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Page 1: 2.2.46. Chromatographic Separation Techniques

EUROPEAN PHARMACOPOEIA 5.0 2.2.46. Chromatographic separation techniques

(capillary columns). A capillary column has a maximuminternal diameter (Ø) of 100 µm.

Mobile phases

Usually the mobile phase is carbon-dioxide which may containa polar modifier such as methanol, 2-propanol or acetonitrile.The 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 toa defined programme (gradient elution of the modifier,pressure (density), temperature or flow rate).

Detectors

Ultraviolet/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 used.

METHOD

Prepare the test solution(s) and the reference solution(s) asprescribed. The solutions must be free from solid particles.

Criteria for assessing the suitability of the system aredescribed in the chapter on Chromatographic separationtechniques (2.2.46). The extent to which adjustments ofparameters of the chromatographic system can be madeto satisfy the criteria of system suitability are also given inthis chapter.

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2.2.46. CHROMATOGRAPHICSEPARATION TECHNIQUES

Chromatographic separation techniques are multi-stageseparation methods in which the components of a sampleare distributed between 2 phases, one of which is stationary,while the other is mobile. The stationary phase may be asolid or a liquid supported on a solid or a gel. The stationaryphase may be packed in a column, spread as a layer, ordistributed as a film, etc. The mobile phase may be gaseousor liquid or supercritical fluid. The separation may be basedon adsorption, mass distribution (partition), ion exchange,etc., or may be based on differences in the physico-chemicalproperties of the molecules such as size, mass, volume, etc.

This chapter contains definitions and calculations ofcommon parameters and generally applicable requirementsfor system suitability. Principles of separation, apparatusand methods are given in the following general methods :

— paper chromatography (2.2.26),

— thin-layer chromatography (2.2.27),

— gas chromatography (2.2.28),

— liquid chromatography (2.2.29),

— size-exclusion chromatography (2.2.30),

— supercritical fluid chromatography (2.2.45).

DEFINITIONS

The following definitions have been used to calculate thelimits in monographs.

With some equipment, certain parameters, such as thesignal-to-noise ratio, can be calculated using softwareprovided by the manufacturer. It is the responsibilityof the user to ensure that the calculation methods usedin the software are compatible with the requirementsof the European Pharmacopoeia. If not, the necessarycorrections must be made.

Chromatogram

A chromatogram is a graphical or other representationof detector response, effluent concentration or otherquantity used as a measure of effluent concentration, versustime, volume or distance. Idealised chromatograms arerepresented as a sequence of gaussian peaks on a baseline.

RETENTION DATA

Retention time and retention volume

Retention measurements in elution chromatography maybe given as the retention time (tR) directly defined by theposition of the maximum of the peak in the chromatogram.From the retention time, the retention volume (VR) may becalculated.

tR = retention time or distance along the baselinefrom the point of injection to the perpendiculardropped from the maximum of the peakcorresponding to the component,

v = flow rate of the mobile phase.

Mass distribution ratio

The mass distribution ratio (Dm) (also known as the capacityfactor k’ or retention factor k) is defined as :

KC = equilibrium distribution coefficient (also knownas distribution constant),

VS = volume of the stationary phase,

VM = volume of the mobile phase.

The mass distribution ratio of a component may bedetermined from the chromatogram using the expression :

tR = retention time (or volume) or distance alongthe baseline from the point of injection to theperpendicular dropped from the maximum of thepeak corresponding to the component,

tM = hold-up time (or volume) : time (or volume) ordistance along the baseline from the point ofinjection to the perpendicular dropped fromthe maximum of the peak corresponding to anunretained component.

Distribution coefficient

The elution characteristics of a component in a particularcolumn, in size-exclusion chromatography, may be given by

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the distribution coefficient (Ko) which is calculated from theexpression :

tR = retention time (or volume) or distance alongthe baseline from the point of injection to theperpendicular dropped from the maximum of thepeak corresponding to the component,

to = hold-up time (or volume) : time (or volume) ordistance along the baseline from the point ofinjection to the perpendicular dropped fromthe maximum of the peak corresponding to anunretained component,

tt = retention time (or volume) or distance alongthe baseline from the point of injection to theperpendicular dropped from the maximum of thepeak corresponding to a component which hasfull access to the pores of the stationary phase.

Retardation factor

The retardation factor (RF) (also known as retentionfactor Rf), used in planar chromatography, is the ratio of thedistance from the point of application to the centre of thespot and the distance travelled by the solvent front from thepoint of application.

b = migration distance of the analyte,a = migration distance of the solvent front.

CHROMATOGRAPHIC DATAThe peak may be defined by the peak area (A) or the peakheight (h) and the peak width at half- height (wh) or thepeak height (h) and the peak width between the points ofinflection (wi). In gaussian peaks (Figure 2.2.46.-1) there isthe relationship :

Figure 2.2.46.-1.

Symmetry factor

The symmetry factor (As) (or tailing factor) of a peak(Figure 2.2.46.-2) is calculated from the expression :

w0.05 = width of the peak at one-twentieth of the peakheight,

d = distance between the perpendicular dropped fromthe peak maximum and the leading edge of thepeak at one-twentieth of the peak height.

A value of 1.0 signifies complete (ideal) symmetry.

Figure 2.2.46.-2.

Column performance and apparent number of theoreticalplates

The column performance (apparent efficiency) may becalculated from data obtained under either isothermal,isocratic or isodense conditions, depending on the technique,as the apparent number of theoretical plates (N) from thefollowing expression, where the values of tR and wh have tobe expressed in the same units (time, volume or distance).

tR = retention time (or volume) or distance alongthe baseline from the point of injection to theperpendicular dropped from the maximum of thepeak corresponding to the component,

wh = width of the peak at half-height.

The apparent number of theoretical plates varies with thecomponent as well as with the column and the retention time.

SEPARATION DATA

Resolution

The resolution (Rs) between peaks of 2 components may becalculated from the expression :

tR1and tR2 = retention times or distances along thebaseline from the point of injection to theperpendiculars dropped from the maximaof 2 adjacent peaks,

wh1 and wh2 = peak widths at half-height.

A resolution of greater than 1.5 corresponds to baselineseparation.

The expression given above may not be applicable if thepeaks are not baseline separated.

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In quantitative planar chromatography, the migrationdistances are used instead of retention times and theresolution may be calculated using the expression :

RF1 and RF2 = ratios of the distances from the point ofapplication to the centres of the spots andthe distance travelled by the solvent frontfrom the point of application (retardationfactor),

wh1 and wh2 = peak widths at half-height,a = migration distance of the solvent front.

Peak-to-valley ratio

The peak-to-valley ratio (p/v) may be employed as asystem suitability requirement in a test for relatedsubstances when baseline separation between 2 peaks is notreached (Figure 2.2.46.-3).

Hp = height above the extrapolated baseline of theminor peak,

Hv = height above the extrapolated baseline at thelowest point of the curve separating the minorand major peaks.

Figure 2.2.46.-3.

Relative retention

The relative retention (r) is calculated as an estimate fromthe expression :

tR2 = retention time of the peak of interest,

tR1 = retention time of the reference peak (usuallythe peak corresponding to the substance to beexamined),

tM = hold-up time : time or distance along the baselinefrom the point of injection to the perpendiculardropped from the maximum of the peakcorresponding to an unretained component.

The unadjusted relative retention (rG) is calculated from theexpression :

Unless otherwise indicated, values for relative retentionstated in monographs correspond to unadjusted relativeretention.

In planar chromatography, the retardation factors RF2 andRF1 are used instead of tR2 and tR1.

PRECISION OF QUANTIFICATION

Signal-to-noise ratio

The signal-to-noise ratio (S/N) influences the precision ofquantification and is calculated from the equation :

H = height of the peak (Figure 2.2.46.-4) correspondingto the component concerned, in the chromatogramobtained with the prescribed reference solution,measured from the maximum of the peak tothe extrapolated baseline of the signal observedover a distance equal to 20 times the width athalf-height,

h = range of the background noise in a chromatogramobtained after injection or application of a blank,observed over a distance equal to 20 timesthe width at half-height of the peak in thechromatogram obtained with the prescribedreference solution and, if possible, situatedequally around the place where this peak wouldbe found.

Figure 2.2.46.-4.

Repeatability

The repeatability of response is expressed as an estimatedpercentage relative standard deviation (RSD%) of aconsecutive series of measurement of injections or

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applications of a reference solution and is calculated fromthe expression :

yi = individual values expressed as peak area,peak height, or ratio of areas by the internalstandardisation method,

= mean of individual values,n = number of individual values.

The maximal permitted relative standard deviation (RSDmax)is calculated for a series of injections of the referencesolution for defined limits using the following expression :

K = constant (0.349), obtained from the expressionin which represents the

required RSD after 6 injections for B = 1.0,

B = upper limit given in the definition of theindividual monograph minus 100 per cent,

n = number of replicate injections of the referencesolution (3 ≤ n ≤ 6),

t90%,n–1 = Student’s t at the 90 per cent probability level(double sided) with n − 1 degrees of freedom.

SYSTEM SUITABILITYThe system suitability tests represent an integral part ofthe method and are used to ensure adequate performanceof the chromatographic system. Apparent efficiency, massdistribution ratio, resolution, relative retention and thesymmetry factor are the parameters which are usuallyemployed in assessing the performance of the column.Factors which may affect the chromatographic behaviourinclude the composition, ionic strength, temperatureand apparent pH of the mobile phase, flow rate, columnlength, temperature and pressure, and stationary phasecharacteristics including porosity, particle size, typeof particles, specific surface area and, in the case ofreverse-phase supports, the extent of chemical modification(as expressed by end-capping, carbon loading etc.).The various components of the equipment employed must bequalified and be capable of achieving the precision requiredto conduct the test or assay.

The following requirements are to be fulfilled unlessotherwise stated in the monograph.

— The symmetry factor of the principal peak is to bebetween 0.8 and 1.5 unless otherwise stated in themonograph. This requirement has general applicability totests or assays described in the monographs.

— Maximal permitted relative standard deviation forreplicate injections of the prescribed reference solutiondo not exceed the values given in Table 2.2.46.-1. Thisrequirement is applicable to assays for content only anddoes not apply to the test for related substances.

— The limit of detection of the peak (corresponding to asignal-to-noise ratio of 3) is below the disregard limit ofthe test for related substances.

— The limit of quantitation of the peak (corresponding toa signal-to-noise ratio of 10) is equal to or less than thedisregard limit of the test for related substances.

Table 2.2.46.-1. — Repeatability requirements

Number of individual injections

3 4 5 6

B(per cent)

Maximal permitted relative standard deviation

2.0 0.41 0.59 0.73 0.85

2.5 0.52 0.74 0.92 1.06

3.0 0.62 0.89 1.10 1.27

ADJUSTMENT OF CHROMATOGRAPHIC CONDITIONSThe extent to which the various parameters of achromatographic test may be adjusted to satisfy thesystem suitability criteria without fundamentally modifyingthe methods are listed below for information. Thechromatographic conditions described have been validatedduring the elaboration of the monograph. The systemsuitability tests are included to ensure the separationrequired for satisfactory performance of the test or assay.Nonetheless, since the stationary phases are describedin a general way and there is such a variety availablecommercially, with differences in chromatographicbehaviour, some adjustments of the chromatographicconditions may be necessary to achieve the prescribedsystem suitability requirements. With reverse-phase liquidchromatographic methods, in particular, adjustment of thevarious parameters will not always result in satisfactorychromatography. In that case, it may be necessary to replacethe column with another of the same type (e.g. octadecylsilylsilica gel) which exhibits the desired chromatographicbehaviour.For critical parameters the adjustments are defined clearlyin the monograph to ensure the system suitability.

Multiple adjustments which may have a cumulative effect inthe performance of the system are to be avoided.

Thin-layer chromatography and paper chromatography

Composition of the mobile phase : the amount of the minorsolvent component may be adjusted by ± 30 per cent relativeor ± 2 per cent absolute, whichever is the larger ; for a minorcomponent at 10 per cent of the mobile phase, a 30 per centrelative adjustment allows a range of 7-13 per cent whereasa 2 per cent absolute adjustment allows a range of 8-12 percent, the relative value being therefore the larger ; for aminor component at 5 per cent of the mobile phase, a 30 percent relative adjustment allows a range of 3.5-6.5 per centwhereas a 2 per cent absolute adjustment allows a range of3-7 per cent, the absolute value being in this case 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,unless otherwise stated in the monograph, or ± 1.0 pH whenneutral substances are to be examined.

Concentration of salts in the buffer component of a mobilephase : ± 10 per cent.

Application volume : 10-20 per cent of the prescribed volumeif using fine particle size plates (2-10 µm).

Migration distance of the solvent front is to be not less than50 mm or 30 mm on high-performance plates.

Liquid chromatography

Composition of the mobile phase : the amount of the minorsolvent component may be adjusted by ± 30 per cent relativeor ± 2 per cent absolute, whichever is the larger (see exampleabove). No other component is altered by more than 10 percent absolute.

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pH of the aqueous component of the mobile phase : ± 0.2 pH,unless otherwise stated in the monograph, or ± 1.0 pH whenneutral substances are to be examined.

Concentration of salts in the buffer component of a mobilephase : ± 10 per cent.

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, noincrease permitted.

Flow rate : ± 50 per cent. When in a monograph the retentiontime of the principle peak is indicated, the flow rate has to beadjusted if the column internal diameter has been changed.No decrease of flow rate is permitted if the monograph usesapparent number of theoretical plates in the qualificationsection.

Temperature : ± 10 per cent, to a maximum of 60 °C.

Injection volume : may be decreased, provided detection andrepeatability of the peak(s) to be determined are satisfactory.

Gradient elution : the configuration of the equipmentemployed may significantly alter the resolution, retentiontime and relative retentions described in the method. Shouldthis occur, it may be due to excessive dwell volume which isthe volume between the point at which the 2 eluants meetand the top of the column.

Gas chromatography

Stationary phase :

— column length : ± 70 per cent,

— column internal diameter : ± 50 per cent,

— particle size : maximal reduction of 50 per cent, noincrease permitted,

— film thickness : −50 per cent to + 100 per cent.

Flow rate : ± 50 per cent.

Temperature : ± 10 per cent.

Injection volume : may be decreased, provided detection andrepeatability are satisfactory.

Supercritical fluid chromatography

Composition of the mobile phase : for packed columns, theamount of the minor solvent component may be adjusted by± 30 per cent relative or ± 2 per cent absolute, whichever isthe larger. No adjustment is permitted for a capillary columnsystem.

Detector wavelength : no adjustment permitted.

Stationary phase :

— column length : ± 70 per cent,

— column internal diameter :

± 25 per cent (packed columns),

± 50 per cent (capillary columns),

— particle size : maximal reduction of 50 per cent, noincrease permitted (packed columns).

Flow rate : ± 50 per cent.

Temperature : ± 10 per cent.

Injection volume : may be decreased, provided detection andrepeatability are satisfactory.

QUANTIFICATION

— Detector response. The detector sensitivity is the signaloutput per unit concentration or unit mass of a substancein the mobile phase entering the detector. The relativedetector response factor, commonly referred to asresponse factor, expresses the sensitivity of a detectorrelative to a standard substance. The correction factor isthe reciprocal of the response factor.

— External standard method. The concentration of thecomponent(s) to be analysed is determined by comparingthe response(s) (peak(s)) obtained with the test solutionto the response(s) (peak(s)) obtained with a referencesolution.

— Internal standard method. Equal amounts of acomponent that is resolved from the substance to beexamined (the internal standard) is introduced into thetest solution and a reference solution. The internalstandard should not react with the substance to beexamined ; it must be stable and must not containimpurities with a retention time similar to that of thesubstance to be examined. The concentration of thesubstance to be examined is determined by comparingthe ratio of the peak areas or peak heights due to thesubstance to be examined and the internal standard inthe test solution with the ratio of the peak areas or peakheights due to the substance to be examined and theinternal standard in the reference solution.

— Normalisation procedure. The percentage content of oneor more components of the substance to be examinedis calculated by determining the area of the peak orpeaks as a percentage of the total area of all the peaks,excluding those due to solvents or any added reagentsand those below the disregard limit.

— Calibration procedure. The relationship betweenthe measured or evaluated signal (y) and the amount(concentration, mass, etc.) of substance (x) is determinedand the calibration function is calculated. The analyticalresults are calculated from the measured signal orevaluated signal of the analyte by means of the inversefunction.

For assays and for quantitative determination of componentsthe external standard method, the internal standardmethod or the calibration procedure may be described inthe monograph, and the normalisation procedure is notnormally applied. In tests for related substances, either theexternal standard method with a single reference solution orthe normalisation procedure is generally applied. However,with both the normalisation procedure or the externalstandard method, when a dilution of the test solution is usedfor comparison, the responses of the related substances aresimilar to the substance itself (response factor of 0.8 to 1.2),otherwise correction factors are included in the text.

When the related substances test prescribes the summationof impurities or there is quantitative determination of animpurity, it is important to choose an appropriate thresholdsetting and appropriate conditions for the integration ofthe peak areas. In such tests the disregard limit, e.g. theareas of peaks whose areas are below the limit are not takeninto account, is generally 0.05 per cent. Thus, the thresholdsetting of the data collection system corresponds to, at least,half of the disregard limit. Integration of the peak areasof the impurities, which are not completely separated fromthe main peak, are preferably performed by valley-to-valleyextrapolation (tangential skim). Peaks due to the solvent(s)used to dissolve the sample are also to be disregarded.

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