USP 34 Physical Tests /...

USP 34 Physical Tests / ⟨621⟩ Chromatography 243

Figure 3.

Compressibility Index—Calculate by the formula: ary phase may be packed in a column, spread as a layer,distributed as a film, or applied by other techniques. The

100(V0 − VF)/V0 mobile phase may be gaseous or liquid or supercritical fluid.The separation may be based on adsorption, mass distribu-

V0 = unsettled apparent volume tion (partition), or ion exchange; or it may be based onVF = final tapped volume differences among the physicochemical properties of the

molecules, such as size, mass, and volume. This chapterHausner Ratio—contains general procedures, definitions, and calculations of

V0/VF common parameters and describes general requirements forsystem suitability. The types of chromatography useful in

Depending on the material, the compressibility index can qualitative and quantitative analysis employed in USP proce-be determined using V10 instead of V0. ✦[NOTE—If V10 is dures are column, gas, paper, thin-layer (including high-per-used, it will be clearly stated in the results.]✦ formance thin-layer chromatography), and pressurized liquid

chromatography (commonly called high-pressure or high-performance liquid chromatography).

GENERAL PROCEDURESChange to read: This section describes the basic procedures used when a

chromatographic method is described in a monograph. Thefollowing procedures are followed unless otherwise indicated⟨621⟩ CHROMATOGRAPHY in the individual monograph.

Paper Chromatography▲INTRODUCTION Stationary Phase: The stationary phase is a sheet of

paper of suitable texture and thickness. Development mayChromatographic separation techniques are multistage be ascending, in which the solvent is carried up the paper

separation methods in which the components of a sample by capillary forces, or descending, in which the solvent floware distributed between two phases, of which one is station- is also assisted by gravitational force. The orientation ofary and the other mobile. The stationary phase may be a paper grain with respect to solvent flow is to be kept con-solid or a liquid supported on a solid or a gel. The station-

Official from August 1, 2011Copyright (c) 2011 The United States Pharmacopeial Convention. All rights reserved.

Accessed from 8.7.228.252 by novartis2 on Mon Sep 19 19:07:10 EDT 2011

244 ⟨621⟩ Chromatography / Physical Tests USP 34

stant in a series of chromatograms. (The machine direction Apparatus: A chromatographic chamber made of in-is usually designated by the manufacturer.) ert, transparent material and having the following specifica-

tions is used: a flat-bottom or twin trough, a tightly fittedApparatus: The essential equipment for paper chro-lid, and a size suitable for the plates. The chamber is linedmatography consists of a vapor-tight chamber with inlets foron at least one wall with filter paper. Sufficient mobileaddition of solvent and a rack of corrosion-resistant materialphase or developing solvent is added to the chamber that,about 5 cm shorter than the inside height of the chamber.after impregnation of the filter paper, a depth appropriateThe rack serves as a support for solvent troughs and forto the dimensions of the plate used is available. The chro-antisiphon rods that, in turn, hold up the chromatographicmatographic chamber is closed and allowed to equilibrate.sheets. The bottom of the chamber is covered with the pre-[NOTE—Unless otherwise indicated, the chromatographicscribed solvent system or mobile phase. Saturation of theseparations are performed in a saturated chamber.]chamber with solvent vapor is facilitated by lining the inside

walls with paper wetted with the prescribed solvent system. Detection/Visualization: An ultraviolet (UV) lightsource suitable for observations under short- (254 nm) andSpotting: The substance or substances analyzed arelong- (365 nm) wavelength UV light and a variety of otherdissolved in a suitable solvent. Convenient volumes, deliv-spray reagents used to make spots visible are often used.ered from suitable micropipets, of the resulting solution,

normally containing 1–20 µg of the compound, are placed Spotting: Solutions are spotted on the surface of thein 6- to 10-mm spots not less than 3 cm apart. stationary phase (plate) at the prescribed volume in suffi-

ciently small portions to obtain circular spots of 2–5 mm inDescending Paper Chromatography Procedurediameter (1–2 mm on HPTLC plates) or bands of 10–20 mm(1) A spotted chromatographic sheet is suspended in the× 1–2 mm (5–10 mm × 0.5–1 mm on HPTLC plates) at anapparatus, using the antisiphon rod to hold the upperappropriate distance from the lower edge of and sides ofend of the sheet in the solvent trough. [NOTE—Ensurethe plate. [NOTE—During development, the application posi-that the portion of the sheet hanging below the rodstion must be at least 5 mm (TLC) or 3 mm (HPTLC) aboveis freely suspended in the chamber without touchingthe level of the mobile phase.] The solutions are applied onthe rack, the chamber walls, or the fluid in thea line parallel to the lower edge of the plate with an intervalchamber.]of at least 10 mm (5 mm on HPTLC plates) between the(2) The chamber is sealed to allow equilibration (satura-centers of spots, or 4 mm (2 mm on HPTLC plates) betweention) of the chamber and the paper with the solventthe edges of bands, then allowed to dry.vapor. Any excess pressure is released as necessary.

(3) After equilibration of the chamber, the prepared mo- Procedurebile phase is introduced into the trough through the (1) Place the plate in the chamber, ensuring that theinlet. spots or bands are above the surface of the mobile

(4) The inlet is closed, and the mobile solvent phase is phase.allowed to travel the desired distance down the (2) Close the chamber.paper. (3) Allow the mobile phase to ascend the plate until the

(5) The sheet is removed from the chamber. solvent front has traveled three-quarters of the length(6) The location of the solvent front is quickly marked, of the plate, or the distance prescribed in the

and the sheet is dried. monograph.(7) The chromatogram is observed and measured directly (4) Remove the plate, mark the solvent front with a pen-

or after suitable development to reveal the location of cil, and allow to dry.the spots of the isolated drug or drugs. (5) Visualize the chromatograms as prescribed.

(6) Determine the chromatographic retardation factor (RF)Ascending Paper Chromatography Procedurevalues for the principal spots or zones.(1) The mobile phase is added to the bottom of the

(7) Presumptive identification can be made by observa-chamber.tion of spots or zones of identical RF value and about(2) The chamber is sealed to allow equilibration (satura-equal magnitude obtained, respectively, with an un-tion) of the chamber and the paper with the solventknown and a standard chromatographed on the samevapor. Any excess pressure is released as necessary.plate. A visual comparison of the size or intensity of(3) The lower edge of the stationary phase is dipped intothe spots or zones may serve for semiquantitative esti-the mobile phase to permit the mobile phase to risemation. Quantitative measurements are possible byon the chromatographic sheet by capillary action.means of densitometry (absorbence or fluorescence(4) When the solvent front has reached the desiredmeasurements).height, the chamber is opened, the sheet is removed,

the location of the solvent front is quickly marked,and the sheet is dried. Column Chromatography(5) The chromatogram is observed and measured directlyor after suitable development to reveal the location of

Solid Support: Purified siliceous earth is used for nor-the spots of the isolated drug or drugs.mal-phase separation. Silanized chromatographic siliceousearth is used for reverse-phase partition chromatography.

Thin-Layer Chromatography Stationary Phase: The solid support is modified bythe addition of a stationary phase specified in the individual

Stationary Phase: The stationary phase is a relatively monograph. If a mixture of liquids is used as the stationarythin, uniform layer of dry, finely powdered material applied phase, mix the liquids before the introduction of the solidto a glass, plastic, or metal sheet or plate (typically called support.the plate). The stationary phase of TLC plates has an aver- Mobile Phase: The mobile phase is specified in theage particle size of 10–15 µm, and that of high-performance individual monograph. If the stationary phase is an aqueousTLC (HPTLC) plates has an average particle size of 5 µm. solution, equilibrate with water. If the stationary phase is aCommercial plates with a preadsorbent zone can be used if polar organic fluid, equilibrate with that fluid.they are specified in a monograph. Sample applied to the Apparatus: Unless otherwise specified in the individ-preadsorbent region develops into sharp, narrow bands at ual monograph, the chromatographic tube is about 22 mmthe preadsorbent–sorbent interface. The separations in inside diameter and 200–300 mm long. Attached to it isachieved may be based on adsorption, partition, or a com- a delivery tube, without stopcock, about 4 mm in inside di-bination of both effects, depending on the particular type of ameter and about 50 mm long.stationary phase.




APPARATUS PREPARATION: Pack a pledget of fine glass wool detector used depends on the nature of the compoundsin the base of the tube. Combine the specified volume of analyzed and is specified in the individual monograph. De-stationary phase and the specified amount of solid support tector output is recorded as a function of time, and theto produce a homogeneous, fluffy mixture. Transfer this instrument response, measured as peak area or peak height,mixture to the chromatographic tube, and tamp, using gen- is a function of the amount present.tle pressure, to obtain a uniform mass. If the specified Temperature Program: The length and quality of aamount of solid support is more than 3 g, transfer the mix- GC separation can be controlled by altering the temperatureture to the column in portions of approximately 2 g, and of the chromatographic column. When a temperature pro-tamp each portion. If the assay or test requires a multiseg- gram is necessary, the individual monograph indicates thement column with a different stationary phase specified for conditions in table format. The table indicates the initialeach segment, tamp after the addition of each segment, temperature, rate of temperature change (ramp), final tem-and add each succeeding segment directly to the previous perature, and hold time at the final temperature.one. Pack a pledget of fine glass wool above the completed Procedurecolumn packing. [NOTE—The mobile phase should flow (1) Equilibrate the column, injector, and detector withthrough a properly packed column as a moderate stream or, flowing carrier gas until a constant signal is received.if reverse-phase chromatography is applied, as a slow (2) Inject a sample through the injector septum, or usetrickle.] an autosampler.If a solution of the analyte is incorporated into the sta- (3) Begin the temperature program.tionary phase, complete the quantitative transfer to the (4) Record the chromatogram.chromatographic tube by scrubbing the beaker used for the (5) Analyze as indicated in the monograph.preparation of the test mixture with a mixture of about 1 gof Solid Support and several drops of the solvent used toprepare the sample solution before adding the final portion Liquid Chromatography (LC)of glass wool.

Procedure The term liquid chromatography, as used in the compendia,(1) Transfer the mobile phase to the column space above is synonymous with high-pressure liquid chromatography

the column packing, and allow it to flow through the and high-performance liquid chromatography. LC is a sepa-column under the influence of gravity. ration technique based on a solid stationary phase and a

(2) Rinse the tip of the chromatographic column with liquid mobile phase.about 1 mL of mobile phase before each change in Stationary Phase: Separations are achieved by parti-composition of mobile phase and after completion of tion, adsorption, or ion-exchange processes, depending onthe elution. the type of stationary phase used. The most commonly used(3) If the analyte is introduced into the column as a solu- stationary phases are modified silica or polymeric beads. Thetion in the mobile phase, allow it to pass completely beads are modified by the addition of long-chain hydrocar-into the column packing, then add mobile phase in bons. The specific type of packing needed to complete anseveral small portions, allowing each to drain com- analysis is indicated by the “L” designation in the individualpletely, before adding the bulk of the mobile phase. monograph (see also the section Chromatographic Columns,(4) Where the procedure indicates the use of multiple below). The size of the beads is often described in the mon-chromatographic columns mounted in series and the ograph as well. Changes in the packing type and size areaddition of mobile phase in divided portions is speci- covered in the System Suitability section of this chapter.fied, allow each portion to drain completely through

Chromatographic Column: The term column in-each column, and rinse the tip of each with mobilecludes stainless steel, lined stainless steel, and polymeric col-phase before the addition of each succeeding portion.umns, packed with a stationary phase. The length and innerdiameter of the column affects the separation, and thereforetypical column dimensions are included in the individualGas Chromatography (GC)monograph. Changes to column dimensions are discussedin the System Suitability section of this chapter. CompendialLiquid Stationary Phase: This type of phase is availa-monographs do not include the name of appropriate col-ble in packed or capillary columns.umns; this omission avoids the appearance of endorsementPacked Column GC: The liquid stationary phase is of a vendor’s product and natural changes in the market-deposited on a finely divided, inert solid support, such as place. See the section Chromatographic Columns for morediatomaceous earth, porous polymer, or graphitized carbon, information.which is packed into a column that is typically 2–4 mm in

Mobile Phase: The mobile phase is a solvent or ainternal diameter and 1–3 m in length.mixture of solvents, as defined in the individual monograph.Capillary Column GC: In capillary columns, which

Apparatus: A liquid chromatograph consists of a res-contain no packed solid support, the liquid stationary phaseervoir containing the mobile phase, a pump to force theis deposited on the inner surface of the column and may bemobile phase through the system at high pressure, an injec-chemically bonded to it.tor to introduce the sample into the mobile phase, a chro-Solid Stationary Phase: This type of phase is availa- matographic column, a detector, and a data collectionble only in packed columns. In these columns the solid device.phase is an active adsorbent, such as alumina, silica, or car-

Gradient Elution: The technique of continuouslybon, packed into a column. Polyaromatic porous resins,changing the solvent composition during the chromato-which are sometimes used in packed columns, are notgraphic run is called gradient elution or solvent program-coated with a liquid phase. [NOTE—Packed and capillary col-ming. The gradient elution profile is presented in the indi-umns must be conditioned before use until the baseline andvidual monograph as a gradient table, which lists the timeother characteristics are stable. The column or packing ma-and proportional composition of the mobile phase at theterial supplier provides instructions for the recommendedstated time.conditioning procedure.]

ProcedureApparatus: A gas chromatograph consists of a carrier(1) Equilibrate the column and detector with mobilegas source, injection port, column, detector, and recording

phase at the specified flow rate until a constant signaldevice. The injection port, column, and detector are tem-is received.perature controlled and may be varied as part of the analy-

(2) Inject a sample through the injector, or use ansis. The typical carrier gas is helium, nitrogen, or hydrogen,autosampler.depending on the column and detector in use. The type of




Figure 1. Chromatographic separation of two substances.

(3) Begin the gradient program. Number of Theoretical Plates (N)1: N is a measure(4) Record the chromatogram. of column efficiency. For Gaussian peaks, it is calculated by:(5) Analyze as directed in the monograph.

N = 16(tR/W)2

CHROMATOGRAPHIC COLUMNS where tR is the retention time of the substance, and W is thepeak width at its base, obtained by extrapolating the rela-

A complete list of packings (L), phases (G), and supports tively straight sides of the peak to the baseline. The value of(S) used in USP–NF tests and assays is located in USP–NF N depends upon the substance being chromatographed asand PF, Reagents, Indicators, and Solutions—Chromatographic well as the operating conditions, such as the flow rate andColumns. This list is intended to be a convenient reference temperature of the mobile phase or carrier gas, the qualityfor the chromatographer in identifying the pertinent chro- of the packing, the uniformity of the packing within thematographic column specified in the individual monograph. column, and, for capillary columns, the thickness of the sta-

tionary phase film and the internal diameter and length ofthe column.DEFINITIONS AND INTERPRETATION OF Where electronic integrators are used, it may be conven-

CHROMATOGRAMS ient to determine the number of theorical plates, by theequation:

Chromatogram: A chromatogram is a graphical rep-resentation of the detector response, concentration ofanalyte in the effluent, or other quantity used as a measureof effluent concentration versus effluent volume or time. Inplanar chromatography, chromatogram may refer to the pa-per or layer with the separated zones.

where Wh/2 is the peak width at half-height. However, in theFigure 1 represents a typical chromatographic separationevent of dispute, only equations based on peak width atof two substances, 1 and 2. tR1 and tR2 are the respectivebaseline are to be used.retention times; and h is the height, h/2 the half-height,

and Wh/2 the width at half-height, for peak 1. W1 and W2 Peak: The peak is the portion of the chromatographicare the respective widths of peaks 1 and 2 at the baseline. recording of the detector response when a single compo-Air peaks are a feature of gas chromatograms and corre- nent is eluted from the column. If separation is incomplete,spond to the solvent front in LC. The retention time of two or more components may be eluted as one unresolvedthese air peaks, or unretained components, is designated as peak.tM. Peak-to-Valley Ratio (p/v): The p/v may be em-

Dwell Volume (D): The dwell volume (also known as ployed as a system suitability criterion in a test for relatedgradient delay volume) is the volume between the point at substances when baseline separation between two peaks iswhich the eluents meet and the top of the column. not achieved. Figure 2 represents a partial separation of two

substances, where Hp is the height above the extrapolatedHold-Up Time (tM): The hold-up time is the time re-baseline of the minor peak and Hv is the height above thequired for elution of an unretained component (see Figure 1,

shown as an air or unretained solvent peak, with the base- 1The parameters k, N, r, and rG were developed for isothermal GCline scale in min). separations and isocratic HPLC separations. Because these terms are

thermodynamic parameters, they are valid only for separations made atHold-Up Volume (VM): The hold-up volume is the constant temperature, mobile phase composition, and flow rate. However, forvolume of mobile phase required for elution of an un- separations made with a temperature program or solvent gradient, theseretained component. It may be calculated from the hold-up parameters may be used simply as comparative means to ensure that

adequate chromatographic conditions exist to perform the methods astime and the flow rate F, in mm/min:intended in the monographs.

VM = tM × F

In size exclusion chromatography, the symbol VO is used.




extrapolated baseline at the lowest point of the curve sepa- made in terms of unadjusted relative retention, unless other-rating the minor and major peaks: wise indicated.

p/v = Hp/Hv RRT = tR2/tR1

The symbol rG is also used to designate unadjusted relativeretention values.

Relative Standard Deviation in Percentage

Retardation Factor (RF): The retardation factor is theratio of the distance traveled by the center of the spot tothe distance simultaneously traveled by the mobile phaseand is used in planar chromatography. Using the symbols inFigure 3:

RF = b/a

Retention Factor (k)1: The retention factor is alsoknown as the capacity factor (k′). Defined as:

Figure 2. Peak-to-valley ratio determination.

Relative Retardation (Rret): The relative retardation is orthe ratio of the distance traveled by the analyte to the dis-tance simultaneously traveled by a reference compound (seeFigure 3) and is used in planar chromatography.

Rrel = b / c

The retention factor of a component may be determinedfrom the chromatogram:

k = (tR − tM)/tM

Retention Time (tR): In liquid chromatography andgas chromatography, the retention time, tR, is defined as thetime elapsed between the injection of the sample and theappearance of the maximum peak response of the elutedsample zone. tR may be used as a parameter for identifica-tion. Chromatographic retention times are characteristic ofthe compounds they represent but are not unique. Coinci-dence of retention times of a sample and a reference sub-stance can be used as a partial criterion in construction ofan identity profile but may not be sufficient on its own toestablish identity. Absolute retention times of a given com-pound may vary from one chromatogram to the next.Figure 3. Typical planar chromatography.

Retention Volume (VR): The retention volume is thevolume of mobile phase required for elution of a compo-nent. It may be calculated from the retention time and theRelative Retention (r)1: Is the ratio of the adjustedflow rate in mL/min:retention time of a component relative to that of another

used as a reference obtained under identical conditions:VR = tR × F

r = tR2 − tM/tR1 − tM

Resolution (RS): The resolution is the separation ofwhere tR2 is the retention time measured from the point of two components in a mixture, calculated by:injection of the compound of interest; tR1 is the retentiontime measured from the point of injection of the compound RS = 2(tR2 − tR1)/(W1 + W2)used as reference; and tM is the retention time of anonretained marker defined in the procedure, all deter- where tR2 and tR1 are the retention times of the two compo-mined under identical experimental conditions on the same nents; and W2 and W1 are the corresponding widths at thecolumn. bases of the peaks obtained by extrapolating the relatively

straight sides of the peaks to the baseline.Relative Retention Time (RRT): Also known as unad-justed relative retention. Comparisons in USP are normally




Where electronic integrators are used, it may be conven- factor, α, and the capacity factor, k. [NOTE—All terms andient to determine the resolution, by the equation: symbols are defined in the preceding section Definitions and

Interpretation of Chromatograms.] For a given stationaryRS = 1.18(tR2 − tR1)/(W1,h/2 + W2,h/2) phase and mobile phase, N may be specified to ensure that

closely eluting compounds are resolved from each other, toestablish the general resolving power of the system, and toSeparation Factor (α): The separation factor is theensure that internal standards are resolved from the drug.relative retention calculated for two adjacent peaks (by con-This is a less reliable means to ensure resolution than is di-vention, the value of the separation factor is always >1):rect measurement. Column efficiency is, in part, a reflection

α = k2/k1 of peak sharpness, which is important for the detection oftrace components.

Replicate injections of a standard preparation or otherSymmetry Factor (AS)2: The symmetry factor (alsostandard solutions are compared to ascertain whether re-known as the tailing factor) of a peak (see Figure 4) is calcu-quirements for precision are met. Unless otherwise specifiedlated by:in the individual monograph, data from five replicate injec-tions of the analyte are used to calculate the relative stan-AS = W0.05/2fdard deviation, %RSD, if the requirement is 2.0% or less;data from six replicate injections are used if the relativewhere W0.05 is the width of the peak at 5% height and f isstandard deviation requirement is more than 2.0%.the distance from the peak maximum to the leading edge

For the Assay in a drug substance monograph, where theof the peak, the distance being measured at a point 5% ofvalue is 100% for the pure substance, and no maximumthe peak height from the baseline.relative standard deviation is stated, the maximum permit-ted %RSD is calculated for a series of injections of the refer-ence solution:

%RSD = KB√n/t90%, n − 1

where K is a constant (0.349), obtained from the expressionK = (0.6/√2) × (t 90%,5/√6), in which 0.6/√2 represents therequired percentage relative standard deviation after six in-jections for B = 1.0; B is the upper limit given in the defini-tion of the individual monograph minus 100%; n is thenumber of replicate injections of the reference solution (3≤n ≤ 6); and t90%, n −1 is the Student’s t at the 90%probability level (double sided) with n − 1 degrees offreedom.Figure 4. Asymmetrical chromatographic peak.

Unless otherwise prescribed, the maximum permitted rela-tive standard deviation does not exceed the appropriate

Tailing Factor (T): See Symmetry Factor. value given in the table of repeatability requirements. Thisrequirement does not apply to tests for related substances.

SYSTEM SUITABILITYRelative Standard Deviation Requirements

System suitability tests are an integral part of gas andNumber of Individual Injectionsliquid chromatographic methods. These tests are used to

3 4 5 6verify that the chromatographic system is adequate for theintended analysis. B (%) Maximum Permitted RSD

The tests are based on the concept that the equipment, 2.0 0.41 0.59 0.73 0.85electronics, analytical operations, and samples analyzed con- 2.5 0.52 0.74 0.92 1.06stitute an integral system that can be evaluated as such.

3.0 0.62 0.89 1.10 1.27Factors that may affect chromatographic behavior includethe following: The symmetry factor, AS, a measure of peak symmetry, is• Composition, ionic strength, temperature, and apparent unity for perfectly symmetrical peaks; and its value increasespH of the mobile phase as tailing becomes more pronounced (see Figure 4). In some• Flow rate, column dimensions, column temperature, cases, values less than unity may be observed. As peak sym-and pressure metry moves away from values of 1, integration, and hence• Stationary phase characteristics, including type of chro- precision, become less reliable.matographic support (particle-based or monolithic), The signal-to-noise ratio (S/N) is a useful system suitabilityparticle or macropore size, porosity, and specific surface parameter. The S/N is calculated as follows:area

• Reverse-phase and other surface modification of the sta- S/N = 2H/htionary phases, the extent of chemical modification (asexpressed by end-capping, carbon loading, etc.) where H is the height of the peak measured from the peak

The resolution, RS, is a function of the number of theoreti- apex to a baseline extrapolated over a distance ≥5 times thecal plates, N (also referred to as efficiency), the separation peak width at its half-height; and h is the difference be-

tween the largest and smallest noise values observed over a2 It is also a common practice to measure the Asymmetry Factor as the ratioof the distance between the vertical line connecting the peak apex with the distance ≥5 times the width at the half-height of the peakinterpolated baseline and the peak front, and the distance between that line and, if possible, situated equally around the peak of interestand the peak back measured at 10% of the peak height (see Figure 4), would (see Figure 5).be (W0.10 − f0.10)/f0.10. However, for the purposes of USP, only the formula (As)as presented here is valid.




Ternary MixturesSPECIFIED RATIO OF 60 : 35 : 5: For the second component,

30% of 35 is 10.5% absolute, which exceeds the maximumpermitted change of ±10% absolute in any component.Therefore the second component may be adjusted onlywithin the range of 25% to 45% absolute. For the thirdcomponent, 30% of 5 is 1.5% absolute. In all cases, a suffi-cient quantity of the first component is used to give a totalof 100%. Therefore, mixture ranges of 50 : 45 : 5 to70 : 25 : 5 or 58.5 : 35 : 6.5 to 61.5 : 35 : 3.5 would meetthe requirement.

Wavelength of UV-Visible Detector (HPLC): Devia-tions from the wavelengths specified in the procedure arenot permitted. The procedure specified by the detectorFigure 5. Noise and chromatographic peak, components of manufacturer, or another validated procedure, is used tothe S/N ratio. verify that error in the detector wavelength is, at most,±3 nm.

These system suitability tests are performed by collecting Stationary Phasedata from replicate injections of standard or other solutions COLUMN LENGTH (GC, HPLC): Can be adjusted by as muchas specified in the individual monograph. as ±70%.

The specification of definitive parameters in a monographCOLUMN INNER DIAMETER (HPLC): Can be adjusted if the lin-does not preclude the use of other suitable operating condi- ear velocity is kept constant. See Flow Rate (HPLC) below.tions. Adjustments are permitted only whenCOLUMN INNER DIAMETER (GC)—Can be adjusted by as much• Suitable standards (including Reference Standards) are

as ±50% for GC.available for all compounds used in the suitability test;and FILM THICKNESS (CAPILLARY CG)—Can be adjusted by as much

• Those standards show that the adjustments improved as −50% to 100%.the quality of the chromatography with respect to the Particle Size (HPLC): The particle size can be re-system suitability requirements. duced by as much as 50%, but cannot be increased.

Adjustments to chromatographic systems performed in or- Particle Size (GC): Changing from a larger to ader to comply with system suitability requirements are not smaller or from a smaller to a larger particle size GC meshto be made in order to compensate for column failure or support is acceptable if the chromatography meets the re-system malfunction. quirements of system suitability and the same particle sizeIf adjustments of operating conditions are necessary in or- range ratio is maintained. The particle size range ratio isder to meet system suitability requirements, each of the defined as the diameter of the largest particle divided byitems in the following list is the maximum variation that can the diameter of the smallest particle.be considered, unless otherwise directed in the monograph;Flow Rate (GC): The flow rate can be adjusted by asthese changes may require additional validation data. To

much as ±50%.verify the suitability of the method under the new condi-Flow Rate (HPLC): When column dimensions havetions, assess the relevant analytical performance characteris-

been modified, the flow rate can be adjusted using:tics potentially affected by the change. Multiple adjustmentscan have a cumulative effect on the performance of thesystem and are to be considered carefully before implemen-tation. Adjustments to the composition of the mobile phasein gradient elution are not recommended. If adjustments arenecessary, only column changes (same packing material) ordwell volume adjustments are recommended. in which F1 is the flow rate indicated in the monograph, in

pH of Mobile Phase (HPLC): The pH of the aqueous mL/min; F2 is the adjusted flow rate, in mL/min; l1 is thebuffer used in the preparation of the mobile phase can be length of the column indicated in the monograph; l2 is theadjusted to within ±0.2 units of the value or range specified. length of the column used; d1 is the column inner diameter

indicated in the monograph; and d2 is the internal diameterConcentration of Salts in Buffer (HPLC): The con-of the column used. Additionally, the flow rate can be ad-centration of the salts used in the preparation of the aque-justed by ±50%.ous buffer employed in the mobile phase can be adjusted to

within ±10% if the permitted pH variation (see above) is Injection Volume (HPLC): The injection volume canmet. be reduced as far as is consistent with accepted precision

and detection limits; no increase is permitted.Ratio of Components in Mobile Phase (HPLC): Thefollowing adjustment limits apply to minor components of Injection Volume and Split Volume (GC): The injec-the mobile phase (specified at 50% or less). The amounts of tion volume and split volume may be adjusted if detectionthese components can be adjusted by ±30% relative. How- and repeatability are satisfactory.ever, the change in any component cannot exceed ±10% Column Temperature (HPLC): The column tempera-absolute (i.e., in relation to the total mobile phase). Adjust- ture can be adjusted by as much as ±10°. Column thermo-ment can be made to one minor component in a ternary stating is recommended to improve control and reproduc-mixture. Examples of adjustments for binary and ternary ibility of retention time.mixtures are given below. Oven Temperature (GC): The oven temperature can

Binary Mixtures be adjusted by as much as ±10%.SPECIFIED RATIO OF 50 : 50: 30% of 50 is 15% absolute, but Oven Temperature Program (GC): Adjustment of

this exceeds the maximum permitted change of ±10% abso- temperatures is permitted as stated above. When the speci-lute in either component. Therefore, the mobile phase ratio fied temperature must be maintained or when the tempera-may be adjusted only within the range of 40 : 60 to 60 : 40. ture must be changed from one value to another, an adjust-

SPECIFIED RATIO OF 2 : 98: 30% of 2 is 0.6% absolute. ment of up to ±20% is permitted.Therefore the maximum allowed adjustment is within the Unless otherwise directed in the monograph, system suit-range of 1.4 : 98.6 to 2.6 : 97.4. ability parameters are determined from the analyte peak.




Measured values of Rr or RF or tR for the sample substance Calibration Procedure: The relationship between thedo not deviate from the values obtained for the reference measured or evaluated signal y and the quantity (e.g., con-compound and mixture by more than the statistically deter- centration, mass) of substance x is determined, and the cali-mined reliability estimates from replicate assays of the refer- bration function is calculated. The analytical results are cal-ence compound. Relative retention times may be provided culated from the measured signal or evaluated signal of thein monographs for informational purposes only to aid in analyte and its position on the calibration curve.peak identification. There are no acceptance criteria applied In tests for impurities for both the External Standardto relative retention times. Method, when a dilution of the sample solution is used for

Suitability testing is used to ascertain the effectiveness of comparison, and the Normalization Procedure, any correctionthe final operating system, which should be subjected to factors indicated in the monograph are applied (e.g., whenthis testing. Make injections of the appropriate prepara- the response factor is outside the range 0.8–1.2).tion(s) as required in order to demonstrate adequate system When the impurity test prescribes the total of impuritiessuitability (as described in the Chromatographic system sec- or there is a quantitative determination of an impurity,tion of the method in a monograph) throughout the run. choice of an appropriate threshold setting and appropriate

The preparation can be a standard preparation or a solu- conditions for the integration of the peak areas is important.tion containing a known amount of analyte and any addi- In such tests the limit at or below which a peak is disre-tional materials (e.g., excipients or impurities) useful in con- garded is generally 0.05%. Thus, the threshold setting oftrolling the analytical system. Whenever there is a significant the data collection system corresponds to at least half of thischange in the chromatographic system (equipment, mobile limit. Integrate the peak area of any impurity that is notphase component, or other components) or in a critical rea- completely separated from the principal peak, preferably bygent, system suitability is to be reestablished. No sample valley-to-valley extrapolation (tangential skim).▲USP34

analysis is acceptable unless the suitability of the system hasbeen demonstrated.

QUANTITATION

During quantitation, disregard peaks caused by solvents ⟨631⟩ COLOR ANDand reagents or arising from the mobile phase or the sam-ple matrix. ACHROMICITY

In the linear range, peak areas and peak heights are usu-ally proportional to the quantity of compound eluting. Thepeak areas and peak heights are commonly measured by Definition—For the purposes of this chapter, color mayelectronic integrators but may be determined by more class- be defined as the perception or subjective response by anical approaches. Peak areas are generally used but may be observer to the objective stimulus of radiant energy in theless accurate if peak interference occurs. The components visible spectrum extending over the range 400 nm tomeasured are separated from any interfering components. 700 nm in wavelength. Perceived color is a function of threePeak tailing and fronting is minimized, and the measure- variables: spectral properties of the object, both absorptivement of peaks on tails of other peaks are avoided when and reflective; spectral properties of the source of illumina-possible. tion; and visual characteristics of the observer.

Although comparison of impurity peaks with those in the Two objects are said to have a color match for a particularchromatogram of a standard at a similar concentration is source of illumination when an observer cannot detect apreferred, impurity tests may be based on the measurement color difference. Where a pair of objects exhibit a colorof the peak response due to impurities and expressed as a match for one source of illumination and not another, theypercentage of the area of the drug peak. The standard may constitute a metameric pair. Color matches of two objectsbe the drug itself at a level corresponding to, for example, occur for all sources of illumination if the absorption and0.5% impurity, assuming similar peak responses. When im- reflectance spectra of the two objects are identical.purities must be determined with greater certainty, use a Achromicity or colorlessness is one extreme of any colorstandard of the impurity itself or apply a correction factor scale for transmission of light. It implies the complete ab-based on the response of the impurity relative to that of the sence of color, and therefore the visible spectrum of themain component. object lacks absorbances. For practical purposes, the ob-

External Standard Method: The concentration of server in this case perceives little if any absorption takingthe component(s) quantified is determined by comparing place in the visible spectrum.the response(s) obtained with the sample solution to the Color Attributes—Because the sensation of color hasresponse(s) obtained with a standard solution. both a subjective and an objective part, color cannot be

Internal Standard Method: Equal amounts of the in- described solely in spectrophotometric terms. The commonternal standard are introduced into the sample solution and attributes of color therefore cannot be given a one-to-onea standard solution. The internal standard is chosen so that correspondence with spectral terminology.it does not react with the test material, is stable, is resolved Three attributes are commonly used to identify a color:from the component(s) quantified (analytes), and does not (1) hue, or the quality by which one color family is distin-contain impurities with the same retention time as that of guished from another, such as red, yellow, blue, green, andthe analytes. The concentrations of the analytes are deter- intermediate terms; (2) value, or the quality that distin-mined by comparing the ratios of their peak areas or peak guishes a light color from a dark one; and (3) chroma, orheights and the internal standard in the sample solution the quality that distinguishes a strong color from a weakwith the ratios of their peak areas or peak heights and the one, or the extent to which a color differs from a gray ofinternal standard in the standard solution. the same value.

The three attributes of color may be used to define aNormalization Procedure: The percentage contentthree-dimensional color space in which any color is locatedof a component of the test material is calculated by deter-by its coordinates. The color space chosen is a visually uni-mining the area of the corresponding peak as a percentageform one if the geometric distance between two colors inof the total area of all the peaks, excluding those due tothe color space is directly a measure of the color distancesolvents or reagents or arising from the mobile phase or thebetween them. Cylindrical coordinates are often conven-sample matrix and those at or below the limit at which theyiently chosen. Points along the long axis represent valuecan be disregarded.



USP 34 Physical Tests /...

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