Hplc.pptx

PHARMACEUTICAL ANALYSIS

1. Hplc high performance liquid chromatography Tenzin palmo Md Fayazuddin ( M.pharm ) Al-ameen college of pharmacy

2. contents INTRODUCTION PRINCIPLE TYPES OF HPLC TECHNIQUES INSTRUMENTATION PARAMETERS USED IN HPLC ADVANTAGES OF HPLC DISAVANTAGES OF HPLC APLLICATIONS

3. Introduction HPLC was developed in early 1970’s from a knowledge of already established chromatographic techniques. Originally , HPLC was referred to as High Pressure Liquid Chromatography because , high pressure were used to increase the efficiency of separation . But now-a-days , the term High Performance Liquid Chromatography is preferred as it describes the characteristic of the chromatography . Earlier , the separation of component by liquid chromatography was dependent on gravity and it took lot of time to separate components . This problem was overcome by HPLC in which the separation is assisted by means of increasing the force of mobile phase at high rate .

4. PRINCIPLE The principle of high performance liquid chromatography is based on adsorption as well as partition chromatography depending on the nature of stationary phase . If the stationary phase is solid , principle is based on adsorption chromatography . If the stationary phase is liquid , principle is based on partition chromatography .

5. TYPES OF HPLC TECHNIQUES : A. Based on modes of chromatography 1.Normal phase mode 2. Reverse phase mode B. Based on principle of separation 1. Adsorption chromatography 2. Ion exchange chromatography 3. Ion pair chromatography 4. Size exclusion (or) Gel permeation chromatography 5. Affinity chromatography 6.Chiral phase chromatography

6. C. Based on elution technique 1. Isocratic separation 2. Gradient separation D. Based on the scale of operation 1. Analytical HPLC 2. Preparative HPLC E. Based on the type on analysis 1. Qualitative analysis 2. Quantitative analysis

7. TYPES OF HPLC TECHNIQUES A. Based on modes of Chromatography : This depends on the polarity of stationary and mobile phase. Normal phase mode : In this mode, the stationary phase is polar in nature and the mobile phase is non-polar . In this technique, the non-polar compounds travel faster and are eluted first, which is due to its less affinity between solute and stationary phase . The polar compounds are retained for a longer time in the column because of more affinity towards stationary phase and takes more time to be eluted from the column. This technique is not advantageous in pharmaceutical applications, since most of the drug substance are polar in nature and takes longer time t be eluted and detected, and hence not widely used. Reverse phase mode : In this mode, the stationary phase is non-polar and mobile phase is polar in nature. In this technique, the polar compounds get eluted first and the non-polar compounds re retained for a longer time, and hence this technique is widely used. B. Based on Elution technique : Isocratic separation: In this technique the same mobile phase (single solvent or combination of solvents) is used throughout the process of separation i.e. the same polarity or elution strength is maintained throughout the process. Gradient separation : In this technique, a mobile phase combination of lower polarity or elution strength is used followed by gradually increasing the polarity or elution strength. The separation efficiency is greatly enhanced by gradient elution, here the proportion of two or more solvents is greatly varied in a programmed way, sometimes continuously or in a series of steps.

8. C. Based on scale of Oparation : Analytical HPLC: In this technique only analysis of sample is done, recovery of samples for reusing is normally not done, since the sample used is very low. Preparative HPLC: In this technique, the individual fractions of pure compounds can be collected using fraction collector. The collected samples can be reused or analysed . D. Based on Type of Analysis: Qualitative analysis: It is used to identify the compounds, detect the presence of impurities i.e. by using the retention time. Quantitative analysis : It is done to determine the quantity of individual or several components in a mixture i.e. by comparing the peak area of standard and sample. E. Based on principle of separation: Adsorption Chromatography Ion Exchange Chromatography Size Exclusion or Gel Permeation Chromatography Affinity Chromatography Ion pair Chromatography Chiral Phase Chromatography

9. INSTRUMENTation of hplc In HPLC, the mobile phase from the solvent reservoir is filtered, pressurized and pumped through the chromatographic column. A mixture of solute injected at the top of the column is separated into components on travelling down the column and the individual solutes are monitored by the detector and recorded as peaks on the chart recorder.

10. The basic components of HPLC Chromatograph are: Solvent delivery system or Solvent reservoirs-----Mobile phase Pumping system or pumps Sample injection systems Column-Guard and Analytical columns----Stationary phase Detectors Recorders Solvent delivery system or Solvent Reservoir: Solvent delivery system consist of solvent reservoirs, inlet filters, degassing facilities and mixing units which work in conjugation with vacuum pumps and flow controls. Solvent reservoirs : HPLC system is equipped with one or more glass or stainless steel reservoirs, each of which can hold 500ml or more of the solvent. The reservoirs are often equipped with a means of removing dissolved gases usually oxygen and nitrogen, which interferes by forming bubbles in the columns and detector systems by causing band spreading.

11. Degassers ---- which are used to remove the dissolved gases in the mobile solvents several gases are soluble in organic solvents, and when solvents are pumped under high pressure, gas bubbles are formed which interferes with the separation process , steady baseline and shape of the peak. Degassing can be carried out by any one technique: Vacuum filtration – It is the filteration of solvent using a vacuum pump. Helium purging —It is by passing the helium gas through the solvent in which the dissolved gases are swept out of the solution by fine bubbles of inert gas of low solubility. Distillation system —In this the solvent is distilled out and used. Ultrasonication —by using sonicator , it converts ultra high frequency to mechanical vibrations, which causes the removal of air bubbles. Heating and Stirring the solvents Filters ---They are used for removing dust and particulate matter from the solvent . The solvents are usually filtered through a Millipore filter (2 μ m-0.45 μ m) under vacuum before introduction into the solvent reservoir . this treatment removes gases as well as suspended matter. Mixing unit —is used to mix the solvents and pass through the column. There are two types of mixing units, low pressure mixing unit(using inert gas helium) and high pressure mixing unit(by using static or dynamic mixers or stirrers and operates under high pressure).

12. PUMPING SYSTEMS OR PUMPS Pumps are used to pass the mobile phase through the column at high pressure and at controlled flow rate. Properties of pumps--- It should Generate pressure upto 6000psi Have flow rates ranging from 0.1ml-5ml per minute. Have flow control and flow rate reproducibility of ±0.5% Should be composition resistant and give a pulse free output. Easy to change from one mobile phase to another. Easy to dismantle and repair. It must be resistant to corrosion by a variety of solvents The high generated pressure by the pumps should not led to any kind of explosion hazards. Check valves- ---are used to control the flow rate of the solvent and back pressure.

13. There are three kinds of pumps: 1.Constant displacement pumps or Syringe pumps 2.Reciprocating pumps 3.Constant pressure pumps or Pneumatic pumps 1.Constant displacement pumps or Syringe pumps It consist of a large syringe like chamber equipped with a plunger that is activated by a screw driven mechanism powered by a stepping motor. This works on the principle of positive solvent displacement by a piston mechanically driven at constant rate in a piston chamber with the generation of pulse less flow with high pressure capability. Double syringe pumps have also been developed in which one is delivering solvent to the column while the other is refilled from the reservoir. Advantages: It is pulse free and flow rate is controlled. Flow rate is independent of viscosity and column back pressure. Disadvantages: Lack of solvent capacity(limited to 200-500ml) Solvent changing is inconvenient.

14. Constand displacement pump:

15. 2. Reciprocating pumps: A reciprocating pump is driven electrically, the driven piston pressurizes the eluent directly or hydraulic oil, which is acting by via a diaphragm, pressurizes the liquid from the solvent reservoir. The movement of a plunger piston causes suction of the eluent during the backward stroke and the delivery of the pressurized solvent during the forward stroke. The pump is equipped with ball check valves to ensure the positive direction of flow. Pulse dampers are used in this type of pumps as the back and forth motion of the piston causes pulses that will affect the detection. A simple pulse damping method involves a compressible gas is capped upright portion of the tea tube to take up some of the portion of the pulsation energy. When the pump refills, this energy is resealed to help smooth the pressure pulsation. Advantages: Small internal volume(35-400μL) High output pressure(up to 10000psi) Readily used for gradient elution. Constant flow rates that are independent of column back pressure and solvent viscosity. Disadvantages : It produces pulses, so pulse dampers are used, otherwise they produce a baseline noise on the chromatogram. Pulse dampers- --are used to dampen the pulses observed from the wavy baseline caused by the pumps.

16. Reciprocating pump:

17. 3. Pnematic pumps or Constant pressure pumps They use a source of pressurized gas(or pressurized by a compressed gas) to drive the liquid mobile phase to the column either directly in holding coil or a stainless steel cylinder or by means of flexible or moving separator in order to prevent substantial dissolution of the gas in upper part of the liquid. Advantages: Simple, inexpensive and pulse free. Disadvantages: Limited capacity and pressure output only upto 2000psi. Gradient elution not possible. Flow rate depends on solvent viscosity column back pressure.

18. Pneumatic pump:

19. Sample injection system It is a device that is used to introduce the sample into the HPLC setup. The volume to be injected is determined by the concentration of the components present in the sample. The overloading of sample in the column causes band broadening. The sample is usually injected at the head of the column with minimum disturbance of the column material. A good injection device should have: The smallest possible contribution to peak broadening. Should be convenient to use. Able to operate at high pressure. Chemically inert with the eluent and the sample. reproducible. Two important types of sample injectors used are: Syringe injection Variable volume valve injection or Stop flow injection

20. Syringe injection This is the earliest and simplest technique, here the syringes are used to inject the sample and are designed to withstand high pressure. Fixed volume is introduced by making use of the fixed volume injector. The sample devices are six port rotary valve. The sample is loaded by means of a loading syringe. Excess sample is used in order to ensure that the storage line is completely filled. This line may either be an external loop with adequate volume is to be changed or an external cavity or fixed volume grooved at the surface of the rotor or drilled into the rotor.

21. 2 . Variable volume valve injection or Stopflow injection: A variable volume Is introduced by making use of an injection valve. Variable volume injectors contain a needle port, which can be closed or sealed at high pressure to insert the syringe. This too is a syringe injection but here the solvent flow is stopped momentarily. A fitting at the column head is removed and sample is injected directly into the head pf the column packing at atmospheric pressure, then the fitting is replaced and the system is again pressurized. Then solvent line switching on causes the sample to be injected into the column.

22. Guard columns or precolumns : A guard column is placed between injector and press. Pump to protect the HPLC column from damage and loss of efficiency caused by particulate matter and strong adsorbant in solvent. In HPLC, guard columns serve to saturate the mobile phase with the stationary phase so that loses of stationary phase from analytical column is minimised. The composition of the guard column should be similar to that of the analytical column. The particle size is usually large, to minimise the pressure drop. It is mainly used to remove the impurities from the solvent and thus prevent contamination of the analytical column. Analytical columns: Column chromatography columns are constructed from smooth bore stainless steel tubing, or heavily walled glass tubing. Column length ranges from 10-100cm and internal diameter of 2-6mm for analytical column and ID of 6mm and more for preparative columns. Column packaging have a particle size of 3-10 μ m. Recently micro columns are available with ID of 1-4.6mm, particle size of 3-5 μ m and length of 3-7.5cm which works with high speed and gives high resolution and minimal solvent consumption. Types of columns: Standard columns Radial compression column Narrow-bore columns Short and fast columns

23. Standard columns: HPLC are done on columns with an internal diameter of usually 4-5mm. They provide a good compromise between efficiency, sample capacity and the amount of packing and solvent required. Column packing materials are uniformly sized and mechanically stable 2. Radial compression columns: In the radial column the packing takes the form of a cylinder and the flow of mobile phase passes from the outside of the cylinder, through the packing to the outside of the cylinder, the separation taking place on the way through. The packing is supported between two cylindrical frits and the gap between represents the bed height or column length. The outer frit is the column inlet and consequently the sample initially has a large area of stationary phase with which to interact. The sample is injected into a radial column that disperses the sample over the total peripheral area of the external frit. This type of column has a low resolution but high loading capacity and is not suitable for separating complex mixtures. It can be used effectively with gradient elution development techniques. Radial columns are slurry packed. 3. Narrow-bore columns: Bore size or the internal diameter of the column is decreased, then there is an increase in the signal of the sample component. When bore size is decreased the volume of solvent requires is decreased. Mobile phase of high purity is used. In these columns there is better homogeneity in packing density and smaller temperature gradient across the column.

24. 4. Short and fast columns: A short, conventional bore column packed with small particles can save solvent cost, increase sample throughput, and deliver higher sensitivity than conventional length columns. Time required is 15-120secomds for isocratic elution and 1-4minutes for gradient elution. These columns are used where analytical speed is essential i.e. in QC dept. Types of column packing material in HPLC: Pellicular or Superficially porous material : it consist of a core hat, surrounded by a crust or a layer of material that is chromatographically active. The core is usually a non-porous spherical glass beads or polymer beads, and the outer layer consist of porous particles. The particle size ranges from 30-40 μ m. Limitations of this is that there is reduction or decrease in the surface area available in the column. E.g. : perisorb A- contains silica, perisorbRP - contains silinised silica. Microparticulate packing or small porous particles : this packing material is totally porous packing material, and the particle size ranges from 3-10 μ m. Usually silica, alumina or an ion exchange resins are used. Mainly silica particles are used with small particle size and spherical shapes. The limitation is that silica is not chemically stable, it reacts with many compounds and also dissolves away in the mobile phase. Silica also requires to use buffer solutions to maintain the Ph and prevent the stationary phase from degrading. Eg : adsorbosphere -silica, IBM-silica. Bonded phase packing : the bonded phase HPLC packings consist of a silica support with an organic moiety bonded to it through a silicon carbon a covalent bonding system. This bonding is obtained by chemical bonding reactions, and it is used in HPLC-partition chromatography.

25. Requirements of Stationary phase The pore size of the stationary phase should be small. The optimum pore size should be ranged between 100 to 1000 angstrom for small molecules. The most frequently used pore size is 300 angstroms for proteins and peptides and 100 angstrom for small molecules. Particle size of the stationary phase is also an important factor as it effects the pressure development. Larger particle size generates less pressure but small particle size gives more pressure. The particle size range used is from 3-5 μ m and most frequently used is 5 μ m. The stationary phase is required to be mechanically stable (especially to high pressure and liquid shear forces) and it should be chemically inert under chromatography conditions. In HPLC using partition as principle bonded phase supports should be used as stationary phases, here the stationary phase is covalently bonded to the support particles or the inside wall of the column tubing. This is done to prevent bleaching of stationary phase.

26. Requirement of Mobile phase: Polarity – it is an important criterion for mobile phase selection. The solvent system chose should have neither high polarity nor should it have low polarity, the polarity of the solvent should be moderate, in normal phase chromatography the solvent system used is non-polar while in reverse phase it is polar solvent. one other criterion is that the polarity of the solvent is also decided on the functional group of the analyte to be separated, in better words solvent can be selected by matching the relative polarity of the solvent to that of the sample component or a solvent is chosen to match the most polar functional group in the sample. Eg : alcohols for hydroxyl group and ketones, acetates for carbonyl group etc. The solvent system should be able to carry loosely held substances or weakly soluble substances and carry out the process of separation efficiently. Mobile phase used must be compatible to the detector that is used for the specific HPLC process. Viscosity is the major consideration and requirement for the development of high column efficiencies. Grade of solvent should be selected for HPLC which is recently constant physical and chemical characteristics. High grade solvents or analytical grade solvents are used. Solvent system containing buffers are also used in HPLC, where the pH should be maintained. Mainly the buffering solutions that are used in the range pf 4-8 Ph.

27. Detectors in HPLC: Detectors are devices that will identify a particular substance and show a specific response that indicates this substance. Criteria for HPLC detectors: Good sensitivity to all eluting compounds. Should not degrade the separation obtained in the column. Reliable and easy to operate. Good reproducibility. There are mainly two types of detectors Bulk property detectors - which compare an overall change in physical property of the mobile phase with and without an eluting solute. eg : refractive index detector, conductivity detector Solute property detector -respond to physical property of the solute that is not exhibited by the pure mobile phase. It gives a good sensitivity and detects even the smallest quantity of sample. Eg : ultra violet, visible adsorption , fluorescent and electrochemical detectors.

28. Refractive index detector The detection is based on the fact that the refractive index of the mobile phase may be different from that of mobile phase containing a solute and the differences is directly proportional to the concentration. Detection occurs when light is bent due to sample eluting from the column, and this is read as a disparity between the two channels. Types of refractive index detectors Deflection refractometer : it measures the deflection of a beam of monochromatic light by a double prism. Eluent passes through half of the prism, pure mobile phase passes through or fills the other half. The two compartment are separated by a glass plate mounted at an angle such that bending of the incident beam occurs if the two solutions differ in refractive index. The resulting displacement of the beam with respect to the photosensitive surface of detector causes variation in the output signal, which when amplified and recorded, provides the chromatogram. Advantages: They respond nearly to all solutes. They are reliable. They are unaffected by flow rate.

29. Disadvantages They lack in high sensitivity. They are not much suitable for gradient elution. They need strict temperature control to be operated at their higher sensitivity.

30. Reflection type refractometer / Frensel refractometer This type of detector measure the change in percentage of reflected light at a glass liquid interface as the refractive index of the liquid changes. In this detector the column, the column mobile phase as well as the reference flow of solvents are passed through small cells on the back surface of the prism. When the two are liquids are identical there is no difference between the two beams reaching the photocell. A change in the amount of light transmitted to the photocell, if the mobile phase containing solute passes through the cell, and as a result a signal is produced.

31. Ultraviolet detectors It works on the principle of adsorption of UV-visible light as the effluent from the column is passed through small flow cell held in the radiation beam. The UV detector measure the change in the UV adsorption as the solute passes through a flow cell (usually 10 μ l in volume) In a UV transparent solvent. A light source delivers a monochromatic parallel light beam which passes through a cell swept by a column effluent, and fall on the photo cell. A signal proportional to the amount of light received is measured and recorded. The source of light of the sample UV photometer is generally a mercury lamp providing work at 200 to 400nm.

32. UV detectors are of three types: Fixed wavelength detectors - uses a light source that emits maximum light intensity at one or several wavelength that are isolated by appropriate filters. HPLC detectors which do not allow changing the wavelength of the radiation that is at fixed wavelength is called Fixed wavelength detectors. A fixed wavelength detectors uses a light source that emits maximum light intensity at one or several discrete wavelength that are isolated by appropriated filters. Such a detector offers a minimum of noise but no free choice of wavelength. Using a medium pressure mercury lamp, wavelength of 254, 280, 313, 334 and 365nm can be selected by the use of narrow band pass interference filters.

33. 2. Variable wavelength detectors- It offers a wide selection of UV and visible wavelengths, but at an increased cost. To obtain a complete spectrum, the eluent flow must be stopped to trap the component of interest in the detector cell while the UV- visible spectrum region is scanned.

34. 3. Scanning wavelength detectors- To obtain a real time spectrum for each solute as it elutes , solid state arrays are required like PHOTODIODE ARRAY DETECTORS: light from continuous source example: deuterium lamp passes through a lens system which focuses polychromatic light onto the flow cell (containing sample). The transmitted light then falls on a holographic gratings, where it is dispersed into a photodiode array(PDA). ADVANTAGES OF PDA DETECTORS It provides spectra of each peak and can be used for peak purity analysis. A very selective detector which will detect only such solutes that specifically absorb UV/visible radiation e.g. alkenes, aromatic and compounds having multiple bonds between C, O, N , S. DISADVANTAGES OF PDA DETECTOR It is less sensitive and more expensive than single wavelength detector

35. Fluorescence detector The fluorescence detectors are based on the principle that a UV beam of light is focused in a detector cell, swept by the column eluent. Light emitted in perpendicular direction is collected on the photocell and its intensity measured a s a function of concentration elute in the effluent stream. Its easy to combine a fluorescence detector to a UV photometer. It is a specific detector that sense only those substances that fluoresce. A flow cell is used as a sensor through which the excitation light passes axially. A photocell is situated at the side of the cell to receive a radially emitted light. The cell wall is made of Pyrex glass to prevent the excitation light from reaching the photocell. When the solute that fluoresces in the excitation light is situated in the cell, the fluorescent light passes through the walls of the cell onto the photocell, the output of which is electronically processed and passed to a computer. The excitation light may be UV at 254nm produced by the mercury lamp or it may be light of any wavelength selected from the light produced by the deuterium lamp using a monochromator. Advantages It has higher detection sensitivity than UV detection. Lower detection limits. Less sensitive to fluctuations

36. Disadvantages Careful choice of mobile phase, Ph and mobile phase composition eg : aniline is cation at acidic Ph and do not fluorescent but in pH range of 7-12 it exist as a neutral species and fluorescent. Recorders The signals from the detector are recorded as deviations from the baseline. The peak position along the curve(retention time) denotes a particular compound and a peak area or height is a measure of amount of component in the sample.

37. Parameters used in hplc : 1. Retention time: Retention time is the difference in time between the point of injection and appearance of peak maxima . It is defined as the time required for 50% of the components to be eluted from a column . Its is measured in minutes and seconds . 2. Retention volume : Retention volume is the volume of carrier gas required to elute 50% of the components from the column . It is the product of retention time and flow rate . Retention volume = Retention time x flow rate

38. 3. Resolution : Resolution is the measure of extent of separation of 2 components and the base line separation achieved . Rs = 2(Rt 1 -Rt 2 )/w 1 +w 2 4. Separation factor : Separation factor is the ability of the chromatography system to chemically distinguish between sample components . It is usually measured as the ratio of the retention factor of the two peaks in the compositions.

39. 5. Height equivalent to a theoretical plate (HETP) : The theoretical plate is a imaginary or hypothetical unit of a column where distribution of solute between stationary phase and mobile phase has attained equilibrium . It can also be called as a functional unit of the column . 6. Asymmetry factor : The asymmetry factor is a measure of peak tailing . It is defined as the distance from the centre line of the peak to the backslope divided by the distance from the centre line of the peak to the front slope , with all measurements made at 10% of the maximum peak height.

40. Advantages of hplc Separation fast and efficient (high resolution power). Continues monitoring of the column effluent . It can be applied to the separation and analysis of very complex mixture . Accurate quantitative measurements . Repetitive and reproducible analysis using the same column Adsorption , partition , ion exchange and exclusion column separations are excellently made . High separation capacity , enabling the batch analysis of multiple components .

41. 8. Superior quantitative capability and reproducibility . 9. Moderate analytical condition . 10. Generally high sensitivity 11. Low sample consumption . 12. Easy preparative separation and purification of samples . 13. HPLC has no restriction to volatile and thermally stable solute and the choice of mobile and stationary phase is much wider . 14. Both aqueous and non aqueous sample can be analysed with little or no sample pre treatment . 15. A variety of solvents and column packings are available , providing a high degree of selectivity for specific analyses . 16. It provides means for determination of multiple components in single analysis .

42. Disadvantages of hplc Complexity , need a skill person to run the instrument . Solvent consumption is more . High cost . Coelution Adsorbed compounds .

43. Application of hplc The main applications of HPLC in pharmacy are: Natural products: HPLC is an ideal method for the separation of various components in plant extracts which resemble in structure like analysis of digitalis, cinchona, liquorice and ergot extracts. Stability studies: HPLC is used for ascertaining the stability of various pharmaceutical substance and products. I.e in impurity profiling and analysis of various degradation products. HPLC can be used to complement bioassays, which are very costly, gives poor precision, requires more time and necessitates replication. HPLC is for analysis of penicillin, chloramphenicol, sulpha drugs, peptide hormones, clotrimoxazole etc. HPLC has been used in the design of pharmaceutical dosage form by studying the pharmacokinetics of the drugs. HPLC has been used for analysis of various categories of compounds like alprazolam, captopril, diclofenac, haloperidol, folic acid, norfloxacin, omeprazole etc. The other applications are: Qualitative analysis: identification of compounds. This is done by comparing the retention time of the sample as well as the standard. Checking the purity of a compound: by comparing the chromatogram of the standard and the sample, the purity of the compound can be found.

44. Continued….. Presence of impurities- this can be found out by the additional peaks in the chromatogram when compared with a reference material. Quantitative analysis Direct comparison method: by injecting a sample and standard separately and comparing their peak areas, the quantity of the sample can be determined. Calibration curve method- a calibration curve of peak area Vs concentration of the drug is plotted. From the peak area of unknown sample, by interpolation, the concentration of the sample can be determined. Internal standard method: a known concentration of the internal standard is added to sample solution whose conc. Is not known. A compound with similar retention characteristics is used. Isolation and identification of drugs or metabolites in biological fluids like urine, plasma, serum etc can be carried out. Isolation and identification of mixture of components of natural or synthetic origin. Purification of some compounds of natural or synthetic origin on preparative scale.

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