Melting-Point Reference Substances

Bull. Org. mond. Sante 1963, 28, 175-188Bull. Wld Hlth Org.

WHO Melting-Point Reference SubstancesH. BERVENMARK,1 N. A. DIDING I & B. OHRNER 1

Batches of 13 highly purified chemicals, intendedfor use as reference substances in thecalibration of apparatus for melting-point determinations, have been subjected to a colla-borative assay by 15 laboratories in 13 countries. All the laboratories performed melting-point determinations by the capillary methods described in the proposed text for the secondedition of the Pharmacopoea Intemationalis and some, in addition, carried out determina-tions by the microscope hot stage (Kofler) method, using both the " going-through " and the"equilibrium " technique.

Statistical analysis of the data obtained by the capillary method showed that the within-laboratory variation was small and that the between-laboratory variation, though constitutingthe greatest part of the whole variance, was not such as to warrant the exclusion of anylaboratory from the evaluation of the results. The average values of the melting-pointsobtained by the laboratories can therefore be used as constantsfor the substances in question,which have accordingly been established as WHO Melting-Point Reference Substancesand included in the WHO collection of authentic chemical substances.

As to the microscope hot stage method, analysis of the results indicated that the valuesobtained by the " going-through " technique did not differ significantly from those obtainedby the capillary method, but the values obtained by the " equilibrium " technique weremostly significantly lower.

At the meeting of the WHO Expert Committee onthe Intemational Pharmacopoeia in October 1957 itwas suggested that a study should be made of the in-clusion of international reference substances suitablefor checking melting-points in the collection ofauthentic chemical substances.2 The WHO Centrefor Authentic Chemical Substances. Apotekenskontrollaboratorium, Stockholm, was authorized topropose the organization of the work and, in areport from that laboratory in 1959,5 it was suggestedthat a set of 13 substances, which had been found to

be suitable for the purpose, should be adopted asmelting-point reference standards.TheWHO Expert Committee on Specifications for

Pharmaceutical Preparations agreed at its meeting inNovember 1959 4 that the work should be carried outalong the lines suggested by the Apotekens kontrol-laboratorium. In a preliminary report 6 an accountwas given of the procuring of the substances and alsodetails about the organization of the collaborativetesting. The present paper is the final report preparedafter completion of the collaborative study.

THE REFERENCE SUBSTANCES

The WHO set of Melting-Point Reference Sub-stances contains the following 13 substances:

Substance Melting-temperature

1. Azobenzene 690C2. Vanillin 830C3. Benzil 960C4. Acetanilide 1160C

"WHO Centre for Authentic Chemical Substances,Apotekens kontrollaboratorium, Stockholm, Sweden.

Unpublished working document WHO/Pharm/330.' Unpublished working document WHO/Pharm/371.

Substance Melting-temperature

5. Phenacetin 1360C6. Benzanilide 1650C7. Sulfanilamide 1660C8. Salophen 1920C9. Sulfapyridine 1930C

10. Dicyandiamide 2100C11. Saccharin 2290C12. Caffeine 2370C13. Phenolphthalein 2630C

'Unpublished working document WHO/Pharm/237.'Unpublished working document WHO/Pharm/385.

1223 -175-

H. BERVENMARK, N. A. DIDING & B. OHRNER

Substances 2, 4, 5, 7, 9 and 12 are those recom-mended in the first edition of the PharmacopoeaInternationalis (Ph. I.) as suitable for the purpose ofchecking the accuracy of different melting-pointmethods. These six substances also constitute theMelting Point Reference Standards of the UnitedStates Pharmacopeia (USP). Substances 1, 3, 4, 5,6, 8, 10, 11 and 13 are recommended by Kofler et al.(1954) for calibrating purposes and for determiningeutectic melting-temperatures with the microscopehot stage and the hot bar. Substances 8 and 10 areactually included in the WHO set primarily for thepurpose of determining eutectic melting-tempera-tures.The substances were purchased after examination

of about 35 samples from 9 different suppliers. In

most cases it was possible to obtain substances ofsufficient purity from commercially available sources.Only substance 6, benzanilide, and substance 11,saccharin, were subjected to further purification.The substances were assayed and checked for

chemical purity at the Apotekens kontrollabora-torium, and the results are shown in Table 1.

Samples of each substance were taken from fourdifferent parts of the container and analysed withrespect to the melting-range. In no case was asignificant difference, indicating lack of homogeneityof the material, observed. The four samples of eachsubstance were then carefully mixed to make grosssamples, from which subsamples were taken fordistribution to the participants in the internationalcollaborative assay.

THE INTERNATIONAL COLLABORATIVE ASSAY

The names and addresses of the laboratoriesthat took part in the international collaborativeassay are listed in the Annex (see page 187).Throughout this report the participating laboratoriesare referred to by arbitrary numbers, which have noconnexion with the order of listing of the laboratoriesin the Annex.Together with the samples of the 13 substances,

the following general directions for the assay weresent to the participants:

" Principally the melting-point determinations shouldbe carried out according to the capillary method proposedfor the second edition of the Ph. I. (a copy of the relevanttext is included). If other apparatus or methods thanthose outlined in the Ph. l. are used, a short descriptionof the essential parts of the apparatus (method) shouldbe given together with the results.For all the substances the melting-range, as defined

in the Ph. I., should be determined and for each substanceat least two determinations should be made. The follow-ing data should be reported:

1. Uncorrected temperature readings.2. Corrections applied:

(a) correction for deviation of the standard thermo-meter;(b) emergent-stem correction.

3. Melting-range in corrected temperatures.

If possible, it would be of very great value if youcould also carry out melting-point determinations onthese substances with a microscope hot stage. For such

determinations we suggest that the following data arereported:

1. Melting-range from " going-through " determina-tions and the approximate rate of temperature rise atthe melting-temperature.

2. Observed micro melting-point, using the " equi-librium " technique as described, for instance, by Kofleret al. (1954) in Thermo-Mikro-Methoden, pages 17 and 18."

CAPILLARY METHOD

Statistical analysis and resultsThe temperature at which the substances begin to

collapse as well as the temperature at which theyare completely molten were determined, in accord-ance with the text of the Ph. I. (2nd ed.), by all thelaboratories except one, which only determined themelting-temperatures.

In most laboratories the melting-point apparatuswas constructed in accordance with the directionsin the Ph. I. (2nd ed.). In three laboratories,electrically heated blocks of aluminium or copperwere used. Since the Ph. I. accepts this type ofapparatus, provided that it gives the same accuracyin the determinations as the prescribed type, it hasbeen possible to use the results from these threelaboratories too.One of the participants distributed the substances

between six different laboratories in his country.Most of these laboratories determined the melting-points of only 5 substances. To facilitate the

176

WHO MELTING-POINT REFERENCE SUBSTANCES

TABLE 1

RESULTS OF ASSAY AND CHECKINGOF CHEMICAL PURITY

Substance Assay (Method) f Purity

Azobenzene 100.1 % (Kjeldahl) Residue on Ignition:nil

Vanillin 100.3 % (Non- Conforms to purityaqueous titra- tests in drafttion with lithium monograph ofmethoxide) Pharmacopoea

Nordica

Benzil 100.8 % (Ketone Residue on ignition:determination nilwith hydroxyl-amine)

Acetanilide 99.9 % (Non- Conforms to purityaqueous titra- tests in drafttion with per- monograph ofchloric acid) Pharmacopoea

Nordica

Phenacetin 100.2 % (Refluxing Conforms to puritywith hydrochlo- tests in draftric acid; non- monograph ofaqueous titra- Pharmacopoeation with per- Nordicachloric acid)

Benzanilide 99.1 % (Kieldahl) Residue on ignition:nil

Sulfanilamide 100.5 % (Bromo- Conforms to puritymetric) tests in draft

monograph ofPharmacopoeaNordica

Salophen 99.2 % (Kjeldahl) Residue on ignition:nil

Sulfapyridine 100.7 % (Potentio- Conforms to puritymetric, with tests in USP XVnitrite)

Dicyandiamide 99.7 % (Kieldahl) Residue on ignition:nil

Saccharin 99.3 % (Acidi- Conforms to puritymetric) tests in USP XVI

Caffeine 100.6 % (Non- Conforms to purityaqueous titra- tests in drafttion with per- monograph ofchloric acid) Pharmacopoea

Nordica

Phenolphthalein 100.3 % (lodo- Conforms to puritymetric) tests in draft

monograph ofPharmacopoeaNordica

statistical evaluation in this investigation the resultsfrom these laboratories have been summarized intwo groups. As the type of apparatus and the pro-cedure of determination was the same,in all labora-tories, this manipulation was considered justified.The results from one laboratory have not been

included because they were received after thestatistical analysis had been finished. The resultsfrom this laboratory, however, agree well with thosefrom the others and their inclusion in the statisticalanalysis would not have changed the conclusionsreached in this study.The statistical evaluation has been carried out

according to the model designed by Mandel (1959)and by Mandel & Lashof (1959) The " collapse "points and melting-points have been dealt withseparately. Tables 2 and 3 show the averagesobtained by each laboratory for each substance(after correction for the deviation of the standardthermometer and for the emergent stem). Each suchaverage makes one "cell ". The tables thereforeconsist of 13 x 14 and 13 x 15 " cells ". The averagefor each substance and laboratory and the standarddeviations and ranges for each substance have beencalculated. The averages of the within-cell standarddeviations have been calculated according to Tippet(1925). The average of all determinations in alllaboratories has also been computed-i.e., the"grand " average (i).

If the within-cell standard deviation shows anorder-of-magnitude relationship with the tempera-ture measurements, the cell averages have to beadjusted accordingly. It can be seen from Tables 2and 3, however, that the within-cell standard devia-tions do not show any relationship of this kind.Therefore, the following calculations can be carriedout on the original cell averages.The purpose of the evaluation is not only to

show the discrepancies between the laboratories butalso to give numerical expression to the variationswithin the laboratories. This last-mentioned within-laboratory variability, -q, consists broadly speakingof two components, one of them, w, due to thenormal replication error when making more thanone determination and the other, A, due to, forexample, different accuracy in the calibration of thescale of an instrument. With regard to the variationsbetween laboratories, see the accompanying figure.From the figure it is seen that an ideal laboratory

ought to have a line with the ordinate of the cen-troid M= C and with the slope Pi= 1. (Under theseconditions the line must pass through the origin.)

-177


TABLE 2" COLLAPSE" POINT DATA, AVERAGES, STANDARD DEVIATIONS AND RANGES

Substance1 2 3 4 5 6 7

Lab.I II

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Average

Standarddeviation

Range

67.4

68.7

67.9

68.0

67.9

67.8

68.0

67.4

67.5

67.3

67.3

68.0

68.2

67.7

67.8

0.4

67.3-68.7

81.6

82.9

81.6

81.7

81.8

82.0

80.0

82.1

82.9

80.9

81.6

82.1

81.6

81.5

81.7

0.7

94.7

95.6

94.6

95.2

94.0

94.5

93.5

95.1

96.0

93.8

94.6

95.8

95.2

94.7

94.8

0.7

114.2

114.6

114.6

115.4

113.9

114.4

114.5

114.5

115.4

114.0

114.1

114.5

114.5

113.5

114.4

0.5

80.0- 93.5- 113.5-82.9 95.8 1 15.4

134.6

135.1

135.0

135.7

134.2

134.9

134.0

135.7

136.3

132.5

134.3

134.8

134.7

133.7

134.7

0.8

132.5-136.3

163.1

164.1

164.2

164.5

163.1

164.0

163.0

163.0

164.8

162.1

162.9

164.0

163.7

162.3

163.5

0.8

162.1 -

164.8

164.0

164.9

165.0

165.5

164.2

165.0

163.0

165.3

166.8

164.1

164.4

165.0

165.1

163.4

8

190.9

191.6

188.6

192.5

191.0

192.2

189.5

191.4

191.9

188.6

188.1

191.8

192.1

190.5

9 10

191.1

191.5

191.5

192.2

190.5

191.6

191.0

192.4

194.8

191.1

191.2

192.2

191.5

190.5

164.7 190.8 191.7

0.9 1.5 1.1

163.0- 188.1 - 190.5-166.8 192.5 194.8

209.0

209.0

208.3

210.2

208.2

209.0

206.5

209.9

210.9

208.6

209.2

210.0

209.8

207.6

209.0

1.1

11

227.5

227.7

228.4

228.4

226.5

227.8

228.0

228.8

227.3

226.6

224.6

226.7

228.1

224.4

227.2

1.3

12

235.0

236.7

235.3

237.1

235.5

236.5

236.5

236.8

235.2

233.7

235.4

236.4

237.0

234.2

13

261.0

263.5

262.4

263.8

261.1

262.4

262.0

261.8

261.7

259.3

258.1

261.5

262.4

259.7

235.8 261.5

1.1 1.6

-206.5 224.4 233.7- 258.1 -

210.9 228.8 237.1 263.8

178

Average

164.2

165.1

164.4

165.4

164.0

164.8

163.8

164.9

165.5

163.3

163.5

164.8

164.9

163.4

164.4 = x


TABLE 3MELTING-POINT DATA, AVERAGES, STANDARD DEVIATIONS AND RANGES

Substance

Lab.

2

3

4

5

6

7

8

9

10

11

12

13

14

15

1

68.2

69.6

69.1

68.7

68.6

68.6

69.0

68.2

68.5

68.8

69.7

68.7

68.8

69.1

68.5

2

82.6

83.6

82.7

82.6

82.8

82.4

83.0

83.1

84.9

82.9

83.4

82.5

82.5

82.5

83.0

3

96.0

96.5

95.4

96.3

95.5

95.5

95.0

95.7

98.0

95.8

97.1

96.7

96.1

95.6

95.5

4 5 6 7 8 9 10 11 12 13 Average

115.2

115.7

115.8

116.3

115.4

115.3

116.0

115.3

117.1

116.0

116.6

116.1

115.4

114.9

115.0

136.1

136.1

135.8

136.6

135.6

135.8

135.0

135.9

137.5

135.0

136.5

135.9

135.9

135.2

136.0

164.4

164.7

164.7

165.2

164.6

164.6

164.0

164.2

166.8

163.6

165.5

165.0

164.9

163.5

165.0

165.4

165.7

165.5

166.4

165.9

165.9

165.0

165.9

167.8

165.6

166.5

166.7

165.8

164.7

165.5

192.2

192.2

189.5

193.3

192.3

192.8

190.5

192.2

194.9

190.6

191.2

193.2

193.5

191.5

192.0

192.0

192.4

192.1

193.0

191.7

192.3

192.0

193.2

195.8

193.1

192.5

193.2

192.4

191.2

193.0

210.8

209.9

209.2

210.9

209.2

210.0

208.0

210.7

211.9

210.1

210.5

211.0

211.1

208.9

211.0

229.3

229.4

229.6

230.2

228.3

229.3

229.0

229.4

229.8

230.1

228.8

229.6

229.6

227.5

230.0

236.5

237.1

236.6

237.9

236.6

237.2

237.0

237.6

237.2

235.7

237.4

237.5

238.0

235.4

237.5

262.5

264.9

263.0

264.6

262.6

263.2

263.5

262.6

263.7

261.8

260.7

263.9

263.2

260.7

264.5

165.5

166.0

165.3

166.3

165.3

165.6

165.2

165.7

167.2

165.3

165.9

166.2

165.9

164.7

165.9

Average 68.8 83.0 96.0 115.7 135.9 164.7 165.9 192.1 192.7 210.2 229.3 237.0 263.0 165.7 = x

Standarddeviation 0.4 0.6 0.8 0.6 0.6 0.8 0.7 1.4 1.1 1.0 0.7 0.7 1.3

Range 68.2- 82.4- 95.0- 114.9- 135.0- 163.5- 164.7- 189.5- 191.2- 208.0- 227.5- 235.4- 260.7-69.7 84.9 98.0 117.1 137.5 166.8 167.8 194.9 195.8 211.9 230.2 238.0 264.9

Average of thewithin-cellstandarddeviation 0.2 0.1 0.2 0.1 0.3 0.2 0.3 0.3 0.2 0.3 0.3 0.2 0.3

179


Each line in the figure is characterized by a loca-tion parameter p (in this case M1C and M2C) andby its slope (81 and [,?). This means that the between-laboratory variability consists of two components,one characterized by ,u and one by ,B. This last com-ponent may also be separated into two parts, the

COMPONENTS OF VARIABILITY BETWEEN AND WITHINTWO LABORATORIES

first one related to the correlation between yt and ,Band the second to that part of the variability of ,Bthat is unrelated to ,u, hereafter called S.

The departure of an experimental point obtainedby laboratory 2 from its response line PP'=27 is thesame as the above-mentioned within-laboratoryvariability consisting of co and A.

Thus, according to Mandel's model, the varianceV (y) of a " collapse " point or melting-point valuemay be considered as being composed of a numberof components, namely, V(CL), V(A), V(,u) and V(S).Each of these variances has been calculated in themanner described below.When deriving the response line for each labora-

tory, the least-square formulae for linear regressionwere used. The following quantities were first com-puted:

X = (L'X12) -b2()X=(x)-bX2 .................. (1)Zi = (2zij2)-b2 .............. (2)Pi = (Xxjzjj)-bpi.. ............. (3)

where xj = average of the melting-points for sub-stance j

b = number of substancesx = "grand " average

Zi = melting-point of a substance in cellP = average of the melting-points obtained

by laboratory i

TABLE 4VALUES OF X, Zi AND Pi OBTAINED FROM EQUATIONS (1), (2) AND (3)

The values from Table 2 give: The values from Table 3 give:Lab.

X zi Pi X Zi Pi

1 46 431.14 46 069.77 46 249.60 46 544.50 46 453.49 46 498.252 46 253.56 46 340.92 46 439.04 46 489.783 46 424.76 46 423.63 46 311.73 46 424.614 47 055.54 46 741.42 47 151.23 46 846.555 46 089.95 46 259.37 46 341.40 46 442.056 46 601.19 46 515.11 46 831.69 46 687.367 46 768.53 46 593.14 46 153.98 46 345.138 46 921.53 46 674.29 46 726.77 46 634.689 46 170.22 46 294.81 46 510.91 46 521.6510 45 763.31 46 093.00 46 237.36 46 387.7511 45 414.45 45 914.54 45 382.47 45 958.25

12 46 365.00 46 396.90 46 686.12 46 614.49

13 46 829.82 46 629.11 47 103.61 46 822.12

14 45 225.49 45 823.75 45 423.44 45 980.52

15 47 141.72 46 840.96

Melting-point averog*sfor one lborciory

A

Laboraky 1

Msltinagpointtag *s far G Il

foboratoriesC - vero of allImelting-points forall laboatories = I

me 2lA

180


The calculated values of X, Zi and Pi are shownin Table 4.The slope of the response line for laboratory

i(=#i) was then obtained by using the followingformula:

.i(4)fiX *****-**ev............................The centroids M1C, M2C etc. (=pi) have already

been computed in Tables 2 and 3, but they are alsogiven in Table 5. This table also shows the values ofVi (7t)-i.e., the within-laboratory variance con-

sisting of the two components V(w) and V(A)-computed by the formula:

a 1 Pj2f(

a-l b-2 X....... .(5)

where a = number of laboratoriesb = number of substances

At this point in the calculations it is of value tostudy the results obtained. The average of ,6 oughtto be 1.000. As can be seen from Table 5 theaverages obtained are close to this value and thedifferences are negligible. If Vi (71) for any labora-tory is much greater than the average, it is advisableto calculate the individual estimates of 7, in order todetect discrepant individual points. In such cases,the laboratory may be omitted from the evaluation,but then it is necessary to recalculate the values offP and Vi (ti) for the remaining laboratories; I.s willbe the same, but the over-all average x must also berecomputed. In the present data the differences areso small that no omission was considered necessary.Observe that V&q) only shows the extent of deviationof the experimental points found by one laboratoryfrom that laboratory's response line and not fromthe response line for an " ideal " laboratory.The average of V*rq) was then used to give the

two components V(w) and V(A). The formula usedwas:

V(A) = V(-q)-I/n * V(co) ............... (6)where n = number of determinations within a cell

V(Bq) = average of V&q)V(Z) was calculated according to Mandel's

method (Technometrics, 1959, vol. 1, p. 256), usingthe values for within-cell standard deviation inTables 2 and 3. V(co) then becomes 0.058 for the" collapse" points and 0.053 for the melting-points.Generally, the average of three determinations hasbeen given within the cells. Therefore the values ofV(A) will be, respectively:

0.058 0.0530.468 = 0.449 and 0.335 ~=0.317.

TABLE 5

PARAMETERS OF THE STRAIGHT LINESCORRESPONDING TO THE VARIOUS LABORATORIES

Lab. Vi (X7)

"Collapse" points

1 0.996 164.2 0.098

2 0.998 165.1 0.262

3 1.000 164.4 0.846

4 1.007 165.4 0.173

5 0.996 164.0 0.167

6 1.002 164.8 0.192

7 1.003 163.8 1.255

8 1.005 164.9 0.276

9 0.997 165.5 1.110

10 0.993 163.3 0.586

11 0.989 163.5 1.053

12 0.999 164.8 0.226

13 1.004 164.9 0.186

14 0.987 163.4 0.115

Average J 0.998 164.4 0.468

Melting-points

1 0.999 165.5 0.141

2 0.999 166.0 0.372

3 0.997 165.3 0.654

4 1.006 166.3 0.065

5 0.998 165.3 0.154

6 1.003 165.6 0.101

7 0.996 165.2 0.718

8 1.002 165.7 0.169

9 1.000 167.2 1.179

10 0.997 165.3 0.569

11 0.987 165.9 0.301

12 1.002 166.2 0.150

13 1.006 165.9 0.216

14 0.989 164.7 0.007

15 1.006 165.9 0.236

Average 0.999 165.7 { 0.335

181


The next step was to calculate V(,), V(f) andV(8), i.e., the between-laboratory variances. Ananalysis of the variance was carried out according tothe usual procedure on the data presented in Tables 2and 3. The results are shown in Tables 6 and 7,which were drawn up on the following basis:Sources of Degrees of Sums of Mean Ratio ofvariation freedom squares squares variancesBetween

laboratories a-I SL ML FBetween

substances b-I SM MMError (a-I) (b-1) SLM MLM

Total ab-1 STFrom tables of the F distribution - see, for

example, Bauer (1960) - it is seen that in this caseF= 2.1 at a 99% significance level. The value of 12.7obtained (Table 6) therefore means that, with regardto the determination of the " collapse " points, thereis a significant difference between the laboratories,with a probability of being wrong less than once inone hundred times.

TABLE 6ANALYSIS OF VARIANCE OF DATAFOR THE "COLLAPSE" POINTS

Sources of Degrees Sums of IMean Ratio ofvariation freedom squares squares variances

Betweenlaboratories 13 93.63 7.202 12.7

Betweensubstances 12 647 994.72 54 000

Error 156 88.46 0.567

Total [ 181 648176.81

TABLE 7ANALYSIS OF VARIANCE OF DATA

FOR THE MELTING-POINTS

Sources of Degrees Sums of Mean Ratio ofvariation freedom squares squares variances

Betweenlaboratories 14 64.43 4.602 10.6

Betweensubstances 12 697 218.49 58102

Error 168 72.63 0.432

Total [ 194 697355.55

From Table 7 it is obvious that there is the same

significant difference between the laboratories inrespect of the determination of the melting-points.But in both cases the level of significance was notconsidered to be high enough to justify the exclusionof any laboratory.The values of the mean squares were used for

computing V(M1) and V@), as follows:

V( = V(K)b ..................... .(7)

=a(Mjm - V(71)MM-V(r).

.................. (8)

For the " collapse " points the following values wereobtained:

V() = 0.518 V($) = 0.0000257The corresponding values for the melting-pointswere:

V(,u) = 0.328 V(g?) = 0.0000250Formulae (9) and (10) were used for calculating V(8):

V(8) = V(fP)-a2V(,u) ................... (9)a b (244Pt- X2'Pj)

SLSM (10)

Since the equations yielded negative values in bothcases, the variance of 8 was then taken to be zero(Table 8).

V(A) is, as pointed out earlier, a measure of thevariations due to different accuracy in the calibrationof the instrument scales. This variance is indepen-dent of the number of measurements, unlike V(Z),which is affected by replication because it is thereplication error. It is very difficult to get the samemelting-point on repeat determinations. This meansthat a better result is obtained with an increasingnumber of determinations. But replication is usefulonly to the point of making V(co)/n small withrespect to V(A). Table 8 shows that the replicationerror is small within the laboratories. There would

TABLE 8NUMERICAL VALUES OF THE DIFFERENT TYPES

OF VARIANCE

V(A V(Wo V(-)/nl V(8) V(y) V(R)

"Collapse"points 0.449 0.058 0.019 0 0.518 0.0000257

Melting-points 0.317 0.053 0.018 0 0.328 0.0000250

182

WHO MELTING-POINT REFERENCE SUBSTANCES 183

be nothing to gain with more determinations persubstance, since, as can also be seen in Table 8,V(A) is higher than V(w)). This indicates inaccuratecalibration of the thermometers used. Another andmore probable explanation is that some of thelaboratories estimated their thermometer readings tothe nearest half-degree and these estimations mayhave given results which were too high in one part ofthe thermometer scale and too low in another. (It isnot always possible to recognize these approximatereadings in Tables 2 and 3 because the emergent-stem correction in some cases is made with anaccuracy of 0.10.)

Table 9 shows the relative importance of thevarious sources of variability for some of the" collapse " points and melting-points. The equationused for this breakdown of the total variability ofany value of y is:VWy)= V(ow)+ V(A)+[l +a(y=x)]2. VQLi)+(y-x)2. V(8)(I1)The values obtained confirm the conclusions made

above concerning the sources of the total variabilityof the " collapse " points and melting-points. Themost important source was ,, i.e., the variabilitybetween laboratories.The values of , and ,B in Table 5 show that the

agreement between laboratories was fairly good.,u and ,B are, as stated above, parameters in theequation for a straight line. An ideal laboratory will

TABLE 9RELATIVE IMPORTANCE OF THE DIFFERENT

SOURCES OF VARIATION

Sources of variation

Sub- Average Within Betweenstance laboratories laboratories

V(W) V(A) V(;,) V(8)

"Collapse " points

1 67.8 0.00 0.04 0.96 0

4 114.4 0.02 0.17 0.81 0

9 191.7 0.01 0.11 0.88 0

13 261.5 0.00 0.02 0.98 0

Melting-points

1 68.8 0.00 0.01 0.99 0

4 115.7 0.01 0.05 0.94 0

9 192.7 0.01 0.06 0.93 0

13 263.0 0.00 0.01 0.99 0

have a straight line passing through the origin at anangle of 45°. If laboratories with such values of 6as those shown by lab. 11 and 14 are eliminated,there will be little or no change in the average of the" collapse " points or the melting-points. Moreover, itis intended to give the melting-points of the referencesubstances to the nearest whole number of degrees.The same applies to the average value of ,, whichought to be 1.000 and was, as Table 5 shows, 0.998for the " collapse " points and 0.999 for the melting-points. These values can both be accepted. V(,u)was also low and that will enable us to take allvalues of ,u as they are. The discrepancies betweenthe individual values of ,u and , are perhaps due tothe use of different kinds of apparatus (all typesused, however, are permitted by the Ph. I.) and to thedifferent accuracy of calibration of the thermo-meters. The method of treatment (pulverization anddrying) before the determination is certainly of someimportance, as is the speed of heating during thedetermination (if, for example, one laboratory heatsat the rate of 0.50 per minute and another at 1.5°per minute, differences will arise, because it has beenshown that temperature equilibrium between theoutside and the inside of the capillary tube is notreached until about 30 seconds have passed).'

V(S) is that component of the between-laboratoryvariance which is associated with that part of fiwhich is not correlated with ,u (the greatest com-ponent of V(P) is usually the one correlated withV(p)). V(8) was in this case zero, which is to be lookedupon as an advantage.

Finally, for all the substances but one, the standarddeviation values (Tables 2 and 3) were surprisinglylow (the exception was salophen, a substance that willbe used only for determining eutectic melting-temperatures).As a final conclusion we can state that the averages

of the different melting-temperatures obtained in thecollaborative assay by the capillary method can beused as constants for the WHO collection of Melting-Point Reference Substances.

MICROSCOPE HOT STAGE METHOD

Some of the laboratories also determined the"collapse" points and melting-points according toKofler's microscope hot stage method. The resultsfrom nine laboratories that made determinations bythe "going-through" technique are shown in Table 10,together with the averages, standard deviations and

1 Unpublished working document WHO/Pharm./Ed.Sec./63 Add.l.

4


" COLLAPSE" POINTS ANDTABLE

MELTING-POINTS

Substance\ 1 2 3 4 5 6

Lab. \

1 68.3-69.3 82.0-83.0 94.8-95.8 114.3-115.3 134.8-135.5 163.0-164.0

2 69.8-70.2 82.3-83.3 96.2-96.5 115.8-116.2 134.4-136.0 164.3-164.6

3 68.3-68.6 81.6-82.3 93.9-95.4 114.7-116.1 134.5-135.2 164.6-164.8

4 68.1-68.4 81.0-81.6 94.9-95.2 114.4-114.8 134.9-135.3 163.7-163.9

5 68.0-69.0 82.5-83.0 94.5-95.0 115.0-116.0 134.5-135.0 162.8-163.0

6 67.9-68.9 82.6-83.2 96.2-96.6 115.2-116.0 135.5-136.0 167.2-167.7

7 x-68.6 x-83.0 x-95.2 x x x

8 x 81.0-81.5 x 114.5-115.5 x 164.0-165.0

9 66.0-67.5 80.5-82.0 93.5-95.0 113.0-113.5 134.0-134.5 x-163.0

Average 68.1 68.8 81.7 82.5 94.9 95.6 114.6 115.4 134.7 135.4 - 164.2 164.5

Standarddeviation 1.1 0.8 0.8 0.7 1.0 0.7 0.8 0.7 0.4 0.5 1.5 1.5

Ranges 66.0- 67.5- 80.5- 81.5- 93.5- 95.0- 113.0- 113.5- 134.0- 134.5- 162.8- 163.0-(R, and Ru) 69.8 70.2 82.6 83.3 96.2 96.6 115.8 116.2 135.5 136.0 167.2 167.7

a RI = lower range (" collapse " points); Ru = upper range (melting-points); x = not determined.

ranges. Seven laboratories determined the melting-points according to the " equilibrium " technique;these results can be seen in Table 11. It is not goodstatistical practice to calculate averages and standarddeviations on such a small number of values as inTables 10 and 11, but as a means of obtaining anidea about the differences between the methods it isjustifiable. When comparing the standard deviationsof the melting-points shown in Tables 3, 10 and 11(see Table 12), it is obvious that the standard devia-tion was generally greater with the " going-through "technique than with the capillary method or the "equi-librium" technique and that the last-mentioned tech-nique on the whole gave the lowest values for thestandard deviation. This confirms our theoreticalconclusions about the three methods with respect tothe different rates of heating, etc.From a comparison of the average melting-points

obtained by the " going-through " method (completemelting) and by the capillary method, it can be seenthat the latter method generally gave higher melting-points. This might have been due to the shorter timeof heating applied in the Ph. I. (capillary) method.

In order to test this hypothesis, the melting-tem-peratures of some of the substances were determinedby the capillary method but with varying heating-times. It was found, however, that there was nosignificant difference between the melting-tempera-tures obtained with the longer heating-times and thoseobtained with the heating-time of about 5 minutesused in the Ph. I. method. Another possible explana-tion of the differences might lie in the calibration ofthe thermometers of the microscope hot stages. Suchcalibration is accomplished by means of certain testsubstances with " known" melting-temperatures.This results in what might be termed a " circulardefinition ", since the melting-temperatures obtainedwith microscope hot stages are dependent on howthe melting-points of the test substances wereoriginally determined and laid down. So far as weknow, no details of the method of determinationhave been published.

In order to find out whether the differencesbetween the melting-points obtained by the capillaryand " going-through " methods were significant, amodified t-test was carried out by the method of

184

WHO MELTING-POINT REFERENCE SUBSTANCES 185

10OBTAINED BY THE " GOING-THROUGH " TECHNIQUE a

7 8 9 10 11 12 13

164.3-165.5 188.0-190.0 189.5-191.3 208.3-210.0 226.8-228.0 235.0-236.3 257.8-259.8

167.5-168.0 188.0-189.8 191.4-192.0 209.6-211.2 228.3-229.4 239.2-239.7 260.7-265.2

165.3-166.3 189.0-190.8 192.2-193.2 209.2-211.7 227.9-229.9 sublimated 262.3-265.9

163.9-164.4 188.0-190.2 190.3-190.8 209.6-210.6 227.6-228.5 234.9-235.0 x

165.0-166.0 190.0-193.0 x-193.0 210.0-210.5 sublimated sublimated sublimated

167.5-168.1 190.8-193.0 192.8-193.5 209.9-211.6 229.0-230.3 x-238.0 262.8-264.0

x-165.2 x x-194.0 x-21 0.0 x-228.0 x x-260.5

x x x 210.0-211.5 x x x

163.0-164.0 188.0-189.0 191 .0-191 .5 209.0-210.0 226.5-227.3 234.0-235.5 259.5-262.5

165.2 165.9 188.8 190.8 191.2 192.4 209.5 210.8 227.7 228.8 235.8 236.9 260.6 263.0

1.5 1.5 1.2 1.6 1.2 1.2 0.6 0.9 0.9 1.1 2.3 1.9 2.1 2.5

163.0- 164.0- 188.0- 189.0- 189.5- 190.8- 208.3- 210.0- 226.5- 227.3- 234.0- 235.0- 257.8- 259.8167.5 I 168.1 190.8 193.0 192.8 194.0 210.0 211.7 229.0 230.3 239.2 I 239.7 I 262.8 265.9

TABLE 11MELTING-POINTS OBTAINED BY THE "EQUILIBRIUM" TECHNIQUEa

Substance1 2 3 [4 5 [6 7 8 9 10 11 12 13

Lab.I

1 69.0 82.8 95.0 114.6 135.5 163.5 164.8 189.0 190.0 208.8 227.5 236.0 259.3

2 69.3 82.9 96.0 115.4 136.0 164.9 165.6 192.5 193.1 211.6 231.0 subl. 266.2

3 68.5 81.8 95.3 115.0 135.0 163.4 164.9 189.3 190.8 210.0 229.9 235.5 261.5

4 68.2 81.5 94.9 114.8 135.2 162.5 163.0 188.2 188.0 209.5 228.0 236.0 261.0

5 x 81.6 96.2 115.6 135.3 163.0 165.1 191.1 191.6 209.7 229.0 237.5 261.9

6 x x x 117.0 136.0 164.6 x 190.0 x x x x x

7 66.5 80.0 94.0 112.5 133.5 162.5 162.5 187.0 189.0 208.0 226.5 234.5 260.5

Average 68.3 81.8 95.2 115.0 135.2 163.5 164.3 189.6 190.4 209.6 228.7 235.9 261.7

Standarddeviation 0.5 0.5 0.4 0.6 0.4 0.4 0.6 0.7 0.8 0.5 0.7 0.5 1.1

Range 66.5- 80.0- 94.0- 112.5- 133.5- 162.5- 162.5- 187.0- 188.0- 208.0- 226.5- 234.5- 259.3-69.3 82.9 ] 96.2 [ 117.0 J 136.0 164.9 j 165.6 [ 192.5 ] 193.1 [ 211.6 J 231.0 [ 237.5 ] 266.2

a x = not determined.


TABLE 12STANDARD DEVIATION VALUES FROM TABLES 3, 10 AND 11

Substance 1 2 3 4 5 6 7 8 {9 |10 11 12 13

Capillary method 0.4 0.6 0.8 0.6 0.6 0.8 0.7 1.4 1.1 1.0 0.7 0.7 1.3

"Going-through " method 0.8 0.7 0.7 0.7 0.5 1.5 1.5 1.6 1.2 0.9 1.1 1.9 2.5

"Equilibrium" method 0.5 0.5 0.4 0.6 0.4 0.4 0.6 0.7 0.8 0.5 0.7 0.5 1.1

Moore (1957). This test is based on the distributionof the following quantity:

Ri,. ... (12)Rx + Ry

where XI = average of the melting-points of sub-stance j determined by the capillarymethod

Yj = average of the melting-points of sub-stance j determined by the "going-through" method

Rx = range of the melting-point valuesobtained with the capillary method(=" Range" in Table 3)

Ry = range of the melting-point valuesobtained with the " going-through"method (= R. in Table 10)

The calculated values ofM are shown in Table 13.

From Table 13 it can be seen that the null hypo-thesis is acceptable in 12 cases out of 13, and very

nearly acceptable in the 13th case. This means thatall the differences in melting-points between thecapillary and the " going-through " methods can beconsidered as due to chance alone.The corresponding values of M obtained in a

comparison of the averages of the capillary melting-points with those of the " equilibrium " points arepresented in Table 14.

It can be seen from Table 14 that the capillarymelting-points are mostly significantly higher thanthe " equilibrium " ones, which means they are twodifferent constants applicable to the substances. The"equilibrium" points, however, agree fairly wellwith the values given for the test substances suppliedwith the microscope hot stages, so that the WHOset of Melting-Point Reference Substances can alsobe used for determining " equilibrium" points bythe microscope technique.

TABLE 13

VALUES OF M OBTAINED IN A COMPARISON OF THE CAPILLARY AND THE " GOING-THROUGH"METHODS USING EQUATION (12)

Substance 1 2 3 4 5 6 7 8 9 10 11 12 13

j 68.8 83.0 96.0 115.7 135.9 164.7 165.9 192.1 192.7 210.2 229.3 237.0 263.0

Yi 68.8 82.5 95.6 115.4 135.4 164.5 165.9 190.8 192.4 210.8 228.8 236.9 263.0

Rx 1.5 2.5 3.0 2.2 2.5 3.3 3.1 5.4 4.6 3.9 2.7 2.6 4.2

RV 2.7 1.8 1.6 2.7 1.5 4.7 4.1 4.0 3.2 1.7 3.0 4.7 6.1

M 0 0.11 0.08 0.06 0.12 0.02 0 0.13 0.03 -0.10 0.09 0.01 0

Acceptance of the null hypothesis I IlIl lat the probability level 0.05 Yes Yes Yes Yes Yes Yes Yes| No Yes Yes Yes Yes Yes

186


TABLE 14VALUES OF M OBTAINED IN A COMPARISON OF THE CAPILLARY AND THE " EQUILIBRIUM" METHODS

USING EQUATION (12)

Substance 1 | 2 3 4 1 5 6 } 7 8 T 9 J 10 11 12 13

xi 68.8 83.0 96.0 115.7 135.9 164.7 165.9 192.1 192.7 210.2 229.3 237.0 263.0

rj 68.3 81.8 95.2 115.0 135.2 163.5 164.3 189.6 190.4 209.6 228.7 235.9 261.7

Rx 1.5 2.5 3.0 2.2 2.5 3.3 3.1 5.4 4.6 3.9 2.7 2.6 4.2

Ry 2.8 2.9 2.2 4.5 2.5 2.4 3.1 5.5 5.1 3.6 4.5 3.0 6.9

M 0.12 0.22 0.15 0.10 0.14 0.21 0.25 0.22 0.23 0.08 0.08 0.19 0.12

Acceptance of thenull hypothesis |level0.05_

Annex

LABORATORIES PARTICIPATING IN THE COLLABORATIVE ASSAY

AusTRIADr M. LaschDirektor der Bundesanstalt fur chemische und pharma-

zeutische UntersuchungenVienna

CANADADr M. PernarowskiPharmaceutical Chemistry SectionDepartment of National Health and WelfareOttawa, Ont.

DENMARKDr P. TerpDAK-laboratorietCopenhagen

FINLANDDr Karl-Erik BonnApteekkitavaraintarkastuslaboratorio OyHelsinki

FRANCEProfessor L. DomangeLaboratoire national de la Sant6 publiqueSection des MedicamentsParis 6e

INDIADr D. GhoshCentral Drugs LaboratoryCalcutta 16

ITALYProfessor A. MarianiIstituto Superiore di SanitaRome

JAPANDr T. ItaiChief, Department of Drug ResearchNational Institute of Hygienic SciencesSetagaya, Tokyo

NETHERLANDSProfessor E. H. VogelenzangFarmaceutisch Laboratorium van de Rijksuniversiteit

te LeidenLeiden

NORWAYDr Toralf StokkeNorges ApotekerforeningOslo

SWEDENDr N. A. DidingApotekens kontrol-

laboratoriumStockholm

Professor H. HellbergStatens FarmacevtiskaLaboratorium

Stockholm

UNITED KINGDOM OF GREAT BRITAINAND NORTHERN IRELANDMr T. C. DenstonSecretary of the British Pharmacopoeia ComniissionGeneral Medical Council OfficeLondon, W. 1

UNIrED STATES OF AMERICAMr Wilbur S. Felker Professor Albert M. MattocksDirector of Quality College of Pharmacy

Control Division University ofSchering Corporation MichiganBloomfield, N. J. Ann Arbor, Mich.

187

188 H. BERVENMARK, N. A. DIDING & B. OHRNER

RESUME

Dans le cadre de l'activit6 du Centre OMS des subs-tances de r6f6rences, a Stockholm, un essai comparatifde substances de r6ference pour la determination destemp6ratures de fusion a ete organise. I1 a port6 sur13 substances, dont les points de fusion ont ete determinesdans 15 laboratoires de 13 pays, soit par la methode dutube capillaire, soit par celle du microscope 'a platinechauffante.

L'analyse statistique des resultats obtenus par lapremiere m6thode montra que la moyenne des divers

laboratoires pouvait servir de constante pour chacunedes substances en question. Celles-ci ont 6t6 etabliescomme substances de references OMS pour la d6termina-tion des points de fusion, et feront partie de la collectiondes substances chimiques de r6ference.La methode du microscope a platine chauffante a donne

des resultats analogues A ceux de la precedente, par latechnique de la fusion complte (going-through), maisde valeur inf6rieure par la technique de 1'# equilibre *.

REFERENCES

Bauer, E. L. (1960) A statistical manual for chemists,New York & London, Academic Press, Table VlIIb

Kofler et al. (1954) Thermo-Mikro-Methoden, Wein-heim/Bergstrasse, Verlag Chemie

Mandel, J. (1959) Technometrics, 1, 251

Mandel, J. & Lashof,T.W. (1959)ASTMBull., No. 239, p. 53Moore, P. G. (1957) Biometrika, 44, 482Tippett, L. H. C. (1925) Biometrika, 17, 364World Health Organization (1951) Pharmacopoea

Internationalis, Geneva, vol. I, p. 290

Documents

Melting-Point Reference Substances