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2.2.46. CHROMATOGRAPHIC SEPARATION …

Supercritical fluid chromatographyEUROPEAN PHARMACOPOEIA solutions. In addition to theTOC water control,prepare suitable blank solutions or other solutions neededfor establishing the baseline or for calibration adjustmentsfollowing the manufacturer s instructions; run the suitability. Run the following solutions and recordthe responses:TOC water(rw);standard solution(rs);systemsuitability solution(rss). Calculate the percentage responseefficiency using the expression:Thesystemissuitableiftherespo nseefficiencyisnotlessthan85 per cent and not more than 115 per cent of the (ru).The test solution complies with the test ifruis not greater thanrs method can also be applied using on-line instrumentationthat has been adequately calibrated and shown to haveacceptable system suitability. The location of instrumentationmust be chosen to ensure that the responses are representativeof the water SUPERCRITICAL FLUIDCHROMATOGRAPHYS upercritical fluid chromatography (SFC) is a method ofchromatographic SEPARATION in which the mobile phase is afluid in a supercritical or a subcritical state.

2.2.45. Supercritical fluid chromatography EUROPEAN PHARMACOPOEIA 7.0 Control solutions.In addition to the TOC water control, prepare suitable blank solutions or other solutions needed for establishing the baseline or for calibration adjustments

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Transcription of 2.2.46. CHROMATOGRAPHIC SEPARATION …

1 Supercritical fluid chromatographyEUROPEAN PHARMACOPOEIA solutions. In addition to theTOC water control,prepare suitable blank solutions or other solutions neededfor establishing the baseline or for calibration adjustmentsfollowing the manufacturer s instructions; run the suitability. Run the following solutions and recordthe responses:TOC water(rw);standard solution(rs);systemsuitability solution(rss). Calculate the percentage responseefficiency using the expression:Thesystemissuitableiftherespo nseefficiencyisnotlessthan85 per cent and not more than 115 per cent of the (ru).The test solution complies with the test ifruis not greater thanrs method can also be applied using on-line instrumentationthat has been adequately calibrated and shown to haveacceptable system suitability. The location of instrumentationmust be chosen to ensure that the responses are representativeof the water SUPERCRITICAL FLUIDCHROMATOGRAPHYS upercritical fluid chromatography (SFC) is a method ofchromatographic SEPARATION in which the mobile phase is afluid in a supercritical or a subcritical state.

2 The stationaryphase, contained in a column, consists of either finely dividedsolid particles, such as a silica or porous graphite, a chemicallymodified stationary phase, as used in liquid chromatography ,or, for capillary columns, a cross-linked liquid film evenly is based on mechanisms of adsorption or mass apparatus usually consists of a cooled pumping system, aninjector, a CHROMATOGRAPHIC column, contained in an oven, adetector, a pressure regulator and a data acquisition device (oran integrator or a chart recorder).Pumping systemPumping systems are required to deliver the mobile phase at aconstant flow rate. Pressure fluctuations are to be minimised, by passing the pressurised solvent through a pulse-dampingdevice. Tubing and connections are capable of withstanding thepressures developed by the pumping controlled systems are capable of accuratelydelivering a mobile phase in either constant or varyingconditions, according to a defined programme.

3 In the case ofgradient elution, pumping systems which deliver solvent(s)from several reservoirs are available and solvent mixing can beachieved on either the low or high-pressure side of the pump(s).InjectorsInjection may be carried out directly at the head of the columnusing a phasesStationary phases are contained in columns which have beendescribed in the chapters onLiquid chromatography ( )(packed columns) andGas chromatography ( )(capillarycolumns). A capillary column has a maximum internal diameter( ) of 100 phasesUsuallythemobilephaseiscarbon-diox idewhichmaycontaina polar modifier such as methanol, 2-propanol or composition, pressure (density), temperature and flowrate of the prescribed mobile phase may either be constantthroughout the whole CHROMATOGRAPHIC procedure (isocratic,isodense, isothermic elution) or may vary according to a definedprogramme (gradient elution of the modifier, pressure (density),temperature or flow rate).

4 DetectorsUltraviolet/visible (UV/Vis) spectrophotometers and flameionisation detectors are the most commonly employeddetectors. Light scattering detectors, infrared absorptionspectrophotometers, thermal conductivity detectors or otherspecial detectors may be the test solution(s) and the reference solution(s) asprescribed. The solutions must be free from solid for assessing the suitability of the system are describedin the chapter onChromatographic SEPARATION techniques( ). The extent to which adjustments of parameters of thechromatographic system can be made to satisfy the criteria ofsystem suitability are also given in this CHROMATOGRAPHICSEPARATION TECHNIQUESC hromatographic SEPARATION techniques are multi-stageseparation methods in which the components of a sample aredistributed between 2 phases, one of which is stationary, whilethe other is mobile.

5 The stationary phase may be a solid ora liquid supported on a solid or a gel. The stationary phasemay be packed in a column, spread as a layer, or distributedas a film, etc. The mobile phase may be gaseous or liquid orsupercritical fluid. The SEPARATION may be based on adsorption,mass distribution (partition), ion exchange, etc., or may bebased on differences in the physico-chemical properties of themolecules such as size, mass, volume, chapter contains definitions and calculations of commonparameters and generally applicable requirements for systemsuitability. Principles of SEPARATION , apparatus and methods aregiven in the following general methods: paperchromatography( ); thin-layer chromatography ( ); gas chromatography ( ); liquidchromatography( ); size-exclusion chromatography ( ); supercritical fluid chromatography ( ).DEFINITIONSThe system suitability and acceptance criteria in monographshave been set using parameters as defined below.

6 With someequipment, certain parameters, such as the signal-to-noiseratio and resolution, can be calculated using softwareprovided by the manufacturer. It is the responsibility ofthe user to ensure that the calculation methods used in thesoftware are equivalent to the requirements of the EuropeanPharmacopoeia and to make any necessary corrections if thisis not the graphical or other representation of detector response,effluent concentration or other quantity used as a measureof effluent concentration, versus time or volume. Idealisedchromatograms are represented as a sequence of Gaussianpeaks on a baseline (Figure ).PeakThe portion of a chromatogram recording the detector responsewhen a single component (or 2 or more unresolved components)is eluted from the the information section on general monographs (cover pages)EUROPEAN PHARMACOPOEIA CHROMATOGRAPHIC SEPARATION techniquesFigure peak may be defined by the peak area, or the peakheight (h) and the peak width at half-height (wh), or the peakheight (h) and the peak width between the points of inflection(wi).

7 In Gaussian peaks (Figure ) there is the followingrelationship:Retention time (tR)Time required for elution of a component (Figure ,baseline scale being in minutes).Retention volume (VR)Volume of the mobile phase required for elution of a may be calculated from the retention time and the flowrate (F) in millilitres per minute using the following equation:Hold-uptime(tM)Time required for elution of an unretained component(Figure , baseline scale being in minutes). Insize-exclusion chromatography , the symbolt0(see below) volume (VM)Volume of the mobile phase required for elution of anunretained component. It may be calculated from the hold-uptime and the flow rate (F) in millilitres per minute using thefollowing equation:In size-exclusion chromatography , the symbolV0(see below)is factor (k)The retention factor (also known as mass distribution ratio (Dm)or capacity factor (k )) is defined as:amount of component in stationary phaseamount of component in mobile phaseKC=distribution constant (also known as equilibriumdistribution coefficient);VS=volume of the stationary phase;VM=volume of the mobile retention factor of a component may be determined fromthe chromatogram using the following equation:Total mobile phase time (tt)In size-exclusion chromatography , retention time of acomponent whose molecules are smaller than the smallest gelpores (Figure ).

8 Totalmobilephasevolume(Vt)In size-exclusion chromatography , retention volume of acomponent whose molecules are smaller than the smallest gelpores. It may be calculated from the total mobile phase time andtheflowrate(F) in millilitres per minute using the followingequation:Retention time of an unretained compound (t0)In size-exclusion chromatography , retention time of acomponent whose molecules are larger than the largest gelpores (Figure ).Retention volume of an unretained compound (V0)In size-exclusion chromatography , retention volume of acomponent whose molecules are larger than the largest gelpores. It may be calculated from the retention time of anGeneral Notices (1) apply to all monographs and other CHROMATOGRAPHIC SEPARATION techniquesEUROPEAN PHARMACOPOEIA compound and the flow rate (F) in millilitres perminute using the following equation:Distribution constant (K0)In size-exclusion chromatography , the elution characteristicsof a component in a particular column may be given bythe distribution constant (also referred to as distributioncoefficient), which is calculated using the following equation:Retardation factor (RF)The retardation factor (also known as retention factor (Rf)), usedin planar chromatography , is the ratio of the distance from thepoint of application to the centre of the spot and the distancetravelled by the solvent front from the point of application(Figure ).

9 B=migration distance of the component;a=migration distance of the solvent mobile phase frontB. spotC. line of applicationFigure number (N)Thecolumn performance (apparent efficiency) may be calculatedfrom data obtained under either isothermal, isocratic orisodense conditions, depending on the technique, as the platenumber (also referred to as number of theoretical plates), using72 See the information section on general monographs (cover pages)EUROPEAN PHARMACOPOEIA CHROMATOGRAPHIC SEPARATION techniquesthe following equation, the values oftRandwhbeing expressedinthesameunits:tR=retention time of the peak corresponding to thecomponent;wh=width of the peak at plate number varies with the component as well as withthecolumn,thecolumntemperature,themo bilephaseandtheretention volume (D)The dwell volume (also known as gradient delay volume) is thevolume between the point at which the eluents meet and thetop of the column.

10 It can be determined using the : replace the CHROMATOGRAPHIC column by an appropriatecapillary tubing ( 1 m mm).Mobile phase: mobile phase A:water R; mobile phase ofacetone R;Time(min)Mobile phase A(per centV/V)Mobile phase B(per centV/V)0-20100 00 10020 - 300100 Flow rate: set to obtain sufficient back-pressure ( 2 mL/min).Detection: spectrophotometer at 265 the time ( ) in minutes when the absorbance hasincreased by 50 per cent (Figure ).tD= (in minutes);tG=pre-defined gradient time (= 20 min);F=flow rate (in millilitres per minute).Figure factor (As)The symmetry factor of a peak (Figure ) is calculatedusing the following of the peak at one-twentieth of the peakheight;d=distance between the perpendicular dropped fromthe peak maximum and the leading edge of the peakat one-twentieth of the peak of signifies symmetry.


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