0 Bewertungen0% fanden dieses Dokument nützlich (0 Abstimmungen)
28 Ansichten9 Seiten
Liquid chromatography was Developed in last 1960s and early 1970s. It is used for purifying,identifying and quantifying the individual components of the mixture. -Based on conventional liquid column chromatography.
Liquid chromatography was Developed in last 1960s and early 1970s. It is used for purifying,identifying and quantifying the individual components of the mixture. -Based on conventional liquid column chromatography.
Liquid chromatography was Developed in last 1960s and early 1970s. It is used for purifying,identifying and quantifying the individual components of the mixture. -Based on conventional liquid column 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. 2)Liquid-liquid
3)Ion exchange. 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) in contrast to earlier(30-70m) . ii)Design of chromatographic equipments. -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. 2
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: -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
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-(CH 2 ) 3 -O-CH 2 -CH-CH 2 , Licrosorb Diol.
OH OH Si-(CH 2 ) 3 -CN e.g, Bondpak CN
Si-(CH 2 ) 3 -NH 2 , e.g., Polygosil NH 2
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.
5
-Where, R= C 6 H 13 (hexyl), C 8 H 17 (Octyl), C 18 H 37 (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 6
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.
7
-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, however-
i) 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). 8
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
9
b)Stability studies.
c)Therapeutical monitoring, drug metabolism, and pharmacokinetic studies. d)Increasingly is being used for the assay of medicines in natural products, e.g. antibiotics & 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