HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase).

1. High Performance Liquid Chromatography Prepared By D.Mahendra,M.Pharm ,( Ph.D ),. Dept of Ph.Analysis & Quality Assurance Research Scholar at PARUL University, Asst.Professor at NCOP_JNTUK Mail- [email protected] Mobile- 9912360343

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7.  INTRODUCTION  TYPES OF HPLC TECHNIQUES  PRINCIPLE  INSTRUMENTATON  PARAMETERS USED IN HPLC  ADVANTAGES OF HPLC  DERIVATISATION IN HPLC  APPLICATIONS CONTENTS

8. Introduction  HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution.  HPLC is characterized by the use of high pressure to push a mobile phase solution through a column of stationary phase allowing separation of complex mixtures with high resolution.  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.

9. TYPES OF HPLC TECHNIQUES: Based on modes of chromatography Normal phase mode 2.Reverse phase mode Based on principle of separation Adsorption chromatography Ion exchange chromatography Ion pair chromatography Size exclusion(or)Gel permeation chromatography Affinity chromatography Chiral phase chromatography

10. Based on elution technique Isocratic separation Gradient separation Based on the scale of operation Analytical HPLC Preparative HPLC Based on the type of analysis Qualitative analysis Quantitative analysis

11. PRINCIPLE  The principle of separation in normal phase mode and reverse phase mode is adsorption.  When a mixture of components are introduced into a HPLC 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 faster. Since no 2 components have the same affinity towards the stationary phase, the components are separated

12. The principle of HPLC are based on Van Deemter equation which relates the efficiency of the chromatographic column to the particle size of the column, molecular diffusion and thickness of stationary phase. The Van Deemter Equation is given as H or HETP = A + B + C υ υ where, A= represents eddy diffusion B= represents molecular diffusion C =represents rate of mass transfer υ =represents flow rate

13.  Separation is based on the analyte’s relative solubility between two liquid phases Stationary Phase Mobile Phase Solvent Bonded Phase

14. Reversed Phase Stationary phase Polar (silica gel) Non-polar (C18) Mobile phase Non-polar (organic solvents) Polar (aqueous/organic) Sample movement Non-polar fastest Polar fastest Separation based on Different polarities (functionality) Different hydrocarbon content H PLC - Modes Normal Phase. - Polar-Stationary phase -Nonpolar- Solvent(Mobile phase) Reverse Phase . - Non-polar- Stationary phase - Polar - Mobile phase (solvent). Normal Phase

15.  Methanol C H 3 O H CH 3 CN Acetonitrile T etr ah y d r o f ur a n W a t e r H 2 O

16.  Solid Support - Backbone for bonded phases. Usually 10µ, 5µ or 3µ silica or polymeric particles.  Bonded Phases - Functional groups firmly linked (chemically bound) to the solid support. Extremely stable Reproducible  Guard - Protects the analytical column: Particles Interferences Prolongs the life of the analytical column Analytical - Performs the separation.

17.  C-2 Ethyl Silyl -Si-CH 2 -CH 3 C N Cyanopropyl Silyl -Si-(CH 2 ) 3 -CN C - 1 8 Octadecyl Silyl -Si-(CH 2 ) 17 -CH 3 C - 8 Octyl Silyl -Si-(CH 2 ) 7 -CH 3

18. Bonding Phases onto the Silica Support CH 3 | -Si- CH 3 | C H 3 O H + C l S i li c a CH 3 | -0-Si- CH 3 | CH 3 + H C l S i li c a Could be many different functional groups here

19. Chromatography Stationary Phases relatively polar surface normal phase O O O | | |  O  S i  O  S i  O  S i  O  H | | | O O O | | |  O  S i  O  S i  O  S i  O  H | | | O O O bulk (SiO 2 ) x surface relatively nonpolar surface reversed phase Silica Gel  O  O x O O O | | |  S i  O  S i  O  S i  O  R | | | O O O | | |  S i  O  S i  O  S i  O  R | | | O O O bulk (SiO 2 ) su rface Derivatized Silica Gel Where R = C 18 H 37 hydrocarbon chain (octadecylsilyl deriv. silica or “C18”)

20. The Surface of Silica For adsorption chromatography, the silica is used without modification, but for most other types of HPLC, the silica is coated with one of many types of stationary phases. More commonly, in modern chromatography columns, the stationary phase is chemically bonded to the silica

21. The Surface of Silica Bonded with C18

22. P u m p I njector C o l u m n Detector Mobile Phases Gradient C on t r o ll er •

23. HPLC system

24. HPLC system  Solvent Reservoir  Degasser  Solvent Delivery System (Pump)  Injector  Column &oven Detectors   Recorder (Data Collection)

25. INSTRUMENTATON: Solvent delivery system Pumps Sample injection system Column Detectors Recorders and Integrators 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. The choice of mobile phase is very important in HPLC and the eluting power of the mobile phase is determined by its overall polarity, the polarity of the stationary phase and the nature of the sample components.

26.  They are low pressure mixing chamber and high pressure mixing chamber. Mixing of solvents is done either with a static mixer or a dynamic mixer.  In an isocratic separation , mobile phase is prepared by using solvent of same eluting power or polarity.  But in gradient elution technique , the polarity of the solvent is gradually increased and hence the solvent composition has to be changed. Hence a gradient controller is used when 2 or more solvent pumps are used for such separations.  Mixing unit is used to mix the solvents in different proportions and pass through the column. There are 2 types of mixing units.

27.  Several gases are soluble in organic solvents.  When solvents are pumped under high pressure, gas bubbles are formed which will interfere with the separation process, steady base line and the shape of the peak.  Hence degassing of solvent is important. This can be done by using Vacuum filtration , Helium purging, Ultrasonication.  In normal phase chromatography hexane, iso octane, di ethyl ether are the mobile phases. In reverse phase chromatography water, methanol, acetonitrile are the mobile phases.

28. Degasser  P r o b l e m s c a u s e d b y d i s s o l v e d a i r ( O 2 , N 2 ) i n m o b il e phase Unstable delivery in pump Bigger noise and large baseline-drift in detector cell  I n o r d e r t o a v oi d c a u s in g th e p r ob l e ms , mobile phase should be degassed . vacuum pumping systems distillation system a system for heating and stirring the solvents sparging system - bubbles an inert gas of low solubility through the solvent

29. C om p o n e nt s Three basic types of LC Pumps are: Pneumatic pumps Motor driven syringe type pumps Reciprocating pumps Pumping systems:      Requirement: high Pressure (6kpsi), Pulse-free, Constant Flow(0.1 10mL/min.), Reproducibility(0.5%), Resistant to corrosion

30. Reciprocating Pumps  Advantages small internal volume high output pressures (up to 10,000 psi) readily adaptable to gradient elution “unlimited” solvent reservoir  Disadvantages produces a pulsed flow expensive

31. Reciprocating pump :  Most widely used.  Small internal volume (35 ~ 400 μL),  High-pressure (105 psi),  Gradient elution,  Constant flow. Need pulse damper.

32. Analytical Research and Developement Laboratory - I,FDC L i m i t e d , M u m b a i 3 2

33. – Displacement pump (Screw-driven syringe pump): Pulse free, Small capacity (250 mL), No gradient elution . Limited mobile phase capacity .

34. Solvent Delivery System Requirements ability to mix solvents and vary polarity of mobile phase during run “unlimited” solvent reservoir generation of pressures up to 6000 psi flow rates ranging from 0.1 to 10 mL/min flow reproducibility’s of 0.5 % or better resistance to corrosion by a variety of solvents pulse-free output

35. Inj e c t o rs Sample Injection System sample valve syringe Sample Injection Systems       For injecting the solvent through the column Minimize possible flow disturbances Limiting factor in precision of liquid chromatographic measurement Volumes must be small .1-500  L Sampling loops  interchangeable loops (5-500  L at pressures up to 7000 psi)

36. Direct injection auto sampler from Pu m p from P u m p to Column V ial Nee dle Measuring Pump to Column LOAD INJECT

37. SAMPLE INJECTION SYSTEM : Several devices are available either for manual or auto injection of the sample. Different devices are: 1.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. Limit of precision of HPLC Sample size: 0.5 ~ 500 μL No interference with the pressure Based on a sample loop, 1 ~ 100 μL, Reproducibility: 0.1%, P < 7000 psi Auto sampler: inject continuously variable volume 1 μL – 1 mL Controlled temperature environment for derivatization reaction.

38. Precolumn remove impurities from solvent saturates mobile phase with liquid of stationary phase before the analytical column Column straight, 15 to 150 cm in length; 2 to 3 mm i.d. packing - silica gel, alumina, Celite

39. Analytical Research and Developement Laboratory - I,FDC L i m i t e d , M u m b a i 4 4

40. Picture of HPLC instrument

41.  UV Single wavelength (filter)] Variable wavelength (monochromator) Multiple wavelengths (PDA)  Fluorescence  Electrochemical  Mass Spectrometric

42. DETECTORS : Detectors used depends upon the property of the compounds to be separated. Different detectors available are: Refractive index detectors U.V detectors Fluorescence detectors Electro chemical detectors Evaporative light scattering detectors IR detectors Photo diode array detector:

43. 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. Retention time for all the peaks can be found out from such recordings, but the area of individual peaks cannot be known. 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. Integrators provide more information on peaks than recorders. Now a days computers and printers are used for recording and processing the obtained data and for controlling several operations. PARAMETERS USED IN HPLC : 1.Retention time 2.Retention volume 3.Seperation factor Resolution Height Equivalent to a Theoretical Plate (HETP) Efficiency Asymmetry factor

44. APPLICATIONS: H P LC i s on e o f th e m ost wi de l y a p p lied analytical separation techniques . Pharmaceutical: Tablet dissolution of pharmaceutical dosages. S h e l f l if e de t e r m i n a t i o n s of p h a r m ace uti ca l products. Identification of counterfeit drug products. Pharmaceutical quality control.

45. Environmental Phenols in Drinking Water. Identification of diphenhydramine in sediment samples. Biomonitoring of PAH pollution in high-altitude mountain lakes through the analysis of fish bile. Estrogens in coastal waters - The sewage source. Toxicity of tetracyclines and tetracycline degradation products to environmentally relevant bacteria. Assessment of TNT toxicity in sediment. Forensics A mobile HPLC apparatus at dance parties - on-site identification and quantification of the drug Ecstasy. Identification of anabolic steroids in serum, urine, sweat and hair. Forensic analysis of textile dyes. Determination of cocaine and metabolites in meconium. Simultaneous quantification of psychotherapeutic drugs in human plasma.

46. Clinical Quantification of DEET in Human Urine. Analysis of antibiotics. Increased urinary excretion of aquaporin 2 in patients with liver cirrhosis. Detection of endogenous neuropeptides in brain extracellular fluids. Food and Flavor Ensuring soft drink consistency and quality. Analysis of vicinal diketones in beer. Sugar analysis in fruit juices. Polycyclic aromatic hydrocarbons in Brazilian vegetables and fruits. Trace analysis of military high explosives in agricultural crops. Stability of aspartame in the presence of glucose and vanillin.

47. USES & APPLICATIONS Separation and analysis of mixed components non-volatile compounds (pharmaceuticals, salts , polymers, heavy hydrocarbons) OR, thermally-unstable ) compounds ( trinitrotoluene(TNT), enzymes) Qualitative analysis The identification (ID) of individual compounds in the sample

48. 3- Quantitative analysis The measurement of the amount of a compound in a sample (concentration); 4-Preparation of Pure Compounds a pure substance can be prepared for later use (e.g. organic synthesis, clinical studies, toxicology studies, etc.). This methodology is called preparative chromatography . USES & APPLICATIONS

49. 6 7 Chemical Pharmaceuticals Consumer Products Soft drink a n t i ox i d a n t s sugars Clinical amino acids vitamins n e u r o p ep t i d e Environmental polyaromatic hydrocarbons Phenol in drinking water herbicides po l y s t y r e n es dyes phthalates tetracyclines corticosteroids an t i d e p r e s s a n ts barbiturates Bioscience proteins peptides nu c l e o t i de s USES & APPLICATIONS

50. Pharmaceutical Research: all areas including early identification of clinically relevant molecules to large-scale processing & purification. • • • • • • • Tablet dissolution Shelf life determinations Pharmaceutical quality control Therapeutic drug monitoring Metabolites (substrates, inhibitors) Forensics (drugs of abuse) Toxicology e.g. paracetamol poisoning USES & APPLICATIONS

51. ADVANTAGES & DISADVANTAGES HPLC ADVANTAGES:  Speed(minutes)  High resolution  Sensitivity  Accuracy  Automation HPLC DISADVANTAGES  Cost  Complexity

52. Importance of HPLC in Pharmaceuticals & Medicines High performance liquid chromatography (HPLC) is used in the pharmaceutical and medicine manufacturing industry to test products, and the ingredients used to make them. This testing is often performed by a pharmaceutical company's quality control (QC) laboratory. Chemists employed by these manufacturers will run samples of raw materials or finished goods through HPLC machines and then analyze the results.

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