USP 36 Physical Tests / 621 Chromatography 1

(2) The chamber is sealed to allow equilibration (satura-tion) of the chamber and the paper with the solvent621 CHROMATOGRAPHY vapor. Any excess pressure is released as necessary.

(3) After equilibration of the chamber, the prepared mo-bile phase is introduced into the trough through theinlet.

(4) The inlet is closed, and the mobile solvent phase isINTRODUCTION allowed to travel the desired distance down the

paper.Chromatographic separation techniques are multistage (5) The sheet is removed from the chamber.

separation methods in which the components of a sample (6) The location of the solvent front is quickly marked,are distributed between two phases, of which one is station- and the sheet is dried.ary and the other mobile. The stationary phase may be a (7) The chromatogram is observed and measured directlysolid or a liquid supported on a solid or a gel. The station- or after suitable development to reveal the location ofary phase may be packed in a column, spread as a layer, the spots of the isolated drug or drugs.distributed as a film, or applied by other techniques. The Ascending Paper Chromatography Proceduremobile phase may be gaseous or liquid or supercritical fluid. (1) The mobile phase is added to the bottom of theThe separation may be based on adsorption, mass distribu- chamber.tion (partition), or ion exchange; or it may be based on (2) The chamber is sealed to allow equilibration (satura-differences among the physicochemical properties of the tion) of the chamber and the paper with the solventmolecules, such as size, mass, and volume. This chapter vapor. Any excess pressure is released as necessary.contains general procedures, definitions, and calculations of (3) The lower edge of the stationary phase is dipped intocommon parameters and describes general requirements for the mobile phase to permit the mobile phase to risesystem suitability. The types of chromatography useful in on the chromatographic sheet by capillary action.qualitative and quantitative analysis employed in USP proce- (4) When the solvent front has reached the desireddures are column, gas, paper, thin-layer (including high-per- height, the chamber is opened, the sheet is removed,formance thin-layer chromatography), and pressurized liquid the location of the solvent front is quickly marked,chromatography (commonly called high-pressure or high- and the sheet is dried.performance liquid chromatography). (5) The chromatogram is observed and measured directly

or after suitable development to reveal the location ofthe spots of the isolated drug or drugs.GENERAL PROCEDURES

This section describes the basic procedures used when a Thin-Layer Chromatographychromatographic method is described in a monograph. Thefollowing procedures are followed unless otherwise indicated Stationary Phase: The stationary phase is a relativelyin the individual monograph. thin, uniform layer of dry, finely powdered material applied

to a glass, plastic, or metal sheet or plate (typically calledthe plate). The stationary phase of TLC plates has an aver-Paper Chromatographyage particle size of 1015 m, and that of high-performanceTLC (HPTLC) plates has an average particle size of 5 m.Stationary Phase: The stationary phase is a sheet of pa- Commercial plates with a preadsorbent zone can be used ifper of suitable texture and thickness. Development may be they are specified in a monograph. Sample applied to theascending, in which the solvent is carried up the paper by preadsorbent region develops into sharp, narrow bands atcapillary forces, or descending, in which the solvent flow is the preadsorbentsorbent interface. The separationsalso assisted by gravitational force. The orientation of paper achieved may be based on adsorption, partition, or a com-grain with respect to solvent flow is to be kept constant in a bination of both effects, depending on the particular type ofseries of chromatograms. (The machine direction is usually stationary phase.designated by the manufacturer.)

Apparatus: A chromatographic chamber made of inert,Apparatus: The essential equipment for paper chroma- transparent material and having the following specificationstography consists of a vapor-tight chamber with inlets for is used: a flat-bottom or twin trough, a tightly fitted lid, andaddition of solvent and a rack of corrosion-resistant material a size suitable for the plates. The chamber is lined on atabout 5 cm shorter than the inside height of the chamber. least one wall with filter paper. Sufficient mobile phase orThe rack serves as a support for solvent troughs and for developing solvent is added to the chamber that, after im-antisiphon rods that, in turn, hold up the chromatographic pregnation of the filter paper, a depth appropriate to thesheets. The bottom of the chamber is covered with the pre- dimensions of the plate used is available. The chromato-scribed solvent system or mobile phase. Saturation of the graphic chamber is closed and allowed to equilibrate.chamber with solvent vapor is facilitated by lining the inside [NOTEUnless otherwise indicated, the chromatographicwalls with paper wetted with the prescribed solvent system. separations are performed in a saturated chamber.]Spotting: The substance or substances analyzed are dis- Detection/Visualization: An ultraviolet (UV) lightsolved in a suitable solvent. Convenient volumes, delivered source suitable for observations under short- (254 nm) andfrom suitable micropipets, of the resulting solution, normally long- (365 nm) wavelength UV light and a variety of othercontaining 120 g of the compound, are placed in 6- to spray reagents used to make spots visible are often used.10-mm spots not less than 3 cm apart.

Spotting: Solutions are spotted on the surface of theDescending Paper Chromatography Procedure stationary phase (plate) at the prescribed volume in suffi-(1) A spotted chromatographic sheet is suspended in the ciently small portions to obtain circular spots of 25 mm inapparatus, using the antisiphon rod to hold the upper diameter (12 mm on HPTLC plates) or bands of 1020 mmend of the sheet in the solvent trough. [NOTEEnsure 12 mm (510 mm 0.51 mm on HPTLC plates) at anthat the portion of the sheet hanging below the rods appropriate distance from the lower edge of and sides ofis freely suspended in the chamber without touching the plate. [NOTEDuring development, the application posi-the rack, the chamber walls, or the fluid in the tion must be at least 5 mm (TLC) or 3 mm (HPTLC) abovechamber.] the level of the mobile phase.] The solutions are applied ona line parallel to the lower edge of the plate with an intervalof at least 10 mm (5 mm on HPTLC plates) between the

2 621 Chromatography / Physical Tests USP 36

centers of spots, or 4 mm (2 mm on HPTLC plates) between (2) Rinse the tip of the chromatographic column withthe edges of bands, then allowed to dry. about 1 mL of mobile phase before each change in

composition of mobile phase and after completion ofProcedurethe elution.(1) Place the plate in the chamber, ensuring that the

(3) If the analyte is introduced into the column as a solu-spots or bands are above the surface of the mobiletion in the mobile phase, allow it to pass completelyphase.into the column packing, then add mobile phase in(2) Close the chamber.several small portions, allowing each to drain com-(3) Allow the mobile phase to ascend the plate until thepletely, before adding the bulk of the mobile phase.solvent front has traveled three-quarters of the length

(4) Where the procedure indicates the use of multipleof the plate, or the distance prescribed in thechromatographic columns mounted in series and themonograph.addition of mobile phase in divided portions is speci-(4) Remove the plate, mark the solvent front with a pen-fied, allow each portion to drain completely throughcil, and allow to dry.each column, and rinse the tip of each with mobile(5) Visualize the chromatograms as prescribed.phase before the addition of each succeeding portion.(6) Determine the chromatographic retardation factor (RF)

values for the principal spots or zones.(7) Presumptive identification can be made by observa- Gas Chromatography (GC)

tion of spots or zones of identical RF value and aboutequal magnitude obtained, respectively, with an un- Liquid Stationary Phase: This type of phase is availableknown and a standard chromatographed on the same in packed or capillary columns.plate. A visual comparison of the size or intensity of

Packed Column GC: The liquid stationary phase is de-the spots or zones may serve for semiquantitative esti-posited on a finely divided, inert solid support, such as dia-mation. Quantitative measurements are possible bytomaceous earth, porous polymer, or graphitized carbon,means of densitometry (absorbence or fluorescencewhich is packed into a column that is typically 24 mm inmeasurements).internal diameter and 13 m in length.

Capillary Column GC: In capillary columns, which con-Column Chromatography tain no packed solid support, the liquid stationary phase isdeposited on the inner surface of the column and may be

Solid Support: Purified siliceous earth is used for nor- chemically bonded to it.mal-phase separation. Silanized chromatographic siliceous Solid Stationary Phase: This type of phase is availableearth is used for reverse-phase partition chromatography. only in packed columns. In these columns the solid phase is

Stationary Phase: The solid support is modified by the an active adsorbent, such as alumina, silica, or carbon,addition of a stationary phase specified in the individual packed into a column. Polyaromatic porous resins, whichmonograph. If a mixture of liquids is used as the stationary are sometimes used in packed columns, are not coated withphase, mix the liquids before the introduction of the solid a liquid phase. [NOTEPacked and capillary columns mustsupport. be conditioned before use until the baseline and other char-

Mobile Phase: The mobile phase is specified in the in- acteristics are stable. The column or packing material sup-dividual monograph. If the stationary phase is an aqueous plier provides instructions for the recommended condition-solution, equilibrate with water. If the stationary phase is a ing procedure.]polar organic fluid, equilibrate with that fluid. Apparatus: A gas chromatograph consists of a carrier

Apparatus: Unless otherwise specified in the individual gas source, injection port, column, detector, and recordingmonograph, the chromatographic tube is about 22 mm in device. The injection port, column, and detector are tem-inside diameter and 200300 mm long. Attached to it is a perature controlled and may be varied as part of the analy-delivery tube, without stopcock, about 4 mm in inside diam- sis. The typical carrier gas is helium, nitrogen, or hydrogen,eter and about 50 mm long. depending on the column and detector in use. The type of

detector used depends on the nature of the compoundsAPPARATUS PREPARATION: Pack a pledget of fine glass woolanalyzed and is specified in the individual monograph. De-in the base of the tube. Combine the specified volume oftector output is recorded as a function of time, and thestationary phase and the specified amount of solid supportinstrument response, measured as peak area or peak height,to produce a homogeneous, fluffy mixture. Transfer thisis a function of the amount present.mixture to the chromatographic tube, and tamp, using gen-

tle pressure, to obtain a uniform mass. If the specified Temperature Program: The length and quality of a GCamount of solid support is more than 3 g, transfer the mix- separation can be controlled by altering the temperature ofture to the column in portions of approximately 2 g, and the chromatographic column. When a temperature programtamp each portion. If the assay or test requires a multiseg- is necessary, the individual monograph indicates the condi-ment column with a different stationary phase specified for tions in table format. The table indicates the initial tempera-each segment, tamp after the addition of each segment, ture, rate of temperature change (ramp), final temperature,and add each succeeding segment directly to the previous and hold time at the final temperature.one. Pack a pledget of fine glass wool above the completed Procedurecolumn packing. [NOTEThe 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 to

Liquid Chromatography (LC)prepare the sample solution before adding the final portionof glass wool.

The term liquid chromatography, as used in the compen-Procedure dia, is synonymous with high-pressure liquid chromatogra-(1) Transfer the mobile phase to the column space above phy and high-performance liquid chromatography. LC is athe column packing, and allow it to flow through thecolumn under the influence of gravity.

USP 36 Physical Tests / 621 Chromatography 3

Figure 1. Chromatographic separation of two substances.

separation technique based on a solid stationary phase and for the chromatographer in identifying the pertinent chro-a liquid mobile phase. matographic column specified in the individual monograph.

Stationary Phase: Separations are achieved by parti-tion, adsorption, or ion-exchange processes, depending on DEFINITIONS AND INTERPRETATION OFthe type of stationary phase used. The most commonly used

CHROMATOGRAMSstationary phases are modified silica or polymeric beads. Thebeads are modified by the addition of long-chain hydrocar-bons. The specific type of packing needed to complete an Chromatogram: A chromatogram is a graphical repre-analysis is indicated by the L designation in the individual sentation of the detector response, concentration of analytemonograph (see also the section Chromatographic Columns, in the effluent, or other quantity used as a measure of efflu-below). The size of the beads is often described in the mon- ent concentration versus effluent volume or time. In planarograph as well. Changes in the packing type and size are chromatography, chromatogram may refer to the paper orcovered in the System Suitability section of this chapter. layer with the separated zones.

Figure 1 represents a typical chromatographic separationChromatographic Column: The term column includesof two substances, 1 and 2. tR1 and tR2 are the respectivestainless steel, lined stainless steel, and polymeric columns,retention times; and h is the height, h/2 the half-height,packed with a stationary phase. The length and inner diam-and Wh/2 the width at half-height, for peak 1. W1 and W2eter of the column affects the separation, and therefore typi-are the respective widths of peaks 1 and 2 at the baseline.cal column dimensions are included in the individual mono-Air peaks are a feature of gas chromatograms and corre-graph. Changes to column dimensions are discussed in thespond to the solvent front in LC. The retention time ofSystem Suitability section of this chapter. Compendial mono-these air peaks, or unretained components, is designated asgraphs do not include the name of appropriate columns;tM. this omission avoids the appearance of endorsement of a

vendor's product and natural changes in the marketplace. Dwell Volume (D): The dwell volume (also known asSee the section Chromatographic Columns for more informa- gradient delay volume) is the volume between the point attion. which the eluents meet and the top of the column.

Mobile Phase: The mobile phase is a solvent or a mix- Hold-Up Time (tM): The hold-up time is the time re-ture of solvents, as defined in the individual monograph. quired for elution of an unretained component (see Figure 1,

shown as an air or unretained solvent peak, with the base-Apparatus: A liquid chromatograph consists of a reser-line scale in min).voir containing the mobile phase, a pump to force the mo-

bile phase through the system at high pressure, an injector Hold-Up Volume (VM): The hold-up volume is the vol-to introduce the sample into the mobile phase, a chromato- ume of mobile phase required for elution of an unretainedgraphic column, a detector, and a data collection device. component. It may be calculated from the hold-up time

and the flow rate F, in mL/min:Gradient Elution: The technique of continuouslychanging the solvent composition during the chromato-

VM = tM Fgraphic run is called gradient elution or solvent program-ming. The gradient elution profile is presented in the indi-

In size exclusion chromatography, the symbol VO is used.vidual monograph as a gradient table, which lists the timeNumber of Theoretical Plates (N)1: N is a measure ofand proportional composition of the mobile phase at the

column efficiency. For Gaussian peaks, it is calculated by:stated time.Procedure N = 16(tR/W)2(1) Equilibrate the column and detector with mobile

phase at the specified flow rate until a constant signal where tR is the retention time of the substance, and W is theis received. peak width at its base, obtained by extrapolating the rela-(2) Inject a sample through the injector, or use an tively straight sides of the peak to the baseline. The value ofautosampler. N depends upon the substance being chromatographed as(3) Begin the gradient program. well as the operating conditions, such as the flow rate and(4) Record the chromatogram. temperature of the mobile phase or carrier gas, the quality(5) Analyze as directed in the monograph. of the packing, the uniformity of the packing within the1The parameters k, N, r, and rG were developed for isothermal GCCHROMATOGRAPHIC COLUMNS separations and isocratic HPLC separations. Because these terms arethermodynamic parameters, they are valid only for separations made atconstant temperature, mobile phase composition, and flow rate. However, forA complete list of packings (L), phases (G), and supports separations made with a temperature program or solvent gradient, these

(S) used in USPNF tests and assays is located in USPNF parameters may be used simply as comparative means to ensure thatadequate chromatographic conditions exist to perform the methods asand PF, Reagents, Indicators, and SolutionsChromatographicintended in the monographs.Columns. This list is intended to be a convenient reference

4 621 Chromatography / Physical Tests USP 36

column, and, for capillary columns, the thickness of the sta- tance simultaneously traveled by a reference compound (seetionary phase film and the internal diameter and length of Figure 3) and is used in planar chromatography.the column.

Rret = b / cWhere electronic integrators are used, it may be conven-ient to determine the number of theorical plates, by theequation:

where Wh/2 is the peak width at half-height. However, in theevent of dispute, only equations based on peak width atbaseline are to be used.

Peak: The peak is the portion of the chromatographicrecording of the detector response when a single compo-nent is eluted from the column. If separation is incomplete,two or more components may be eluted as one unresolvedpeak.

Peak-to-Valley Ratio (p/v): The p/v may be employed Figure 3. Typical planar chromatography.as a system suitability criterion in a test for related sub-stances when baseline separation between two peaks is notachieved. Figure 2 represents a partial separation of two sub- Relative Retention (r)1: Is the ratio of the adjusted re-stances, where Hp is the height above the extrapolated tention time of a component relative to that of anotherbaseline of the minor peak and Hv is the height above the used as a reference obtained under identical conditions:extrapolated baseline at the lowest point of the curve sepa-rating the minor and major peaks: r = tR2 tM/tR1 tM

p/v = Hp/Hv where tR2 is the retention time measured from the point ofinjection of the compound of interest; tR1 is the retentiontime measured from the point of injection of the compoundused as reference; and tM is the retention time of anonretained marker defined in the procedure, all deter-mined under identical experimental conditions on the samecolumn.

Relative Retention Time (RRT): Also known as unad-justed relative retention. Comparisons in USP are normallymade in terms of unadjusted relative retention, unless other-wise indicated.

RRT = tR2/tR1

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

Relative Standard Deviation in Percentage

Figure 2. Peak-to-valley ratio determination.

Retardation Factor (RF): The retardation factor is theRelative Retardation (Rret): The relative retardation is ratio of the distance traveled by the center of the spot tothe ratio of the distance traveled by the analyte to the dis-the 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:

or

USP 36 Physical Tests / 621 Chromatography 5

The retention factor of a component may be determined Tailing Factor (T): See Symmetry Factor.from the chromatogram:

SYSTEM SUITABILITYk = (tR tM)/tMSystem suitability tests are an integral part of gas andRetention Time (tR): In liquid chromatography and gas liquid chromatographic methods. These tests are used tochromatography, the retention time, tR, is defined as the verify that the chromatographic system is adequate for thetime elapsed between the injection of the sample and the

intended analysis.appearance of the maximum peak response of the elutedThe tests are based on the concept that the equipment,sample zone. tR may be used as a parameter for identifica- electronics, analytical operations, and samples analyzed con-tion. Chromatographic retention times are characteristic of

stitute an integral system that can be evaluated as such.the compounds they represent but are not unique. Coinci-Factors that may affect chromatographic behavior includedence of retention times of a sample and a reference sub-

the following:stance can be used as a partial criterion in construction of Composition, ionic strength, temperature, and apparentan identity profile but may not be sufficient on its own to

pH of the mobile phaseestablish identity. Absolute retention times of a given com- Flow rate, column dimensions, column temperature,pound may vary from one chromatogram to the next.

and pressureRetention Volume (VR): The retention volume is the Stationary phase characteristics, including type of chro-volume of mobile phase required for elution of a compo- matographic support (particle-based or monolithic),nent. It may be calculated from the retention time and the particle or macropore size, porosity, and specific surfaceflow rate in mL/min: area Reverse-phase and other surface modification of the sta-VR = tR F tionary phases, the extent of chemical modification (as

expressed by end-capping, carbon loading, etc.)Resolution (RS): The resolution is the separation of two The resolution, RS, is a function of the number of theoreti-components in a mixture, calculated by: cal plates, N (also referred to as efficiency), the separation

factor, , and the capacity factor, k. [NOTEAll terms andRS = 2(tR2 tR1)/(W1 + W2) symbols are defined in the preceding section Definitions andInterpretation of Chromatograms.] For a given stationarywhere tR2 and tR1 are the retention times of the two compo- phase and mobile phase, N may be specified to ensure thatnents; and W2 and W1 are the corresponding widths at the closely eluting compounds are resolved from each other, tobases of the peaks obtained by extrapolating the relatively establish the general resolving power of the system, and tostraight sides of the peaks to the baseline. ensure that internal standards are resolved from the drug.Where electronic integrators are used, it may be conven- This is a less reliable means to ensure resolution than is di-ient to determine the resolution, by the equation: rect measurement. Column efficiency is, in part, a reflectionof peak sharpness, which is important for the detection ofRS = 1.18(tR2 tR1)/(W1,h/2 + W2,h/2) trace components.

Replicate injections of a standard preparation or otherSeparation Factor (): The separation factor is the rela- standard solutions are compared to ascertain whether re-

tive retention calculated for two adjacent peaks (by conven- quirements for precision are met. Unless otherwise specifiedtion, the value of the separation factor is always >1): in the individual monograph, data from five replicate injec-

tions of the analyte are used to calculate the relative stan- = k2/k1 dard deviation, %RSD, if the requirement is 2.0% or less;

data from six replicate injections are used if the relativeSymmetry Factor (AS)2: The symmetry factor (also standard deviation requirement is more than 2.0%.

known as the tailing factor) of a peak (see Figure 4) is calcu- For the Assay in a drug substance monograph, where thelated by: value is 100% for the pure substance, and no maximum

relative standard deviation is stated, the maximum permit-AS = W0.05/2f ted %RSD is calculated for a series of injections of the refer-

ence solution:where W0.05 is the width of the peak at 5% height and f isthe distance from the peak maximum to the leading edge %RSD = KBn/t90%, n 1of the peak, the distance being measured at a point 5% ofthe peak height from the baseline. where K is a constant (0.349), obtained from the expression

K = (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 of free-dom.

Unless otherwise prescribed, the maximum permitted rela-tive standard deviation does not exceed the appropriatevalue given in the table of repeatability requirements. Thisrequirement does not apply to tests for related substances.

Figure 4. Asymmetrical chromatographic peak.2 It is also a common practice to measure the Asymmetry Factor as the ratio Relative Standard Deviation Requirementsof the distance between the vertical line connecting the peak apex with theinterpolated baseline and the peak front, and the distance between that line Number of Individual Injectionsand the peak back measured at 10% of the peak height (see Figure 4), would 3 4 5 6be (W0.10 f0.10)/f0.10. However, for the purposes of USP, only the formula (As)as presented here is valid. B (%) Maximum Permitted RSD

2.0 0.41 0.59 0.73 0.85

6 621 Chromatography / Physical Tests USP 36

Relative Standard Deviation Requirements (Continued) Concentration of Salts in Buffer (HPLC): The concen-tration of the salts used in the preparation of the aqueousNumber of Individual Injections buffer employed in the mobile phase can be adjusted to

2.5 0.52 0.74 0.92 1.06 within 10% if the permitted pH variation (see above) is3.0 0.62 0.89 1.10 1.27 met.

Ratio of Components in Mobile Phase (HPLC): TheThe symmetry factor, AS, a measure of peak symmetry, is following adjustment limits apply to minor components ofunity for perfectly symmetrical peaks; and its value increasesthe mobile phase (specified at 50% or less). The amounts ofas tailing becomes more pronounced (see Figure 4). In somethese components can be adjusted by 30% relative. How-cases, values less than unity may be observed. As peak sym-ever, the change in any component cannot exceed 10%metry moves away from values of 1, integration, and henceabsolute (i.e., in relation to the total mobile phase). Adjust-precision, become less reliable.ment can be made to one minor component in a ternaryThe signal-to-noise ratio (S/N) is a useful system suitabilitymixture. Examples of adjustments for binary and ternaryparameter. The S/N is calculated as follows:mixtures are given below.

Binary MixturesS/N = 2H/hSPECIFIED RATIO OF 50:50: 30% of 50 is 15% absolute, but

where H is the height of the peak measured from the peak this exceeds the maximum permitted change of 10% abso-apex to a baseline extrapolated over a distance 5 times the lute in either component. Therefore, the mobile phase ratiopeak width at its half-height; and h is the difference be- may be adjusted only within the range of 40:60 to 60:40.tween the largest and smallest noise values observed over a SPECIFIED RATIO OF 2:98: 30% of 2 is 0.6% absolute. There-distance 5 times the width at the half-height of the peak fore the maximum allowed adjustment is within the rangeand, if possible, situated equally around the peak of interest of 1.4:98.6 to 2.6:97.4.(see Figure 5).

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 to 70:25:5or 58.5:35:6.5 to 61.5:35:3.5 would meet the requirement.

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

Stationary PhaseCOLUMN LENGTH (GC, HPLC): Can be adjusted by as much

These system suitability tests are performed by collecting as 70%.data from replicate injections of standard or other solutions

COLUMN INNER DIAMETER (HPLC): Can be adjusted if the lin-as specified in the individual monograph.ear velocity is kept constant. See Flow Rate (HPLC) below.The specification of definitive parameters in a monograph

COLUMN INNER DIAMETER (GC)Can be adjusted by as muchdoes not preclude the use of other suitable operating condi-as 50% for GC.tions. Adjustments are permitted only when

Suitable standards (including Reference Standards) are FILM THICKNESS (CAPILLARY CG)Can be adjusted by as muchavailable for all compounds used in the suitability test; as 50% to 100%.and Particle Size (HPLC): The particle size can be reduced

Those standards show that the adjustments improved by as much as 50%, but cannot be increased.the quality of the chromatography with respect to the Particle Size (GC): Changing from a larger to a smallersystem suitability requirements. or from a smaller to a larger particle size GC mesh supportAdjustments to chromatographic systems performed in or- is acceptable if the chromatography meets the requirementsder to comply with system suitability requirements are not of system suitability and the same particle size range ratio isto be made in order to compensate for column failure or maintained. The particle size range ratio is defined as thesystem malfunction. diameter of the largest particle divided by the diameter ofIf adjustments of operating conditions are necessary in or- the smallest particle.der to meet system suitability requirements, each of the

Flow Rate (GC): The flow rate can be adjusted by asitems in the following list is the maximum variation that canmuch as 50%.be considered, unless otherwise directed in the monograph;

Flow Rate (HPLC): When column dimensions havethese changes may require additional validation data. Tobeen modified, the flow rate can be adjusted using:verify the suitability of the method under the new condi-

tions, assess the relevant analytical performance characteris-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 are in which F1 is the flow rate indicated in the monograph, innecessary, only column changes (same packing material) or mL/min; F2 is the adjusted flow rate, in mL/min; l1 is thedwell volume adjustments are recommended. length of the column indicated in the monograph; l2 is the

pH of Mobile Phase (HPLC): The pH of the aqueous length of the column used; d1 is the column inner diameterbuffer used in the preparation of the mobile phase can be indicated in the monograph; and d2 is the internal diameteradjusted to within 0.2 units of the value or range specified.

USP 36 Physical Tests / 621 Chromatography 7

of the column used. Additionally, the flow rate can be ad- Peak tailing and fronting is minimized, and the measure-justed by 50%. ment of peaks on tails of other peaks are avoided when

possible.Injection Volume (HPLC): The injection volume can beAlthough comparison of impurity peaks with those in thereduced as far as is consistent with accepted precision and

chromatogram of a standard at a similar concentration isdetection limits; no increase is permitted.preferred, impurity tests may be based on the measurementInjection Volume and Split Volume (GC): The injec- of the peak response due to impurities and expressed as ation volume and split volume may be adjusted if detection percentage of the area of the drug peak. The standard mayand repeatability are satisfactory. be the drug itself at a level corresponding to, for example,

Column Temperature (HPLC): The column tempera- 0.5% impurity, assuming similar peak responses. When im-ture can be adjusted by as much as 10. Column thermo- purities must be determined with greater certainty, use astating is recommended to improve control and reproduc- standard of the impurity itself or apply a correction factoribility of retention time. based on the response of the impurity relative to that of the

Oven Temperature (GC): The oven temperature can main component.be adjusted by as much as 10%. External Standard Method: The concentration of the

Oven Temperature Program (GC): Adjustment of tem- component(s) quantified is determined by comparing theperatures is permitted as stated above. When the specified response(s) obtained with the sample solution to the re-temperature must be maintained or when the temperature sponse(s) obtained with a standard solution.must be changed from one value to another, an adjustment Internal Standard Method: Equal amounts of the in-of up to 20% is permitted. ternal standard are introduced into the sample solution and

Unless otherwise directed in the monograph, system suit- a standard solution. The internal standard is chosen so thatability parameters are determined from the analyte peak. it does not react with the test material, is stable, is resolved

Measured values of Rr or RF or tR for the sample substance from the component(s) quantified (analytes), and does notdo not deviate from the values obtained for the reference contain impurities with the same retention time as that ofcompound and mixture by more than the statistically deter- the analytes. The concentrations of the analytes are deter-mined reliability estimates from replicate assays of the refer- mined by comparing the ratios of their peak areas or peakence compound. Relative retention times may be provided heights and the internal standard in the sample solutionin monographs for informational purposes only to aid in with the ratios of their peak areas or peak heights and thepeak identification. There are no acceptance criteria applied internal standard in the standard solution.to relative retention times. Normalization Procedure: The percentage content of aSuitability testing is used to ascertain the effectiveness of component of the test material is calculated by determiningthe final operating system, which should be subjected to the area of the corresponding peak as a percentage of thethis testing. Make injections of the appropriate prepara- total area of all the peaks, excluding those due to solventstion(s) as required in order to demonstrate adequate system or reagents or arising from the mobile phase or the samplesuitability (as described in the Chromatographic system sec- matrix and those at or below the limit at which they can betion of the method in a monograph) throughout the run. disregarded.The preparation can be a standard preparation or a solu-

Calibration Procedure: The relationship between thetion containing a known amount of analyte and any addi-measured or evaluated signal y and the quantity (e.g., con-tional materials (e.g., excipients or impurities) useful in con-centration, mass) of substance x is determined, and the cali-trolling the analytical system. Whenever there is a significantbration function is calculated. The analytical results are cal-change in the chromatographic system (equipment, mobileculated from the measured signal or evaluated signal of thephase component, or other components) or in a critical rea-analyte and its position on the calibration curve.gent, system suitability is to be reestablished. No sample

In tests for impurities for both the External Standardanalysis is acceptable unless the suitability of the system hasMethod, when a dilution of the sample solution is used forbeen demonstrated.comparison, and the Normalization Procedure, any correctionfactors indicated in the monograph are applied (e.g., when

QUANTITATION the response factor is outside the range 0.81.2).When the impurity test prescribes the total of impurities

During quantitation, disregard peaks caused by solvents or there is a quantitative determination of an impurity,and reagents or arising from the mobile phase or the sam- choice of an appropriate threshold setting and appropriateple matrix. conditions for the integration of the peak areas is important.

In the linear range, peak areas and peak heights are usu- In such tests the limit at or below which a peak is disre-ally proportional to the quantity of compound eluting. The garded is generally 0.05%. Thus, the threshold setting ofpeak areas and peak heights are commonly measured by the data collection system corresponds to at least half of thiselectronic integrators but may be determined by more class- limit. Integrate the peak area of any impurity that is notical approaches. Peak areas are generally used but may be completely separated from the principal peak, preferably byless accurate if peak interference occurs. The components valley-to-valley extrapolation (tangential skim).measured are separated from any interfering components.