Department of Pharmacy (Pharmaceutics) | Sagar savale | HIGH PERFORMANCE LIQUID CHROMATOGRAPHY [HPLC] Mr. Sagar Kishor savale [Department of Pharmacy (Pharmaceuties)| avengersagar 1 6(@gmall.com e 22-12-2015 |CHROMATOGRAPHY Itis define as, it is analytical method in which separation of active constituent in complex mixture, and the mixture was distributed in two phases i.e. stationary phase and mobile phase is known as chromatography. It is technique is used for separation, purification, Identification and extraction of compound. It is method it can consist of two phases stationary phase and mobile phase. © Stationary phase is constant phase or column packaging material. © Mobile phase is moveable phase. o The basic principle of chromatography is based on Adsorption and partition chromatography. Adsorption chromatography - The affinity of molecules towards stationary phase is known as Adsorption chromatography. Partition chromatography - The molecule can moves in two phases of liquid is known as partition chromatography. itis important for qualitative and quantitative analysis. aTYPES OF CHROMATOGRAPHY Based on modes of chromatography 1. Normal phase mode 2. Reverse phase miode Based on principle of separation 1. Adsor ion chromatography 2. Jon exchange chromatography 3. Partition chromatography 4, Based on elution technique jize exclusion 1. Isocratic separation 2. Gradient separation Based on the scale of operation 1. Analytical HPLC 2. Preparative HPLC Based on the type of analysis 1. Qualitative analysis 2, Quantitative analysisPRINCIPLES OF LIQUID CHROMATOGRAPHY ii ou iz ‘ HyTYPES OF LIQUID CHROMATOGRAPHY LC mode Normal phase chromatography Packing materials Mobile phase Silica gel n-Hexane/IPE Reversed phase chromatography Silica-C18(ODS) MeOH/Water Size exclusion chromatography lon exchange chromatography Affinity chromatography Porous polymer THF lon exchange gel Buffer sol. Packings with ligand Buffer sol. Interaction Adsorption Hydrophobic Gel permeation lon exchange AffinityOo 0 0 © 0 Oo oo 0 0 HPLC HPLC is a High Performance liquid Chromatography. High Pressure Liquid Chromatography. High Priced Liquid Chromatography. It is column chromatography, It is Liquid Chromatography. It is modified from of gas chromatography, it is applicable for both Volatile as well as Non volatile compound. It can mainly divided by two types 1. Normal phase HPLC 2. Reversed Phase HPLC, It is having a high resolution and separation capaci Itis used as qualitative as well as quantitative analysis. High performance liquid chromatography (HPLC) is a chromatographic technique used to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying or purifying the individual components of the mixture. rPRINCIPLE High Performance Liquid Chromatography [HPLC] is principle is based on adsorption as well as partition chromatography is depending on the nature: of stationary phase, if stationary phase is solid principle is based on adsorption chromatography and if stationary phase is liquid principle is based on partition chromatography. itis important for determination of volatile and non volatile compounds. itis important for determination qualitative and quantitative analysis. It is important for determination of Retention Time (the time is required , after sample injection maximum angle peak reaches to detector)ooec00000000000 a0 ADVANTAGES It is simple, rapid , reproducible. High sensitivity. High performance. Rapid process and hence time saving. It is having a high resolution and separation capacity. Accuracy and Precision. Stationary phase was chemically innert, Wide varities of stationary phase. Mobile phase was chemically innert. Less requirement of mobile phase in developing chamber. Early recovery of separated component. Easy visualization of separated components. It is having Good reproducibility and repeatability. Tt is analytical technique is important for validation of product, quality control studies of product. It is important for qualitative and quantitative analysis. 6 It is used for both analytical and preparative purpose.TYPES OF HPLC SEPARATIONS Normal Phase: Separation of polar analytes by partitioning onto-a polar, bonded stationary phase. Reversed Phase: Separation of non-polar analytes by partitioning onto a non-polar, bonded stationary phase. Adsorption: In Between Normal and Reversed. Separation of moderately polar analytes using adsorption onto a pure stationary phase (e.g. alumina or silica) Ton Chromatography: Separation of organic and inorganic ions by their partitioning onto ionic stationary phases bonded to a solid support. Size Exclusion Chromatography: Separation of large molecules based in the paths they take through a “maze” of tunnels in the stationary phaseHPLC METHODS o Parameter Group Method Compounds * SDW05.23000's EPA 555 Cl-PhenoxyAcids * WPP05.06000’s EPA 605 Benzidines * WPP05.13000’s EPA 610 PAHs * SHW06.26000's SW-846 8316 Acrylics * SHW06.28000’s SW-846 8330's Explosives * SHW07.06000’s SW-846 8325 Benzidines and N- PesticidesMODES OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY High Molecular Weight Compounds PolymersHPLC ConraAIN © Stationary Phase - It is non polar and The stationary solid surface is coated with a 2nd liquid (the Stationary Phase) which is immiscible with mobile phase. © Mobile Phase - A polar mobile phase(ACN, MeOH, WATER + BUFFER). Bonded Phases - oC-2 Ethyl Silyl *C8 Octyl Silyl * C-18 Octadecyl Silyl *CN Cyanopropyl Silyl -Si-CH,-CH; -Si(CH,);-CH; -Si-(CH,),-CH, -Si-(CH,),-CNBLOCK DIAGRAM OF HPLC = r| Reservoir Reservoir [ Recorder | Pum Gradient ' controller ‘ Fraction Detector i collector column chamber ‘ Solvent ; i I |__| Conditioning njEctor Precolumn columnWHY USE HPLC « Simultaneous analysis ~ High resolution + High sensitivity + Good repeatability + Moderate analysis condition + Easy to fractionate and purify + Not destructiveINSTRUMENTATION OF HPLC o Solvent storage bottle o Gradient controller and mixing unit o De-gassing of solvents o Pump o Pressure gauge o Pre-column o Sample introduction system o Column o Detector o RecorderRegulated helium source —= Priming syringeHPLC System Solvent HPLC Column Injector PumpInjector Reservoir Data SystemColumn Detector Interface Data station =| Mim =V/ yi = e z Mobile phase (eluent) eoOUTLINE OF LC-2010 System Controller Auto sampler Column Oven | Degassing Unit| UV detector,HPLC SYSTEM 9060 Polychrom Computer (Diode Array) Detector Workstation = 9050 Variable SS 010Solvenr Me UV/Vis Detector HPLC Solvent (™ Delivery System Reservoirs| MOBILE PHASE RESERVOIRS 0 Stainless steel should be avoided for use with solvents containing halide ions.DEGASSING & FILTRATION OF MOBILE PHASE © In many cases, aqueous solvents & some organic solvents are degassed prior to use able to carry sufficient volumePUMP © The solvents or mobile phase must be passed through a column at high pressures at up to 6000 psi(Ib/in?) or 414 bar. @ As the particle size of stationary phase is smaller (5 to 101) the resistance to the flow of solvent will be high. © That is, smaller the particle size of the stationary phase the greater is the resistance to the flow of solvents. © Hence high pressure is recommended.+ Generation of pressure of about 6000 psi. + Pulse free output & all materials in he pump should be chemic: lly resistant to solvents. _& Flow rates ranging from 0.1 to 10 m of taking th than one reservoir containing different solvents simultaneously. in ‘+ Pumps should be capal vent from a single reservoir or moreTypes of pumps used in HPLC DISPLACEMENT RECIPROCATING PNEUMATIC PUMPS PUMPS. PUMPSsyringe a (papiaes Reciprocating pump a | pump) i Single piston Double piston reciprocati | | reciprocating ng pump pump Direct pressure pump Reciprocating diaphragm pumpFeats & eee + Disadvantages:- It has a limited solvent capacity(~250) & considerably inconvenient when solyents must be changed.Te Column Piston Drive Motor Mobile phase reservoir | Fast refill motor Driver Control <——____ Manual rewindRECIPROCATING PUMPS o This pump transmits alternative pressure to the solvent via a flexible diaphragm ,which in turn is hydraulically pumped by a reciprocating pump. o Disadvantages Produces a pulsed flow which is damped because pulses appear as baseline noise on the chromatograph. This can be overcome by use of dual pump heads or elliptical cams to minimize such pulsations. 6pump head moter and cam plunger # \check valve tin Mobile phase plunger seal° ° ° Solvent is pumped back and forth by a motor driven piston Two ball check valves which open & close which controls the flow The piston is in direct contact with the solvent Small internal volume 35-400)L High output pressure up to 10,000 psi Ready adaptability to gradient elution and constant flow rate valves Reciprocating SolventFLOWRATE ft LS A A. Single-piston pump with slow filling cycle C. A dual-piston pump with rapid filling cycles and oferate 180° out of phase. TIME “ Schematic representation of solvent flow from© Advantages: ° Have small eral volume of 35-400uL © Higher output pressures up to 10,000 psi. © Adaptability to gradient elution. © Large solvent capacities & constant flow rates. © Largely independent of column back pressure & solvent viscosity.PNEUMATIC PUMPS © In this pumps, the mobile phase is driven through the column with the use of pressure produced from a gas cylinder. © Ithas limited capacity of solvent © Due to solvent viscosity back pressure may develop. REUY y From Solvam Resevair To Sampie vane = Non-Retun vane senn vane‘SAMPLE INJECTOR SYSTEM ‘ices are available either for manual or auto (i) Septum Injector (ii) Stop Flow Injector (iii) Rheodyne InjectorSEPTUM INJECTOR: ° These. are: used \jecting the sample through a rubber septum. - commonly used , since the se ii. Stop Flow « In this type the flow of mobile phase is stopped for a while & the sample is injected through a valve. Hi WAIl, RHEODYNE INJECTOR © It is the most popular injector and is widely used. © This has a fixed volume of loop, for holding sample until its injected into the column, like 204L, 504 or more. © Through an injector the sample is introduced into the column. © The injector is positioned just before the inlet of the column.| FIGURE 28-6 A sampling loop for LC. With the valve handie as shown on the left, the loop Is filled from the syringe, and the mobile phase flows from pump to col- umn. When the valve Is placed In the position on the right, the loop Is inserted between the pump and the column so ‘that the mobile phase sweeps the sample onto the col- wrmn. (Courtesy of Bectonan-Coutlter, Inc.)HPLC Auto INJECTORS Inside of SIL-20ACCOLUMN 1, Precolumn 1.1 It contains a packing chemically identical to that in analytical column. 1.2 Mainly used to remove the impurities from the solvent and thus prevents contamination of the analytic column, it can protect analytical column. ‘enrence 1.3 It is also called as guard column or protective column. it is having large particle size. awa 1.5 It is having short length of 2 10 10 om, so does not affect separation. ‘ani coerce oarernh ae 2. Analytical column = 2.1 The success or failure of analysis depends upon choice of column, hiieace 2.2 Actual separation is carried out here, 2.3 Stainless -steel tube 2.4 size — length -25 to 100 cm 2.5 Internal diameter — 2 to 4.6 mm 2.6 Column is filled with small particles 5 — 10 micron. The solid support can be silica gel, alumina. 2.7 The separation is result of different components adhering to or diffusion into the packing particles whi the mobile phase is forced through column.2.8 C8 and C18 columns are considered as examples of reversed phase liquid. chromatography (RP). 2.9 The stationary phase here is seen as a thin film of non-polar liquid phase that has been designed to be chemically similar to an inert material (Silica gel particles). (0 The non-polar layer is chemically linked to the silica particles rea with the polar silanol groups on the stationary phase surface and so rendering them less polar or non-polar. 2.11 The difference between the two. columns will be in the length of the carbon- chain attached to the silica surface. 212. Accordingly C8 hple columns have packing material composed of silica particles attached to C8 carbon units. 18 will, of course, have packing materials coated with C18 hydrophobic 2.14 Categorically both are reversed phase but C18 columns will definitely be ‘more "hydrophobic rather than the C8 columns. eowhich a potential difference solute) + Conduc Light scattering + Mass spectrometry (HPLC-MS)SELECTION OF DETECTORS Detectors Type of compounds can be detected La Compounds with chromophores, such as aromatic rings or aN multiple alternating double bonds. RE Fluorescent compounds, usually with fused rings or highly i conjugated planar system. Priel Charged compounds, such as inorganic ions and organic i acid. ECD For easily oxidized compounds like quinones or amines. RID & ELSD For compounds that do not show characteristics usable by the other detectors, e.g. polymers, saccharides.TYPES OF HPLC DETECTORS L Name | Advantage Disadvantage Works w/all molecules Non-specific; comy samples; absorption wavelength DAD Works for ail wavelengths | High LOD Fluorescence | Very specific;low LOD Not everything uoresces. | ) IR Works w/all molecules Many solvents IR active Refractive Index | Works w/nearly all Temperature sensitive; molecules high LOD ‘Scattering Uniform response; Non-specific; interference 5ng/25mL LOD from solvent _Electrochemical Commercially available Rented high LOD Low LOD; analyte identificationIDEAL DETECTOR PROPERTIES > High Sensitivity > Un a, isin or predictable. specific icity perature & mobile phase ion | ® Reliable and ‘easy to use » No single detector fits all these criteria3 Fixed Du Aes tos Selon | Aitcrie s evaree 1 provide approprite wavelengthTo waste Quartz windowsREFRACTIVE INDEX (RI) DETECTOR 9 Detection occurs when the light is bent due to samples eluting from the columns, and this is read as a disparity b/w the two channels. & [tis not much used for analytical applications because of low sensitivity & specificity. a When a solute is in the sample compartment, refractive index changes will shift the light beam from the detector, f S Mirror Sample ew —- Amplifier and power supply Referencea a PHOTODIODE ARRAY (PDA) A photodiode array (PDA) is a linear array of discrete photodiodes on an integrated cireuit (IC) chip. Allows a range of wavelengths to be detected simultaneously. In this regard it can be thought of as an electronic version of photographic film. Array detectors are especially useful for recording the full Uv- vis is a absorption spectra of samples that are rapidly passing through a sample flow cell, such as in an HPLC detector. PDAs work on the same principle as simple, Photovoltaic detector similar to UV detector, non destructive 190-600 nm for quantization & identification Spectra is 3D, Response vs time vs WL. Light SensorsFLUORIMETRIC DETECTORSSample cell Ecita tion monochromator monochromator Photo detectorLight Source i 1 on 1 ' ‘ eee ieee SS ae | / PhotodetectorRECORDERS AND INTEGRATORS Recorders are used to record responses obtained from the detectors after amplification, if necessary. ‘They record the baseline & all the peaks obtained with respect tot time. Retention time can be found out from this recordings, but area under curve cannot be determined. ‘The Integrators are improved versions Pe “recorders with some data processing capabi ities. ‘They can record the individual peaks with retention time, height, width of peaks, peak yercentage area, etc. i ides more | information on peaks than recorders. we days computers and printers are used for recording and processing the obtained data & for controlling several operations.COMPARISION BETWEEN HPTLC AND HPLC aiked a 1 | High Performance Thin —_ Layer | High Performance Liquid | Chromatography Chromatography 2 | | Planar Chromatography ‘| Column Chromatography ig | Principle. based on Adsorption | Principle is based on Adsorption and | Chromatography Partition Chromatography | 4 | Simultaneous method for test as well as | Not simultaneous method for test as reference material well as reference material 5 jit is simple, rapid, reproducible method | itis Tedious method ‘| Sample preparation is simple Sample preparation is complex 6 7 [Limited Fiexibitty "| Extreme Flexibility | 8 | Semiautomatic Technique ‘Automatic (Instrumental) Technique 9 il Delehtinalion of Sutace Area "| Determination of Retention TimeCOMPARISION BETWEEN GC AND HPLC |e resolton High resolution i Determination of Volatile inati compounds Volatile CompoundsCOMPARISION BETWEEN HPLC AND UPLC Pe cy a meee UPLC Assay AQUITY UPLC BEH count 'XTerra,C18,50 « 4.6mm 18,50 *2.1mm Particle size 4m particles 1.7um particles Flow rate ) 3.0 ml per min 0.6 ml per min Injection volume 20 pl |3 HI partial loop fill or 5 l full loop fill Total run time 40min 4.5 min Theoretical Plate | 7500 | 2000Lele s ed heey ‘UPLC Assay Lower limit of | 0.2 ug/mi 0.054,I/m! 2 ee Total solvent | Acetonitrile:10.5ml, Acetonitrile:0.53ml, | consumption water:21ml water:0.66ml Delay volume 1720 yl oul | Column temperature |30 ot 65 °C Maximum back 35-40 Mpa 103.5 Mpa pressure jless more | Resolution | Less High ‘ii development | High Loweo tb te oe ee tee ee + APPLICATION Drug Discovery Clinical Analysis Proteomics Forensic Chemistry Drug Metabolism study Environmental chemistry Diagnostic studi Cosmetic analysis Determination of Green Florescent Protein Structural Determination Pharmaceutical Applications Identification of Bile Acid Metabolite Clinical Applications Biochemical Genetics qualitative and quantitative analysis ‘Therapeutic Drug Monitoring* + * REFERENCE Knox JH, Done JN, Fell AF et al, High-Performance Liquid Chromatography. Edinburgh: Edinburgh University Press: 1978 Simpson CF, Practical High-Performance Liquid Chromatography. London: leyden and Son; 1976. Pungor E. A Practical Guide to Instrumental Analysis. Boca Raton: CRC Press; 1995. Moffat AC, Osselton MD, Widdop B. Clarke's Analysis of Drugs and Poisons. London: Pharmace utical Press; 2004. Y. Shen and R.D, Smith, Electrophoresis 23 (2002) pp. 3106-3124. D.A. Skoog and D.M. West, Fundamentals of Analytical Chemistry, 3" edit., pp. 643-649. D. Sievers, ME, Swartz and B.J. Murphy, G.1.T. Laboratory Journal (2004) pp.43-45.(2004) pp. 503-504 L.A. Colon, J.M. Cintron, J.-A. Anspach, A.M. Fermier and K.A. Swinney, The Analyst 129 LR. Snyder, J.J. Kirkland and J.L. Glajeh, Practical HPLC Method development, 2nd edit., pp. 41 43. J.C. Giddings, Dynamics of Chromatography: Part I Principles and Theory (Marcel Dekker, Inc., New York: 1965) pp. 25-26, 229-230, J.M. Cintron and L.A. Colon, The Analyst 127 (2002) pp, 701-704. A.D. Jerkovieh, 1S. ‘oO and J.W. Jorgenson, LOGC 21 (uly 2003) pp. 600-611,