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High Performance Liquid Chromatography
High Performance Liquid Chromatography (HPLC) is a separation technique that involves injecting a small volume of liquid sample into a column packed with tiny particles. Individual components of the sample are then transported through the column by a mobile phase and separated based on interactions with the stationary phase. These separated components exit the column and are detected, providing a chromatogram. HPLC uses small particle sizes, high column pressures up to 6000-9000 psi, and flow rates of 1-3 mL/min to achieve fast, efficient, and high resolution separations of both volatile and non-volatile compounds.
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High Performance Liquid Chromatography
- 1. High Performance Liquid Chromatography(HPLC) Dr. Ashwani Dhingra Associate Professor GGSCOP, Yamunanagar
- 2. Chromatography Chromatography is aseparation 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.
- 3. Principle • The processinvolves 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 includes :- • Polarity • Charge • Molecular weight • Present of functional group
- 4. Introduction • HPLC isa 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.
- 5. Liquid Chromatography • Liquidchromatography 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".
- 6. Principles of LiquidChromatography
- 7. Principles of HPLC •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 Trendline: Column length No. of theoretical plates per unit area Resolving power Column length Particle size Surface area
- 8. What is HPLC? HPLC isa 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.
- 9. HPLC system Flow chartof HPLC mechanism
- 10. COMPOSITION OF ALIQUID CHROMATOGRAPH SYSTEM • Solvent • Solvent Delivery System (Pump) Injector • Sample Column Detector • Waste Collector • Recorder (Data Collection)
- 11. Instrumentation of HPLC (Describingthe 5 major components and their functions) Solvent reservoirs and degassing 1 – Pump; 2 – Injector; 3 – Column; 4 – Detector; 5 – Computer 1 5 Not shown 2 here 3 4
- 12. Pump • The roleof 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).
- 13. Pump Module–types • Isocraticpump - 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
- 14. Sample Injection Manual Injector: Usermanually loads sample into the injector using a syringe 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: User loads vials filled with sample solution into the auto sampler tray (100 samples) 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
- 15. Manual Injectors Inject Front ViewRear View Sample Loop Load - Inject
- 16. Automatic Injectors Step 1Step 2 Step 3
- 17. • 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. Column
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- 19. Normal phase In this columntype, 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
- 20. STATIONARY PHASES (NORMAL POLARITY) Mostpolar…….Least polar Components elute in increasing order of polarity. O Polar Group HO Si O Silica or alumina possess polar sites that interact with polar molecules. silica
- 21. Reverse phase • Inthis column the packing material is relatively non-polar 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
- 22. STATIONARY PHASES (REVERSE POLARITY) Mostnon-polar…….Least non-polar C g omponents elute in decreasin order of polarity. C18 phase If the polar sites on silica or alumina are capped with non-polar groups, they interact strongly with non-polar molecules. silica Me Me Si O Si O O
- 23. Size exclusion In size exclusionthe 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.
- 24. 25 STATIONARY PHASES (SIZE EXCLUSION) Sizeexclusion 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)
- 25. Ion exchange In this columntype 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.
- 26. STATIONARY PHASES (CATION EXCHANGE) Most+ve…….Least+ve +ve charged species adhere to the support and are later eluted with acid (H+) Silica is substituted with anionic residues that interact strongly with cationic species (+ve charged) Cations exchange Na+ silica O Na O S O
- 27. Silica is substitutedwith 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 Me Me N CH2 Me Cl STATIONARY PHASES (ANION EXCHANGE)
- 28. Types of CompoundsMode Stationary Phase Mobile Phase Neutrals Weak Acids WeakBases Reversed Phase C18, C8, C4 cyano, amino Water/Organic Modifiers Ionics, Bases, Acids Ion Pair C-18, C-8 Water/Organic Ion-Pair Reagent Compounds not soluble in water Normal Phase Silica, Amino, Cyano, Diol Organics Ionics Inorganic Ions Ion Exchange Anion or Cation Exchange Resin Aqueous/Buffer Counter Ion High Molecular Weight Compounds Polymers Size Exclusion Polystyrene Silica Gel Filtration- Aqueous Gel Permeation- Organic Modes of HPLC
- 29. Separations Separation in basedupon 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. Injector Detector Column Solvents Mixer Pumps High Performance Liquid Chromatograph Waste
- 30. Injector Detector Column Solvents Mixer Pumps Chromatogram Start Injection mAU time High PerformanceLiquid Chromatograph Separations
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- 46. Computer • Frequently calledthe 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: • determine the time of elution (retention time) of the sample components (qualitative analysis) and • the amount of sample (quantitative analysis).
- 47. Capillary Column • Itis also known as micro columns. • It has a diameter much less than a millimeter and there 3 types: Open tubular, Partially packed, and Tightly packed. • They allow the user to work with nanoliter sample volume, decreased flow rate and decreased solvent usage volume, led to cost effectiveness. • Guard column is used to remove particular matter and contamination, it protect the analytical column and contains similar packing its temperature is controlled at < 150°C±0.1 °C • As mention before, columns are divided into different types according to their functions (see types of HPLC).
- 48. Preparatory Column USED WHENOBJECTIVE 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
- 49. Detector • The detectorcan 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).
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- 51. Common HPLC Detectors •UV-VIS •DiodeArray •MultipleWavelength •Variable Wavelength •Mass Spectrometers •Refractive Index •Fluorescence •Light Scattering •Electrochemical •Radioactivity •Conductivity
- 52. UV-Vis Detectors Principles: Thefraction of light transmitted through the detector cell is related to the solute concentration according to Beer’s Law. b Log I0 = A = abc I Characteristics: Specific, Concentration Sensitive, good stability, gradient capability. Special: UV-Vis Spectral capability (Diode Array Technology). I0 Detector Flow Cell c I
- 54. Electrochemical Detectors • Goldfor carbohydrates. • Platinum for chlorite, sulfate, hydrazine, etc. • Carbon for phenols, amines. • Silver for chloride, bromide, cyanide.
- 55. Applications Separation and analysisof non-volatile or thermally unstable compounds 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
- 56. PARAMETERS Retention time (RT) Ina chromatogram, different peaks correspond to different components of the separated mixture. Time elapsed between sample introduction and maximum of response, it is the characteristic time it takes for a particular analyte to pass through the system Time taken for the analyte to travel from the column inlet to the point of detection(maximum peak)
- 57. • For assaythe main peak(main component) is considered • For purity test all the peaks are considered Area of peak A= height × base/2 Peak area gives us the concentration of the analyte
- 58. 2.Retention volume Retention volumeis the volume of carrier gas required to elute 50% of the component from the column. It is the product of retention time and flow rate. Retention volume = Retention time × flow rate 3.Seperation factor: Separation factor is the ratio of partition coefficient of the two components to be separated. S=Ka/Kb=(tb-to)/(ta-to) Where to = Retention time of unretained substance Ka, Kb = Partition coefficients of a, b ta, tb = Retention time of substance a, b If there is a big difference in partition coefficient between 2 compounds, the peaks are far apart and the separation factor is more. If the partition coefficient of 2 compounds are similar, then the peaks are closer and the separation factor is less.
- 59. 4. Resolution: Resolution isthe measure of extent of separation of 2 components and the base line separation achieved. Rs = 2 (Rt1-Rt2) / w1+w2 5. Height Equivalent to a Theoretical Plate (HETP): A theoretical plate is an imaginary or hypothetical unit of a column where distri bution of solute between stationary phase and mobile phase has attained equilib rium. It can also be called as a functional unit of the column. A theoretical plate can be of any height, which describes the efficiency of separation. If HETP is less, the column is more efficient. If HETP is more, the column is less efficient.
- 60. • HETP =length of the column/no. of theoretical plates • HETP is given by Van-deemter equation HETP =A+B/u+Cu Where A = Eddy diffusion term or multiple path diffusion which arises due to uniformity of the packing of the column. This ca n be minimized by packing. • B = Longitudinal diffusion term or molecular diffusion. • C = Effect of mass transfer. • u = flow rate or velocity of the mobile phase.
- 61. Efficiency • Efficiency ofa column is expressed by the theoretical plates. n = 16 Rt2/ w2 Where n = no of theoretical plates Rt= retention time w = peak width at base • Rt and w are measured in common units (cm or mm, min or sec). • No of theoretical plates is high, the column is said to be highly efficient. • For GLC, a value of 600/metre is sufficient. But in HPLC, high values like 40,000 to 70,000/ meter are recommended.
- 62. • A chromatographicpeak should be symmetrical about its centre and said to follow Gaussian distribution. But in practice due to some factors, the peak is not symmetrical and shows tailing or fronting. • Fronting is due to saturation of stationary phase and can be avoided by using less quantity of sample. Tailing is due to more active adsorption sites and can be eliminated by support pretreatment. • Asymmetry factor (0.95 to 1.05) can be calculated by AF = b/a (b, a calculated by 5% or 10% of the peak height). • Broad peaks occur due to the more conc. of sample, large injection volume, column deterioration. • Ghost peaks occur due to the contamination of the column, compound from earlier injections. Asymmetry factor
- 63. Chromatogram to - elutiontime of unretained peak tR- retention time - determines sample identity tR mAU Area or height is proportional to the quantity of analyte. Injection time to tR
- 64. HPLC used forQualitative Analysis
- 65. HPLC used forQuantitative Analysis
- 66. Uses This technique isused for - • chemistry and biochemistry research analysis of 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.
- 67. HPLC Applications Chemical polystyrene dyes phthalates Environmental polyaromatic hydrocarbons Inorganicions herbicides Pharmaceuticals tetracyclines corticosteroids antidepressants barbiturates Bioscience proteins peptides nucleotides Consumer Products lipids antioxidants sugars Clinical amino acids vitamins homocysteine
- 68. Dr. Ashwani Dhingra AssociateProfessor GGSCOP, Yamunanagar THANK YOU!