HPLC Principle,Instrumentation and Application


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  • High Performance Liquid Chromatography ALAKESH PRADHAN COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY School of Industrial Fisheries M.Sc IInd Sem.
  • Overview: Chromatography and its principle Liquid chromatography High Performance Liquid Chromatography ( HPLC ) The components of the high performance liquid chromatograph (HPLC). The separation process. The chromatogram.
  • Latest instrument
  • Background Chromatography and its Principle Chromatography is a separation technique which is used to separate a mixture of compounds into its individual components based on certain physical and chemical properties.
  • Some important terms: Mobile phase: The solvent system which carries the mixture to be separated. Stationary phase: Immobile surface which is particulate in nature. This is the region over which the compound gets separated.
  • Principle: The process involves the interaction of the compounds in the analyte (which travels along with a mobile phase) across an immobile surface (stationary phase). The compounds bind at specific regions of stationary phase based on certain physical and chemical properties. These bound molecules are then eluted with a suitable buffer and the same are collected with time. These are – Polarity Charge Molecular weight Present of functional group
  • Introduction HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution. 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.
  • Continued… What is Liquid Chromatography? Liquid chromatography is a separation technique that involves: • the placement (injection) of a small volume of liquid sample • into a tube packed with porous particles (stationary phase) • where individual components of the sample are transported along the packed tube (column) by a liquid moved by gravity. The components of the sample are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles. The separated components are collected at the exit of this column and identified by an external measurement technique , such as a spectrophotometer that measures the intensity of the color , or by another device that can measure their amount.  Note: The modern form of liquid chromatography is now referred to as “flash chromatography
  • Principles of Liquid Chromatography
  • Terminologies for HPLC HPLC : High Performance Liquid Chromatography : High Pressure LC Now, before we go in depth of principle, lets have a basic look at few terms as follows: Resolving Power: The extent of separation of the compounds present in the mixture across the column. Theoretical plates : An imaginary division of the column into equilength plates.
  • Principles of HPLC Principle: The table shows relation between various parameters of HPLC. Trendline: Stationary phase have small particulate size and high surface areas. Columns: 20 cm or less Mobile phase pumped at high pressures of 200Bar, 3000 psi. Flow rates: 1-3 cm3 per min Column length No. of theoretical plates per unit area Resolving power Column length Particle size Surface area
  • What is HPLC? HPLC is a separation technique that involves: •the injection of a small volume of liquid sample •into a tube packed with tiny particles (3 to 5 micron ( μm ) in diameter called the stationary phase) •where individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump. These components are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles. These separated components are detected at the exit of this tube (column) by a flow-through device (detector) that measures their amount. An output from this detector is called a “liquid chromatogram”.  In principle, LC and HPLC work the same way except the speed , efficiency, sensitivity and ease of operation of HPLC is vastly superior. s
  • HPLC system Flow chart of HPLC mechanism
  • Varian 9010 Solvent Delivery System Rheodyne Injector %A %B %C Flow Rate Pressure {H2O} {MeOH} (mL/min) (atmos.) Ready Ternary Pump A C B from solvent reservoir Column to detector to column through pulse dampener to injector through pump load inject
  • Picture of HPLC instrument
  • COMPOSITION OF A LIQUID CHROMATOGRAPH SYSTEM Solvent Solvent Delivery System (Pump) Injector Sample Column Detectors Waste Collector Recorder (Data Collection)
  • Instrumentation of HPLC ( Describing the 5 major components and their functions….) 1 2 3 4 5 Solvent reservoirs and degassing Not shown here 1 – Pump 2 – Injector 3 – Column 4 – Detector 5 – Computer
  • Pump: •The role of the pump is to force a liquid (called the mobile phase) through the liquid chromatograph at a specific flow rate, expressed in milliliters per min (mL /min). •Normal flow rates in HPLC are in the 1-to 2-mL/min range. •Typical pumps can reach pressures in the range of 6000-9000 psi (400-to 600-bar). •During the chromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition (gradient).
  • Pump Module–types: Isocratic pump - Delivers constant mobile phase composition; •solvent must be pre-mixed; •lowest cost pump Gradient pump - Delivers variable mobile phase composition; •can be used to mix and deliver an isocratic mobile phase or a gradient mobile phase –Binary gradient pump –delivers two solvents –Quaternary gradient pump –four solvents
  • Injector: •The injector serves to introduce the liquid sample into the flow stream of the mobile phase. •Typical sample volumes are 5-to 20-microliters (μL). •The injector must also be able to withstand the high pressures of the liquid system. •An auto sampler is the automatic version for when the user has many samples to analyze or when manual injection is not practical .
  • Sample Injection ……how is a sample actually put into an LC system Manual Injector: 1.User manually loads sample into the injector using a syringe 2.and then turns the handle to inject sample into the flowing mobile phase… which transports the sample into the beginning (head) of the column, which is at high pressure Auto sampler: 1.User loads vials filled with sample solution into the auto sampler tray (100 samples) 2.and the auto sampler automatically a. measures the appropriate sample volume, b. injects the sample, c. then flushes the injector to be ready for the next sample, etc., until all sample vials are processed …… …….for unattended automatic operation
  • * Manual Injectors Front View Inject Rear View Load - Inject Sample Loop
  • * Automatic Injectors
  • Column: • Considered the “heart of the chromatograph” the column’s stationary phase separates the sample components of interest using various physical and chemical parameters. •The small particles inside the column are what cause the high back pressure at normal flow rates. •The pump must push hard to move the mobile phase through the column and this resistance causes a high pressure within the chromatograph.
  • Several Column Types ( can be classified as) Normal phase Reverse phase Size exclusion Ion exchange
  • Normal phase In this column type, the retention is governed by the interaction of the polar parts of the stationary phase and solute. For retention to occur in normal phase, the packing must be more polar than the mobile phase with respect to the sample
  • * STATIONARY PHASES (NORMAL POLARITY) Silica or alumina possess polar sites that interact with polar molecules. Most polar…….Least polar Components elute in increasing order of polarity. Polar Group silica
  • Reverse phase In this column the packing material is relatively nonpolar and the solvent is polar with respect to the sample. Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase. Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water. Common Reverse Phase Solvents – Methanol Acetonitrile Tetrahydrofuran Water CH3OH CH3CN H2O
  • * STATIONARY PHASES (REVERSE POLARITY) If the polar sites on silica or alumina are capped with non-polar groups, they interact strongly with non-polar molecules. Most non-polar…….Least non-polar Components elute in decreasing order of polarity. C18 phase silica
  • Size exclusion In size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size. Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores. The large molecules elute before the smaller molecules.
  • * STATIONARY PHASES (SIZE EXCLUSION) Size exclusion gels separate on the basis of molecular size. Individual gel beads have pores of set size, that restrict entry to molecules of a minium size. Larger molecules…….Smaller molecules Large molecules elute fast (restricted path), while small molecules elute slowly (long path length)
  • Ion exchange In this column type the sample components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase. Separations are made between a polar mobile liquid, usually water containing salts or small amounts of alcohols, and a stationary phase containing either acidic or basic fixed sites.
  • * STATIONARY PHASES (CATION EXCHANGE) Silica is substituted with anionic residues that interact strongly with cationic species (+ve charged) Most +ve…….Least +ve +ve charged species adhere to the support and are later eluted with acid (H+) Cations exchange Na+ silica
  • * STATIONARY PHASES (ANION EXCHANGE) Silica is substituted with cationic residues that interact strongly with anionic species (-ve charged) Most -ve…….Least -ve -ve charged species adhere to the support and are later eluted with acid (H+) Anions exchange Cl- silica
  • HPLC Columns Within the Column is where separation occurs. Key Point –Proper choice of column is critical for success in HPLC Materials of construction for the tubing Stainless steel (the most popular; gives high pressure capabilities) Glass (mostly for biomolecules) PEEK polymer (biocompatible and chemically inert to most solvents Packing material: 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. According to the mode of HPLC , they are available in different size , diameters, pore size or they can have special materials attached ( such as antigen or antibody ) for immuno affinity chromatography.
  • * Modes of High Performance Liquid Chromatography
  • Types of columns in HPLC: Guard Column Fast Column Preparative(i.d. > 4.6 mm; lengths 50 –250 mm) Capillary(i.d. 0.1 -1.0 mm; various lengths) Nano(i.d. < 0.1 mm, or sometimes stated as < 100 μm) Analytical[internal diameter (i.d.) 1.0 -4.6-mm; lengths 15 –250 mm]
  • Guard Column These are placed anterior to the separating column. This serves as protective factor. They are dependable columns designed to filter or remove : Particles that clog the separation column Compounds and ions that could ultimately cause “ Baseline drift ”, decreased resolution, decreased sensitivity and create false peaks. These columns must be changed on a regular basis in order to optimize their protective function.
  • Fast Column One of the primary reasons for using these column is to obtain improved sample output ( amount of compound per unit time). Fast column are designed to decrease the time of chromatographic analysis Here internal diameter is same but length is short and packed with smaller particles , that are 3 μm diameter. Advantages- Increased sensitivity Decreased analysis time Decreased mobile phase usage Increase reproducibility
  • Capillary Column It is also known as micro columns It has a diameter much less than a millimeter and there 3 types: Open tubular Partially packed Tightly packed They allow the user to work with nanoliter sample volume , decreased flow rate and decreased solvent usage volume , led to cost effectiveness
  • Preparatory Column Used when objective is to prepare bulk ( milligrams) of sample for laboratory preparatory application. It has usually a large column diameter , which is designed to facilitate large volume injections into the HPLC system
  • Detector: • The detector can see (detect) the individual molecules that come out (elute) from the column. •A detector serves to measure the amount of those molecules so that the chemist can quantitatively analyze the sample components. •The detector provides an output to a recorder or computer that results in the liquid chromatogram(i.e., the graph of the detector response).
  • HPLC Detectors
  • * Common HPLC Detectors UV-VIS Diode Array Multiple Wavelength Variable Wavelength Mass Spectrometers Refractive Index Fluorescence Light Scattering Electrochemical Radioactivity Conductivity
  • * UV-Vis Detectors Principles: The fraction of light transmitted through the detector cell is related to the solute concentration according to Beer’s Law. Characteristics: Specific, Concentration Sensitive, good stability, gradient capability. Special: UV-Vis Spectral capability (Diode Array Technology ).
  • * Fluorescence Detection
  • * Electrochemical Detectors Gold for carbohydrates. Platinum for chlorite, sulfate, hydrazine, etc. Carbon for phenols, amines. Silver for chloride, bromide, cyanide.
  • Variable UV/Vis Detector ABS AUFS l RunTime EndTime 0.001 2.000 238 0.00 min 10.0 min Ready
  • Computer: • Frequently called the data system, The computer not only controls all the modules of the HPLC instrument but it takes the signal from the detector and uses it to: 1. determine the time of elution (retention time) of the sample components (qualitative analysis) and 2. the amount of sample ( quantitative analysis) .
  • Varian 9060 Polychrom Detector UV Spectrum Chromatogram Reset Ready UV Spectrum {shows full UV abs.} Chromatogram {shows peaks, Rt} ABS. ABS. UVmax UVmax Rt Rt
  • HPLC is optimum for the separation of chemical and biological compounds that are non-volatile . NOTE: If a compound is volatile (i.e. a gas, fragrance, hydrocarbon in gasoline, etc.), gas chromatography is a better separation technique . Typical non-volatile compounds are:  Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol)  Salts like sodium chloride and potassium phosphate  Proteins like egg white or blood protein  Organic chemicals like polymers (e.g. polystyrene, polyethylene)  Heavy hydrocarbons like asphalt or motor oil  Many natural products such as ginseng, herbal medicines, plant extracts  Thermally unstable compounds such as trinitrotoluene (TNT), enzymes What is HPLC used for ? Separation and analysis of non-volatile or thermally unstable compounds
  • Separation Technique
  • * How can We Analyze the Sample? For example: Carbohydrates 1. fructose 2. Glucose 3. Saccharose 4. Palatinose 5. Trehalulose 6. isomaltose
  • * Separations Separation in based upon differential migration between the stationary and mobile phases. Stationary Phase - the phase which remains fixed in the column, e.g. C18, Silica Mobile Phase - carries the sample through the stationary phase as it moves through the column.
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  • * The Chromatogram
  • HPLC used for Qualitative Analysis
  • HPLC used for Quantitative Analysis
  • HPLC uses This technique is used for - chemistry and biochemistry research analyzing complex mixtures purifying chemical compounds developing processes for synthesizing chemical compounds isolating natural products, or predicting physical properties. It is also used in quality control to ensure the purity of raw materials, to control and improve process yields, to quantify assays of final products, or to evaluate product stability and monitor degradation.
  • In addition, it is used for analyzing air and water pollutants, for monitoring materials that may jeopardize occupational safety or health, and for monitoring pesticide levels in the environment.
  • * HPLC Applications
  • References HPLC instrumentation – Agilent Technologies Introduction to HPLC – Agilent Technologies Principles and Technique of Biochemistry and Moleculer Biology – Wilson.Keith and Walker. John HPLC THEORY INTRODUCTION AND INSTRUMENTATION HARDWARE 6th October 2008. L1 - Dr Cristina Legido-Quigley, Lecturer in Pharmaceutical Chemistry (Separation Science) at KCL WEB REFERENCES Skoog, Holler, and Neiman. Principles of Instrumental Analysis. 5th ed. Orlando: Harcourt Brace & Co., 1998. http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htm http://www.chemistry.nmsu.edu/Instrumentation/Lqd_Chroma.html http://weather.nmsu.edu/Teaching_Material/SOIL698/Student_Material/HPLCHP1090/HPLCINJ.HTM http://test-equipment.globalspec.com/LearnMore/Labware_Scientific_Instruments/Analytical_Instruments/Chromatographs/HPLC_Columns
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