Working document QAS/21.

905
February 2022
Draft for comments

2 1.14.1 CHROMATOGRAPHY

3 Draft proposal for inclusion in The International Pharmacopoeia

4 (February 2022)

5 DRAFT FOR COMMENTS

Please send any comments you may have on this draft working document to Dr Herbert Schmidt,
Technical Officer, Norms and Standards for Pharmaceuticals, Technical Standards and Specifications
(email: [email protected]), with a copy to Ms Sinéad Jones (email: [email protected]) by 25 March 2022.

Our working documents are sent out electronically and they will be placed on the WHO Medicines website
(https://www.who.int/teams/health-product-and-policy-standards/standards-and-
specifications/pharmaceuticals/current-projects) for comments under the “Working documents in public
consultation” link. If you wish to receive our draft guidelines, please send your e-mail address to
[email protected] and your name will be added to our electronic mailing list.

7 .
8 © World Health Organization 2022
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10 All rights reserved.
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12 This is a draft. The content of this document is not final, and the text may be subject to revisions before publication. The
13 document may not be reviewed, abstracted, quoted, reproduced, transmitted, distributed, translated or adapted, in part or in
14 whole, in any form or by any means without the permission of the World Health Organization.

15 Please send any request for permission to:

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17 Ms Sinéad Jones, Norms and Standards for Pharmaceuticals, Technical Standards and Specifications, Department of Health
18 Products Policy and Standards, World Health Organization, CH-1211 Geneva 27, Switzerland, email: [email protected].
19
20 The designations employed and the presentation of the material in this draft do not imply the expression of any opinion
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22 of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate
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35 This draft does not necessarily represent the decisions or the stated policy of the World Health Organization.
 Working document QAS/21.905
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36 SCHEDULE FOR THE ADOPTION PROCESS OF DOCUMENT QAS/21.905

37 1.14.1 CHROMATOGRAPHY

38

Description Date

Preparation of first draft working document based on the

internationally-harmonized text developed by the February 2022
Pharmacopoeial Discussion Group (PDG).

Document sent out for comment to the members of the

advisory panel on International Pharmacopoeia and February-March 2022
Pharmaceutical Preparations.

Presentation to the 56th meeting of the Expert

25 April to 2 May
Committee on Specifications for Pharmaceutical
2022
Preparations.

Further follow-up action as required.

39

40

41 [Note from the Secretariat. It is proposed to include the internationally-harmonized

42 text on chromatography (G20), developed by the Pharmacopoeial Discussion Group
43 (PDG), in The International Pharmacopoeia. The new chapter 1.14.1 Chromatography
44 shall replace the chapters 1.14.1 Thin-layer chromatography, 1.14.4 High-performance
45 liquid chromatography and 1.14.5 Gas Chromatography. Current references to the
46 chapters 1.14.1, 1.14.4 and 1.14.5 will be updated accordingly.]

47
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48 1.14.1 CHROMATOGRAPHY

49 This text is based on the internationally-harmonized text developed by the

50 Pharmacopoeial Discussion Group (PDG). It has been developed in line with the style
51 and requirements of The International Pharmacopoeia.

52 INTRODUCTION

53 Chromatographic separation techniques are multi-stage separation procedures in which

54 the components of a sample are distributed between two phases, one of which is
55 stationary whilst the other is mobile. The stationary phase may be a solid or a liquid
56 supported on a solid or a gel. The stationary phase may be packed in a column, spread
57 as a layer or distributed as a film. The mobile phase may be gaseous or liquid. The
58 separation may be based on adsorption, mass distribution (partition), ion exchange or
59 may be based on differences in the physico-chemical properties of the molecules such
60 as size, mass, volume, or others.

61 This chapter contains definitions and calculations of common parameters and generally
62 applicable requirements for system suitability. The principles of separation, apparatus
63 and methods are given in the corresponding general chapters.

64 DEFINITIONS

65 The system suitability and acceptance criteria in monographs have been set using
66 parameters as defined below. With some equipment, certain parameters, such as the
67 signal-to-noise ratio and resolution, can be calculated using software provided by the
68 manufacturer. It is the responsibility of the user to ensure that the calculation methods
69 used in the software are equivalent to the requirements of The International
70 Pharmacopoeia and to make any necessary corrections if this is not the case.

71

72
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73 Chromatogram

74 A graphical or other representation of detector response, effluent concentration or

75 other quantity used as a measure of effluent concentration, versus time or volume.
76 Idealised chromatograms are represented as a sequence of Gaussian peaks on a
77 baseline (Figure 1).

78

79 Figure 1

80 VM = hold-up volume;

81 tM = hold-up time;

82 VR1 = retention volume of peak 1;

83 tR1 = retention time of peak 1;

84 VR2 = retention volume of peak 2;

85 tR2 = retention time of peak 2;

86 Wh = peak width at half-height;

87 Wi = peak width at the inflexion point;

88 h = height of the peak;

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89 h/2 = half-height of the peak.

90 Distribution constant (K0)

91 In size-exclusion chromatography, the elution characteristics of a component in a

92 particular column may be given by the distribution constant (also referred to as
93 distribution coefficient) which is calculated using the following equation:

𝑡𝑅 − 𝑡0
94 𝐾0 =
𝑡𝑡 − 𝑡0

95 tR = retention time;

96 t0 = retention time of an unretained compound;

97 tt = total mobile phase time.

98 Dwell volume (D) (also referred to as VD)

99 The dwell volume (also known as gradient delay volume) is the volume between the
100 point at which the eluents meet and the inlet of the column. It can be determined using
101 the following procedure:

102 Column: replace the chromatographic column by an appropriate capillary tubing (e.g. 1
103 m × 0.12 mm).

104 Mobile phase:

105 − mobile phase A: water;

106 − mobile phase B: 0.1 per cent (V/V) solution of acetone R in water R.

Time Mobile phase A Mobile phase B

(min) (per cent V/V) (per cent V/V)
0 – 20 100 → 0 0 → 100

20 - 30 0 100
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107 Flow rate: set to obtain sufficient back-pressure (e.g. 2 mL/min).

108 Detection: spectrophotometer at 265 nm.

109 Determine the time (t0.5) in minutes when the absorbance has increased by 50 per cent
110 (see Figure 2).

111 𝐷 = 𝑡𝐷 × 𝐹

112 tD = t0.5 − 0.5tG, in minutes;

113 tG = pre-defined gradient time (= 20 min);

114 F = flow rate, in millilitres per minute.

115

116 Figure 2

117 Note: where applicable, this measurement is performed with the autosampler in the
118 inject position so as to include the injection loop volume in the dwell volume.

119 Hold-up time (tM)

120 Time required for elution of an unretained component (Figure 1, baseline scale being in
121 minutes or seconds).

122 In size-exclusion chromatography, the term retention time of an unretained compound

123 (t0) is used.

124
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125 Hold-up volume (VM)

126 The volume of the mobile phase required for elution of an unretained component. It
127 may be calculated from the hold-up time and the flow rate (F) in millilitres per minute
128 using the following equation:

129 𝑉𝑀 = 𝑡𝑀 × 𝐹

130 In size-exclusion chromatography, the term retention volume of an unretained

131 compound (V0) is used.

132 Peak

133 The portion of a chromatogram recording the detector response when a single
134 component (or 2 or more unresolved components) is eluted from the column.

135 The peak response may be represented by the peak area or the peak height (h).

136 Peak-to-valley ratio (p/v)

137 The peak-to-valley ratio may be employed as a system suitability criterion when the
138 baseline separation between two peaks is not achieved (Figure 3).

139

140 Figure 3
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𝑝 𝐻𝑝
141 =
𝑣 𝐻𝑣

142 Hp = height above the extrapolated baseline of the minor peak;

143 Hv = height above the extrapolated baseline at the lowest point of the curve separating
144 the minor and major peaks.

145 Plate height (H) (synonym: height equivalent to one theoretical plate {HETP})

146 Ratio of the column length (L), in micrometers, to the plate number (N):

𝐿
147 H=
𝑁

148 Plate number (N) (synonym: number of theoretical plates)

149 A number indicative of column performance (column efficiency). It can only be

150 calculated from data obtained under either isothermal, isocratic or isodense conditions
151 - depending on the technique - as the plate number, using the following equation, the
152 values of tR and wh being expressed in the same units:

𝑡𝑅 2
153 𝑁 = 5.54 ( )
𝑊ℎ

154 tR = retention time of the peak corresponding to the component;

155 wh = peak width at half-height (h/2).

156 The plate number varies with the component as well as with the column, the column
157 temperature, the mobile phase and the retention time.

158 Reduced plate height (h)

159 The ratio of the plate height (H), in micrometers, to the particle diameter (dp) in
160 micrometers:
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𝐻
161 h=
𝑑𝑝

162 Relative retardation (R rel)

163 The relative retardation, used in thin-layer chromatography, is calculated as the ratio of
164 the distances travelled by the spot of the compound of interest and a reference
165 compound (Figure 4).

166 R rel= b/c

167 Figure 4

168 a = migration distance of the mobile phase;

169 b = migration distance of the compound of interest;

170 c = migration distance of the reference compound.

171 Relative retention (r)

172 The relative retention is calculated as an estimate using the following equation:

𝑡𝑅𝑖 − 𝑡𝑀
173 𝑟=
𝑡𝑅𝑠𝑡 − 𝑡𝑀

174 tRi = retention time of the peak of interest;

175 tRst = retention time of the reference peak (usually the peak corresponding to the
176 substance to be examined);
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177 tM = hold-up time.

178 Relative retention, unadjusted (rG) or RRT

179 Unadjusted relative retention is calculated using the following equation:

𝑡𝑅𝑖
180 𝑟𝐺 =
𝑡𝑅𝑠𝑡

181 Unless otherwise indicated, the values for relative retention stated in monographs
182 correspond to unadjusted relative retention.

183 Relative retention time (RRT): see Relative retention, unadjusted.

184 Resolution (Rs)

185 The resolution between peaks of 2 components (Figure 1) may be calculated using the
186 following equation:

1.18(𝑡𝑅2 − 𝑡𝑅1 )
187 𝑅𝑠 =
𝑤ℎ1 + 𝑤ℎ2

188 tR2 > tR1

189 tR1, tR2 = retention times of the peaks;

190 wh1, wh2 = peak widths at half-height.

191 In quantitative thin-layer chromatography, using densitometry, the migration distances

192 are used instead of retention times and the resolution between peaks of 2 components
193 may be calculated using the following equation:

1.18𝑎(𝑅𝐹2 − 𝑅𝐹1 )
194 𝑅𝑠 =
𝑤ℎ1 + 𝑤ℎ2

195 RF2 > RF1

196 RF1, RF2 = retardation factors of the peaks;

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197 wh1, wh2 = peak widths at half-height;

198 a = migration distance of the solvent front.

199 Retardation factor (RF)

200 The retardation factor, used in thin-layer chromatography, is the ratio of the distance
201 from the point of application to the centre of the spot and the distance simultaneously
202 travelled by the solvent front from the point of application (Figure 4).

𝑏
203 𝑅𝐹 =
𝑎

204 b = migration distance of the compound of interest;

205 a = migration distance of the solvent front.

206 Retention factor (k)

207 The retention factor (also known as mass distribution ratio (Dm) or capacity factor (k′))
208 is defined as:

𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 𝑖𝑛 𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦 𝑝ℎ𝑎𝑠𝑒 𝑉𝑆

209 𝑘= = 𝐾𝐶
𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 𝑖𝑛 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒 𝑉𝑀

210 KC = distribution constant (also known as equilibrium distribution coefficient);

211 VS = volume of the stationary phase;

212 VM = volume of the mobile phase.

213 The retention factor of a component may be determined from the chromatogram using
214 the following equation:

𝑡𝑅 − 𝑡𝑀
215 𝑘=
𝑡𝑀

216 tR = retention time;

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217 tM = hold-up time.

218 Retention time (tR)

219 The time elapsed between the injection of the sample and the appearance of the
220 maximum peak response of the eluted sample zone (Figure 1, baseline scale being in
221 minutes or seconds).

222 Retention volume (VR)

223 The volume of the mobile phase required for elution of a compound. It may be
224 calculated from the retention time (tR) and the flow rate (F) in millilitres per minute
225 using the following equation:

226 𝑉𝑅 = 𝑡𝑅 × 𝐹

227 Retention time of an unretained compound (t0)

228 In size-exclusion chromatography, the retention time of a component whose molecules

229 are larger than the largest gel pores (Figure 5).
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230

231 Figure 5

232 Retention volume of an unretained compound (V0)

233 In size-exclusion chromatography, the retention volume of a component whose

234 molecules are larger than the largest gel pores. It may be calculated from the retention
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235 time of an unretained compound (t0) and the flow rate (F) in millilitres per minute using
236 the following equation:

237 𝑉0 = 𝑡0 × 𝐹

238 Separation factor (α)

239 The relative retention calculated for two adjacent peaks (by convention, the value of the
240 separation factor is always > 1):

241 α = k2/k1

242 k1 = retention factor of the first peak;

243 k2 = retention factor of the second peak.

244 Signal-to-noise ratio (S/N)

245 The short-term noise influences the precision and accuracy of quantitation. The signal-
246 to-noise ratio is calculated using the following equation:

2𝐻
247 𝑆/𝑁 =
ℎ

H = height of the peak (Figure 6) corresponding to the component concerned,

in the chromatogram obtained with the prescribed reference solution,
measured from the maximum of the peak to the extrapolated baseline of
the signal observed over a distance equal to 20 times the width at half-
height;
h = range of the noise in a chromatogram obtained after injection of a blank
(Figure 7), observed over a distance equal to 20 times the width at half-
height of the peak in the chromatogram obtained with the prescribed
reference solution and, if possible, situated equally around the place where
this peak would be found.
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248

249 Figure 6. Chromatogram of the reference solution

250

251 Figure 7. Chromatogram of a blank

252 If a baseline of 20 times the width at half-height is not obtainable because of peaks due
253 to the solvents or reagents, or arising from the mobile phase or the sample matrix, or
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254 due to the gas chromatographic temperature programme, a baseline of at least 5 times
255 the width at half-height is permitted.

256 Symmetry factor (As)

257 The symmetry factor of a peak (also known as the asymmetry factor or tailing factor)
258 (Figure 8) is calculated using the following equation:

𝑤0.05
259 𝐴𝑠 =
2𝑑

260 w0.05 = width of the peak at one-twentieth of the peak height;

261 d = distance between the perpendicular dropped from the peak maximum and the
262 leading edge of the peak at one-twentieth of the peak height.

263 An As value of 1.0 signifies symmetry. When As > 1.0, the peak is tailing. When As <
264 1.0, the peak is fronting.

265

266 Figure 8

267

268
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269 System repeatability

270 The repeatability of response is expressed as an estimated percentage relative standard

271 deviation (%RSD) of a consecutive series of measurements for not fewer than 3
272 injections or applications of a reference solution, and is calculated using the following
273 equation:

100 ∑(𝑦𝑖 − 𝑦̅)2

274 %RSD = √
𝑦̅ 𝑛−1

275 𝑦𝑖 = individual values expressed as peak area, peak height or ratio of areas by the
276 internal standardisation method;

277 𝑦̅ = mean of individual values;

278 n = number of individual values.

279 Total mobile phase time (tt)

280 In size-exclusion chromatography, the retention time of a component whose molecules

281 are smaller than the smallest gel pores (Figure 5).

282 Total mobile phase volume (Vt)

283 In size-exclusion chromatography, the retention volume of a component whose

284 molecules are smaller than the smallest gel pores. It may be calculated from the total
285 mobile phase time and the flow rate (F) in millilitres per minute using the following
286 equation:

287 𝑉𝑡 = 𝑡𝑡 × 𝐹

288 SYSTEM SUITABILITY

289 This section only covers liquid chromatography and gas chromatography.
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290 The various components of the equipment employed must be qualified and capable of
291 achieving the performance required to conduct the test or assay.

292 The system suitability tests represent an integral part of the analytical procedure and
293 are used to ensure the adequate performance of the chromatographic system. The
294 column plate number, retention factor (mass distribution ratio), system repeatability,
295 signal-to-noise, symmetry factor and resolution/peak-to-valley ratio are the parameters
296 that may be employed in assessing the performance of the chromatographic system.
297 When stated in the individual monograph, in cases of complex chromatographic
298 profiles (e.g. for biotechnological/biological products), visual comparison of the
299 profiles can be used as a system suitability test.

300 Factors that may affect the chromatographic behaviour include:

301 • composition and temperature of the mobile phase;

302 • ionic strength and pH of the aqueous component of the mobile phase;
303 • flow rate, column dimensions, column temperature and pressure;
304 • stationary phase characteristics, including type of chromatographic support
305 (particle-based or monolithic), particle or pore size, porosity and specific surface
306 area;
307 • reversed phase and other surface-modification of the stationary phases, the
308 extent of chemical modification (as expressed by end-capping, carbon loading,
309 etc.).

310 Retention times and relative retentions may be provided in monographs for information
311 purposes only, unless otherwise stated in the monograph. There are no acceptance
312 criteria applied to relative retentions.

313 Compliance with the system suitability criteria is required throughout the
314 chromatographic procedure. No sample analysis is acceptable unless the suitability of
315 the system has been demonstrated.
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316 The following requirements are to be fulfilled, in addition to any other system
317 suitability criteria stated in the monograph. When specific requirements are stated in
318 the monograph, they supersede the requirements mentioned in this chapter:

319 System repeatability – assay of an active substance or an excipient

320 In an assay of an active substance or an excipient, where the target value is 100 per cent
321 for a pure substance, and a system repeatability requirement is not specified, the
322 maximum permitted relative standard deviation (%RSDmax) for the defined limits is
323 calculated for a series (n = 3 to 6) of injections of the reference solution. The maximum
324 permitted relative standard deviation of the peak response does not exceed the
325 appropriate value given in Table 1.

𝐾𝐵√𝑛
326 %RSDmax =
𝑡90%, 𝑛−1

327 K = constant (0.349), obtained from the expression:

0.6 𝑡90 .5
330 𝐾= 𝑥
√2 √6
0.6
328 in which represents the required percentage relative standard deviation determined
√2

329 on 6 injections for B = 1.0;

331 B = upper limit given in the definition of the individual monograph minus 100 per cent;

332 n = number of replicate injections of the reference solution (3 ≤ n ≤ 6);

333 t90%,n-1 = Student’s t at the 90 per cent probability level (double sided) with n−1 degrees
334 of freedom.
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335 Table 1 – Maximum permitted relative standard deviation (assay)

Number of individual injections n

3 4 5 6
B (per cent) Maximum permitted relative standard deviation (per cent)
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

336 B = upper limit of content given in the individual monograph minus 100 per cent.

337 System sensitivity

338 The signal-to-noise ratio is used to define the system sensitivity. The limit of
339 quantitation (corresponding to a signal-to-noise ratio of 10) is equal to or less than the
340 reporting threshold.

341 Peak symmetry

342 Unless otherwise stated, in a test or assay, the symmetry factor (tailing factor) of the
343 peak used for quantitation is 0.8 to 1.8.

344 ADJUSTMENT OF CHROMATOGRAPHIC CONDITIONS

345 The chromatographic conditions described have been validated during the elaboration
346 of the monograph.

347 The extent to which the various parameters of a chromatographic test may be adjusted
348 without fundamentally modifying the pharmacopoeial analytical procedures are listed
349 below. Changes other than those indicated require revalidation of the procedure.

350 Multiple adjustments can have a cumulative effect on the performance of the system
351 and are to be properly evaluated by the users. This is particularly important in cases
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352 where the separation pattern is described as a profile. In those cases, a risk assessment
353 has to be carried out.

354 Any adjustments must be made on the basis of the pharmacopoeial procedure.

355 If adjustments are made to a pharmacopoeial procedure, additional verification tests

356 may be required. To verify the suitability of the adjusted pharmacopoeial procedure,
357 assess the relevant analytical performance characteristics potentially affected by the
358 change.

359 When a pharmacopoeial analytical procedure has been adjusted according to the
360 requirements stated below, no further adjustments are allowed without appropriate
361 revalidation.

362 Compliance with the system suitability criteria is required to verify that conditions for
363 satisfactory performance of the test or assay are achieved.

364 The adjustment of conditions with gradient elution (HPLC) or temperature

365 programming (GC) is more critical than with isocratic (HPLC) or isothermal (GC)
366 elution since it may shift some peaks to a different step of the gradient or to different
367 elution temperatures, potentially causing partial or complete coelution of adjacent peaks
368 or peak inversion, and thus leading to the incorrect assignment of peaks and to the
369 masking of peaks or a shift such that elution occurs beyond the prescribed elution time.

370 For some parameters, the adjustments are explicitly defined in the monograph to ensure
371 the system suitability.

372 Thin-layer chromatography

373 Composition of the mobile phase: the amount of the minor solvent components may be
374 adjusted by ± 30 per cent relative or ± 2 per cent absolute, whichever is the larger; no
375 other component is altered by more than 10 per cent absolute. A minor component
376 comprises less than or equal to (100/n) per cent, n being the total number of components
 Working document QAS/21.905
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377 of the mobile phase. For a minor component at 10 per cent of the mobile phase, a 30
378 per cent relative adjustment allows a range of 7-13 per cent whereas a 2 per cent absolute
379 adjustment allows a range of 8-12 per cent, the relative value therefore being the larger;
380 for a minor component at 5 per cent of the mobile phase, a 30 per cent relative
381 adjustment allows a range of 3.5-6.5 per cent whereas a 2 per cent absolute adjustment
382 allows a range of 3-7 per cent, the absolute value being the larger in this case.

383 pH of the aqueous component of the mobile phase: ± 0.2 pH units, unless otherwise
384 prescribed.

385 Concentration of salts in the buffer component of a mobile phase: ± 10 per cent.

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

388 Liquid chromatography: isocratic elution

389 Column parameters and flow rate

390 • Stationary phase: no change of the identity of the substituent (e.g. no

391 replacement of C18 by C8); the other physico-chemical characteristics of the
392 stationary phase (i.e. chromatographic support, surface modification and
393 extent of chemical modification must be similar); a change from Totally
394 Porous Particle (TPP) columns to Superficially Porous Particle (SPP)
395 columns is allowed provided the above-mentioned requirements are met.
396 • Column dimensions (particle size, length): the particle size and/or length of
397 the column may be modified provided that the ratio of the column length (L)
398 to the particle size (dp) remains constant or in the range between − 25 per cent
399 to + 50 per cent of the prescribed L/dp ratio.
400 • Adjustments from totally porous to superficially porous particles: for the
401 application of particle-size adjustment from totally porous to superficially
402 porous particles, other combinations of L and dp can be used provided that
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403 the plate number (N) is within – 25 per cent to + 50 per cent, relative to the
404 prescribed column. These changes are acceptable provided the system
405 suitability criteria are fulfilled, and selectivity and elution order of the
406 specified impurities to be controlled are demonstrated to be equivalent.
407 • Internal diameter: in absence of a change in particle size and/or length, the
408 internal diameter of the column may be adjusted.

409 Caution is necessary when the adjustment results in smaller peak volumes, due to a
410 smaller particle size or a smaller internal diameter, a situation which may require
411 adjustments to minimize extra-column band broadening by factors such as instrument
412 connections, detector cell volume and sampling rate, and injection volume.

413 When the particle size is changed, the flow rate requires adjustment because smaller-
414 particle columns will require higher linear velocities for the same performance (as
415 measured by reduced plate height). The flow rate is adjusted for both the change in
416 column diameter and particle size using the following equation:

417 F2 = F1 × [(dc22 × dp1)/(dc12 × dp2)]

F1 = flow rate indicated in the monograph, in millilitres per minute;

F2 = adjusted flow rate, in millilitres per minute;

418 dc1 = internal diameter of the column indicated in the monograph, in millimetres;

419 dc2 = internal diameter of the column used, in millimetres;

420 dp1 = particle size indicated in the monograph, in micrometres;

421 dp2 = particle size of the column used, in micrometres.

422 When a change is made from ≥ 3-µm to < 3-µm particles in isocratic separations, an
423 additional increase in linear velocity (by adjusting the flow rate) may be justified,
424 provided that the column performance does not drop by more than 20 per cent.
425 Similarly, when a change is made from < 3-µm to ≥ 3-µm particles, an additional
 Working document QAS/21.905
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426 reduction of linear velocity (flow rate) may be justified to avoid reduction in column
427 performance by more than 20 per cent.

428 After an adjustment due to a change in column dimensions, an additional change in flow
429 rate of ± 50 per cent is permitted.

430 • Column temperature: ± 10 °C, where the operating temperature is specified,

431 unless otherwise prescribed.

432 Further adjustments in analytical procedure conditions (mobile phase, temperature, pH,
433 etc.) may be required, within the permitted ranges described under System Suitability
434 and Adjustment of chromatographic conditions in this chapter.

435 Mobile phase

436 • Composition: the amount of the minor solvent components may be adjusted
437 by ± 30 per cent relative (see examples under Thin-layer chromatography);
438 no component is altered by more than 10 per cent absolute. A minor
439 component comprises less than or equal to (100/n) per cent, n being the total
440 number of components of the mobile phase;
441 • pH of the aqueous component of the mobile phase: ± 0.2 pH units, unless
442 otherwise prescribed;
443 • Concentration of salts in the buffer component of a mobile phase: ± 10 per
444 cent;
445 • Flow rate: in absence of a change in column dimensions, an adjustment of the
446 flow rate by ± 50 per cent is permitted.

447 Detector wavelength

448 No adjustment permitted.

449
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450 Injection volume

451 When the column dimensions are changed, the following equation may be used for
452 adjusting the injection volume:

453 Vinj2 = Vinj1 (L2 dc22) / (L1 dc12)

454 Vinj1 = injection volume indicated in the monograph, in microlitres;

455 Vinj2 = adjusted injection volume, in microlitres;

456 L1 = column length indicated in the monograph, in millimetres;

457 L2 = new column length, in millimetres;

458 dc1 = column internal diameter indicated in the monograph, in millimetres;

459 dc2 = new column internal diameter, in millimetres.

460 This equation may not be applicable to changes from TPP columns to SPP columns.

461 Even in the absence of any column dimension change, the injection volume may be
462 varied provided System Suitability criteria remain within their established acceptability
463 limits. When the injection volume is decreased, special attention is given to (the limit
464 of) detection and repeatability of the peak response(s) to be determined. An increase is
465 permitted provided, in particular, the linearity and resolution of the peak(s) to be
466 determined remain satisfactory.

467 Liquid chromatography: gradient elution

468 The adjustment of chromatographic conditions for gradient systems requires greater
469 caution than for isocratic systems.

470 Column parameters and flow rate

471 • Stationary phase: no change of the identity of the substituent (e.g. no

472 replacement of C18 by C8); the other physico-chemical characteristics of the
 Working document QAS/21.905
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473 stationary phase (i.e. chromatographic support, surface modification and extent
474 of chemical modification must be similar); a change from Totally Porous Particle
475 (TPP) columns to Superficially Porous Particle (SPP) columns is allowed
476 provided the above-mentioned requirements are met.
477 • Column dimensions (particle size, length): the particle size and/or length of the
478 column may be modified provided that the ratio of the column length (L) to the
479 particle size (dp) remains constant or in the range between − 25 per cent to +50
480 per cent of the prescribed L/dp ratio.
481 • Adjustments from totally porous to superficially porous particles: for the
482 application of particle-size adjustment from totally porous to superficially
483 porous particles, other combinations of L and dp can be used provided that the
484 ratio (tR/wh)2 is within – 25 per cent to + 50 per cent, relative to the prescribed
485 column, for each peak used to check the system suitability, as stated in this
486 chapter and the individual monograph. These changes are acceptable provided
487 system suitability criteria are fulfilled, and selectivity and elution order of the
488 specified impurities to be controlled are demonstrated to be equivalent.
489 • Internal diameter: in the absence of a change in particle size and/or length, the
490 internal diameter of the column may be adjusted.

491 Caution is necessary when the adjustment results in smaller peak volumes due to a
492 smaller particle size or a smaller internal diameter, a situation which may require
493 adjustments to minimize extra-column band broadening by factors such as instrument
494 connections, detector cell volume and sampling rate, and injection volume.

495 When the particle size is changed, the flow rate requires adjustment because smaller-
496 particle columns will require higher linear velocities for the same performance (as
497 measured by reduced plate height). The flow rate is adjusted for both the change in
498 column diameter and particle size using the following equation:

499 F2 = F1 × [(dc22 × dp1)/(dc12 × dp2)]

 Working document QAS/21.905
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500 F1 = flow rate indicated in the monograph, in millilitres per minute;

501 F2 = adjusted flow rate, in millilitres per minute;

502 dc1 = internal diameter of the column indicated in the monograph, in millimetres;

503 dc2 = internal diameter of the column used, in millimetres;

504 dp1 = particle size indicated in the monograph, in micrometres;

505 dp2. = particle size of the column used, in micrometres.

506 A change in column dimensions, and thus in column volume, impacts the gradient
507 volume which controls selectivity. Gradients are adjusted to the column volume by
508 changing the gradient volume in proportion to the column volume. This applies to every
509 gradient segment volume. Since the gradient volume is the gradient time, tG, multiplied
510 by the flow rate, F, the gradient time for each gradient segment needs to be adjusted to
511 maintain a constant ratio of the gradient volume to the column volume (expressed as L
512 x dc2). Thus, the new gradient time, tG2 can be calculated from the original gradient
513 time, tG1, the flow rate(s) and the column dimensions as follows:

514 tG2 = tG1 × (F1 / F2) [(L2 × dc22) / (L1 × dc12)]

515 Thus, the change in conditions for gradient elution requires three steps:

516 (1) adjust the column length and particle size according to L/dp;
517 (2) adjust the flow rate for changes in particle size and column diameter; and
518 (3) adjust the gradient time of each segment for changes in column length, diameter
519 and flow rate. The example below illustrates this process.
 Working document QAS/21.905
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520 Table 2

Variable Original Adjusted Comment

Conditions Conditions
Column length (L) in mm 150 100 User’s choice
Column diameter (dc) in 4.6 2.1 User’s choice
mm
Particle size (dp) in µm 5 3 User’s choice
L/dp 30.0 33.3 (1)
Flow rate in mL/min 2.0 0.7 (2)
Gradient adjustment 0.4 (3)
factor (tG2/tG1)
Gradient conditions
B (per cent) Time (min) Time (min)
30 0 0
30 3 (3x0.4)=1.2
70 13 [1.2+(10x0.4)]=5.2
30 16 [5.2+(3x0.4)]=6.4

521 (1) 11 per cent increase within allowed L/dp change of – 25 per cent to + 50 per cent.

522 (2) calculated using F2= F1 [(dc22 x dp1) / (dc12 x dp2)].

523 (3) calculated using tG2 = tG1 x (F1 / F2) [(L2 x dc22) / (L1 x dc12)].

524 • Column temperature: ± 5 °C, where the operating temperature is specified,

525 unless otherwise prescribed.

526 Further adjustments in analytical procedure conditions (mobile phase, temperature, pH,
527 etc.) may be required, within the permitted ranges described under System Suitability
528 and Adjustment of Chromatographic Conditions in this chapter.

529 Mobile phase

530 • Composition/gradient: adjustments of the composition of the mobile phase and

531 the gradient are acceptable, provided that:
532 o the system suitability criteria are fulfilled;
 Working document QAS/21.905
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533 o the principal peak(s) elute(s) within ± 15 per cent of the retention time(s)
534 obtained with the original conditions; this requirement does not apply
535 when the column dimensions are changed; and
536 o the composition of the mobile phase and the gradient are such that the
537 first peaks are sufficiently retained and the last peaks are eluted.
538 • pH of the aqueous component of the mobile phase: ± 0.2 pH units, unless
539 otherwise prescribed.
540 • Concentration of salts in the buffer component of a mobile phase: ± 10 per cent.

541 Where compliance with the system suitability criteria cannot be achieved, it is often
542 preferable to consider the dwell volume or to change the column.

543 Dwell volume

544 The configuration of the equipment employed may significantly alter the resolution,
545 retention time and relative retentions described. Should this occur, it may be due to a
546 change in dwell volume. Monographs preferably include an isocratic step before the
547 start of the gradient programme so that an adaptation can be made to the gradient time
548 points to take account of differences in dwell volume between the system used for
549 analytical procedure development and that actually used. It is the user’s responsibility
550 to adapt the length of the isocratic step to the analytical equipment used. If the dwell
551 volume used during the elaboration of the monograph is given in the monograph, the
552 time points (t min) stated in the gradient table may be replaced by adapted time points
553 (tc min), calculated using the following equation:

(𝐷 − 𝐷0 )
554 𝑡𝑐 = 𝑡 −
𝐹

555 D = dwell volume, in millilitres;

556 D0 = dwell volume used for development of the analytical procedure, in millilitres;

557 F = flow rate, in millilitres per minute.

 Working document QAS/21.905
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558 The isocratic step introduced for this purpose may be omitted if validation data for
559 application of the analytical procedure without this step is available.

560 Detector wavelength

561 No adjustment permitted.

562 Injection volume

563 When the column dimensions are changed, the following equation may be used for
564 adjusting the injection volume:

565 Vinj2 = Vinj1 (L2 dc22) / (L1 dc12)

566 Vinj1 = injection volume indicated in the monograph, in microlitres;

567 Vinj2 = adjusted injection volume, in microliters;

568 L1 = column length indicated in the monograph, in millimetres;

569 L2 = new column length, in millimetres;

570 dc1 = column internal diameter indicated in the monograph, in millimetres;

571 dc2 = new column internal diameter, in millimetres.

572 This equation may not be applicable to changes from TPP columns to SPP columns.

573 Even in the absence of any column dimension change, the injection volume may be
574 varied provided system suitability criteria remain within their established acceptability
575 limits. When the injection volume is decreased, special attention is given to (the limit
576 of) detection and repeatability of the peak response(s) to be determined. An increase is
577 permitted provided, in particular, the linearity and resolution of the peak(s) to be
578 determined remain satisfactory.

579
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580 Gas chromatography

581 Column parameters

582 ➢ Stationary phase:

583 − particle size: maximum reduction of 50 per cent; no increase permitted
584 (packed columns);
585 − film thickness: − 50 per cent to + 100 per cent (capillary columns).
586 ➢ Column dimensions:
587 − length: − 70 per cent to + 100 per cent;
588 − internal diameter: ± 50 per cent.
589 ➢ Column temperature: ± 10 per cent.
590 ➢ Temperature programme: adjustment of temperatures is permitted as stated
591 above; adjustment of ramp rates and hold times of up to ± 20 per cent is
592 permitted.

593 Flow rate

594 ± 50 per cent.

595 The above changes are acceptable provided system suitability criteria are fulfilled and
596 selectivity and elution order of the specified impurities to be controlled are
597 demonstrated to be equivalent.

598 Injection volume and split ratio

599 May be varied provided system suitability criteria remain within their established
600 acceptability limits. When the injection volume is decreased, or the split ratio is
601 increased, special attention is given to (the limit of) detection and repeatability of the
602 peak response(s) to be determined. An increase in injection volume or a decrease in
603 split ratio is permitted provided, in particular, the linearity and resolution of the peak(s)
604 to be determined remain satisfactory.
 Working document QAS/21.905
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605 Injection port temperature and transfer-line temperature in static head-space

606 conditions

607 ± 10 °C, provided no decomposition or condensation occurs.

608 QUANTITATION

609 The following quantitation approaches may be used in general texts or monographs:
610

611 External standard method.

612 • using a calibration function

613 Standard solutions with several graded amounts of a reference standard of the
614 compound to be analysed are prepared in a range that has been demonstrated to give a
615 linear response, and a fixed volume of these standard solutions is injected. With the
616 chromatograms obtained, a calibration function is prepared by plotting the peak areas
617 or peak heights on the ordinate against the amount of reference standard on the abscissa.
618 The calibration function is generally obtained by linear regression. Then, a sample
619 solution is prepared according to the procedure specified in the individual monograph.
620 The chromatography is performed under the same operating conditions as for the
621 preparation of the calibration function, the peak area or peak height of the compound to
622 be analysed is measured, and the amount of the compound is read out or calculated from
623 the calibration function.

624 • using one-point calibration

625 In an individual monograph, generally one of the standard solutions with a

626 concentration within the linear range of the calibration function and a sample solution
627 with a concentration close to that of the standard solution are prepared, and the
628 chromatography is performed under fixed conditions to obtain the amount of the
 Working document QAS/21.905
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629 compound by comparing the responses obtained. In this method, all procedures, such
630 as the injection, must be carried out under constant conditions.

631 Internal standard method.

632 • using a calibration function

633 In the internal standard method, a stable compound is chosen as an internal standard
634 which shows a retention time close to that of the compound to be analysed, and whose
635 peak is well separated from all other peaks in the chromatogram. Several standard
636 solutions containing a fixed amount of the internal standard and graded amounts of a
637 reference standard of the compound to be analysed are prepared. Based on the
638 chromatograms obtained by injection of a fixed volume of individual standard solutions,
639 the ratio of peak area or peak height of the reference standard to that of the internal
640 standard is calculated. A calibration function by plotting these ratios on the ordinate
641 against the amount of the reference standard or the ratio of the amount of reference
642 standard to that of the internal standard on the abscissa is prepared. The calibration
643 function is generally obtained by linear regression. Then, a sample solution containing
644 the internal standard in the same amount as in the standard solutions used for the
645 preparation of the calibration function is prepared according to the procedure specified
646 in the individual monograph. The chromatography is performed under the same
647 operating conditions as for the preparation of the calibration function. The ratio of the
648 peak area or peak height of the compound to be analysed to that of the internal standard
649 is calculated, and the amount of the compound is read out or calculated from the
650 calibration function.

651 • using one point calibration

652 In an individual monograph, generally one of the standard solutions with a

653 concentration within the linear range of the calibration function and a sample solution
654 with a concentration close to that of the standard solution, both containing a fixed
655 amount of the internal standard, are prepared, and the chromatography is performed
 Working document QAS/21.905
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656 under fixed conditions to determine the amount of the compound to be analysed by
657 comparing the ratios obtained.

658 Normalisation procedure

659 Provided linearity of the peaks has been demonstrated, individual monographs may
660 prescribe that the percentage content of a component of the substance to be examined
661 is calculated by determining the area of the corresponding peak as a percentage of the
662 total area of all the peaks, excluding those due to solvents or reagents or arising from
663 the mobile phase or the sample matrix, and those at or below the disregard limit or
664 reporting threshold.

665 OTHER CONSIDERATIONS

666 Detector response

667 The detector sensitivity is the signal output per unit concentration or unit mass of a
668 substance in the mobile phase entering the detector. The relative detector response
669 factor, commonly referred to as response factor, expresses the sensitivity of a detector
670 for a given substance relative to a standard substance. The correction factor is the
671 reciprocal of the response factor.

672 In tests for related substances, any correction factors indicated in the monograph are
673 applied (i.e. when the response factor is outside the range 0.8-1.2).

674 Interfering peaks

675 Peaks due to solvents and reagents or arising from the mobile phase or the sample matrix
676 are disregarded.

677 Measurement of peaks

678 Integration of the peak area of any impurity that is not completely separated from the
679 principal peak is preferably performed by tangential skim (Figure 9).
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680

681 Figure 9

682 Reporting threshold.

683 When the related substances test prescribes a limit for the total of impurities or a
684 quantitative determination of an impurity, it is important to choose an appropriate
685 reporting threshold and appropriate conditions for the integration of the peak areas.

686 In such tests, the reporting threshold (i.e. the limit above which a peak is reported) is
687 generally 0.05 per cent.

688

689 ***