TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1  
DRAFT TANZANIA STANDARD  
 
 
 
 
 
 
Specification for plastic materials for food contact applications part 3: 
colourants  
   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
TANZANIA BUREAU OF STANDARDS  
 
                              TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 0 Foreword   
The packaging of foodstuffs is essential for their conservation, tra nsport and handling. It also avoids 
contamination and maintain hygienic conditions. There is always a possibility of transfer of part of 
packaging materials to the packed food or foodstuffs due to intimate contact of each other. Therefore, 
formulation of t he packaging materials must be selected to ensure that any such transfer is at a minimum 
and a substance which do migrate from package to the packed food cause no toxic hazards on 
consumption. These considerations are equally important in case of processin g equipment and utensils 
which come in contact with foods and foodstuffs.  
This standard is expected to serve as a basic guide for selection of colouring matters which are 
considered toxicologically safe for use in plastics and not on the manner of their ac tual processing or use.  
In the preparation of this standard assistance has been derived from: IS 9833:2018 list of colourants for 
use in plastics in contacts with Foodstuffs and Pharmaceuticals published by Bureau of Indian Standards 
(BIS).  
In reporting th e result of a test or analysis made in accordance with this standard, if the final values 
observed or calculated, is to be rounded off it shall be done in accordance with TZS 4 Rounding off 
numerical values . 
 
1 Scope   
This standard list permitted coloura nts for use in plastics that may be reg arded as safe for use in contact 
with foodstuffs.  
2 Normative  reference s 
The following referenced standards referred to in the text in such a way that some or all of their content 
constitutes requirements of this st andard.   For dated references, only the  edition cited applies. For 
undated references, the latest edition of the referenced document (including any amendments) applies.  
TZS 4 : Rounding off numerical values . 
AFDC 2(229)  CD2: Determination of overall migrat ion of constituents of plastics materia ls and articles  
              intended to come in contact with foodstuffs - Method of analysis  
3.   Terms and definitions  
For the purposes of this standard the following definitions shall apply:  
3.1   colourant  
ingredient s that alone or in combinatio n with other ingredients impart or alter the colour of the product.   
 NOTE :  
 1. Colourants comprise pigments (see 3.3) which are insoluble in the medium as well as dyestuffs  (see 3.2) 
     which are soluble  in the medi um  
 2. A pigment may contain the pure chemical substance and/or a surface treatment and/or additives  
 3. a colourant may also contains traces of impurities, which may originate from raw materials and/or the  
    production process                               TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1  4. In orde r to improve  applic ation properties, a colourant may contain additives.  
3.2    dyestuffs  
Colourants soluble in the application medium  
3.3    pigments  
Colourants consisting of particles, insoluble in the application medium (for ex ample coating material or 
plastic)  
 NOTE: p igments can be further described on the basis of their chemical composition, their optical or  
             technical properties, for example inorganic pigments, organic pigments coloured pigment,   
             white pigment , effect pigment, corros ion inh ibition -inhibiting pigment, magnetic pigment.  
3.4   colourant Products  
 It refers to the commercially available colo urant available in the market. These commercially available 
products contain  the components resp onsible for the colour  as well as the  quanti ties of other substances 
(generally referred to as additives) designed to improve the application properties of the product., such as 
the dispensability colouristics, flow and flocculation resistance of pigments.  Dyestuffs often contain 
significant amounts  of diluents.  
3.5 Essential colourants  
The component of colourant product responsible for colour excluding additive s  
4.    Requirements  
4.1 materials  
Material shall be manufactured in compliance with good man ufacturing practices so that under no rmal or  
foreseeable condition of use they do not transfer their constituents to foodstuffs in quantities which could 
endanger human health; bring about an unacceptable change  in the composition of foodstuffs or bring 
about deterioration in the organoleptic  charac teristics thereof.  
4.2 when tested in accordance with AFDC 2( 229) CD1 , colourants used should not show visible bleeding 
or migration from the finished product and shall show no sign of instability or degradation during 
processing.  
Colourants shall be suff iciently integrated within plastic material so as to preclude any visible migration in 
a foodstuff or under normal condition of use . 
4.3 colourants used shall have a high degree of purity. It shall also comply wi th the requirements given in 
Table 1 when te sted in accordance with the methods given in col 4  
Table 1: Requirements for Pigment and Colourants  
  SI 
No. Characteristics  Requirements  Methods of test  
i.  Lead , percent by mass, Max  0.01 annex G  
ii.  Arsenic , percent by mass, Max  0.005  annex G  
iii.  Mercury , percent by mass, Max  0.005  annex G  
iv.  Cadmium,  percent by mass, Max  0.010  annex G  
v.  Zinc, percent by mass, Max  0.05 annex G  
vi.  Selenium,  percent by mass, Max  0.01 annex G  
vii.  Barium , percent by mass, Max  0.01 annex  G 
viii.  Chromium , percent by mass, Max  0.025 annex G                               TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 ix.  Antimony , percent by mass, Max  0.025  annex G  
x.  Total primary aromatic amine , Percent by mass, 
Max, (calculated as aniline equivalent) ,   0.05 Annex B 
xi.  Sulphonated aromatic amine , Percent by mass, 
Max (calculated as aniline sulphonic acid)  0.05 Annex B  
xii.  Polychlorinated biphenyl , max (mg/kg) reported 
as decachloro biphenyl  25 Anne x C 
xiii.  Carcinogenic amine listed in Annex A, max 
(mg/kg)  10 IS 14816  
 
4.4 carbon black, if used, shall not be more than 2.5  percent ( m/m). the UV absorbance of cy clohexan e 
extract at 386 nm shall be less than 0.02 AU for a 5cm cell, it shall comply with the requirement given in 
table 2.  
Table 2 carbon black  
  SI 
No. Characteristics  Requirements  Methods of test  
i.  Toluene ex tract, percent by mass, Max 0.1  Annex D 
ii.  3,4-Benzopyrene, ppm, Max 0.01 Annex E  
  
5.  List of Colourants for Plastics in Contacts with Foodstuffs  
5.1 Organic Pigments  
Table 3: List of organic pigments for Plastics in Contacts with Foodstuffs  
  SI 
No. CAS No.  C.I. No  C.I. Name  
1.  2512 -29-0 11680 Pigment Yellow 1  
2.  6486 -23-3 11710  Pigment Yellow 3  
3.  5979 -28-2 20040  Pigment Yellow 16  
4.  6370 -75-8 65405  Pigment Yellow 12  
5.  12286 -66-7 13940  Pigment Yellow 62  
6.  5580 -58-5 20038  Pigment Yellow 94  
7.  5280 -80-8 20034 Pigment Yellow 95  
8.  5590 -18-1 56280  Pigment Yellow 110  
9.  29920 -31-8 11738  Pigment Yellow 120  
10.  79953 -85-8 20037  Pigment Yellow 128  
11.  30125 -47-4 56300  Pigment Yellow 138  
12.  36888 -99-0 56298  Pigment Yellow 139  
13.  71832 -85-4 13960  Pigment Yellow 168  
14.  96352 -23-7 56160  Pigment Yellow 173  
15.  77804 -81-0 2129 0 Pigment Yellow 180  
16.  74441 -05-7 11777  Pigment Yellow 181  
17.  67906 -31-4 12830  Pigment Yellow 182  
18.  65212 -77-3 18792  Pigment Yellow 183  
19.  129433 -54-7 18795  Pigment Yellow 191  
20.  3627 -47-2 65410  Vat Yellow 26  
21.  12236 -62-3 11780  Pigment Orange 36  
22.  4424-06-0 71105  Pigment Orange 43                               TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 23.  40716 -47-0 11265  Pigment Orange 61  
24.  72102 -84-2 12760  Pigment Orange 64  
25.  35869 -64-8 20060  Pigment Brown 23  
26.  68516 -75-6  Pigment Brown 41  
27.  6041 -94-7 12310  Pigment Red 2  
28.  2425 -85-6  12120  Pigment Red 3  
29.  2814 -77-9 12085  Pigment Red 4  
30.  6410 -41-9 12490  Pigment Red 5  
31.  6471 -51-8 12420  Pigment Red 7  
32.  6410 -30-6 12335  Pigment Red 8  
33.  6410 -38-4 12460  Pigment Red 9  
34.  6410 -35-1 12440  Pigment Red 10  
35.  6410 -32-8 12385  Pigment Red 12  
36.  3564 -22-5 12350  Pigment Red 18  
37.  6883 -91-6 21205 Pigm ent Red 37  
38.  6358 -87-8 21120  Pigment Red 38  
39.  7023 -61-2 15865:2  Pigment Red 48:2  
40.  15782 -05-5 15865:3  Pigment Red 48:3  
41.  1103 -39-5 15630:2  Pigment Red 49:2  
42.  17852 -99-2 15860:1  Pigment Red 52:1  
43.  4/9/5281  1585 0:1 Pigment Red 57:1  
44.  6417 -83-0 15880: 1 Pigmen t Red 63:1  
45.  5850 -80-6 15525  Pigment Red 68  
46.  72-48-0 58000:1  Pigment Red 83  
47.  14295 -43-3 73312  Pigment Red 88  
48.  6409 -74-1 60745  Pigment Red 89  
49.  6535 -46-2 12370  Pigment Red 112  
50.  980-26-7 73915  Pigment Red 12 2 
51.  5280 -78-4 20735  Pigment Red 144  
52.  5280-68-2 12485  Pigment Red  146 
53.  4948 -15-6 71137  Pigment Red  149  
54.  56396 -10-2 12290  Pigment Red  150  
55.  3905 -19-9 20730  Pigment Red  166 
56.  2786 -76-7 12475  Pigment Red  170 
57.  4051 -63-2 65300  Pigment Red  177  
58.  5521 -31-3 71130  Pigment Red  179 
59.  77804 -81-0 2129 0 Pigmen t Red  180 
60.  2379 -74-0 73360  Pigment Red  181 
61.  59847 -23-9 12486  Pigment Red  187 
62.  3089 -17-6 73907  Pigment Red  202 
63.  31778 -10-6 12514  Pigment Red  208 
64.  1/1/33573  73905  Pigment Red  209 
65.  40618 -31-3 20066  Pigment Red 214 
66.  68259 -05-2 20055  Pigment Red  220  
67.  71566 -54-6 20065  Pigment Red  221 
68.  52238 -92-3 20067  Pigment Red  242 
69.  43035 -18-3 15915  Pigment Red  247 
70.  84632 -65-5 56110  Pigment Red  254 
71.  120500 -90-5 561050  Pigment Red  257 
72.  70833 -37-3 56270  Pigment Red  256 
73.  88949 -33-1 561300  Pigment Red 264                               TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1  
5.2 Dyestuffs  
Table  4: List of dyestuffs for plastics in contacts with foodstuffs  74.  1047-16-1 73900  Pigment Violet 19  
75.  6358 -30-1 51319  Pigment Violet 23  
76.  81-33-4 71129  Pigment Violet 29  
77.  5462 -29-3 73385  Pigment Violet 36  
78.  2379 -75-1 73395  Pigment Violet 38  
79.  147-14-8 74160  Pigment Blue 15:X  
80.  574-93-6 74100  Pigment Blue 16  
81.  1328 -50-3 74140  Vat Blue 29  
82.  81-77-6 69800  Pigment Blue 60  
83.  482-89-3 73000  Pigment Blue 66  
84.  1328 -53-6 74260  Pigment Green 7  
85.  1330 -37-6 74255  Pigment Green 37  
86.  3183 7-42-0 13980  Pigment Yellow 151  
87.  4118 -16-5 60645  Pigment Y ellow 147  
88.  52238 -92-3 20067  Pigment Re d 242 
89.  250640 -08-5  Pigment Orange 79  
90.  84632 -66-6/61951 -98-2  Pigment Red 272  
91.  154946 -66-4 18759:1  Pigment Yellow 191:1  
  SI 
No. CAS No.  C.I. No  C.I. Name  
1.  6370 -85-0 66510 Vat Yellow 9  
2.  6252 -78-4 73860  Vat Red 45  
3.  6492 -68-8 73305  Vat Red 47  
4.  482-89-3 73000  Vat Blue 1  
5.  130-20-1 69825  Vat Blue 6  
6.  1330 -38-7 74180  Direct Blue 86  
7.  128-80-3 61656  Solvent Green 3  
8.  116-75-6 61568  Solvent Blue 104  
9.  12236 -03-2 61568  Dispe rse Oran ge 47  
10.  17354 -14-2 61554  Solvent Blue 35  
11.  4702 -90-3 48160  Solvent Yellow 93  
12.  4851 -50-7 625580  Solvent Green 28  
13.  61969 -44-6 615290  Solvent Blue 97  
14.  6408 -72-6 615290  Disperse Violet 26  
15.  64696 -98-6 48525  Solvent Brown 53  
16.  6829 -22-7 564150  Solve nt Red 1 79 
17.  61969 -47-9/6925 -69-5 564100  Solvent Orange 60  
18.  75216 -45-4/7576 -65-0 47020  Solvent Yellow 114/Disperse Yellow 54  
19.  81-39-0 68210  Solvent Red 52  
20.  81-48-1 60725  Solvent Violet 13  
21.  83249 -52-9 56280  Dispers e Yellow 241  
22.  80748 -21-6/54079 -53-
7/17772-51-9  Disperse Yellow 201  
23.  20749 -68-2/71902 -17-5 564120  Solvent Red 135  
24.  12226 -78-7/81457 -65-0  Solvent Blue 67                               TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1  
5.3 Inorganic Pigment/Alloys   
Table 5: list of inorganic pigment/alloys for plastics in contacts  with foodstuffs   
  
 25.  6408 -72-6 62025  Solvent Violet 59  
26.  72968 -71-9  Solvent Red 195  
27.  23552 -74-1/37229 -23-5  Solvent Blue 45  
  SI 
No. CAS No.  C.I. No  C.I. Name  
1.  7429 -90-5 77000  Aluminium   
2.  7440 -50-6 77400  Copper  
3.  7440 -22-4 77820  Silver  
4.  7440 -57-5 77480 Gold  
5.  7440 -31-5 77860  Tin  
6.  7440 -06-4 77795 Platinum and platinum group metal  
7.  7440 -50-8 77400  Bronzes of copper  
8.  471-341-1 77220  Whitening (calcium carbonate)  
9.  10101 -41-4 77231  Calcium sulphate (gypsum, plaster of Paris)  
10.  1332 -58-7 77005  Kaoli n  
11.  13463 -67-7 77891  Titan white (tita nium oxi de) 
12.  1344 -28-1 77002  Alumina  
13.  637-12-7  Aluminium stearate  
14.  14807 -96-6 and 8005 -37-6 77718  Talc  
15.  51274 -00-1 77492  Yellow iron oxide  
16.  1345 -27-3 77491  Iron oxide  
17.  57455 -37-5 77007  Ultramarine blue (co mplex silicate of 
aluminium and sodium sulphura ted) 
18.   77437  Egyptian blue (double silicate of copper and 
calcium)  
19.  1345 -16-0 77346  Cobalt blue (cobalt aluminate)  
20.  1333 -86-4 77266  Carbon black  
21.  7727 -43-7 77120  Barytes (barium sulphates)  
22.  64294 -91-3 77492 Sienna (natural ferric oxides)  
23.  12769-96-9 77007  Pigment violet 15  
24.  1308 -38-9 77288  Pigment green 17  
25.  1309 -37-1 77491  Pigment red 101  
26.  1314 -13-2 77975  Pigment white 4  
27.  1314 -98-3 77975  Pigment white 7  
28.  1317 -61-9 77499  Pigment black 11  
29.  57455 -37-5/101357 -30-6 77007  Pigment blue 29  
30.  68187-51-9 77496  Pigment yellow 119  
31.  7727 -43-7 77120  Pigment white 21  
32.  8007 -18-9 77788  Pigment yellow 53  
33.  12001 -26-2 77019  Pigment white 20  
34.  68186 -90-3 77310  Pigment brown 24  
35.  1345 -05-7 77115  Pigment white 5                               TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 Annex A  
[Table 1, SI No. (xiii)]  
List of Carcinog enic Amine  
  
 
 
 
 
 
 
 
 
 
 
 
   SI 
No. CAS No.  Substance  
1.  92-67-1 4-aminobiphenyl  
2.  92-87-5 Benzidine  
3.  95-69-2 4-chloro -o-toluidine  
4.  91-59-8 2-Naphthylamine  
5.  97-56-3 o-aminoazotoluene/4 -amino -2,3-dimethylazobenzene/4 -o-Tolylazo -o-toluidine    
6.  99-55-8 5-Nitro-o-toluidine  
7.  106-47-8 p-Chloroaniline  
8.  615-05-4 2,4-Diamino anisole  
9.  101-77-9 4,4’-Methylenedianiline/4,4’ -Diaminodiphenyl methane  
10.  91-94-1 3,3’-Dichlorobiphenyl -4,4’-xylendiamine  
11.  119-90-4 3,3’-Dimethoxybe nzidine  
12.  119-93-7 3,3’-Dimethylbenzid ine/4,4’ -Bi-o-toluidine  
13.  838-88-0 3,3’-Dimethyl -4,4’-diaminodiphenylmethane  
14.  120-71-8 6-Methoxy -m-toluidine -p-cresidine  
15.  101-14-4 4,4’-Methylene -bis-(2-chloroaniline)/2,2’ -Dichloro -4,4’-methylenedianiline  
16.  101-80-4 4,4’-Oxydianiline  
17.  139-65-1 4,4’-Thiodianil ine  
18.  95-53-4 o-Toluidine/2 -Aminotoluene  
19.  95-80-7 4-Methyl -m-phenylenediamine  
20.  137-17-7 2,4,5 -Trimethylaniline  
21.  90-04-0 o-Anisidine -2-methoxyaniline  
22.  60-09-3 4-Aminoazobenzene                               TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 Annex B  
[Table 1, SI No. (x) and (xi) ] 
Determin ation of  total primary aromatic amines  
B-1 General  
Analysis of primary aromatic amine (PAA) in organic colorants  to ascertain quality for safe use in food 
contact application . 
B-2 Apparatus  
B-2.1 Weighing Balance, ne arest to 0.0001 g . 
B-2.2 Ultrasonic Bat h 
B-2.3 Centrifuge Capable to 3 000 rpm   
B-2.4 HPLC System equipped with Gradient Elution -DAD detector and pump. UV detector can be used 
as optional  
B-2.5 Column -250 mm x 4mm, 5µm or equivalent HPLC column  
B-3 Reage nts  
B-3.1 hydrochloric acid solution -1N 
B-3.2 sodium hydroxide solution -5N 
B-3.3 HPLC Grade Methanol  
B-3.4 pH strips  
B-3.5 Distilled Water  
B-3.6 Analytical Reference standards for amines, to test appropriate individual colourant. For example, 
2,5-dichlo roaniline may be used as analytical ref erence s tandards for Pigment Red 2 . 
 B-3.7 Phosphoric acid – Analytical grade  
B-4 Procedure  
B-4.1 Sample Preparation  
Weigh accurately 0.5g sample in 250mL capacity flask. Add 20mL methanol and 60mL 1N hydrochloric 
acid. Apply sonication for 5min. in ultr asonic b ath at 37 ±2ºC  and subsequently stir it for 25min at 200rpm 
and 37±2ºC. transfer the content to centrifuge tube and apply centrifuge for 5min at 3 000 rpm and decant 
clear layer in 250mL beaker through filter p aper. Add 30mL , 1N hydrochloric acid in  centrif uge tube and 
stir for 25min at 200rpm and 37±2ºC. Centrifuge  this at 3000 rpm and decant clear layer in 250mL beaker 
through filter paper. Wash filter paper with approximately 2mL 1N hydrochloric acid and comb ine the 
filtrate with the first aqueous  phase . Adjust the pH to 7 using 5N sodium hydroxide solution and transfer to 
200mL volumetric flask. Make up the volume to 200mL with distilled water. Inject 10 µL in HPLC directly.  
 
                              TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 B-4.2 Standards Preparation.  
Weigh, to the nearest 0.1mg, 10±1 mg of ea ch aroma tic amine into a 100mL volumetric flask. Add  
methanol/ water 8:2 ( v/v). Place it in an ultrasonic bath for 10min to ensure complete dissolution to ma ke 
standard solution for calibration curve. The stability of the mixed stock standard solution shoul d be 
checked regularly. It should be stable for up to 6 months when stored in cool and dark place (27ºC). 
calculate the response factor (RF). Use minimum three points calibration curve.  
B-4.3 instrument (HPLC) Set UP  
Column: 250 mm x 4 mm, 5µm or equivalen t HPLC c olumn  
Oven temperature: 40ºC  
Mobile phase  
A: 5mmol Diammonium hydrogen phosphate buffered to pH 7 with concentrated phosphoric acid  
B: HPLC grade methanol  
Gradient: 90 percent ( v/v) A: 10 percent (v/v) B, in 30 min 5 percent ( v/v) A and 95 perc ent (v/v) B 
Flow rate: 0.9mL/min  
Injection volume: 10 µL or higher depending upon limit of detection.  
Injection volume: suitable to ensure the limit of detection (LOD) (5 -15µL)  
Run time: 45 min  
Flow: 0.3mL/min  
DAD mode : 240±20 nm  
DAD range 200 to 800 nm  
B-4.4 Calcu lation of response factor for each concentration  
Response Factor (RF) = area of standards/concentration of standards (ppm)  
B-4.5 Calculation of Individual Primary Aromatic Amine  
Individual primary aromatic am ine (PAA), in ppm  
                               = (sample are a x dilution factor ) / (RF x Weight of sample ) (g) 
Recalculate to aniline equivalent in case of all amines other than those listed in Annex A  
 
 
 
 
                              TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 Annex C  
Determination of polychlorinated bipheny l (PCB)  
C-1 General  
This method covers  determ ination of total polychlorinated biphenyl (PCBs) contents in colourant material 
by low resolution gas chromatography coupled to high resolution mass spectrometer (LRGC -HRMS) 
using electron impact (EI) mode.  
Application of LRGC -HRMS ensures separatio n/recog nition of most PCB congeners are separated or 
recognizable at different retention times.  
  Note: in case a specific PCB is to be reported which is different from any congeners directly  
  specified by internal standard, suitable care should be taken  using external window standards for  
  identification.  
C-2 Apparatus  
C-2.1 Low Resolution Gas Chromatography Coupled to High Resolution Mass Spectrometer (LRGC -
HRMS) using Electron Impact (EI) Mode  
C-2.2 Rotary Eva porator  
C-2.3 weighing balance, neares t to 0. 000 1g.  
C-2.4 Micropipette, with disposable pipette tips  
C-2.5 Surgical Hand Gloves  
C-2.6 Glass Columns  
C-2.6.1 Column, 30cm long, 18mm diameter with 250mL top reservoir  
C-2.6.2 Column, 30cm long, 10mm diame ter with 250mL top reservoir.  
C-2.7 Mag netic S tirrer and Magnetic Bar  
C-2.8 Ground Joint Conical Flask -500mL  
C-3 Reagents  
C-3.1 concentrated sulphuric acid – analytical grade  
C-3.2 n -Hexane – analytical reagent grade  
C-3.3 Dichloromethane – analytical gr ade 
C-3.4 Silica –Gel- technical grade,  pore s ize 60 Á, 70 -230 mesh, 63 -200 µm  
C-3.5 PCB Standard Solution (EC 4058) or Equivalent  
C-3.6 n-Nonane  
C-3.7 Celite 545 (CAS No. 68855 -54-9) 
C-3.8 Cesium Hydroxide                               TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 C-3.9 Silver nitrate  
C-3.10 Alumina (ICN alum ina B -Super I (Basic) (50 -200 µm or alt ernativ e)  
C-3.11 sodium sulphate, anhydrous.  
C-3.12 Toluene, analytical reagent grade  
C-3.13 Ethanol, analytical reagent grade  
C-4 Work Conditions   
Dust free environment with positive pressure inside the laborator y by hair handling unit and temperature  
to mai ntained at 22±2ºC  
C-5 procedure  
C-5.1 Preparation of Test Sample  
C-5.1.1 weigh accurately 0.75 -2.5 g sample into a conical flask and add 0.5 -0.9 g ethanol to wet the 
colourant surface  
C-5.1.2 Add 100 µL PCB s tandard (5 times diluted with n -nonane) . Add 1 -2g of phosphoric acid and 
sodium sulphate of approximately same weight as of the sample. Add 40g of 92 -96 percent sulphuric acid 
twice, carefully with repeated shaking. Sonicate the mixture for 30 -100min until  homogenous dark reddish 
or olive colou r solut ion is obtained.  
C-5.2 Liquid –Liquid Extraction  
 C-5.2.1 extract sulphuric acid solution with 200mL n -hexane and rapidly stir it by ultra -sonication for at 
least 30 min. in case of suspected of unusually high  impurity content, a mixture of 50mL n -hexane and 
200mL dichloromethane to be used  
C-5.2.2 Transfer the mixture to a separating funnel and collect the organic phase in a 500mL round 
bottom flask. Repeat the extraction steps 2 times (more if necessary) and  combine all the organic phases 
in a ro und bot tomed flask. In case, if possible, decant organic layer directly. In case, if oily layer found 
then follow the step as given as C -5.2.3. add 1 -2mL n -nonane in organic phase and remove the solvent 
using rotary e vaporator. Reduce the volume up to 1 -2mL in ro und bottom flask, follow the clean -up 
procedure using 1 -2mL remaining solvent  
C-5.2.3 Add 50 percent aqueous potassium hydroxide solution in organic phase to make its pH 7 -8, and 
shake out the aqueous layer twi ce with 100mL n -hexane, in order to con trol th e changes of temperature 
due to heat of neutralization. After reaching pH of 8, transfer the solution into the separation funnel, rinse 
the flask with 10ml of n -hexane. Combine all the clean organic phases in a  round bottom flask and dry 
them by add ing 0.5 g of anhydrous sodium sulphate. Add 1 -2mL n-nonane in organic phase and remove 
the solvent using rotary evaporator. Reduce the volume up to 1 -2mL in round bottom flask. Follow the 
clean -up procedure using 1 -2mL remaining solvent.   
 
                              TBS/AFDC 2 (231) CD2 
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©TBS_202 1 C-5.3 Clean -up  
C-5.3.1 Take the neat and clean glass column (size 30 cm long, 18mm diameter with 250 mL top 
reservoir). Fill the glass column with n -hexane up to 1/3 of top reservoir. Weigh the chemical and transfer 
to column in foll owing sequence. While addition of reag ent sha kes the column externally to avoid any air 
bubbles left in the column for better efficiency.  
C-5.4 Alumina Column  
 
Reagent/chemicals  Mass (g)  
Silica gel  5 
Celite: Sulphuric acid (1:1 mix)  30-33 
Silica gel  5-6 
Anhydrous sodium sulphate  5-7 
 
 Let n -hexane run until top layer reached. Then pre -condition with 200m L hexane: dichloromethane (4:1, 
v/v) and let run off to the top layer again. Discard the elute.  
C-5.3.2 transfers the analyte from round bottom fla sk to column with help of transfer pip ette an d rinse the 
round bottom flask for 5 times with approximately 5 mL hexane: dichloromethane (4:1, v/v) and transfer to 
multi -layer column. Add approximately 150m L n-hexane. Collet the solvent n 500m L round bottom flask. 
Recover the excess n -hexane by rotary evaporator until 5 -7mL solution is left.  
C-5.4 Alumina Column  
C-5.4.1 Take the neat and clean glass column (size 30 cm long, 10 mm diameter with 250 ml top 
reservoir). Fill the glass column with toluene up to 1/ 3rd of top reservoir. Usage of n -hexan e inste ad of 
toluene is also permissible. Weigh the chemicals and transfer to column in following sequence. While 
addition of reagent shakes the column externally to avoid any air bubbles left in the column . 
Reagent/Ch emicals  Mass (g)  
Silica gel  0.3-0.5  
Anhydrous alumina  12.5-13 
Silica gel  0.3  
Anhydrous sodium sulphate  3  
 
C-5.4.2 After filling of the column, remove the toluene/ n -hexane up the just above the top layer of 
reagent.  
Load concentration - Eluate of previous step into the column. R inse th e flask twice (2 times 3 mL 
toluene/n -hexane) and add the solvent to the column.  
Pre-run - Elute off with 40mL toluene into a calibrated cylinder in case of brominated sample matrix. Elute 
with hexane: dichloro methane (98: 2, v/v), adding first 2 m L x 2 m L and finally 76m L of the eluant and 
collect into the same cylinder of the pre -run till reaching a volume of 120mL.  
C-5.4.3 Transfer the extract solution quantitatively to a flask and evaporate to a volume of 5 00 µL, reduce 
to approximately 200 µL by nitrogen blowing and transfer to a standard 1.2mL septum -sealed glass vial.                               TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 The measuring solution is obtained by rinsing the flask 2 times with 25 µL n-nonane and adding the 
volume to the GC -vial. 
 
C-5.5 Determinati on of Analytes  
Refer the instrument m anual f or operation and analysis of PCB by gas chromatography - Mass 
Spectrometry (GCMS) using auto -sampler, injection volume shall be 3 -10 µL.  
NOTE - Perfluorotributylamine (PFTBA) tuning is performed every two month s or according to instrument 
performan ce moni toring requirement  
C-5.6 Calculation and Quantification Procedure  
C-5.6.1 In case, if peaks are not appearing in desire windows then set the time accordingly.  
Integration the M peaks in GC -quadrapole should b e carried out manually in the re -quant ification mode. 
Small peaks and especially those looking significant, but lacking even an approximate isotopic ratio as 
predetermined for the Analyte, response is considered  
100 percent. Chemically and structurally mo st congers are to be ratio -calculated from th e congener 
standard amount. Amount of each determined congener is divided by the sample weight.  
 
C-5.6.2 Lower Detection Limit  
C-5.6.3 Mass Conversion Factors  
PCB below the range of I ppm cannot be measured by  this method, since the internal relat ive sta ndard 
deviation (RSO) will approach 100 percent. Report the sum of decachlorobiphenyl (DeCB) equivalent 
(ppm) from mono - to deca -chlorobiphenyl and apply correction factor, if any, based on the measurement 
of un certainty.  
 
Degree of  
Chlorination  From CB t o 
DeCB  
Equivalent  From DeCB to  
CB Equivalent  
Multiply by  
(1) (2) (3) 
Mono -CB 2.649  0.377 50  
Di-CB 2.244  0.44564  
Tri-CB 1.930  0.518 13  
Tetra -CB 1.706  0.586 17  
Penta -CB 1.528  0.65445  
Hexa -CB 1.391  0.7189 1 
Hepta -CB 1.264  0.791 14  
Octa -CB 1.158 0.86356  
Nona -CB 1.073  0.93197  
Oeca -CB(OeCB)  1.000  1.00000  
 
 
 
 
 
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©TBS_202 1 Annex D  
[Table 2, Sl No. ( i)] 
Determination of toluene extract  
D-1 toluene discoloration method  
D-1.1 General  
This method covers determination of the degree discoloration of toluene by  carbon  black  by means of 
spectrophotometer. This method is not applicable to high extra ct thermal type black.  
D-1.2 Apparatus  
D-1.2.1 spectrophotometer -20nm maximum spectral band pass capable of measuring transmittanc e in 
the 425±5 nm range  
D-1.2.2 Absor ption C ells-with an optical light path  of 10 mm (light path of the toluene and not the outside 
for the cell) for the spectrophotometer  
D-1.2.3 Analytical balance -sensitivity 0.01mg  
D-1.2.4 oven -gravity convection type  capable of temperature regulation of ±1ºC at  105ºC.  
D-1.2.5 Filter paper – whatman No. 41 or equivalent, diameter of 150 mm.  
D-1.2.6 mechanical shaker - capable of 240 strokes/min.  
D-1.2.7 mortar and pestle  
D-1.2.8 glass filtering funnel  
D-1.2.9 beaker  
D-1.2.10 Erlenmeyer flask  
D-1.3 reagen t  
D-1.3.1 toluene  
D-1.4 procedure  
D-1.4.1 standardization of apparatus  
Clean the absorption cell with the clean tissue. Allow the spectrophotometer to warm up at least 10 min 
before standardization. Rinse the cell twice with clean toluene, wipe the out side su rface with clean tissue. 
Adjust the transmission value to 100 percent on the spectrophotometer, using the wavelength of 425 nm.  
D-1.4.2 Crush pelleted samples using mortar and pestle or equivalent. Dry an adequa te amount of 
crushed carbon black samp le at 105ºC for 60min using the oven.  
 NOTE -an infra -red lamp must not be used for drying sample as it could vapourize some of the 
extractible    material  
D-1.4.3 allow sample to cool to room temperature in a closed co ntainer. Weigh 2.00±0.01g of sample an d 
trans fer to a 125 mL Erlenmeyer flask. Add 20 mL of toluene to the sample in the flask stopper. Without                              TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 delay, begin shaking the mixture in the mechanical shaker for 60 seconds . Immediately pour as much of 
the mixtur e as possible into the glass funnel wi th filter paper which has previously been prepared and 
inserted into a 125 mL Erlenmeyer flask . Check standardization of spectrophotometer at 425 nm. Using 
the same cell as used to standardize before testing, rinse the  cell twice with the same filtrate to be test ed. 
Fill the absorption cell with the filtrate and determine the percentage. Transmission on the 
spectrophotometer at 425 nm. If necessary, large quantities of sample and toluene may be used, keeping 
the ratio 1 0 mL of toluene per gram of carbon bla ck unch anged, to a maximum of 5g/50mL of toluene     
D-2 DETERMINATION OF THE TOLUENE EXTRACTABLE FRACTION OF CARBON BLACK  
D-2.1 Principle  
Carbon black is extracted in a Soxhlet apparatus with toluene. Therefore, the solvent is evaporated and 
the residue weighed   
D-2.2 Apparatus  
D-2.2.1 Soxhlet extraction apparatus  
D-2.2.2 extraction vessel of paper fiber, capable of containing 10 of carbon black  
D-2.2.3 laboratory glass ware  
D-2.2.4 oven, adjustable up to 140±5ºC  
D-2.3 solvent  
D-2.3.1 toluene  
D-2.4 procedu re  
Dry a sufficient large sample of Carbon black for one hour at 105±2ºC  place 10 g of dried carbon black 
(weighed with an accuracy of 0.1g) in an extraction vessel previously washed with toluene. Close the 
vessel with cotton previously washed with  toluen e and place the vessel in the Soxhlet apparatus.  
Extract the carbon black for 8h with 150 mL toluene, adjust the heating element of the apparatus in such 
a way that the extraction solvent in the extraction vesse l is replenished approximately 10 time s per h our. 
Allow to cool, remove the extraction vessel from the apparatus and concentrate the extraction to a small 
volume by evaporation. Wash the residue with a little toluene into a weighed glass crucible (weight: a,g) 
and remove the toluene by evapora tion on  a water bath, place the crucible in an oven for two hours at 
140ºC; allow to cool in a desiccator and reweigh the crucible (weight: b,g)  
D-2.5 calculation  
Calculate the extractable fraction E of the carbon bla ck using the following formula:  
                       E= 10 x (b -a) percent   
 
 
 
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©TBS_202 1 Annex E  
[Table 2, SI No. (ii)]  
Determination of 3,4 -Benzopyrene by GC/MS  
E-1 General  
This method is based on gas chromatography combined with mass spectroscopy for detectio n of 3,4 -
benzopropyne at ppb level. Pr ior to the use of this method, appro priate sample extraction techniques 
must be used. In this method, a 1 µL aliquot of the extract is injected into the instrument and 3,4 -
benzopyrene is detected by mass spectroscopy in  ionization mode M/Z -50-400 
E-2 Reagen t  
E-2.1 Dichloromethane  
E-2.2 polycyclic aromatic hydrocarbon (PAH) standard -mix containing naphthalene, acenaphthylene, 
acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, Benz(a)anthracene, chrys ene, 
Benzo(b)fluoranthene, Benzo(k)flu oranthe ne, Benzo(a)pyrene, Benzo(gh)                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  
perylrylene, Dibenz (a) hjan thracen e and indeno (1,2,3,4 -cd) pyrene.  
E-2.3 Internal Standards – Mix containing four isotopically labelled PAHs, namely, D12 -chrysene, D10 -
phenanthrene, D10 -acenaphthrene and D10 -prylene  
E-3 apparatus  
E-3.1 GC/MS , equipped with auto -sampler and a 30m , 0.25I D BD -5 MS fused silica capillary column  
E-3.2 Rotary Evaporator  
E-3.3 helium gas  
E-4 Extraction  
Liquid -liquid extraction -before analysis , 0.5 µg of four internal standards: D10 -phenanthrene, D10 -
acenaphthren e, D12 -chrysene and D10 -prylene are ad ded in the sample and volume is reduced to 1mL.  
E-5 Analysis  
The analysis is done by GC/MS apparatus where helium gas is used as carrier gas. The column head 
pressure is maintained at 10 psi to give flow rate approxim ately 1 mL/min. injector and transfer line ar e 
maintained at 290  ºC and 250  ºC, respectively . All injection volumes are 1 µL in the split less mode. The 
column temperature is initially held at 70 ºC for 4 min, ranged to 300 ºC for 10 min. MS is used in the  
ionization mode M/Z -50-400  
E-6 Prepa ration of Calibration Standards  
Five standard solutions  are prepared by diluting the standard mixtures with dichloromethane to desired 
concentrations. To these, add 0.5µg of four internal standards, namely, D10 -acenap hthene, D10 -
phenanthrene, D12 -chrysene , and D 12-perylene. These are transferred to a capped and sealed vial.  
E-7 Identification and quantification                               TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 Benzo(a) pyrene in the sample is identified by a combination of retention time match and MS match 
against the  calibration standards quantification perform ed by method of internal standardization using 
D12-perylene.  
E-8 Results  
Calibration curve of benzo(a) pyrene is obtained using a series of standards solutions.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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©TBS_202 1 ANNEX F 
TEST FOR POLYC HLOROBIPHENYLES (PCB ) 
F-1 Principle  
The proce dure comprises separation of PCB from the organochlorine pesticides residues by means of 
silica gel adsorption column and determination of its presence by gas -liquid chromatography (GLC).  
F-2 Apparatus  
M-2.1 Gl ass Chromatographic Column, 300 mm lon g, 8 mm  1D with a ground -glass socket at the upper 
end and a stopcock at the lower end.  
F-2.2 Gas Chromatograph  
Equipped with electron capture detector and coupled with printer -plotter -cum-integrator.  
a) Column 1 -1.8m lo ng, 3 mm 1D; Apiezon L grease + 0.15 p er cent  Epikote Resin 1001 on 
chromosorb G (acid -washed, DMCS treated, 60 -80 mesh);  
b) Column 2 - 1.8 m long, 3 mm 1 D; 1.3 per cent silicone gum GE - SE-52+0.15 per cent Epikote 
Resin 1 001, on the same support material ;  
Temperature: Column oven 200°C  
Dete ctor 20 00c  
Injection port 200°C  
F-2.3 Electron Capture Detector  
F-2.4 Kuderna -Danisb Type, Evaporator, with interchangeable 10 ml graduated collection tubes. M -2.5 
Snyder Columns, two -bubble micro -columns, with groun d glass cones to fit the Kudema -Danish  type, 10 
ml collection tubes.  
F-2.6 Syringe - µL capacity.  
F-3 Reagents  
F-3.1 Silica Gel, 60 -100 mesh.  
Heat the gel at 260°C for 4 h and, when it is cooled to 65°C, place it in a desiccator. When it has cooled 
finally to room temperature, weigh the req uired a mount into a glass -stoppered flask and quickly add 2.5 
ml distilled water to each 100g. Immediately stopper the flask and shake it for 1.5 h on a shaker. The 
silica gel is ready for use.                               TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 F-3.2 n-hexane, redistil led from potassium hydroxide pellets. When co ncentrated 100 -fold, a 5µL portion 
should give no significant GCL peaks.  
 F-3.3 Sodium Hydroxide Solution - 5N. 
 F-3.4 Diethyl Ether, chromatography grade.  
F-3.5 Cotton Wool, extracted with hexane and diethyl e ther  
F-3.6 Acetic Acid, glacial, redi stilled.  
F-3.7 Chromium Trioxide, re -crystallized.  
F-4 Separation of PCB from  the more Polar Pesticide Residues   
Weigh 5.0 g of the prepared gel and rapidly transfer it, by using small amounts of hexane, to the 
chrom atographic column, in which a plug of cotton wool has been placed just above the stopcock. The 
stopcock may be moistened with solvent but must not be lubricated with grease. Allow the gel to settle in 
the column and remove and trapped air bubbles by stirri ng with a glass rod. Drain the surplus  hexane  
from the column until its meniscus just touches the surface of the gel. Introduce the cleaned -up sample 
extract as a solution in 1 ml of hexane, and allow the hexane to drain until the meniscus again just 
touch es the surface of the gel. Wash the ve ssel th at contained the extract with two I -ml portions of 
hexane, adding each washing separately to the column' and allowing it to run just into the gel. Place a 
receiver, preferably a Kudema Danish evaporator, beneath  the column and pass 42 ml of hexane t hrough 
the column at a rate not exceeding 0.7 mL/min. Collect the eluate, stopping the elution when the 
meniscus reaches the top of the gel, and label this fraction I. It should contain all the PCB. 
Hexachlorobenzene, aldrin, pp -DDT and pp' -DDE, if present , are a lso eluted in this fraction. Change the 
receiver and pass 50 ml of a 10 percent solution of diethyl ether in hexane through the column. Collect 
the eluate and label it fractions 2. This contains the remainder of  the organochlorine pesticide residues , 
including usually a small amount of pp' -DOE, and can be used for the determination of these. 
Concentrate fraction I to 5 ml in the Kudema -Danish evaporator and examine it by GLC with electron 
capture detection, on at  least two stationary phases of differ ent pol arities. If further concentration is 
necessary, reduce the volume of the solution with a gentle stream of dry air or nitrogen at room 
temperature. Compare the chromatograms from fraction 1 with those given by so lutions of commercial 
PCB preparations  when i njected on the same columns as well as the correspondence between peak 
retention times which give indication of the presence of PCB in the sample.  
F-5 Oxidation of pp'-DDE in the PCB fraction  
F-5.1 Adjust the volume of fraction I to 2 ml, concentr ating, if necessary, as described above. Add 2 ml of 
acetic acid and, after replacing the micro -Snyder  column, heat the tube cautiously in the steam bath until 
all of  the hexane has evaporated, as judged by the reducti on in volume. Introduce 100mg of chrom ium 
trioxide and place the tube in boiling water for 15 -20 min. Cool the mixture and shake it vigorously with 
2.0 m L of hexane (accurately measured) in the stoppered tube. Neutralize the acid with 6 -7 mL of 5 N 
sodium hydroxide, solution. Shake the tube ag ain and  then set it aside until the two layers separate. Keep 
the tube well stoppered to prevent loss of hexane.                               TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 F-6 Detection of PCB  
Inject 5 J.11 of the upper hexane layer of the oxidized mixture on to at least two  GLC columns with 
different liquid pha ses, on e of which should be Apiezon L. Compare the retention times of the peaks so 
obtained with those given by the PCB reference material. Agreement between the retention times of the 
peaks so obtained now indicates t he presence of PCB compounds in the sa mple wi th a greater degree of 
certainty. Any pp' -DDE, which was present in fraction 1, will have been converted into 4, 4' - dichloro -
benzophenone. Under the GLC conditions quoted, this compound has a retention time sim ilar to that of 
heptachlor epoxide and  so giv es a peak on the chromatogram before most of the PCB isomers 
encountered in practice. A comparison of the traces before and after oxidation of fraction 1will show how 
much pp' -DOE was originally present. Adjust the volume of the hexane solution so t hat the  individual 
heights ofthe PCB peaks are within the linear response range of the electron capture detector. Under the 
prescribed GLC conditions for the Apiezon L. column, the last of the PCB isomers normally foun d in 
wildlife tissues emerges in about  120 mi n after injection.  
 
  
  
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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©TBS_202 1 Annex G  
Determination of Metallic Impurities  
G-0. Both instrumental and chemical methods have been given for determination of metallic impurities. 
For certain metallic impurities, namely; antimony, barium, cadmium , zinc and mercury only instrumental 
methods have been given. For lead, arsenic, copper and chromium both instrumental and chemical 
methods have been given. In cases where more than one method has been given, an y of these may be 
used. However, in ca se of d ispute instrumental method shall be used as referee method.  
G-1 Instrumental Methods  
The instrumental methods described in this section may be employed in the quantitative analysis of 
certain metallic impuriti es in food additives.  
Method I is app licable  to substances soluble in dilute acids or mixtures of acids. Method II is used for 
other substances.  
G-1.1 Principle  
The samples are dissolved in acid or digested in a mixture of sulphuric, nitric and in some c ases 
perchloric acids. The barium, cad mium, l ead, copper, chromium, and zinc in solution are determined by 
conventional name atomic absorption spectroscopy. Antimony and arsenic are determined by using a 
hydride generation technique. Alternatively, antimon y may be determined by flame atomic ab sorptio n but 
the hydride generation technique is more sensitive.  
G-1.2 General Precautions  
Because of the minute amounts of metals involved, special care shall be taken to reduce the reagent 
blanks to as Iowa value as  possible and to avoid contamination d uring t he test. All apparatus should be 
thoroughly cleaned with a mixture of hot dilute acids (I part hydrochloric acid, 1 part concentrated nitric 
acid, and 3 parts water) followed by thorough washing with water immed iately before use.  
G-1.3 Apparatus  
G-1.3.1 K jeldahl flasks - of silica or borosilicate glass fitted with an extension to the neck by means of a 
B24 ground joint, as shown in Fig. 4, The extension serves to condense the fumes and carries a tap 
funnel throu gh which the reagents are introduced.  
                              TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 Fig. Modified Kjeldah Flask (Open Type)   
G-1.3.2 Atomic absorption spectrophotometer.  
Any instrument operating in the absorption mode may be used providing it has facilities for the selection 
of the required oxidan t/fuel combination from a choice of ai r, argo n, nitrous oxide, hydrogen and 
acetylene and has a wavelength range from 180 to 600 nm.  
 A hydride generation vessel accessory is also required and is available from all the major commercial 
manufacturers of ato mic absorption equipment. For operatio ns in e mission mode and measurements of 
absorption involving the generation of a gaseous hydride, a potentiometric recorder is necessary, 
preferably a multi -range type covering the range 1 -20 mV.  
G-1.4. Reagents  
13.1.4.0 Reagents shall be of an order of purity higher than accepted analytical reagent grade quality. 
Metal free water (see below) shall be used throughout:  
 a) Nitric acid - sp gr 1.42.  
b) Perchloric acid - 60% (mlm) solution.  
c) Sulphuric acid - 9R%.  
d) Hydrochloric acid - sp gr 1.16 to 1.18 .  
e) Hydrochloric acid - 5 N solution prepared by dilution of reagent (d) with metal -free distilled water.  
f) Water - metal -free. Distilled water may he re -distilled from an all -glass apparatus or may be passed 
down a  column or cation exchange resin, for example , Amberlite IR 120 (H).  
g) Sodium sulphate  
h) Sodium borohydride pellets  
 i) Potassium chloride  
G-1.4.1 Standards  
Use commercially available standard solutions or prepare solutions as follows:  
a)  Standard c opper solution  
Dissolve 3.928g of pur e coppe r sulphate CuSO 4.5H 2O in water. dilute to 1 1000 mL at 20°C with water in 
a one -mark graduated flask. Dilute 10 mL to 100 mL with water in a one -mark graduated flask as 
required. I ml contains 100 ug Cu.  
b) Sta ndard zinc solution                               TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 Dissolve 1.00g of pure zi nc powder in a mixture of 10 mL water and 5 mL hydrochloric acid [special 
reagent (d) (and dilute to 1 000 ml at 20°C with waler, in a one -mark graduated flask. Dilute 10 mL to 10 
100 mL with water in a one mark  graduated flask as required. 1 mL con tains 1 00 µg Zn.  
c) Standard chromium solution  
Dilute 5.80 mL or 0.1 N potassium dichromate solution to 100 mL at 20 °C with water in a one -mark 
graduated flask as required. 1 mL contains 100µg Cr.  
d) Standard antimon y solution  
Dissolve 2.668 g potassium antimon y tartrate K(SbO)C 4H4O6 in distilled water, dilute to 1 000 ml at 20°C 
with water in a one -mark graduated flask. Dilute 10.0 ml to 100 mL with distilled water in a one -mark 
graduated flask as required. I ml cont ains 100 µg Sb.  
e) Standard lead solu tion 
Dissolve 1.60 g of lead nitrate, Pb (NO 3)2 in nitric acid (10 mL of concentrated nitric acid diluted with 20 
mL water, boiled to remove nitrous fumes, and cooled) and dilute to I 000 ml with water in a one -mark 
graduated flask. Dilute 10.0 mL of this solutio n to 500 mL at 20°C with water in a one -mark graduated 
flask as required. I ml contains 20 ug Pb.  
f) Standard barium solution  
Dissolve 1.779 g barium chloride BaCI2.2H2O in distilled water, dilute to 1000 mL at  20°C with water in a 
one-mark graduat ed flas k. Dilute 10.0 mL to 100  mL with water in a one -mark graduated flask as 
required. 1 ml contain. 100 µg Ba,  
g) Standard arsenic solution  
Dissolve 1.320g of arsenous oxide, As 2O3 by warming at a temperature not e xceeding 60°C with 14mL of 
5N sodium h ydroxid e solution in a 100mL beaker. Cool, add 0.2mL of phenolphthalein indicator and 
neutralize with 6N sulphuric acid. Transfer the solution to a 1000mL one -mark graduated flask containing 
10 g of sodium hydrogen car bonate dissolved in water, washing out  the be aker with water. Dilute to the 
mark with water at 20°C and mix. Dilute 5 mL of this solution to 1 000mL at 20°C with water in a one -mark 
graduated flask as required. 1mL contains 5µg As.  
h) Standard cadmium sol ution  
Dissolve 2.282g CdSO4.8H 2O in di stilled water, dilute to 1000mL at 20°C with water in a one -mark 
graduated flask. Dilute 10.0 mL of this solution to 500 mL at 20°C with water in a one -mark graduated 
flask 1 contains 20µg Cd.  
G-1.5 Preparation of Tes t Solutions  
G-1.3.0 Prepare the test solutio n according to Method I in the case of substances soluble in dilute acids. 
Use Method II for other substances.  
G-1.5.1 Method I                               TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 Accurately weigh about 2.5 g of the sample and dissolve in a mixture of 4 ml of s ulphuric acid and 5 ml of 
hydrochloric  acid. Transfer the solution 10 a 50mL one -mark graduated flask. If barium is 10, he 
measured from the solution, add 0.095 4 g of potassium chloride. Dilute to the mark with water. Call this 
'Solution A'.  
G-1.5.2 Meth od II 
Accurately weigh about 2.5g of t he samp le into a 100 -150 m L Kjeldahl flask, and add 5m L of dilute nitric 
acid. As soon as any initial reaction subside heat gently until further vigorous reaction ceases and then 
cool. Add gradually 4m L of concentrated  sulphuric acid at such a rate as not to caus e excessive frothing 
on heating (5 -11 min ar e usually required) and then heat until the liquid darkens appreciably in colour, 
that is, begins to char.  
Add concentrated nitric acid slowly in small portions, heat ing between additions until darkening again 
takes place. Do not heat so strongly that charring is excessive or loss of arsenic may occur; small but not 
excessive amount of free nitric acid should he present throughout. Continue this treatment until the 
solution is only pale yellow in colour an d fails to darken in colour on prolonged heating. If the solution is 
still coloured run in 0.5mL of the perchloric acid solution and a little concentrated nitric acid and heat for 
about 15 minutes, then add a further 0 .5mL of the perchloric acid solution a nd heat  for a few minutes 
longer. Note the total amount of concentrated nitric acid used. Allow to cool somewhat and dilute with 
10mL of water. The solution should be quite colourless (if much iron is present it may be  faintly yellow). 
Boil down gently, ta king ca re to avoid bumping, until while fumes appear. Allow to cool, add a further 5mL 
of water and again boil down gently to fuming. Finally, cool, add 10mL of 5N hydrochloric acid and boil 
gently for a few minutes. C ool and transfer the solution to a 50 ml one -mark graduated flask washing out 
the Kjeldahl flask with small portions of water. Add the washings to the graduated flask and dilute to the 
mark with water. If barium is to be measured from the solution, add bef ore dilution n.095 4g of potassium 
chloride, as an ionizing buffer to prevent ionization of barium. Call this 'Solution A'.  
Prepare a reagent blank using the same quantities of reagents as used in the sample oxidation.  
G-1.6 Measurement of Antimony, Bariu m, Cadmium, Chromium, Copper, Lead and  Zinc b y Atomic 
Absorption   
15.1.6.1 Preparation of calibration curve solutions  
To a series of 100mL one -mark volumetric flasks, pipette 0, 1, 2, 3, 4 and 5mL of the appropriate 
standard solution [standards (a) to (f)  and (h) see G -1.4.11 and dilute 10 ab out 50m L. Add 8m L 
concentrated sulphuric acid [reagent (c)] and 10 mL concentrated hydrochloric acid [reagent (d)]. Shake 
to dissolve. In the case of barium [standard (f)], add 0.191g of potassium chloride as an ioniza tion buffer. 
When solution is complete , dilute to the mark with metal free water.  
These solutions then contain 1.0, 2.0, 3.0, 4.0 and 5.0µg per m L of either barium, copper, zinc, chromium 
or antimony respectively, or 0, 0.2, 0.4, 0.6, 0.8 and 1.0 µg per m L of cadmium or lead.  
 
 
                              TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 G-1.6.2 Inst rumenta l conditions  
Select the wavelength and gases to be used for the particular clement under consideration from the table 
below:  
Element  Wavelength (nm)  Gases  
Antimony  217.6  Air/acetylene  
Barium  553.6  Nitrous o xide/acetylene  
Cadmium  228.8  Air/ace tylene  
Chromium  357.9  Nitrous oxide/acetylene  
Copper  324.8  Air/acetylene  
Lead  283.3  Air/acetylene  
Zinc 213.9  Air/acetylene  
The recommended settings for the various instrumental parameters vary from model to mod el, and 
certain parameters require opt imizati on at the time of use to obtain the best results. Instruments should 
therefore be adjusted as described in the manufacturer's instructions using the type of name and 
wavelength settings specified above.  
15.1.6. 3 Procedure  
Set the atomic' absorptio n spect rophotometer to the appropriate conditions. Aspirate the strongest 
standard containing the clement to be determined and optimize the instrument settings to give full -scale 
or maximum deflection on the "hart reco rder. Measure the absorbances of the o ther st andards and plot a 
graph showing the net absorbance against the concentration of the element in the standard solutions. 
Aspirate the solution A obtained from dissolution or the wet oxidation of the sample and th e 
corresponding blank solution and det ermine the net absorbance. Using the graph prepared above, 
determine the concentration of the clement in the sample solution;   
  
𝐸𝑙𝑒𝑚𝑒𝑛𝑡 𝑖𝑛 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒,𝑚𝑔
𝐾𝑔=𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑒𝑙𝑒𝑚𝑒𝑛𝑡  𝑔
𝑚𝐿  𝑥 50
𝑀𝑎𝑠𝑠 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑡𝑎𝑘𝑒𝑛  𝑔   
 G-1.7 Measurement of Arsenic and Antimony by Atomic Absorption Hydride Technique  
 G-1.7.0 Arsenic and antimony are determine d after preparation of their volatile hydrides which are 
collected either in the generation vessel itself or, in some designs, in a rubber balloon attached to the 
vessel. The gases are then expelled with argon into a h ydrogen flame.  
G-1.7.1 Preparation of  calibr ation curve solution  
Into a series of 100 -mL one -mark volumetric flasks add from a burette, 0, 1,2,3,4 and 5 m L of standard 
arsenic or antimony solution [Standards (g) and (d)] and dilute to about 50 mL with di stilled water. Add 8 
ml concentrated s ulphuri c add [reagent (c)] and 10 ml hydrochloric acid [reagent (d)], Shake to dissolve, 
and when solution is complete, dilute to the mark with distilled water.  
G-1.7.2 Instrumental conditions  
Using the atomic absorp tion spectrophotometer with the approp riate h ollow cathode or electrode less 
discharge lamp, select the wavelength for either arsenic (1937 nm) or antimony (2176 nm)                               TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 G-1.7.3 Procedure  
Measure 5.0 ml of the strongest standard into the generation vessel, a dd 25mL water and 2 mL 5N 
hydrochloric  acid [ reagent (e)]. Stopper the vessel and expel any air as described in the makers 
instructions, filling the apparatus with argon. Isolate the vessel from the atomizer using the by -pass valve. 
Remove the atomizer and  then quickly add 1 pellet of sodium b orohydr ide weighing approximately 0.2 g 
and replace the stopper. Ensure that all the joints are secure.  
When the reaction slows (20 -30 sec) open the appropriate taps to allow argon to drive the generated 
hydride into t he flame. When the hydride has all bee n expel led as shown by the recorder trace, return the 
taps to their original position and empty the vessel.  
Optimize the instrument setting to give full scale deflection for the strongest standard. Measure the other 
standards, the sample and the blank sol ution u sing the same procedure.  
Plot a graph relating peak height on the recorder to concentration of the arsenic or antimony in the 
standards using the net absorbance of the sample, read from the graph the concentrat ion of arsenic or 
antimony in the solu tion. 
15.1.7.4 Calculation  
𝐴𝑟𝑠𝑒𝑛𝑖𝑐 𝑜𝑟 𝐴𝑛𝑡𝑖𝑚𝑜𝑛𝑦 𝑖𝑛 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒,𝑚𝑔
𝐾𝑔=𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑎𝑟𝑠𝑒𝑛𝑖𝑐 𝑜𝑟 𝑎𝑛𝑡𝑖𝑚𝑜𝑛𝑦  𝑔
𝑚𝐿  𝑥 50
𝑀𝑎𝑠𝑠 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑡𝑎𝑘𝑒𝑛  𝑔  
 
G-1.8 Determination of Mercury by atomic Absorption Cold vapour Technique  
G-1.8.0 Principle  
The sample is ashed by heating under reflux with sulphuric and nitric acids. The oxidation is completed by 
addition of potassium per mangana te solution. After succession additions of hydroxylamine hydrochloride 
solution stannous chloride solution, the mercury chloride is measured by cold vapour atomic absorption 
spectrometry.  
G-1.8.1 Special reagent s  
a) Nitric acid – sp gr 1.40  
b) Sulphuric acid -sp gr 1.84  
c) Sulphuric acid – approximately 3.5M. Prepare by diluting 1 volume of concentrated sulphuric acid 
(b), with 4 volume, of water  
d) Suphuric acid – approximately 1M. prepare by diluting 1 volume of 3.5M sul phuric acid (c) with 25 
volumes of wat er  
e) Hydrochloric acid - sp gr 1.18;  
f)  Potassium permanganate solution - 50.0 g/L                               TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 g) Hydroxylamine hydrochloride solution -10.0g/L  
h) Stannous chloride solution - Prepare by dissolving 250g of' stannous chloride (SnCl 2.2H 2O), in 
50mL hydrochloric acid (e). Make u p to 25 0mL with water and bubble nitrogen through the 
solution. Store over a few granules of metallic tin.    
i) Chromic acid mixture - Dissolve 4.0g of potassium dichromate in 300mL of 3.5M sulphuric acid 
(c) and make up  to 1 litre with water  
j) Magnesium perch lorate - in granular form for gas desiccation; and  
k) Mercury chloride.  
G-1.8.2 Standards  
Use commercially available standard solutions, or prepare the standards as follows:  
a) Mercuric chloride solution 0.5mg Hg/m L. Weigh out to the nearest 0.1mg. 0.677 g  of mer cury 
chloride. Dissolve in approximately 250mL 3.5M sulphuric acid in a 1L volumetric flask, add 
approximately 700mL water and then potassium permanganate solution dropwise until a colouration 
persists. Make up to the mark with water and mix well. R enew th is solution every three months.  
b) Mercury chloride solution - 0.02 µg Hg/mL. Dilute the standard mercuric chloride solution 0.5 mg 
Hg/ml (standard (A)] by a factor of 25 000 by successive dilution with sulphur ic acid [special reagent 
(d)], for exa mple. 1 0mL made up to 250mL twice followed by 10mL made up to 400mL. Before 
bringing up to the mark in the final dilution, add potassium permanganate solution [special reagent (f)] 
dropwise until a colouration persists . Renew this solution daily.  
G-1.8.3 Apparat us   
All the glassware must be cleaned with hot nitric acid [special reagent (a)] and washed thoroughly 
with water before use.  
a) Mineralization apparatus – fitted with reflux condenser (see fig. 5)  
b) Bubblers - with a ground glass stopper fitted with t wo tube s to permit entrainment of the mercury 
vapour and with a calibration mark at the required volume for measurement. The capacity of the 
bubbler and position of the mark depend on the atomic absorption spectrophoto meter used  
                              TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 Fig:  Mineralization Appa ratus   
Clean the bubbler successively with chromic acid mixture [special reagent (j)], tap water and double 
distilled water before use  
(c) Water vapour absorption apparatus -containing magnesium perchlorate [special r eagent (k)]  
d) Atomic absorption spec trophot ometer - suitable for the cold vapour determination of mercury in open or 
closed circuit, with recorder  
G-1.8.4 Procedure  
Ashing - Weigh out, to the nearest 2µg, approximately 0.5g sample containing not more t han 0.5µg total 
mercury. Introduce the  sample  into the receiver flask (M), and add a few glass beads. Connect the 
receiver flask to the condensate reservoir (D) and close the stopcock (R)  
Introduce into the reservoir 25mL of nitric acid [special reagent ( a)] followed by 10mL sulphuric acid an d 
[spec ial regent (b)]. Mount the turn on the condenser (A) open the stopcock carefully and allow small 
portions of the mixture of acids to run into the receiver flask. Interrupt the flow of acids If the reaction 
becom es too vigorous.  
Empty the reservoir i nto the  receiver flask, mix the contents of the latter well by careful shaking and leave 
the stopcock open.  
Heat the receiver flask carefully as soon as foaming has ceased, close the stopcock (R), continue heating 
and let the condensate collect in the rese rvoir. 
Discontinue heating when the contents of the receiver flask begin to char Allow a small portion of the 
condensate to run to the receiver flask, close the stopcock again and resume healing the receiver flask 
Repe at this procedure for as long as the c ontents  display charring when healing.  
When charring has ceased, heat and add condensate as soon as white fumes appear. Continue 
alternately heating and adding condensate for one hour. Finally heat the contents of the flask to white 
fumes.  
Stop heating an d allow  to cool to appropriately 40°C Open the stopcock and allow all the condensate to 
run into the receiver flask. Wash the apparatus out from the top of the condenser with 5 -10 mL of water, 
collect the washings in t he receiver flask and disconnect it fr om the reservoir.  
Treatment of the solution - Introduce the potassium permanganate solution [special reagent (f)] dropwise 
into the receiver flask, with agitation, until a pink colouration persists. Note the quantity o f reagent (f) used 
(If this quantity e xceeds 10 ml, repeat the procedure 'Ashing' as above). Heat gently to boiling, then allow 
to cool  
Pour the contents of the receiver flask into a bubbler, wash the receiver flask with water and add the 
washings to the contents of the bubbler.  
Measure the m ercury content (see below) the same day as the treatment of the solution.                               TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 Measurement of mercury content – Introduce 5mL of hydroxylamine hydrochloride [special reagent (g)] 
into the bubbler and make up the mark either  with double distilled water or with s ulphuri c acid [special 
reagent (d)] in the case of standard solutions. Add 5 mL of stannous chloride solution [special reagent 
(g)], assemble the bubbler, connect it to the water vapour absorption apparatus and to the atomic 
absorption spectrophotometer. S et the latter in operation.  
MIX the contents of the bubbler well by gently shaking, pass air or nitrogen through, measure and record. 
Carry out measurements as quickly as possible after the addition of stannous chlorid e If an open -circuit 
system is used, w ait 30 sec before passing air or nitrogen.  
Calibration curve -Introduce respectively 2, 5, 10, 15 and 25mL aliquots of the standard mercury solution 
[standard (b)] into bubblers and 25mL sulphuric acid  [special reagent  (d)] into a sixth bubbler Add 
potassi um perm anganate solution [special reagent (f)] dropwise, with agitation to each bubbler until a 
colouration persists.  
Plot the calibration curve with the measured absorption values as ordinates and the corresponding 
mercury contents in microprograms as abs cissae.  The working standards contain 0, 0.04, 0.10, 0.20, 0.30 
and 0 .50µg of mercury, respectively.  
Method of addition – the method of addition may be used if an open circuit system is used  
Place one of the working st andard solutions in a bubble and add a n aliqu ot portion of the sample solution 
obtained after treatment. The quantity of mercury in the bubbler must lie in the range in which the 
photometer gives a linear response. Measure the mercury content as described above. If necessary, 
carry out several  such d eterminations, using different working standard solutions.  
Blank determination - Carry out all the operations, from ashing to measurement, except for introduction of 
the sample. When treating the solution, add a quantity of potassium permanganate s olution  [special 
reagent (f)] equal to that used for the experimental sample.  
G-1.8.5 Calculation  
Read off from the calibration curve the quantities, in µg, of mercury corresponding to the measured 
absorption values.   
Subtract the quantity of mercury fou nd in t he blank from that found in the sample:  
 
𝐻𝑔 𝑖𝑛 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒,𝑚𝑔
𝐾𝑔=𝑁𝑒𝑡 𝑚𝑎𝑠𝑠 𝑜𝑓 𝑚𝑒𝑟𝑐𝑢𝑟𝑦  µ𝑔
𝑚𝐿 
𝑀𝑎𝑠𝑠 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑡𝑎𝑘𝑒𝑛  𝑔  
 
G-2 Chemical Methods  
G-2.0 Chemical methods have been given for determination of lead (Pb), arsenic (As), Copper (Cu) and 
chromium (Cr).  
15,2.1 Test for Lead                               TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 15.2.1 .1 Apparatus  
a) Digestion Funnel  
b) Separat ory Funnel  
15.2.1.2 Reagents  
a) Nitric acid - 65 percent.  
b) Sulphuric acid - sp gr 1.84.  
e) Ammonium acetate -citrate solution - Dissolve 12.5g of ammonium acetate and 12.5g of ammonium 
citrate in water, add concentrated ammonia until the solutio n is alkaline to thymol blue paper and add 
water to 100mL. Purify with 0.002 percent m/v solution of dithizone in carbon tetrachloride, and finally 
shake the solution with carbon tetrachloride to remove excess of dithi zone.  
d) Ammonia solution - 25 percen t.  
e) Carbon tetrachloride  
f) Ammonium hydroxide - 0.2 N.  
g) Potassium cyanide - 10 percent.  
h) Hydroxylamine hydrochloride solution - 10 percent.  
j) Dithizone solution - 0.1 percent (m/v), purified by the followi ng procedure:  
Dissolve the dithizone in chlo roform and treat it with ammonia. Add mineral acid. Precipitate shall be pure 
dithizone. The aqueous ammoniacal solution obtained from chloroform solution of dithizone should be 
colourless; otherwise, further pu rification as mentioned above should b e carried out.  
k) Buffer pH2 - Add 11.90 mL of 0.2 M hydrochloric acid and 88.10 m L of 0.2M potassium chloride in a 
200-mL volumetric flask and add water to volume.  
G-2.1.3 Procedure  
The limit test described for lead  is designed to show if a sample conta ins mor e than 10 mg/kg or 20 mg/ 
kg of lead. The sample is digested with nitric and sulphuric acids, and a clear solution of the digest is 
prepared.  
Digestion - Weigh 1.0 g of sample for substances with 10 mg/kg limit  or 0.5 g for those with 20 mg/ kg 
limit. Pla ce with 5mL of water, 5mL of 65 percent nitric acid and 5 mL of sulphuric acid in a digestion 
flask. Warm slightly. If foaming becomes excessive, add a little water. Evaporate the mixture. Maintain 
strongly oxid izing conditions in the flask during d igestio n by adding cautiously small quantities of nitric 
acid whenever the mixture begins to turn brown or dark. Continue digestion until organic matter is 
destroyed and sulphuric trioxide fumes are copiously evolved. The final solution should be colourles s or 
at most slight straw colour.                               TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 To remove nitrosyl -sulphuric acid, after partial cooling transfer the residue to a dish, rime with 25mL of 
water, evaporate and heat again to fuming point. Add 25mL of water and evapor ate and heat again. 
Solution of digest  - Allow to cool, add 20mL of ammonium acetate -citrate solution, and allow again to 
cool, neutralize to about pH 7 with 25 percent ammonia and boil if necessary, to dissolve calcium 
sulphate. Cool the clear solution. S hake the solution with 10mL of carbon tetrach loride and discard the 
lower layer.  
Test with dithizone - To a 100m L separatory funnel, add 10mL of 0.2N ammonium hydroxide, 2mL of 10 
percent potassium cyanide, 2mL of 10 percent hydroxylamine hydrochloride so lution and 2 mL of 20 mg 
per litre sol ution o f dithizone in carbon tetrachloride. Shake the separatory funnel for a few minutes and 
discard the carbon tetrachloride layer. Add 2mL of carbon tetrachloride, shake and discard again the 
carbon tetrachloride la yer. Add the solution of digest to the  separa tory funnel. Again,  add 5mL of 
dithizone solution in carbon tetrachloride and shake vigorously for a few minutes. The carbon 
tetrachloride layer becomes red according to the amount of lead present.  
Treat simult aneously a standard solution containin g 10µg of lead in the same manner as the solution of 
the digest. Evaluate the quantity of lead in the digest by comparing the colour of the carbon tetrachloride 
layers.  
Ensure that the red colour is due to lead by shak ing the carbon tetrachloride layer obt ained f rom the 
digest solution with 10 mL of a buffer pH2. The red colour will turn green if it is due to lead.    
G-2.2 Test for Arsenic  
G-2.2.0 Arsenic may be tested by either the method given below for routine purp oses . 
G-2.2.1 Apparatus  
a) Distillation apparat us - as shown in Fig. 6  
 
 
Fig. 6  Fig. 7  
b)  Conical flask - 25 m L close. as shown in Fig. 7  
G-2.2.2 Reagents                               TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 a) Sulphuric acid - sp gr 1.84.  
b) Potassium permanganate solution - 0.1N.  
c) Ferrous sulphate - freshly powdered.  
d) Hydrochloric acid  - 38 percent.  
e) Potassium bromide solution - 20 percent.  
f) Aluminium strips - 8 mm x 8 mm x I mm.  
g) Tin chloride solution - 2 percent tin chloride in 10 percent hydrochloric acid,  
h) Test paper - Soak strips of f ilter paper in saturated ethanolic sol ution o f mercuric bromide and 
allow to dry.  
G-2.2.3 Procedure  
The limit test prescribed for arsenic shows whether a sample contains more than 3mg/kg of 
arsenic.  
Digestion of 1 g sample and removal of nitrosyl -sulphu ric acid as described in para 3 of G -2.1.3. 
Allow the digest. with 5 mL sulphuric acid. to cool.  
Distillation (according to Snyder) - Pour the digest into the distillation flask (Fig. 6). Rinse the dish 
twice using each time 2.5mL of water and add the wat er to the digest. Cool the solution. A dd 
some  drops of potassium permanganate solution until the red colour persists. Add 0.250g of 
freshly powdered ferrous sulphate, 2.5mL  of 38 percent hydrochloric acid and 0.1mL of 
potassium bromide solution.  
Close the flask, place a reagent tube containing  8 mL o f water in a beaker with water as shown in 
Fig. 6.  
Heat with micro -burner. In the beginning a slow stream of air bubbles appear, followed after 3 to 5 
minutes by hydrochloric acid gas. Heat then a little strong er so that the solution boils. After b ulb A 
has become very hot, boil for 40 seconds. There upon lower the reagent tube and remove the 
flame. The hydrochloric acid content of the distillate shall be 9 to 10 percent only then has arsenic 
been completely dis tilled over; moreover, this acid conce ntratio n is required for the test given 
below;  
Test with mercury bromide paper (Mayercon – Bergerel) - The distillate should be transferred to a 
conical flask of 25 ml capacity. The flask should be dosed with the devic e as shown in Fig. 7 
containing a smal l disk of test paper. Add 3 pieces of aluminium strips, 1 mL of tin chloride 
solution and Immediately close the flask with the stopper. Allow the flask to stand in a water -bath 
of 25° to 30°C for 50 for 60 minutes. At the same time carry out a parallel tes t using  a solution of 
2, 4, 6 or 8µg of arsenic in 10 mL of 10 percent hydrochloric acid in place of the test sample. 
Compare the colour of the test paper for evaluating arsenic content of the sample.                               TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 G-2.3 Determinati on of Copper  
G-2.3.1 Reagents  
a) Cit ric aci d - solid.  
b) Ammonium hydroxide solution - sp gr 0.92 (not less than 27 percent ammonia).  
c) Concentrated hydrochloric acid  
d) Dithizone (diphenyl thiocarbazone] solution - 0.1 percent (m/v) in chloroform.  
e) Concentrated nitric acid  
f) Concen trated sulphuric acid  
 g) Citric acid solution - 5 percent (m/v). aqueous.  
h) Gum arahic solution - one percent.  
 j) Sodium diethyl dithiocarbamate solution - 0.2 percent (mlv). aqueous, freshly prepared.  
k) Standard  strong soluiion of copper - Dissolve 0.392 5 g of pure crystallized copper sulphate 
(CuSO 4. 5H 2O) in water and make up the volume to 100 mL, this solution contains one milligram 
of copper per milliliter.  
m) Standard dilute solution of copper - Dilute one m illilitre of the standard strong solut ion of 
copper to 100 mL in a graduated flask, one millilitre of this solution contains 0.01 µg of copper.  
G-2.3.2 Procedure  
Take a suitable aliquot of the test solution prepared as described above and add 28 of citri c acid. 
Neutralize exactly with the am monium hydroxide solution using a piece of litmus paper and 
acidify with one millilitre of concentrated hydrochloric acid. Cool and transfer to a separating 
funnel. The total volume of the solution should be about 100m L. 
Extract the copper by shaking with three s uccessive portions of 5mL of the solution of dithizone, 
shaking thoroughly for a minute for each extraction. Separate the dithizone layers and wash the 
combined dithizone extracts with about 10 ml of water. Tran sfer the dithizone extract to a tube o f 
heat-resistant glass and evaporate the chloroform on a water -bath.  
Heat the copper -dithizone residue in the test tube with one millilitre or concentrated sulphuric acid 
and a little of nitric acid until all organic m atter is destroyed. Add 5mL of water a nd re-heat to 
fuming stage. Cool, dilute with water and transfer the whole of the solution or a measured volume 
of the solution, depending upon the amount of copper present, to a Nessler cylinder. Add one 
millilitre of  the citric acid solution and 4 mL of the amm onium hydroxide solution followed by 5 mL 
of the gum arabic solution and make up the volume to 50 mL with water. Add 5mL of the sodium 
diethyl dithiocarbarmate solution and match the colour by adding the standar d dilute solution of 
copper [reagent ( m)] to a control cylinder containing the same quantities of reagents as present in                              TBS/AFDC 2 (231) CD2 
                                                  ICS 67:250  
  
 
©TBS_202 1 the test solution. Calculate the copper content of the material in parts per million from the known 
volume of the standard dilute solut ion of copper required for matching.  
G-2.4 D etermination of Chromium  
G-2.4.1 Reagents  
a) Magnesium nitrate solution - 25 percent (m/v).  
b) Strong sulphuric acid solution - 4 N. 
c) Potassium permanganate solution - 0.1 N.  
d) Sodium azide solution - 5 per cent (mlv).  
e) Sodium dihydrophosphat e - 4 M. 
f) Diphenyl -carbazide solution - Dissolve 125mg of diphenyl -carbazide (C 6H5.NH. NH) 3CO] in a 
mixture of 25 mL of acetone and 25 mL of water. This should be prepared immediately before 
use. 
 g) Standard chromiu m solution - Dissolve 0.0566 g of pota ssium d ichromate (K 2Cr3O7) ll one litre 
of water. One millilitre of this solution contains 0.02 mg of chromium,  
h) Sucrose  
 G-2.4.2 Procedure  
Weigh 1.0g of the sample into a quartz dish. Cbar the material. raising th e temperature slowly. 
Allow to cool. a dd 10 m L of the magnesium nitrate solution and evaporate, heating slowly until no 
more nitrous vapour evolves. Heat the material in an oven at 600±2°C until all black particles 
have disappeared (30 to 60 minutes). Diss olve the residue by adding 10 ml of th e stron g 
sulphuric acid solution and 20 ml of water. Heat on a water -bath for about 5 minutes.  
Add 0.5 mL of the potassium permanganate solution, cover with a watch -glass and heat on a 
water -bath for about 20 minutes. Add more potassium permanganate, if th e solut ion decolourizes, 
add sodium azide solution, one drops every 10 seconds, until the excess potassium 
permanganate has been removed. (Avoid excess of sodium azide, 2 drops are usually sufficient.) 
Cool the solutio n in running water and filler, if mang anese d ioxide is evident. Transfer the solution 
to a 50 -mL graduated flask. Add 2.5 mL of sodium dihydrophosphate and 2m L of 
diphenylcarbazide solution and fill to the mark with water. Measure the extinction at 540 nm,  30 
minutes after adding the diphenylc arbazid e solution, A blank with the last two reagents (sodium 
dihydrophosphate and diphenyl -carbazide solutions) should show no colour or only a slight purple 
colour. At the same time, run a parallel test with 1.00 ml of the standard chromium solution and a 
few m illigrams of sucrose placed in a second quartz dish. Treat the mixture exactly as the sample 
and measure the extinction at the same wavelength.  
Calculate the chromium content of the sample from the two extincti on values observed.