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High Pressure/Performance Liquid Chromatography

-Developed in last 1960s & early 1970s. -Most widely used of all analytical separation techniques. -It is used for purifying ,identifying & quantifying the individual components of the mixture. -Based on conventional liquid column chromatography. 1)Liquid-solid. 3)Ion exchange. 2)Liquid-liquid 4)Size exclusion.

Working principle/Theory i)Adsorption. ii)Partition( normal/reversed phase). iii)Ion exchange. iv)Size exclusion. -Facts that brought about the difference: i)Column packing material & particle size(3-5m) ii)Design of chromatographic equipments.

in contrast to earlier(30-70m) .

-Close packing of small particle in column reduces flow rate. -To maintain suitable flow , high pressure should be applied. -Better and faster separation is result. -Hence the name given High performance or High pressure.

Matches with gas chromatography in the following respects: i)High resolving power. ii)Speed of separation. iii)Continuous monitoring of the column efficiency. iv)Accurate/precise quantitative measurement. v)Reproducible analysis using the same column. vi)Automation.

Versatility over gas chromatography: i. Not limited to volatile & thermally stable sample. ii)Wider choice of mobile & stationery phases. Instrumentation: Mode of operations i)Isocratic ii)Gradient i)Isocratic: One particular mobile phase composition is used through out the analysis ii)Gradient: Mobile phase composition is altered gradually to give gradient elution.

1)Solvent reservoir 2)Pumps 3)Sample injection system. 4)The column. 5)The detector. 6)Recorder 7)Data handling device & microprocessor control.

1)Solvent reservoir: -One or more glass reservoirs( 500ml or more capacity). -Provisions are made to remove dissolved gases & dust from the liquids. -Dissolve gas can lead irreproducible flow rates & band spreading. -Both dust ,bubbles interfere performance of column detector. 2)Pumps: -Required to deliver a constant/pulsate free flow of mobile phase. -Should pressurise the mobile phase from 0.1- 55MPa (14.6-8000psi). -400-1500 psi for analytical work. -Flow rate 0.01-10ml min-1 . -Two types of pumps are in use: a)Screw driven syringe type. b)Reciprocating type. b)Reciprocating type. -It is commonly used. (dual & triple head reciprocating type ~ pulse-free flow) -Pulse causes excessive noise at high levels of sensitivity & low pressure may cripple the detection small quantity of sample.

3)Sample injection system: a)Sample is directly injected into column (through a self-sealing septum). (old technique) b)Sample is deposited in a loop(10-20l)then swept by a valving action into column by the mobile phase. & transferred immediately before the column inlet.

4)Column -High quality stainless steel polished internally to a mirror finish. -Standard analytical column: Int.Dia: 4-5mm, L=10-30cm

-Shorter column: Int.Dia.:4-5mm, L= 3-6cm -Packing material size:3-5m Partition HPLC: a)Normal phase: Mobile phase is less polar , hence the least polar solute is eluted first. -Nowadays polar phases are chemically bonded to silica. Si-(CH2)3-O-CH2-CH-CH2 , Licrosorb Diol. OH OH Si-(CH2)3-CN e.g, Bondpak CN Si-(CH2)3-NH2 , e.g., Polygosil NH2 b)Reversed-phase chromatography: (More polar mobile phase), hence the most polar solute is eluted first -Silica is chemically bonded to less polar functional group through (Si-O-Si-C). -Surface silanol gr. of silica reacts with an organochlorosilane reagent.

-Where, R= C6H13 (hexyl), C8H17 (Octyl), C18H37 (Octadecyl). -The most frequenty used bonded phase is C-18 in pharmaceutical analysis. Retention or elution of the solute is controlled by: (in reversed phase partition chromatography) 1)Composition of mobile phase& flow rate. 2)pH of mobile phase(ion suppression). 3)Ion pairing agent. Ion exchange HPLC -Packing materials are based on the cross-linked polystyrene-divinylbenzene resins or ion exchange residues chemically bonded to silica. Size exclusion HPLC(Gel permeation) -Material used are crossed-linked polystyrene divinylbenzene resins or silica microspheres(5-15m diameter). -Solutes are separated according to their molecular size & shape -Larger molecules are eluted first. 5)Detectors: -are to monitor the mobile phase as it emerges from the column. -able to give a linear response over a wide range concentration range. -Detection of solute depends upon : i)Bulk property ii)solute property Refractive index UV/absorption, Fluorescence, Electrochemical

Types of detectors: i)UV detector(Variable wavelength/ photo diode array) -Most widely used in HPLC. -Normally operates in UV region. -Comprises Light source, A dispersing device to select radiation of appropriate wavelength, flow cell to measure absorbance of eluate, Photomultiplier or photo diode array (PDA) detector for the measurement of intensity of transmitted light. -Both single & double beam instruments are available. -In variable wavelength detector any wavelength along 190-360nm can be selected with the help of grating monochromator. -Photodiode array(multichannel) detector is of the very advanced type. -In this, polychromatic light from UV source is passed through the flow cell.

-The emergent radiation is diffracted by a grating & then falls on an array of photodiodes(1042 diodes). -Each diode receives narrow wavelength band. -A microprocessor scans the array of diodes many times a second. -The spectrum so obtained may be displayed on a screen. ii)Fluorescence detectors: -Sensitivity depends upon the fluorescence property of eluate in cell. iii)Refractive index detector(Differential) -It is the closest approach to the universal detector. -It responds to the bulk property i.e. refractive index of the mobile phase as it leaves the column. -The presence of solute changes the refractive index. -It is not applicable in gradient elution type HPLC. -It is suitable for detection of carbohydrates, alcohols other which do not show other specific property for their specific detection. iv)Electrochemical detector: -Based on standard electrochemical principles, i.e amperometry, voltametry & polarography. -Very sensitive for oxidising & reducing substances at the suitable potential. -Useful in the assay of low level of endogenous catecholamine in biological tissues, pesticides, tryptophan derivative & many drugs. 6)Recorder & 7)Data handling device & microprocessor control -Peak area, height vs. retention time displayed automatically as a chromatogram -Peak area Vs time required for evaluation. -The chromatogram can be stored in computer or be printed as appropriate. Application: -It is impossible to review briefly the range of application of HPLC, howeveri) Qualitative ii) Quantitative i)Qualitative; Identification: a)Comparing the retention time of Sp. & standard reference substance for identification. b)If diode array detector is available then it is possible to obtain a spectrum with Abs.Vs wavelength. This further consolidate the proof gathered in (a).

Detection of the impurities & related substances: -4-epianhydrotetracycling, anhydrotetracycline in tetracycline. -Additional substances in ciprofloxacin & in glimepiride. ii)Quantitative -Chromatography is mainly a resolving technique, -However it is useful in quantitative analysis. Peak area/height concentration. -Peak area is preferred over peak height. -Peak area is relatively independent of mobile phase composition.

Techniques of concentration calculation: a.Direct comparison(single point standardisation. -Chromatograms of sample & standard solution are developed one by one. -Sample concentration is calculated comparing the peak area of the standard solution. b.Calibration by external standards. c.Calibration by internal standards. Quantitative pharmaceutical analysis: a)Quality control testing of drugs & medicines for compliance with specifications. e.g. Omeprazol(RP-18/Phosphate Buffer ) at 302nm , Paracetamol(RP-18/Sodium butane sulphonate in water : methanol: Formic acid) at 243nm

b)Stability studies. c)Therapeutical monitoring, drug metabolism, and pharmacokinetic studies. d)Increasingly is being used for the assay of hormones. -It reduces dependence on biological assays. e)It is also effective in the separation of geometrical isomer & racemates, e.g -E-isomer in Tamoxifen citrate. -In the limitation of Diasteroisomers in Fenoterol HBr medicines in natural products, e.g. antibiotics &