HPLC AND ITS PHARMACEUTICAL APPLICATIONS

THIS SLIDES CONTAINS A BRIEF INTRODUCTION ABOUT HPLC ,ITS INSTRUMENTATION AND APPLICATIONS MAINLY IN PHARMACEUTICAL AREAS

1. NEELU MARIAM VARGHESE INTERNSHIP TRAINEE HPLC:INSTRUMENTATION AND APPLICATION IN PHARMACEUTICALS

2. MARTIN SYNGER

3. INTRODUCTION HPLC - High Performance Liquid Chromatography P owerful tool in analysis , yields high performance and high speed compared to traditional columns chromatography because of the forcibly pumped mobile phase Defined as: “ A chromatographic technique used to separate components of mixture for the purpose to identify, quantify or purify the individual components of the mixture ” .

4. Widely used in the field of biochemistry and analytical chemistry. Chromatography : physical method in which separation of components takes place between two phases-a stationary phase and a mobile phase. Stationary phase : The substance on which adsorption of the analyte ( the substance to be separated during chromatography ) takes place . It can be a solid , a gel, or a solid liquid combination. Mobile phase : solvent which carries the analyte (a liquid or a gas)

5. PRINCIPLE OF HPLC Partitioning of the analytes between the solid phase and mobile phase. C hemical/ physical interactions between sample molecules and column They travel according to their relative affinities towards the stationary phase. The component which has more affinity towards the adsorbent , travels slower . The component which has less affinity towards the stationary phase travels fast

6. TYPES OF HPLC Stationary phase: Polar eg:SiO2,Al2O3 Mobile phase : Non-polar eg:heptane ,hexane Polar molecules elutes slowly and non polar molecules elutes quickly Stationary phase: Non- polar eg : (Silica bounded with C-18) Mobile phase : Polar e g:methanol-water Polar molecules elutes quickly and non polar molecules elutes slowly NORMAL PHASE HPLC REVERSE PHASE HPLC NORMAL PHASE HPLC REVERSE PHASE HPLC STATIONARY PHASE POLAR eg:SiO2,Al2O3 NON POLAR eg : ( Silica bonded with C-18) MOBILE PHASE NON POLAR eg : heptane ,hexane POLAR eg : methanol-water AFFINITY Polar molecules elutes slowly and non polar molecules elutes quickly Polar molecules elutes quickly and non polar molecules elutes slowly

7. INSTRUMENTATION OF HPLC Main components are Solvent Reservoir Degassing system Pump Sample Injection Port Column Detector Computer System

8. Schematic representation of HPLC system

9. 1. SOLVENT RESERVOIR A reservoir holds the solvent(mobile phase) Made up of glass or stainless steel Requirements The reservoir and its attachment to the pump should be made of materials that will not contaminate the mobile phase. The vessel should have some cap to prevent particulate matter from contaminating the mobile phase

10. Mobile Phase: water solvent/organic solvent/mixture of both Acts as a carrier for the sample solution S olvent selection depends upon the nature of the compound to be separated. The mobile phase should be of highest purity. Isocratic elution : Composition of mobile phase is constant Gradient elution : Composition of the mobile phase changes during the separation process

11. 2. DEGASSING SYSTEM Several gases are soluble in organic solvents. Dissolved gases in solvents interfere separation process, steady baseline and the shape of the peak . Hence degassing is necessary

12. Degassing Methods  Vacuum filtration :- R emove air bubbles, but it is not always reliable and complete .  Helium purging :- By passing helium through the solvent. This is very effective but expensive .  Ultrasonification :- By using Ultrasonicator, which converts ultra high frequency to mechanical vibrations. This causes the removal of air bubbles.

13. 3. PUMP The role of the pump is to force the mobile phase through the liquid chromatography at a specific flow rate, expressed in milliliters per min (mL/min ) Normal flow rate :: 1-2mL/min range. Pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition (gradient).

14. RECIPROCATING PUMP On the back stroke , the separation column valve is closed , and the piston pulls in solvent from the mobile phase reservoir On the forward stroke ,the pump pushes solvent out of the column from the reservoir Widely used one. A small motor driven piston which moves rapidly back and forth in a hydraulic chamber

15. DISPLACEMENT PUMP / SYRINGE TYPE PUMP Most suitable for small bore columns Pump volume between 250 - 500mL O perates by a motorized lead screw that delivers mobile phase to the column at a constant rate The rate of solvent delivery is controlled by changing the voltage on the motor.

16. 3. PNEUMATIC PUMPS The mobile phase is driven through the column with the use of pressure from the gas cylinder. A low-pressure gas source is needed to generate high liquid pressures. The valving arrangement allows the rapid refill of the solvent chamber whose capacity is about 70mL

17. 4. SAMPLE INJECTION PORT I ntroduce the liquid sample into the flow stream of the mobile phase Different types of injector devices : ( i) Septum Injector ( ii) Stop Flow Injector ( iii) Rheodyne Injector

18. i SEPTUM INJECTOR U sed for injecting the sample through a rubber septum . Cannot be commonly used , since the septum has to withstand high pressures . ii . STOP FLOW INJECTOR F low of mobile phase is stopped for a while when the sample is injected iii. RHEODYNE INJECTOR W idely used injector Two position : LOAD & INJECT

19. Typical sample volumes for manual injector are 5-to 20-microliters( μL )

20. • An auto sampler is the automatic version for when the user has many samples to analyze or when manual injection is not practical . It can continuously i nject variable volume a of 1 μL – 1 mL AUTOSAMPLER

21. 5.COLUMN Heart of the chromatogram T he column stationary phase separates the sample components of interest using various physical and chemical parameter It consist of Guard Column Analytical Column

22. Guard Column Increase the life of analytical column It contains a packing chemically identical to that in analytical column . Mainly used to remove the impurities from the solvent and thus prevents contamination of the analytical column The success or failure of analysis depends upon choice of column . Actual separation is carried out here. Stainless –steel tube size – length -25 to 100 cm Internal diameter – 2 to 4.6 mm ANALYTICAL COLUMN

23. Materials of construction for the tubing PACKING MATERIAL  Stainless steel ( the most popular, gives high pressure capabilities) Glass (mostly for biomolecules)  PEEK (poly ether ether ketone) polymer (biocompatibility and chemically inert to most solvents ).   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 .

24. 6. DETECTOR Detects the individual molecules that elute from the column and convert the data into an electrical signal. Measure the amount of those molecules. Detector is selected based on the analyte or the sample under detection

25. The detectors used in HPLC should have following ideal properties : High sensitivity. Good stability and reproducibility . A linear response to solute. Negligible base line noise. Should be inexpensive. Capable of providing information on the identity of the solute. A short response time independent of flow-rate. High reliability and ease of operation. The detector should be non-destructive. Responses independent of mobile phase composition. A temperature range from room temperature to at least 400º C IDEAL PROPERTIES OF A DETECTOR

26. Commonly used detectors in hplc 1.Ultraviolet(UV)/Visible Detector Measures the ability of solutes to absorb light at a particular wavelength(s) in the ultraviolet (UV) or visible (Vis) wavelength range .   S eparate and identify the principal active components of a mixture . V ersatile , have the best sensitivity and linearity. UV detectors cannot be used for testing substances that are low in chromophores (colorless or virtually colorless) as they cannot absorb light at low range .

27. C ost-effective and widely used U seful for aromatic compounds and other type unsaturated systems . C lassified as : Fixed detectors : employ filter as a source to provide appropriate wavelength. Variable detectors : employ a spectrophotometer to provide dispersion of light and selection of any wavelength

28. Diffraction gratings are frequently used for wavelength dispersion

29. 2. Flourescence Detector I deal detector for those solutes that exhibit molecular fluorescence The excitation source passes through the flow cell to a photo detector while a monochromatic measures the emission wavelengths . Their sensitivity depends on the fluorescence properties of the components in the elute . The light source is usually a broad spectrum deuterium or xenon flash lamp

30. Schematic representation of flourescence detector

31. 3.Photo Diode Array (PDA) Detector: L inear array of discrete photodiodes on an integrated circuit (IC) chip. Light from the broad emission source such as a deuterium lamp is collimated by an achromatic lens system so that the total light passes through the detector cell onto a holographic grating. The array may contain many hundreds of diodes and the output from each diode is stored on a hard disc.

32. 4.Refractive index Detector   Measures the overall ability of the mobile phase and its solutes to refract or bend light. The amount of defection is proportional to the concentration of the solute in the mobile phase . U seful for detecting those compounds that are non-ionic , do not absorb ultraviolet light and do not fluorescent e.g . sugar, alcohol, fatty acid and polymers.

33. 5. Electrochemical Detector   It is based on the measurement of the current resulting from an oxidation/ reduction reaction of the analyte at a suitable electrode. The level of current is directly proportional to the analyte concentration . Three electrodes are employed which are :  Working electrode  Auxiliary electrode  Reference electrode

34. 6. Computer(Data Processing System) Frequently called the data system The concentration of each detected component is calculated from the area or height of the corresponding peak and reported.

35. MANGIFERIN SOLVENTS: ACETONITRILE O-PHOSPHORIC ACID

36. APPLICATIONS OF HPLC

37. HPLC IN PHARMACEUTICALS To control the drug stability Quantity of drug determination from pharmaceutical dosage forms eg:Paracetamol determination in panadol tablet Quantity of drug determination from biological fluids eg : Blood glucose level Therapeutic drug monitoring Purity Checking Toxicology eg:Paracetamol Poisoning

38. HPLC is useful in compound identification and pharmaceutical purification testing , as it is estimated that over 60% of existing molecules have been uniquely identified by HPLC testing. 1.IN DRUG ANALYSIS M ost important analytical method for identification and quantification of drugs. E ither in their active pharmaceutical ingredient or in their formulations during the process of their discovery, development and manufacturing . Discovery Development Manufacturing

39. Example1 : HPLC analysis of the novel antipsychotic drug quetiapine in human plasma R . Mandrioli a, S. Fanali b, A. Ferranti a, M.A. Raggi *Carried out on a C8 (150/4.6 mm i.d. , 5 mm) reversed-phase column *Mixture of acetonitrile,methanol phenoll and pH 1.9 phosphate buffer as the mobile phase; * Triprolidine was used as the internal standard. Example2 : HPLC analysis of raloxifene hydrochloride and its application to drug quality control studies Jurij Trontelj ∗, Tomaˇz Vovk , Marija Bogataj , Aleˇs Mrhar * Raloxifene hydrochloride is a selective estrogen receptor modulator and is currently being used for prevention of osteoporosis in postmenopausal women

40. HPLC method for detection of raloxifene hydrochloride was developed and validated using an ultraviolet (UV) and coulometric detectors. The mobile phase consisted of methanol, acetonitrile, water (28:32:30%, v/v/v) and ammonium acetate (5 mM ) and was delivered isocratically with a flow rate of 0.5 mL min−1 Chromatograms obtained with an UV detector (a) and coulometric detector (b). The main peaks correspond to 1 mg L−1 raloxifene hydrochloride .

41. 2.Quantity of drug determination from pharmaceutical dosage forms Example 1 : A RP-HPLC method for simultaneous estimation of paracetamol and aceclofenac in tablets R. Gopinath , S. Rajan , S. N. Meyyanathan * , N. Krishnaveni and B. Suresh * A simple, selective, rapid, precise and economical reverse phase HPLC method has been developed for the simultaneous estimation of paracetamol and aceclofenac from pharmaceutical dosage forms. * The method was carried out on a Hichrom C18 (25 cm×4.6 mm i.d. , 5 µ) column with a mobile phase consisting of acetonitrile : 20 mM phosphate buffer (pH 5.0) (60:40 v/v) at a flow rate of 0.8 ml/min. * Detection was carried out at 265 nm . * Etoricoxib was used as an internal standard. * The retention time of paracetamol , aceclofenac and etoricoxib was 4.75, 6.44 and 8.83 min, respectively. 

42. Example 2 : . RP-HPLC Estimation of Risperidone in Tablet Dosage Forms S L Baldania , K K Bhatt, R S Mehta   *A Phenomenex Gemini C-18, 5 μ m column having 250×4.6 mm i.d. in isocratic mode *Mobile phase containing methanol: acetonitrile: 50 mM potassium dihydrogen orthophosphate (80:10:10 v/v) was used . *The flow rate was 1.3 ml/min and effluents were monitored at 234 nm . *Clozapine was used as an internal standard .

43. The retention time of risperidone and clozapine were 2.5 min and 3.3 min, respectively . Risperidone (RIS) is psychotropic agent  3.Quantity of drug determination from biological fluids Example: Rapid determination of metformin in human plasma using ion-pair HPLC A . Zarghi a,*, S.M. Foroutan b , A. Shafaati a , A. Khoddam

44. The separation was performed on an analytical 150/4.6 mm i.d. µ bondapak C18 column . The wavelength was set at 235 nm . The mobile phase was 40% acetonitrile, 0.01 M sodium dodecyl sulphate , 0.01 M sodium dihydrogen phosphate, and distilled water to 100%, adjusted to pH 5.1 at a flow rate of 1.5 ml/min. Metformin is an antidiabetic agent which used in the treatment of non-insulin - dependent diabetes phenytoin is the internal standard