CHROMATOGRAPHYChromatography is a separation technique. These separation techniques aremulti-stage separation methods in which the components of a sample aredistributed between 2 phases, one of which is stationary, while the other ismobile.The stationary phase may be a solid or a liquid supported on a solid or a gel. Thestationary phase may be packed in a column, spread as a layer, or distributed asa film, etc.The mobile phase may be gaseous or liquid or supercritical fluid (solution).The separation may be based on adsorption, mass distribution (partition), ionexchange, etc., or may be based on differences in the physico-chemicalproperties of the molecules such as size, mass, volume, etc.The very common and generally applicable Chromatographic methods are asunder:# Paper chromatography# Thin-layer chromatography# Gas chromatography# Liquid chromatography# size-exclusion chromatography:Size-exclusion chromatography is a chromatographic technique which separatesmolecules in solution according to their size. With organic mobile phases, thetechnique is known as gel-permeation chromatography and with aqueous mobilephases, the term gel-filtration chromatography has been used.The sample is introduced into a column, which is filled with a gel or a porousparticle packing material, and is carried by the mobile phase through the column.The size separation takes place by repeated exchange of the solute moleculesbetween the solvent of the mobile phase and the same solvent in the stagnant(inactive) liquid phase (stationary phase) within the pores of the packing material.# Supercritical fluid chromatography:
Supercritical fluid chromatography (SFC) is a method of chromatographicseparation in which the mobile phase is a fluid in a supercritical or a sub criticalstate. The stationary phase, contained in a column, consists of either finelydivided solid particle, such as silica or porous graphite, a chemically modifiedstationary phase, as used in liquid chromatography, or, for capillary columns, across-linked liquid film evenly coated on the walls of the column.SFC is based on mechanisms of adsorption or mass distribution.Stationary phases Stationary phases are contained in columns like Liquid chromatography(Packed Column) and Gas chromatography (capillary columns).Mobile phases Some Common DefinitionsChromatogram: A chromatogram is a graphical or other representation ofdetector response, effluent concentration or other quantity used as ameasure of effluent concentration, versus time, volume or distance.Idealized chromatograms are represented as a sequence of Gaussianpeaks on a baseline.Retention Time: Retention time of a chromatogram is the time in minutescalculated from the point of injection to maximum peak height or theperpendicular dropped from the maximum of the peak corresponding to thecomponent.Retardation Factor: The retardation factor (RF) (also known as retention factorRf), used in paper/TLC chromatography, is the ratio of the distance from thepoint or zone of application to the centre of the spot and the distance traveled bythe solvent front from the point or zone of application.
b = migration distance of the analyte, a = migration distance of the solvent front.Symmetry Factor: The symmetry factor (As) (or tailing factor) of a peak(Figure shown below) is calculated from the expression:Where,w0.05 = width of the peak at one-twentieth of the peak height,d = distance between the perpendicular dropped from the peak maximumand the leading edge of the peak at one-twentieth of the peak height.A value of 1.0 signifies complete (ideal) symmetry.Number of theoretical plates:
The performance of column is very closely related to its apparent numberof theoretical plate. Depending on the separation technique and itschromatographic response, the number of theoretical plates (N) of acolumn can be calculated from the following expression, where the valuesof tR and wh have to be expressed in the same units (time, volume ordistance).tR = retention time (or volume) or distance along the baseline from the point of injection to the perpendicular dropped from the maximum of the peak corresponding to the component, wh = width of the peak at half-height. The apparent number of theoretical plates varies with the component as well as with the column and the retention time.Resolution:The resolution (Rs) between peaks of 2 components may be calculated fromthe expression:tR1 and tR2 = retention times or distances along the baseline from the pointof injection to the perpendiculars dropped from the maxima of 2 adjacentpeaks,wh1 and wh2 = peak widths at half-height.A resolution of greater than 1.5 corresponds to baseline separation.
The expression given above may not be applicable if the peaks are notbaseline separated.Peak to valley ratio:The peak-to-valley ratio (p/v) may be employed as a system suitabilityrequirement in a test for related substances when baseline separationbetween 2 peaks is not reached (Figure 2.2.46.-3).Hp = height above the extrapolated baseline of the minor peak,Hv = height above the extrapolated baseline at the lowest point of the curveseparating the minor and major peaks.
Signal to Noise Ration:The signal-to-noise ratio (S/N) influences the precision of quantificationand is calculated from the equation:Where,H=height of the peak (Figure 2.2.46.-4) corresponding to the componentconcerned, in the chromatogram obtained with the prescribed referencesolution, measured from the maximum of the peak to the extrapolatedbaseline of the signal observed over a distance equal to 20 times the widthat half-height,h=range of the background noise in a chromatogram obtained afterinjection or application of a blank, observed over a distance equal to 20times the width at half-height of the peak in the chromatogram obtainedwith the prescribed reference solution and, if possible, situated equallyaround the place where this peak would be found.
The limit of detection of the peak (corresponding to a signal-to-noise ratioof 3) is below the disregard limit of the test for related substances.The limit of quantization of the peak (corresponding to a signal-to-noiseratio of 10) is equal to or less than the disregard limit of the test for relatedsubstances.System Suitability:The system suitability tests represent an integral part of the method andare used to ensure adequate performance of the many analyticalprocedures mainly for chromatographic separation techniques. The testsare based on the concept that the equipment, electronics, analyticaloperations and samples to be analyzed constitute an integral system thatcan be evaluated as such.System suitability test includes: Repeatability (Area & Retention Time) Tailing Factor/ Symmetry Factor Column Efficiency/ Number of theoretical plate Resolution (Where applicable) Accepted limit: RSD of Repeatability: Not more than 2 % Tailing Factor: Not more than 2 Number of theoretical Plate: Not less than 2000.
Liquid chromatographyHigh-pressure liquid chromatography (HPLC), sometimes called high-performance liquid chromatography, is a separation technique based on asolid stationary phase and a liquid mobile phase. Separations are achievedby partition, adsorption, or ion-exchange processes, depending upon thetype of stationary phase used. HPLC has distinct advantages over gaschromatography for the analysis of organic compounds. Compounds to beanalyzed are dissolved in a suitable solvent, and most separations takeplace at room temperature.Apparatus— A liquid chromatograph consists of(i) A reservoir containing the mobile phase,(ii) A pump to force the mobile phase through the system at high pressure,(iii) An injector to introduce the sample into the mobile phase,(iv) A chromatographic column,(v) A detector, and(vi) A data collection device such as a computer, integrator, or recorder.Pumping Systems— HPLC pumping systems deliver metered amounts ofmobile phase from the solvent reservoirs to the column through high-pressure tubing and fittings. Modern systems consist of one or morecomputer-controlled metering pumps that can be programmed to vary theratio of mobile phase components, as is required for gradientchromatography, or to mix isocratic mobile phases (i.e., mobile phaseshaving a fixed ratio of solvents).
Injectors— After preparation of sample solutions as per procedure, areinjected into the mobile phase, either manually by syringe or loop injectors,or automatically by auto samplers. The latter consist of a carousel or rackto hold sample vials with tops that have a pierce able septum or stopperand an injection device to transfer sample from the vials to a loop fromwhich it is loaded into the chromatograph.Columns— For most pharmaceutical analyses, separation is achieved bypartition of compounds in the test solution between the mobile andstationary phases.A system consisting of polar stationary phases and nonpolar mobilephases are called normal phase chromatography andA system consisting nonpolar stationary phases and polar mobile phasesare called reverse phase chromatography.The affinity of a compound for the stationary phase, and thus its retentiontime on the column, is controlled by making the mobile phase more or lesspolar.Mobile phase polarity can be varied by the addition of a second, andsometimes a third or even a fourth, component.Stationary phases for modern, reverse-phase liquid chromatographytypically consist of an organic phase chemically bound to silica or othermaterials. Particles are usually 3 to 10 µm in diameter, but sizes may rangeup to 50 µm or more for preparative columns.Small particles thinly coated with organic phase provide for low masstransfer resistance and, hence, rapid transfer of compounds between thestationary and mobile phases.Column polarity depends on the polarity of the bound functional groups,which range from relatively nonpolar octadecyl silane to very polar nitrilegroups.
Columns used for analytical separations usually have internal diameters of2 to 5 mm.Columns may be heated to give more efficient separations, but only rarelyare they used at temperatures above 60 because of potential stationaryphase degradation or mobile phase volatility. Commonly used bonded phases are shown below:# Octyl =Si-[CH2]7-CH3, C8# octadecyl=Si-[CH2]17-CH3, C18# phenyl=Si-[CH2]n-C6H5, C6H5# cyanopropyl=Si-[CH2]3-CN, CN# aminopropyl=Si-[CH2]3-NH2, NH2Detectors— Many compendial HPLC methods require the use ofspectrophotometric detectors. Such a detector consists of a flow-throughcell mounted at the end of the column. A beam of UV radiation passesthrough the flow cell and into the detector. As compounds elute from thecolumn, they pass through the cell and absorb the radiation, resulting inmeasurable energy level changes.Fixed, variable, and multi-wavelength detectors are widely available. Fixedwavelength detectors operate at a single wavelength, typically 254 nm,emitted by a low-pressure mercury lamp. Variable wavelength detectorscontain a continuous source, such as a deuterium or high-pressure xenon
lamp, and a monochromator or an interference filter to generatemonochromatic radiation at a wavelength selected by the operator.Multi-wavelength detectors measure absorbance at two or morewavelengths simultaneously. In diode array multi-wavelength detectors,continuous radiation is passed through the sample cell, and then resolved(set on) into its constituent wavelengths, which are individually detected bythe photodiode array. Diode array detectors usually have lower signal-to-noise ratios than fixed or variable wavelength detectors, and thus are lesssuitable for analysis of compounds present at low concentrations.Differential refractometer detectors measure the difference between therefractive index of the mobile phase alone and that of the mobile phasecontaining chromatographed compounds as it emerges from the column.Refractive index detectors are used to detect non-UV absorbingcompounds, but they are less sensitive than UV detectors.Fluorometric detectors are sensitive to compounds that are inherentlyfluorescent (bright/shining) or that can be converted to fluorescentderivatives either by chemical transformation of the compound or bycoupling with fluorescent reagents at specific functional groups.Data Collection Devices— Modern data stations receive and store detectoroutput and print out chromatograms complete with peak heights, peakareas, sample identification, and method variables. They are also used toprogram the liquid chromatograph, controlling most variables andproviding for long periods of unattended operation.The below mentioned adjustment can be done during HPLC methodchoice:
# Composition of the mobile phase: the amount of the minor solventcomponent may be adjusted by ± 30 per cent relative or ± 2 per cent absolute,whichever is the larger. No other component is altered by more than 10 per centabsolute.# pH of the aqueous component of the mobile phase: ± 0.2 pH, unlessotherwise stated in the monograph, or ± 1.0 pH when neutral substances are tobe examined.# Concentration of salts: in the buffer component of a mobile phase: ± 10 percent.# Detector wavelength: no adjustment permitted. Stationary phase:# Column length:: ± 70 per cent,# column internal diameter: ± 25 per cent,# Particle size: maximal reduction of 50 per cent, no increase permitted.# Flow rate: ± 50 per cent. When in a monograph the retention time of theprinciple peak is indicated, the flow rate has to be adjusted if the column internaldiameter has been changed. No decrease of flow rate is permitted if themonograph uses apparent number of theoretical plates in the qualificationsection.# Temperature: ± 10 per cent, to a maximum of 60 °C. # Injection volume: may be decreased, provided detection and repeatability of the peak(s) to be determined are satisfactory.