used followed by gradually increasing the polarity or strength. BASED ON SCALE OF OPERATION Analytical HPLC : where only analysis of the samples are done. Preparative HPLC : where the individual fractions of pure compounds samples are reused. 26-Jun-16 VIKAS COLLEGE OF PHARMACY 7

8. BASED ON THE TYPE OF ANALYSIS Qualitative analysis: which is used to identify the compound, detect the presence of impurities, to find out the number of components. Quantitative analysis : which is done to determine the quantity of the individual or several components in a mixture. This is done by comparing the peak area of the standard and sample. 26-Jun-16 VIKAS COLLEGE OF PHARMACY 8

9. 26-Jun-16 VIKAS COLLEGE OF PHARMACY 9 PRINCIPLE The principle of separation in normal phase mode and reversed phase mode is adsorption. when a mixture of components are introduced in to a HPLC column , they travel according to the relative affinities towards the stationary phase. The component which has more affinity towards stationary phase travels slower and less affinity towards the stationary phase travels faster. Since no two components have the same affinity towards the stationary phase , the components are separated.

10. 26-Jun-16 VIKAS COLLEGE OF PHARMACY 10 Separation Process and Chromatogram Output concentration Time Chromatogram

11. 26-Jun-16 VIKAS COLLEGE OF PHARMACY 11 INSTRUMENTATION Solvent delivery system Pumps Sample injecting system Column Detector Integrators & read out device .

12. High-Performance Liquid Chromatography (HPLC) 26-Jun-16 VIKAS COLLEGE OF PHARMACY 12

13. SOLVENT DELIVERY SYSTEM : The solvents or mobile phases used must be passed through the column at high pressure at about 1000 to 3000 psi. This is because as the particle size of stationary phase is few µ (5-10µ ), The resistance to the flow of solvent is high. Hence such high pressure is recommended. MIXING UNIT: Mixing unit is used to mix the solvents in different proportions and pass through the column. There are 2 types of mixing units. Low pressure mixing unit High pressure mixing unit. 26-Jun-16 VIKAS COLLEGE OF PHARMACY 13

14. SOLVENT DEGASSING Several gasses are soluble in organic solvents.When solvents are pumped under high pressure ,gass bubbles are formed ,which will interfere with the separation process. Hence degassing of solvents is important. This is done by: Vaccum filteration Helium purging Ultrasonication 26-Jun-16 VIKAS COLLEGE OF PHARMACY 14

15. Normal phase chromatography Hexane ; dichloromethane; isopropanol; methanol Increasing strength Reverse phase chromatography water ; methanol; acetonitrile; tetrahydrofuran (THF) The Mobile Phase Increasing strength 26-Jun-16 VIKAS COLLEGE OF PHARMACY 15

16. 26-Jun-16 VIKAS COLLEGE OF PHARMACY 16 HPLC Pumps: Types Reciprocating pumps Syringe pumps Constant pressure pumps RECIPROCATING PUMPS Small head volumes (50 to 250 ml) Short piston stroke Inert pistons (generally sapphire) Continuous use (no refill time) Pulse dampeners

17. Syringe Pumps Constant flow rate pump Non-pulsating flow Low flow rates (1 to 100 mL/min) Isocratic flow only Refill required when reservoir (~50mL) expended 26-Jun-16 VIKAS COLLEGE OF PHARMACY 17

18. SAMPLE INJECTION SYSTEM Several devices are available either for manual or auto injection of the sample. Different devices are: Septum injectors Stop flow injectors Rheodyne injectors (loop valve type) Rheodyne injector is the most popular injector This has a fixed volume loop like 20 μl or 50 μl or more . Injector has 2 modes. Load position and Inject mode. 26-Jun-16 VIKAS COLLEGE OF PHARMACY 18

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20. COLUMNS: Stainless steel tubing for high pressure Heavy-wall glass or PEEK tubing for low P (< 600 psi) TYPES OF COLUMNS: Analytical column : straight, L(5~ 25 cm), d (3 ~ 5 mm) Micro column : L (3 ~ 7.5 cm), d (1 ~ 5 mm). Guard column : remove particulate matter and contamination protect analytical column, similar packing Column packing: silica, alumina , a polystyrene, divinyl benzene synthetic or an ion-exchange resin 26-Jun-16 VIKAS COLLEGE OF PHARMACY 20

21. Picture of an HPLC column 26-Jun-16 VIKAS COLLEGE OF PHARMACY 21

22. DETECTORS: Detectors used depends upon the property of the compounds to be separated. Different detectors available are: 1. Refractive index detectors 2. U.V detectors 3. Fluorescence detectors 4. Electro chemical detectors 5. Evaporative light scattering detectors 6. IR detectors 7. Photo diode array detector : 26-Jun-16 VIKAS COLLEGE OF PHARMACY 22

23. Refractive Index Detector It is a deflection type detector: Two triangular 5- to 10- L compartments where pure solvent or eluate passes; when solute of different refractive index enters the cell, the beam is deflected and the photocell output changes. IR radiation must be removed (avoid heating of sample soln.!) 26-Jun-16 VIKAS COLLEGE OF PHARMACY 23

24. UV Detectors A special type of UV detectors: the p hoto d iode a rray detectors (PDA) which records the spectrum of each solute as it is eluted. The linearity range for this type of detectors is in a 5 orders of magnitude. 26-Jun-16 VIKAS COLLEGE OF PHARMACY 24

25. FLUORESCENCE DETECTOR This detector is based on the flourescent radiation emitted by some class of compounds. This detector has more specificity and selectivity. 26-Jun-16 VIKAS COLLEGE OF PHARMACY 25

26. RECORDERS AND INTEGRATORS: Recorders are used to record the responses obtained from detectors after amplification. They record the base line and all the peaks obtained, with respect to time. Integrators are improved version of recorders with some data processing capabilities. They can record the individual peaks with retention time, height and width of peaks, peak area, percentage of area, etc. Now a days computers and printers are used for recording and processing the obtained data and for controlling several operations. 26-Jun-16 VIKAS COLLEGE OF PHARMACY 26

27. 26-Jun-16 VIKAS COLLEGE OF PHARMACY 27 5. PARAMETERS USED IN HPLC : 1.Retention time 2.Retention volume 3.Seperation factor 4. Resolution 5. Height Equivalent to a Theoretical Plate (HETP) 6. Efficiency 7. Asymmetry factor

28. 26-Jun-16 VIKAS COLLEGE OF PHARMACY 28 ADVANTAGES High resolving power. Continuous monitoring of the column effluent. Accurate quantitative measurement Repetitive and Reproducible analysis using the same column Determination of multiple components in a single analysis Both aqueous and non-aqueous samples can be analysed with little or no sample pre treatment

29. 26-Jun-16 VIKAS COLLEGE OF PHARMACY 29 Using fresh leaves , replicas of leaf surface may be made which are satisfactory for determination of stomatal number and stomatal index. An appropriate 50% gelatin and water gel is liquefied on a water-bath and smeared on a hot slide. The fresh leaf is added , the slide is inverted and cooled under a tap and after about 15-30 min the specimen is stripped off. The imprint on the gelatin gives a clear outline of epidermal cells ,stomata and trichomes .

30. DIFFERENCE B/W HPLC AND HPTLC HPLC: Avg particle size-3-5µm No of samples-1sample Running time-10-30min HPTLC 5-7µm 36-72 on a 20cm plate 3-10 min 26-Jun-16 VIKAS COLLEGE OF PHARMACY 30

31. 26-Jun-16 VIKAS COLLEGE OF PHARMACY 31 APPLICATIONS OF HPLC HPLC is being more widely used in severalfields . Apart from it’s use in pharmaceutical field , it is used in chemical and petrochemical industry , environmental applications ,forensic applications , biochemical separations , biotechnology , food industry etc.

32. Qualitative Analysis Purification of Compounds Identification of Compounds Peaks correspond to individual components Compound Impurity Separation of Mixture Components Authentic Unknown 26-Jun-16 VIKAS COLLEGE OF PHARMACY 32

33. 26-Jun-16 VIKAS COLLEGE OF PHARMACY 33 Quantitative Analysis Concentration Peak hight ratio Calibration curve Internal Standard Method 10 5  g/mL 10 10  g/mL 10 25  g/mL 10 50  g/mL 10 75  g/mL 10 100  g/mL Internal Standard Compound 10 Unknown

34. 26-Jun-16 VIKAS COLLEGE OF PHARMACY 34 Direct comparision method. Calibration curve method. Multi component analysis. Isolation and identification of drugs. Stability studies. Purification of some components. Identification of specific impurities.

35. REFERENCE Instrumental methods of chemical analysis By B.K. sharma (Pg:286-385) Instrumental methods of Chemical Analysis By Gurdeep Chathwal (Pg: 2.624-2.639) WIKIPEDIA. 26-Jun-16 VIKAS COLLEGE OF PHARMACY 35

36. THANK YOU 26-Jun-16 VIKAS COLLEGE OF PHARMACY 36