FINAL DRAFT UGANDA 
STANDARD  
  FDUS EAS 766
First Edition
2011-mm-dd
 
 Reference numbe r
FDUS EAS 766: 2011
© UNBS 2011
  
Antibacterial solid toilet soap — Specification 
 FDUS EAS 766: 2011 
i i                 ©  U N B S  2011 - All rights reserved 
 Compliance with this standard does not, of itself confer immunity from legal obligations 
A Uganda Standard does not purport to include all necessary provisions of a contract. Users are 
responsible for its correct application  
© UNBS 2011 
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilised in any 
form or by any means, electronic or mechanical, in cluding photocopying and microfilm, without prior written 
permission from UNBS. 
Requests for permission to reproduce this document should be addressed to 
The Executive Director 
Uganda National Bureau of Standards 
P.O. Box 6329 
Kampala  
Uganda 
Tel: 256 41 505 995 
Fax: 256 41 286 123 
E-mail: unbs@infocom.co.ug  
Web: www.unbs.go.ug  
 FDUS EAS 766: 2011 
© UNBS  2011 - All rights rese rved                iii National foreword 
Uganda National Bureau of Standards (UNBS) is a parastatal under the Ministry  of Tourism, Trade and 
Industry established under Cap 327, of the Laws of U ganda. UNBS is mandated to co-ordinate the elaboration 
of standards and is  
(a) a member of International Organisation for Standardisation (ISO) and  
(b) a contact point for the WHO/FAO Codex A limentarius Commission on Food Standards, and  
(c) the National Enquiry Point on TBT/SPS Agr eements of the World Trade Organisation (WTO). 
The work of preparing Uganda Standards is carried out through Technical Committees. A Technical 
Committee is established to deliberate on standards in a gi ven field or area and consists of representatives of 
consumers, traders, academician s, manufacturers, governme nt and other stakeholders. 
Draft Uganda Standards adopted by the Technical Committ ee are widely circulated to stakeholders and the 
general public for comments. The committee reviews the comments before recommending the draft standards 
for approval and declaration as Uganda Standards by the National Standards Council. 
This Final Draft Uganda Standard, FDUS EAS 766: 2011 , Antibacterial solid toilet soap  — Specification, is 
identical with  and has been reproduced from an East African Standard, EAS 766: 2011 , Antibacterial solid 
toilet soap — Specification,  and is being proposed for adoption as a Uganda Standard.  
This standard was developed by the Chemicals and env ironment Standards Technical Committee (UNBS/TC 
5).  
Wherever the words, “East Africa Standard" appear, they should be replaced by "Uganda Standard."  EAS 766: 2011 
ICS 71.100.40
© EAS 2011                                                                                                                              First Edition 2011  
  
EAST AFRICAN STANDARD 
Antibacterial solid toil et soap — Specification 
EAST AFRICAN COMMUNITY EAS 766: 2011 
ii © EAC 2011 – All rights reserved
 Copyright notice 
This EAC document is copyright-protected by EA C. While the reproduction of this document by 
participants in the EAC standards development process is permitted without prior permission from EAC, 
neither this document nor any extract from it may be reproduced, stored or transmitted in any form for 
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as shown below or to EAC’s member bo dy in the country of the requester: 
© East African Community 2011 — All rights reserved 
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Reproduction for sales purposes may be subject to royalty payments or a licensing agreement. 
Violators may be persecuted EAS 766: 2011  
© EAC 2011 – All rights reserved  iii
 Foreword 
Development of the East African Standards has been necessitated by the need for harmonizing requirements 
governing quality of products and services in the East African Community. It is envisaged that through 
harmonized standardization, trade barriers that are encountered when goods and services are exchanged 
within the Community will be removed.  
In order to achieve this objectiv e, the Community established an East  African Standards Committee mandated 
to develop and issue East African Standards.   
The Committee is composed of representatives of the National Standards Bodies in Partner States, together 
with the representatives from the pr ivate sectors and consumer organizati ons. Draft East African Standards 
are circulated to stakeholders through the National Standards Bodies in the Partner States. The comments 
received are discussed and incorporated before finalization of standards, in accordance with the procedures 
of the Community. 
East African Standards are subject to review, to keep pace with technological advan ces. Users of the East 
African Standards are therefore expected to ensure that they always have the latest versions of the standards 
they are implementing .  
EAS 766: 2011 was prepared by Te chnical Committee EAS/TC 000, TC title , Subcommittee SC 0, SC title . 
 EAST AFRICAN STANDARD EAS 766: 2011
 
© EAC 2011 – All rights reserved 1
 Antibacterial solid toilet soap — Specification 
1 Scope 
This draft East African standard spec ifies the requirements and methods of sampling and test for antibacterial 
solid toilet soap. 
2 Normative references 
The following referenced documents are indispensable for the application of this document. For dated 
references, only the edition cited applies. For undated references, the latest edition of the referenced  
IS 286, Methods of sampling and test for soaps  
IS 1070, Reagent grade water  
IS 4955, Household laundry detergent powders  
IS 7597, Glossary of terms related to surface active agents  
IS 13424, Safety evaluation of bathing bar and toilet soap - Methods of test  
3 Terms and definitions 
For the purposes of this standard the terms and de finitions given in IS 7597 and the following apply. 
4 Requirements 
4.1 Description 
Antibacterial solid toilet soap shall be a high grade, thoroughly saponified, milled soap or homogenized soap 
or both, white or coloured, perfumed, and compressed in the form of firm and sm ooth cakes, tablets or bars 
and shall possess good cleaning, lathering and antibacterial properties. 
4.2 General requirements  
4.2.1  Perfume, moisture, normal colouring matters, preservatives acceptable in toilet soaps may be 
added; 
4.2.2  The soap shall contain permitted antibacterial agent  as in Annex ..... 
4.2.3  The label shall clearly state  the antibacterial agent used and its level.  
4.2.4.  Antibacterial solid toilet soap shall pass the test for dermatological safety when evaluated using 
IS 13424. EAS 766: 2011 
2 © EAC 2011 – All rights reserved
 4.2.5  The soap shall pass the antibacterial activity test  when determined by the method given in Annex A. 
4.3 Specific chemical requirements 
Antibacterial toilet soap, solid cake shall also comply with the specific  chemical requirements specified in 
Table 1. 
Table 1 — Specific chemical requirement for antibacterial toilet soap, solid cake 
1 Total fatty matter, percent by mass,  min 76.0  
2 Rosin acids, percent by mass, of total fatty 
matter,  max  3.0  
3 Free caustic alkali, as sodium hydroxide 
(NaOH), percent by mass,  max 0.05  
4 Free carbonated alkali, as sodium carbonate 
(Na 2CO 3), percent by mass,  max 1.0  
5 Matter insoluble in alcohol, percent by mass,  
max 2.5  
6 Antibacterial agent TricIosan (TCN) and 
Trichlorocarbanilide (TCC) shall not exceed 1 % by 
mass either singly or in combination Annex B 
7 Chloroaniline content  shall not exceed 10 ppm Annex C 
8 Synthetic detergent Shall be absent IS 4955 
9 Phosphate Shall be absent IS 286 
 
5 Environmental precautions 
Environmental precaution shall be adhered to during production and disposal as per the provisions of the 
relevant national legislations on preventi on and control of pollution of water and air.  
6 Packing and marking 
6.1 Packing 
The product shall be packed in a suitable, well-closed cont ainer, to protect the integrity of the product during 
transportation and sale. 
6.2 Marking 
The packages shall be securely closed and marked with the following particulars: 
a) name and physical address  manufacture, supplier or importer and/or trade mark; b) net weight ; 
c) batch number; and 
d) date of manufacture ;and 
The following identified critical ingredients in descending order of quantity; percent by mass. 
i) Total Fatty Matter (TFM); EAS 766: 2011  
© EAC 2011 – All rights reserved  3
 ii) matter insoluble in alcohol; and 
iii) antibacterial agent. 
7 Sampling 
7.1 Preparation of test samples 
For the purpose general precautions, scale of sampling and preparation of test samples shall be as prescribed 
in IS 286. 
7.2 Number of tests 
7.2.1 Tests for determination of total fatty matter and free caustic alkali and matter insoluble in alcohol shall 
be conducted on each of the individual samples separately. 
7.2.2  Tests for determination of all the remaining characteristics shall be conducted on the composite 
sample. 
7.3 Criteria for conformity 
7.3.1  For each of the characteristics which has bee n determined on the individual samples (see  7.2.1) the 
mean ( X) and the range (R) of the test results shall be calculated as follows: 
Mean (X) = sum of test result/number of test result 
Range (R) = the difference between the maximum and the minimum value of test results. The lot shall be 
deemed as conforming to the requirements given in 7.2.1 if the expression (X - 0.6 R) is greater than or equal 
to minimum value given in Table 1 and (X + 0.6 R) is less than or equal to maximum value given in Table 1. 
7.3.2  For declaring the conformity of a lot to the requi rements of other characteristics determined on the 
composite sample, the test results for each of the ch aracteristics shall satisfy the relevant requirement. EAS 766: 2011 
4 © EAC 2011 – All rights reserved
 Annex A 
(normative)  
 
Determination of antibacterial activity 
A.1 General 
Two methods have been prescribed, namely, serial dilution method and substantivity test. The serial dilution 
test shall be the screening test and the subs tantivity test shall be the absolute test. 
A.2 Serial dilution test 
A.2.1 Outline of the method 
Antibacterial activity is determined by serial diluti on method by comparing the effectiveness of antibacterial 
chemicals present in 10 micrograms of soap per milIilit er specified as the maximum inhibitory concentration. 
A.2.2 Apparatus 
A.2.2.1 Culture tube , rimless, 150 mm x 18 mm 
A.2.2.2 Sterilized pipettes , 10 mL, 5 mL and 1 mL capacities 
A.2.2.3 Loop,  made of stainless steel or platinum wire 
A.2.2.4 Conical flasks , 250 mL capacity. 
A.2.3 Nutrient Broth 
A.2.3.1  Dissolve 5 g of beef extract, 5 g of sodium chlori de, 10 g of peptone in one litre of distilled water 
by warming over a water bath. Cool and adjust the pH to 7.2 to 7.6 with sodium hydroxide solution. Distribute 9 mL each to the culture tubes. Plug the tube with n on-absorbent cotton wool and sterilize in an autoclave for 
half an hour at 1 kg/cm
2 pressure. 
A.2.3.2  Take 99 mL and 90 mL of distilled water in 250-mL conical flasks. Plug them with non-absorbent 
cotton wool and sterilize in an autoclave. 
A.2.3.3  Get a pure stain of Staphylococcus aureus,  ATCC 6538 P. Maintain on nutrient agar medium. 
Transfer to a fresh slant every month and keep in t he cold. Use a 24 h nutrient broth culture for the 
experiment. 
A.2.4 Procedure 
A.2.4.1  Aseptically transfer 1 g of the soap sample to t he flask containing 99 mL of water. Dissolve by 
slight warming not exceeding 60 °C. Transfer 10 mL of th is solution to another flask containing 90 ml of water. 
Take 1 mL of this solution and add 9 mL of nutrient br oth in a culture tube. This gives a concentration of 100 
µg/mL. A.2.4.2 To three tubes containing 9 ml nutrient broth add 1 mL each of the above solution to get a 
concentration of 10 µg/mL soap per ml of nutrient broth in each tube. Inoculate the tubes with a loopful of the 
24 h culture of Staphylococcus aureus  and keep them in an incubator maintained at 37 °C +- 2 °C. Keep a 
control tube of nutrient broth containing the same concentration of soap. 
A.2.4.3  If after 24 h incubation period, the liquid in all the three tubes is as clear as the control, the soap 
sample passes the test. Any turbidity more t han the control shows the growth of bacteria. EAS 766: 2011  
© EAC 2011 – All rights reserved  5
 A.3 Substantivity test 
A.3.1 Basic principles 
For a soap to have antibacterial activity, it shall satisfy two criteria: 
a) it shall show, antibacterial activity on the skin even  after the soap is rinsed away, that is, the germicide 
should be retained on the skin under the conditions of use; and 
b) the antibacterial activity should be retained on the sk in for some period so as to provide protection to 
the skin. 
The test devised gives a measure of both these properties. The test involves application of soap solution on 
the forearm, rinsing it off in running water and allowing it to dry. A mixed culture of skin flora isolated from five 
individuals (see A.3.2.1) is applied immediately in pr escribed areas and assayed by swabbing at 0 and 10 
min. The percent reduction in surviv ors in 10 min is determined. Similarly the soap solution after rinsing is 
allowed to remain on the skin for 2 h. The test micro-organisms are applied to the skin at this time in 
prescribed areas and assayed by swabbing at 0 and 10 min.  The percent reduction in survivors is determined. 
If the reduction in survivors at this  time is greater than 45 %, the germicide is said to be substantive. 
A.3.2 Method 
A.3.2.1 Test micro-organisms 
The test organisms consist of a mixed skin flora, prepa red by collecting washings from the arms and forearms 
of at least five individuals using 50 mL of sterile water in each case. Ten mL aliquot of each washing is 
individually inoculated into flasks containing 90 mL of sterilized nutrient broth. Culture is allowed to grow 
overnight at 30 °C and flask showing turbidity are p ooled together. The mixed culture is transferred through 
broth and grown as above at least three times and finally  maintained as Tryptone-Agar-Glucose Yeast Extract 
(TGYE) agar, Trypticase Soy Agar (TSA), Nutrient Agar (NA) or simila r agar slants. For a test culture, an 
overnight slant culture is suspended into sterile sa line and adjusted to a cell population of 1 x 107 cells per 
mL. 
A.3.2.2 Test procedure 
A.3.2.2.1  A number of 4 cm2 areas (2 cm x 2 cm) are marked out on the innerside of the forearm. 0.1 mL 
aliquot of an 8 % soap solution with germicide is applied onto individual squares and allowed to dry for 1 min. 
The areas are then washed with a gentle flow of tap wa ter for two min, dried by blowing warm air. The 
retentivity of the germicide on skin and its antibacteria l action are then assayed by applying 0.1 mL of mixed 
skin flora (107 cells/mL) onto four such squares at 0 h. Two of the squares are swabbed immediately using 
standard sterile cotton swab on a stic k. Swabs are placed in 5 mL saline solutions. Contents are shaken well 
in a vortex mixer and ten fold dilutions are prepared. Bacterial cells are assayed on TGYE agar, TSA or NA 
plates to determine the initial count. After 10 min, two other squares are swabbed and assayed in a similar 
manner. 
A.3.2.2.2  In another set of tests, soap solutions are applie d to the four more squares, rinsed and dried. 
After allowing 2 h interval, 0.1 mL of culture is app lied as above to four squares. Two of the squares are 
swabbed and assayed at 0 h and remaining two after 10 mi n. Survivals at 0 h and after 2 h are determined. 
A.3.2.2.3  The soap shall be considered to have passed the test  if the percent kill is greater than or equal to 
45 % after two hours challenge. EAS 766: 2011 
6 © EAC 2011 – All rights reserved
 Annex B 
(normative) 
 
Determination of TCC and TCN in soaps by HPLC 
B.1 Principle 
TCC and TCN are antibacterial  agents, which are separated from other  components in soap by normal phase 
or reverse phase liquid chromatography, detected spec trophotometrically and quantified by comparison with 
standard TCC and TCN. The method can estimate  as low as 1 ppm of the above compounds: 
Procedures for both normal and reverse HPLC has bee n described and provide the option to use either 
method whichever is available to the users. Both methods are comparable. 
B.2 Normal phase HPLC 
B.2.1 Reagents 
B.2.1.1 Iso-octane , HPLC grade. 
B.2.1.2 Iso-propanol (2-propanol) , HPLC grade  
B.2.1.3 Hexane , HPLC grade. 
B.2.1.4 Standard TCC , 99 % pure 
B.2.1.5 Standard TCN , 99 % pure 
B.2.2 Apparatus 
B.2.2.1 High Performance Liquid Chromatograph consisting of a pump, a sample injector of fixed 
volume with UV detector having variable wavelengths and a recorder 
B.2.2.2 Standard volumetric flasks  
B.2.2.3 Pipettes B.2.2.4 Magnetic stirrer 
B.2.2.5 Millipore filter apparatus with 0.5 µ filter  
B.2.2.6 Column 
B.2.2.6.1 Silica column , stainless steel 25 cm x 0.46 cm packed wi th Normal phase-silica 5 µ (Lichrosorb 
Si -60) 
B.2.2.6.2 Cyano column , stainless steel 25 cm x 0.40 cm packed with (Lichrospher 100) cyano 5 µ. 
NOTE Either, of the above columns c an be used depending on the availability. 
B.2.2.7 Mobile phase-  EAS 766: 2011  
© EAC 2011 – All rights reserved  7
 B.2.2.7.1 For silica column - Transfer 20 ml of isopropan ol into a 500 ml volumetric flash and make upto 
mark with iso-octane and mix well. Assemble milllipore filt er apparatus and filter the solvent system prior to 
use. 
B.2.2.7.2  For cyano column - Transfer 50 ml of HPLC grade iso-pr opanol (2-propanol) into a 500 ml 
volumetric flask, fill upto the mark with hexane and mix well assemble milllipore filter apparatus and filter the 
solvent system prior to use. 
B.2.2.7.1 For silica column - Transfer 20 mL of iso-propanol into a 500-mL volumetric flask and make up 
to mark with iso-octane and mix well. Assemble millipore filter apparatus and filter the solvent system prior to 
use. 
B.2.2.7.2 For cyano column  - Transfer 50 mL of HPLC grade iso-propanol (2-propanol) into a 500-mL 
volumetric flask, fill up to the mark  with hexane and mix well. Assemble m illipore filter appara tus and filter the 
solvent system prior to use. 
B.2.2.8 HPLC conditions 
Detector wavelength Flow rate: 280 nm 
Flow rate:     0.5 mL/min 
Injection volume:    20 µL  
Retention time Silica column 
 TCN - 7.5 min  
 TCC - 19.2 min Cyano column 
 TCN - 4.0 min  
 TCC - 7.5 min 
B.2.3 Procedure 
B.2.3.1 Standard Preparat ion (see note under B.3.4) 
Weigh accurately 25 mg of triclosan (TCN) and 25 mg of TCC into a 100-mL volumetric flask and make up to 
volume with the mobile phase and mix well. Pipette 1.0 mL of this solution in a 50 mL volumetric flask and 
dilute with mobile phase. Final concentration of TCC and TCN is 250 µg/50 mL (5.0 ppm). 
B.2.3.2 Sample Preparation 
Weigh accurately 1 g of homogenized sample into a 100-mL standard flask, and dilute to the mark with mobile 
phase. Pipette 10 mL of the supernatant liquid to a 50-mL volumetric flask, dilute with mobile phase, to the 
mark, and filter through 0.45 ~m filter. 
B.2.3.3 Chromatography 
Equilibrate the column, maintained at a temperature of 30°C,  with the mobile phase with a flow rate of 0.5 ml 
/min for iso-octane - iso-propanol mobile phase and 1.0 ml/min for Hexane - iso-propanol mobile phase for 30 
min. Set the wavelength at 280 nm. Inject 20 µl of standard solution and then sample solutions. EAS 766: 2011 
8 © EAC 2011 – All rights reserved
 Measure area of the peaks of respective retention time for standard and sample. 
B.2.4 Calculation 
100 x
sample  of   ion Concentratx TCN  standard  of  AreaTCN  standard  of  ion Concentratx TCN  for  sample  of  Areamass by  percent  TCN,=  
100 x
sample  of   ion Concentratx TCC  standard  of  AreaTCC  standard  of  ion Concentratx TCC  for  sample  of  Areamass by  percent  TCC,=
 
B.3 REVERSE PHASE 
B.3.1 Reagents 
B-3.1.1 Methanol - HPLC grade. 
B-3.1.2 Sodium Dihydrogen Phosphate Monohydrate - Chemical grade. 
B-3.1.3  Standard TCC 
B-3.1.4  Standard TCN (TCS) 
B.3.2 Apparatus 
B.3.2.1  Column 
Octyldimethylsilyl (C-DB) 
Supercosil LC-8-DB - 15 cm x 4.6 mm. 5 µ  
B.3.2.2 Mobile Phase 
MeOH/0.01 M Phosphate buffer 62:38 v/v 
0.01 M Phosphate buffer: Dissolve 1.38 g sodium dihy drogen phosphate monohydrate in 1 000 mL of distilled 
water. Prepare to pH 3.0 by 10 % phosphate solutions. 
B.3.3 Procedure 
B.3.3.1 Standard preparation  (see Note under B.3.4) 
B.3.3.1.1  Weigh accurately about 90 mg of TCN. Dissolve in methanol and make up to 1 000 mL volumetric 
flask with methanol. 
B.3.3.1.2  Weigh about 110 mg of TCC, dissolve well with methanol, and make up the volume to 1 000 mL. 
B.3.3.1.3  Accurately pipette 10 mL of the solution prepared in (B.3.3.1.1) into th e (B.3.3.1.2) volumetric 
flask containing TCC. And make up to the volume with me thanol. Then accurately pipette 5 mL of the solution 
into a 50-mL volumetric flask. Make up to the volume with methanol. Filter this standard solution through 0.45 
µm filter. EAS 766: 2011  
© EAC 2011 – All rights reserved  9
 B.3.3.2 Sample preparation 
Weigh accurately about 1.0 g of product, dissolve in methanol and make up to 100 mL in a volumetric flask 
with methanol. Filter this sample solution through 0.45 Jim filter. 
B.3.3.3 HPLC conditions 
Detector wavelength : 280 nm 
Column temperature :  35 ºC 
Flow rate:    1.0 mL/min 
Injection volume:   10 µL 
Prepare the standard solution and the sample solution at the same time. Inject the standard solution three 
times and calculate the average of each ingredients peak count. Inject 10 µg the sample solution and determine each ingredients percent age by the calculation shown. 
B.3.4 Calculations 
()
() 100 t sr s
× ×× ×
M AF A M
TCN, percent by mass =  
()
()TCC, percent by mass =  100 t sr s
× ×× ×
M AF A M 
where 
Ar is the peak area of the test sample, 
As is the averaged peak area of the standard,  
F is the purity of standard (percent). 
Ms is the mass, in grams, of the standard, and 
Mt is the mass , in grams, of the test sample, 
NOTE  Both TCC and TCN are photosensitive, h ence standards should be freshly prepared. EAS 766: 2011 
10 © EAC 2011 – All rights reserved
 Annex C 
(normative) 
 
Determination of zone of inhibition  
C.1 Principle 
The chloroanilines are extracted from soap with dimethyl sulfoxide and diazotized with nitrous acid. The 
reaction products are then coupled with N-1-(naphthy 1)  ethylenediamine hydrochloride to produce co loured 
compounds which are estimated spectrophotometrically. 
C.2 Safety precautions 
Dimethyl sulfoxide (DMSO) is readily absorbed into the skin. Inhalation or skin penetration must be avoided. 
DMSO should never be pi petted using mouth. Always use pipette bulb. The standard chloroanilines and N-1-
(naphthyl)-ethylenediamine hydrochloride must not be allowed to come into contact with the skin. If they should, then wash the contaminated parts thoroughly with soap and water. 
A supply of diluted sodium hypochlorite should be at hand at all times to deal with accidental spillages of 
chloraniline solution. Spillage on laboratory surfac e should be treated immediately with the sodium 
hypochlorite solution, followed by water. 
C.3 Reagents 
C.3.1 Dimethyl Sulphoxide (DMSO) , AR grade 
C.3.2 Hydrochloric Acid , concentrated (speci fic gravity -1.18) 
C.3.3 Sodium Nitrite , 0.4 % w/v  analytical grade, freshly prepared (aqueous) 
C.3.4  Ammonium Sulphamate , 2 % w/v  solution freshly prepared (aqueous) 
C.3.5 N-1-(naphthyl) ethylene , 0.1 % w/v  solution diamine hydrochloride freshly prepared (aqueous) 
C.3.6 Il-Butanol- AR grade 
C.3.7 Sand, a cid purified 40 - 100 micron mesh 
C.3.8 Solvent mixture 
DMSO    5 Volumes 
n-Butanol  2 Volumes 
Distilled water 2 Volumes 
Hydrochloric acid 1 Volume Mix n-butanol, water and HCI.  
Cool the mixture and add DMSO.
 EAS 766: 2011  
© EAC 2011 – All rights reserved  11
 C.3.9 4-Chloroaniline and 3, 4-Dichloroaniline AR grade 
C.4 Apparatus 
C.4.1 Spectrophotometer,  suitable for use at 554 nm 
C.4.2 Cuvettes - Glass (matched pair) 10 mm 
C.4.3 Water bath  - Thermostatically controlled at 25 °C 
C.4.4 Stop watch 
C.4.5 Standard laboratory glassware  
C.4.6 Filter Paper, Whatman No. 541 
C.5 Procedure 
C.5.1 Preparation of Calibration Curve 
C.5.1.1  Dissolve 0.349 8 g of 3, 4-dichloroaniline and 0. 2753 g of 4-chloroaniline in solvent mixture (see  
C.2.8) in a 250 ml amber volumetric flask. 
Dilute to mark with solvent mixture. 1 mL = 2.5 mg mixed chloroanilines (stock solution). 
C.5.1.2  Dilute this stock solution with solvent mixture as given below: 
a) Take 5 mL of stock solution and dilute it to  250 mL with solvent mixture 1 mL = 50 µg mixed 
chloroanilines. 
b) Take 5 mL of the above solution [see  C.5.1.2(a)] and further dilute to 250 mL with solvent mixture. 
1 mL = 1 µg mixed chloroanilines. 
Use this solution for preparation of calibration curve. 
Transfer using a burette 0, I mL, 2 mL, 5 mL, 10 mL , 20 mL, 40 mL into 50 mL amber volumetric flasks. 
C.5.1.3  From a burette, add sufficient solvent mixture to make total volume to 40-mL in each flask. The 
flasks are incubated in a water bath at 25 °C for 20 min: After exactly 20 min, add 2-mL of reagent (see C.3.3) 
into each flask and return them to the water bath for exactly 10 min (measure with a stop watch). 
Then add 2-mL of reagent (see  C.3.4) into each flask and return them  to the water bath for exactly 10 min. 
Swirl the flask occasionally. 
Then add 2-mL of reagent (see  C.3.5) into each flask and remove them from the water bath. Dilute to volume 
with distilled water, mix and allow to stand for 30 min. Measure absorbance at 554 nm against the blank 
solution as prepared in C.5.1.4. 
C.5.1.4  In preparing the blank solution, take 40 ml of solvent mixture in a 50 011 amber volumetric flask. 
Incubate the flask in a water bath at 25 °C for 20 min.  After exactly 20 min, add 2 mL of reagent (see C.3.3) 
into the flask and return it to the water bath for exactl y 10 min. Then add 2 ml of reagent (see C.3.4) into the 
flask and return it to the water bath for exactly 10 min (s wirl the flask occasionally ). Then add 2 011 of reagent 
(see C.3.5) into the flask and remove it from the wate r bath. Dilute to volume with distilled water, mix and 
allow to stand for 30 min. Use this blank solu tion for preparation of calibration curve only. EAS 766: 2011 
12 © EAC 2011 – All rights reserved
 C.5.1.5  Prepare a graph by plotting weight (Jlg) of chlo roanilines contained in each 50 mL flask against 
absorbance. The linear calibration will pass through the origin/or determine the average absorbance (AA) of 1 
Jlg of mixed chloroanilines by dividi ng sum of absorbances of all different  aliquots of the standard by sum of 
Jlg of chloroanilines in all different aliquots of standard. 
C.6 Determination of chloroanilines 
C.6.1  Weigh to the nearest mg 3.0-l5 g of finely grated soap add 10.0 g - 15.0 g of acid purified sand. 
Transfer quantitatively the sample and the sand into a mortar and grind the mixture thoroughly with a pestle to 
give a homogenous mass. Transfer the Ittass to a previ ously weighed 250 011 flat bottom flask quantitatively 
and reweigh. Add DMSO (100 mL), stopper firmly and atta ch the flask to an automatic shaker. Shake for 1 h. 
Filter the DMSO extract through Whatman No. 541 into a 250 mL amber volumetric flask. Wash the flask and 
filter paper with small aliquots of DM SO. Allow the filtrate to drain completely, dilute to volume with DMSO and 
mix. Transfer 20 mL DMSO extract into  a 50 mL amber volumetric flask. Add 20 mL of solvent mixture. The 
flask is incubated in a water bath at 25 °C for 20  min. After exactly 20 min, add 2 mL of reagent (see  C.3.3) 
into the flask and return it to the w'ater bath for exactly 10 min (measure with a stop watch). Then add 2 m I of reagent (see  C.3.4) into the flask and return it to the water bath for exactly 10 min (swirl the flask 
occasionally). Then add 2 mL of reagent (see C-3.5) into the flask and remove it from the water bath. Dilute to 
volume with distilled water, mix and allow to stand for 30 min. Read the absorbance at 554 nm against blank (prepared as below). 
C.6.2 Prepare the blank solution by mixing 20 mL of DMSO  extract of sample and 20 mL of solvent mixture 
in a 50 mL amber volumetric flask. Incubate the flask in a water bath at 25 °C for 20 min. .After exactly 20 min, 
add 2 mL of distilled water into the flask and return it to the water bath for exactly 10 min. Then add 2 mL of 
reagent (see  C.3.4) into the flask and return it to the water bath for exactly 10 min (swirl the flask 
occasionally). Then add 2 mL of reagent (see  C.3.5) into the flask and remove it from the water bath. Dilute to 
volume with distilled water, mix and allow to stand for 30 min. Use this solution as a blank for reading sample 
only. 
C.6.3 Deduce the amount of chloroanilines (µg) from the calibration graph curve. 
NOTE The determination should be completed in one day. 
C.7 Calculations 
Determine the amount of mixed chloroanilines in the aliquot of test solution from the calibration graph. 
Chloroaniline content (in ppm) = 250(M  + M 1)M3/20M 2M 
where 
M is the mass, in grams, of soap 
M1 is the mass, in grams, of sand 
M2  is the mass, in grams, of soap and sand transferred to the flask 
M3 is the mass, in micrograms, (µg) of mixed chloroanilines found from calibration graph/or it can be 
calculated as given below: . 
M3= Mass of the sample/Average absorbance of 1 µg mixed chloroanilines (AA) 
where AA is the sum of the 00 of the standards/Sum of  concentration of standard chloroanilines in µg 
Weight of soap actually used, in g = M2M/ (M + M1) EAS 766: 2011 
 
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