HPLC

HPLC

1. From Liquid Chromatography to High Performance Liquid Chromatography Higher degree of separation!  Refinement of packing material (3 to 10 µ m) Reduction of analysis time!  Delivery of eluent by pump  Demand for special equipment that can withstand high pressures The arrival of high performance liquid chromatography !

2. What is HPLC? HPLC is really the automation of traditional liquid chromatography under conditions which provide for enhanced separations during shorter periods of time, utilizing very small particles, small column diameters, and very high fluid pressures.

3. HPLC principle It is a technique by which a sample mixture is separated into components for identification, quantification and purification of mixtures

4. 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.

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

6. 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 and 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.

7. Column The heart of a HPLC system is the column . The column contains the particles that contains the stationary phase . The mobile phase is pumped through the column by a pump

8. COMPOSITION OF A LIQUID CHROMATOGRAPH SYSTEM Degasser Solvent Reservoir Solvent Delivery System (Pump) Pre gaurd column Sample Injector System Column Detectors (Diode Array) Recorder (Data Collection)and Integrators Waste Collector

9. Pump The role of the pump is to force a liquid (mobile phase) through the liquid chromatograph at a specific flow rate. A pump can deliver a constant mobile phase composition (isocratic) which the mobile phase composition remains unchanged during the analysis. or (gradient ) in which the mobile phase changed during the analysis..

10. Pump 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 (5-10 µ ), 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. Mixing unit is used to mix the solvents in different proportions and pass through the column.

11. Injector • The injector serves to introduce the liquid sample into the flow stream of the mobile phase. May be auto-sampler or manual

12. Septum injector

13. Syringe

14. HPLC columns The column is one of the most important components of the HPLC chromatograph. Because the separation of the sample components is achieved when those components pass through the column.

15. Column has a diameter of 3 to 5mm Normally, columns are filled with silica gel because its particle shape, surface properties, and pore structure help to get a good separation. Silica is wetted by nearly every potential mobile phase, is inert to most compounds and has a high surface activity which can be modified easily with water and other agents. Silica can be used to separate a wide variety of chemical compounds, and its chromatographic behavior is generally predictable and reproducible .

16. Picture of an HPLC column

17. Bonded Phases C-2 Ethyl Silyl -Si-CH 2 -CH 3 CN Cyanopropyl Silyl -Si-(CH 2 ) 3 -CN C-18 Octadecyl Silyl -Si-(CH 2 ) 17 -CH 3 C-8 Octyl Silyl -Si-(CH 2 ) 7 -CH 3

18. 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 Columns Characteristics

19. Selectivity Factor K’ values tell us where bands elute relative to the void volume. These values are unaffected by variables such as flow rate and column dimensions. The value tell us where two peaks elute relative to each other. This is referred to as the selectivity factor or separation factor.

20. Selectivity factor,  α  , which describes the separation of two species ( A and B ) on the column. α  = k 'B / k 'A Where k’ is the retention factor

21. Sample Preparation Dissolve the sample in the mobile phase or in a solvent weaker than the mobile phase . The sample volume should be kept as small as possible. 47 Sample in Mobile Phase Sample in Stronger Solvent

22. U.V detectors : Based on electronic transitions within molecules. Most common type of detector for LC Fixed wavelength, Hg lamp 254 nm (π => π*) Tunable wavelength, selectable for specific wavelengths, monochromators or filters. Still limited to single wavelengths. Solvent limitations with UV- vis abs. Detectors Z-shape, flow-through cell.

23. Solvent UV-Cutoff/Transparency 53 Solvent UV Cutoff (nm) Acetonitrile 190 Water 190 Cyclohexane 195 Hexane 200 Methanol 210 Ethanol 210 Diethyl Ether 220 Dichloromethane 220 Chloroform 240 Carbon Tet 265 Tetrahydrofuran 280 Toluene 285 UV cutoff is the wavelength at which absorbance equals 1, measured in a 1 cm cell with air as a reference.

24. Refractive index detectors: Nearly universal but poor detection limit Passes visible light through 2 compartments, sample &reference. When the solvent composition are the same the light passed through the compartments the light beam that passes through is recorded as zero. When a solute is in the sample compartment, refractive index changes will shift the light beam from the detector. Limit of detection (LOD) 10 ng of solute

25. Fluorescence detectors: Review - based on emission of excited state molecules. Fluorescing species or fluorescent derivatives

26. Electro chemical detectors: Based on amperometric response of analyte to electrode usually held at constant potential. If the analyte is electro active, can be highly sensitive since response is based on a surface phenomenon rather than a solution bulk property (e.g. UV- V is absorbance) simplicity, convenience and wide-spreading application Thin-layer flow cell of Teflon : 50μm thick, 1 ~ 5 μL volume Indictor E: Pt, Au, C Multi-electrode: simultaneous detection or sample purity indication.

27. Evaporative light scattering detectors: Responds to any analyte that is significantly less volatile than the mobile phase. Eluate is mixed with N2(g) and forms a fine mist. Solvent ( m.p .) evaporates leaving fine particles of analyte . The particles themselves are detected by light scattering. Response is proportional to analyte mass.

28. IR detectors: F ilter instrument or FTIR Similar cell as that of UV-Vis Limit: no suitable solvent, special optics FT-IR allows for spectrum records of flowing systems analogous to the diode array system. Water/alcohols can be major interferences to solute detection LOD 100 ng Photo diode array detector: This is a recent one which is similar to U.V detector which operates from 190-600 nm. Allows for the recording of the entire spectrum of each solute as it passed through the diode array detector. The resulting spectra is a 3-D or three dimensional plot of Response Vs Time Vs Wave length.

29. Photo diode array detector

30. WHAT AFFECTS SYSTEM Column Parameters Column Material Deactivation Stationary Phase Coating Material Instrument Parameters Temperature Flow Signal Sample Sensitivity Detector

31. 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. Negative peaks occur if mobile phase absorbance is larger than sample absorbance. Peak doubling occurs due to the co- elution of interfering compound, column over load, channeling in column. Base line spikes occur due to the air bubbles in the mobile phase and/or detector, column deterioration . Peaks:

32. TYPES OF HPLC TECHNIQUES : A. Based on modes of chromatography 1. Normal phase mode 2.Reverse phase mode B. Based on principle of separation 1. Adsorption chromatography 2. Ion exchange chromatography 3. Ion pair chromatography 4.Size exclusion(or)Gel permeation chromatography 5. Affinity chromatography 6. Chiral phase chromatography

33. C. Based on elution technique 1. Isocratic separation 2. Gradient separation D. Based on the scale of operation 1. Analytical HPLC 2. Preparative HPLC E. Based on the type of analysis 1. Qualitative analysis 2. Quantitative analysis

34. Several types of column Normal phase Reverse phase Size exclusion Ion exchange

35. 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

36. 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.

37. Common Reverse Phase Solvents Methanol CH 3 OH • Acetonitrile CH 3 CN • Tetrahydrofuran • Water H 2 O

38. 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.

39. 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.

40. RECORDERS 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.

41. 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. INTEGRATORS

42. PARAMETERS USED IN HPLC 1.Retention time 2.Retention volume 3.Seperation factor 4. Resolution 5. Height Equivalent to a Theoretical Plate (HETP) 6. Asymmetry factor

43. 1.Retention time: Retention time is the difference in time between the point of injection and appearance of peak maxima. It is also defined as time required for 50% of a component to be eluted from a column. It is measured in minutes and seconds. 2.Retention volume : Retention volume is 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

44. 4 . Resolution: Resolution is the measure of extent of separation of 2 components and the base line separation achieved. Rs = 2 (Rt 1 -Rt 2 ) / w 1 +w 2 5. Height Equivalent to a Theoretical Plate (HETP): A theoretical plate is an imaginary or hypothetical unit of a column where distribution of solute between stationary phase and mobile phase has attained equilibrium. It can also be called as a functional unit of the column.

45. 6. Asymmetry factor : A chromatographic peak 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).

46. DERIVATISATION IN HPLC: In order to increase the detectability of various classes of compounds ( for which sensitive detectors are not available ) derivatisation is carried out in HPLC. A good amount of work has been performed on the labelling of compounds with chromophores and flurophores for detection using UV spectrometers and fluorimeters respectively. There are 2 important types of derivatisation . These are, 1. Pre column derivatisation 2. Post column derivatisation

47. PRE COLUMN DERIVATISATION : In pre column derivatisation there are no restrictions on the solvents, reagents, or reaction rates chosen and excess of reagents can be removed before the injection. However , artifact formation, if present, can be checked by positive identification of the eluted peaks. For example, in the derivatisation of a triketone with more than one functional group capable of being derivatised there is a possibility of range of derivatives being formed from one solute.

48. Examples of pre column derivatisation to form UV chromophores include the treatment of ketosteroids with 2,4, DNP Benzoylation of hydroxy steroids Esterification of fatty acids. Similarly, fluorophores have been introduced into amino acids, biogenic amines, and alkaloids by treatment with dansyl chloride .

49. POST COLUMN DERIVATISATION It is carried out on the separated solutes as they emerge from the chromatographic column. In HPLC, this places serious restriction on the derivatisation reactions, because dilution of the eluent peak must be minimized. Consequently, very fast reactions must be used and the reagents and mobile phase must be compatible. Examples of post column derivatisation reactions for use with UV detectors include: Reaction of amino acids with ninhydrin and fluorescamine . Reaction of fatty acid with ortho nitro phenol. Reaction of ketones with 2, 4, DNP. Thermal or acid treatment of carbohydrates. An oxidation detector for the fluorimetric analysis of carbohydrates in body fluids using Ce (III) flourescence has also been reported .

50. Parameters of HPLC 1- Qualitative analysis the most common parameter for compound is retention time (the time it takes for that specific compound to elute from the column after injection)

51. Capacity Factor (k’): Is a measure for the position of a sample peak in the chromatogram. k’ = (t R1 -t o )/t o

52. 2- Quantitative Analysis The measurement of the amount of compound in a sample (concentration) 1.determination of the peak height 2.determination of the peak area

53. Resolution (R S ) Resolution of a column provides a quantitative measure of its ability to separate two analytes R s = 2(T R2 - T R1 ) / W 2 +W 1

54. The factors which influence the HPLC performance Internal diameter of column - the smaller in diameter, the higher in sensitivity 2. Pump pressure - the higher in pressure, the higher in separation 3. Sample size 4. The polarity of sample, solvent and column 5. Temperature - the higher in temperature, the higher in separation

55. Chromatograms A B Conditions: A: 150mm x 4.6mm, 5µ. Flow Rate: 1.5 mL/min Conditions: B: 50mm x 4.6mm, 3µ. Flow Rate: 3.0 mL/min

56. HPLC CHROMATOGRAM

57. Uses of HPLC 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. Federal and state regulatory agencies use HPLC to survey food and drug products, for identifying confiscated narcotics or to check for adherence to label claims.

58. Application of HPLC 1. Pharmaceuticals industry To control the drug stability Quantity of drug determination frompharmaceutical dosage forms, ex. Paracetamol determination in panadol tablet Quantity of drug determination from biological fluids, ex: blood glucose level 2. Analysis of natural contamination - Phenol & Mercury from sea water .

59. 3. Forensic test Determination of steroid in blood, urine & sweat. Detection of psychotropic drug in plasma Forensic analysis of textile dyes. Determination of cocaine and metabolites. 4. Clinical test Monitoring of hepatic cirrhosis patient through aquaporin 2 in the urine. Quantification of DEET in Human Urine. Analysis of antibiotics. Detection of endogenous neuropeptides in brain extracellular fluids.

60. 5. Food and essence manufacture Sweetener analysis in the fruit juice Preservative analysis in sausage. 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 aspartamine in the presence of glucose and vanillin.

61. 6. 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 .

62. ADVANTAGES OF HPLC Separations fast and efficient (high resolution power) Continuous monitoring of the column effluent. It can be applied to the separation and analysis of very complex mixtures. Accurate quantitative measurements. Repetitive and reproducible analysis using the same column. Adsorption, partition, ion exchange and exclusion column separations are excellently made.

63. ADVANTAGES OF HPLC 7. High separation capacity, enabling the batch analysis of multiple components. 8. Superior quantitative capability and reproducibility. 9. Moderate analytical conditions. Unlike GC, the sample does not need to be vaporized. 10. Generally high sensitivity. 11. Low sample consumption. 12. Easy preparative separation and purification of samples. 105

64. 13. HPLC is more versatile than GLC in some aspects , because it has the advantage of not being restricted to volatile and thermally stable solute and the choice of mobile and stationary phases is much wider in HPLC 14. Both aqueous and non aqueous samples can be analyzed with little or no sample pre treatment 15. A variety of solvents and column packings are available, providing a high degree of selectivity for specific analyses. 16. It provide a means for determination of multiple components in a single analysis.

65. Disadvantages 1. Need a skill to run the instrument. 2. Solvents consumption is more.