INTRODUCTION OF AZO DYES

Azo dye stuff and pigment (azo colorant) as a group classified chemically
are among other thing used in dyeing and printing. The seventy percent of
dyestuff, which are available in market, are synthetic dyestuff. The
dyestuffs, which are made by synthesis of chemicals, are known as synthetic
dye.

The certain aromatic amines may be split off from azo colorant by reductive
cleavage process. The aromatic amines are not only splitting from azo dyes
but also from Reactive. Direct, disperse and acid dyes.

Azo dye stuff and azo pigment are referred as collectively as azo colorant.
Azo colorant is a group of chemical compound containing one or more azo
group(s). The group(s) contains a double bond between two Nitrogen atoms.

-------N=N-— (Azo Group)

Azo colorant is subdivided in azo dyestuff, which are soluble in the

application medium, and azo pigment, which are not soluble in the
application medium. Azo dye stuff can also be subdivided in water soluble
and fat-soluble i.e. lipophilic and Hydrophilic.

Azo dyestuff is used for both dyeing and printing where azo pigment is only
used for printing.

Introduction of Synthetic Dyes

The discovery by Sir William Perkin, an English chemist in 1936 that a

mauve coloring matter capable of dyeing silk could be prepared by the
oxidation of aniline started a vast chain of events. It resulted in production of
approximately 8,000 distinctly different dyestuffs being manufactured all
over the world and sold under 40,000 trade names.

i
Class of Synthetic Dyes

1. Basic dyes
2. Direct dyes
3. Vat dyes
4. Reactive dyes
5. Azoic dyes
6. Sulfur dyes
7. Mordant dyes
8. Acid.dyes
9. Disperse dye
10.Oxidation dyes
11. Mineral and pigment dyes

1. BASIC DYES

MAUVENE, the first to be discovered by Perkin, was a basic dye and most
of the dyes which followed, including magenta, malachite green and crystal
violet, were of the same type. Basic dyes dye wool and silk from a dye bath
containing acid but dye cotton fibres only in the presence of a mordant
usually a metallic salt that increases affinity of the fabric for the dye. Basic
dyes include the most brilliant of all the synthetic dyes known, but
unfortunately they have very poor light and wash fastness.

USE: Basic dyes will dye wool and silk from an acid bath and are
used where brightness is of prime consideration. With the introduction
of cotton dyes possessing higher fastness properties their use for
dyeing cotton has diminished. Basic dyes are used extensively for
dyeing cut flowers, dried flowers, also dyeing jute sisal, raffia, coir
and wood (toys). With the introduction of acrylic Fibre a new range of
'modified' basic dyes were perfected for dyeing of this material.

2. DIRECT DYES

These are soluble in water and have direct affinity for all cellulose fibres.
Some will also dye silk and wool. By continuous research this group of dyes

2
has been supplemented with dyes of good fastness to light and washing. As
these dyes, when'dyed without additives, do not exhaust well, an addition of
salt is required to improve the yield of the dye and obtain deeper shades.
Generally, the wash fastness of these dyes is inferior but there are a number
of after treatments available to improve the wash fastness of the dyeing.
Most direct dyes can be stripped of the use of stripping salts (Sodium
Hydrosulphite) without harmful effects on the fibres.

USE; Direct dyes dye all cellulosic fibres, including viscose rayon,
and most of them also dye wool and silk. They do not dye acetate
rayon and synthetic fibres. Direct dyes can be applied well at low
temperatures and are therefore suitable for tie-dyeing and batik work.
Generally these dyes are used where high wash fastness is not
required.
3. VAT DYES.

INDIGO, probably the oldest dye known to man, is one of the most
important members of this group. Natural indigo extracted from the plant
'Indigofera tinctorie' was used by the Egyptians in 200 BC. The first
synthetic indigo was introduced to the textile trade in 1897 and had the
effect of completely replacing the natural product. Although the vat dyes
may be divided into three chemical groups, they are similar in that they are
insoluble in water and become water soluble when reduced in the presence
of an alkali. After dyeing, the fabric is oxidized and the dye again becomes
water insoluble. Because of the time consuming and costly procedure in
reducing vat dye into a water-soluble complex, dye manufacturers have
produced a stabilized water-soluble vat dye. This dye can be applied to
cotton and viscose rayon by the methods used by applying direct cotton
dyes. After the dyeing, a simple treatment restores the vat dye to its normal
insoluble state. Solubilized vat dyes have an affinity for cellulose and animal
fibres.

USE: Vat dyes are used in cotton dyeing where high wash and boil
fastness required. Because of the high alkali concentration in the dye
bath, pure vat dyes cannot be used on animal fibres, (wool, natural
silk, and various hairs). Bright red is absent in vat dye range.
Solubilized vat dyes, not requiring the presence of alkali, can be used
for dyeing on animal fibres. Because they are dyed at low
temperatures, they are used in Indonesian batik dyeing for green
shades.

3
4. REACTIVE DYES.

This is an entirely class of dye introduced to the market in 1956. They react
chemically with the Fibre being dyed and if correctly applied, cannot be
removed by washing or boiling. The main feature of the dyestuff is its low
affinity to cellulose; therefore large amounts of salt are required to force its
deposition on he fabric. After this has been achieved, addition of alkali
causes the deposited dyes to react with the Fibre. Only a successfully
concluded reaction guarantees a fast dyeing. Basically there are two types of
reactive dyes: the cold dyeing and hot dyeing types.

USE: Reactive dyes are used where bright dyeing with high light and
wash fastness is required. Cold dyeing is used extensively in batik
work. Although some reactive dyestuffs have been specially modified
to dye wool, their main usage is in dyeing cotton linen and viscose
rayon.

5. AZOIC DYES.

The word 'Azoic' is the distinguishing name given to insoluble azo dyes that
are not applied directly as dyes, but are actually produced within the Fibre
itself. This is done with impregnating the Fibre with one component of the
dye, followed by treatment in another component, thus forming the dye
within the Fibre. The formation of this insoluble dye within the fabric makes
it very fast to washing. The deposition of the free pigment on the surface of
the dyed fabric produces poor rub fastness, but once the loose pigment is
removed by boiling the fabric in soap the dyeing becomes one of the fastest
available.

6. SULPHUR DYES

The first Sulfur dye was discovered in France in 1873, and further work
done by Raymond Videl enabled the manufacture of 'Videl black". Its
outstanding fastness to light, washing and boiling far surpassed any cotton
black known at that time. The general disadvantage of the Sulfur dyes that
they produce dull shades and lack a red. The main advantage lays in their
cheapness, ease of application and good wash-fastness. In their normal state
SulfUr dyes are insoluble in water but are readily soluble in the solution of

4
Sodium Sulfide. In this form they have high affinity to the all cellulose
fibres.

USE: The use of Sulfur dyes is restricted to dull brown, Khaki and
Navy shades, where a good wash but not boil-fastness is required.
Most Khaki and Navy overalls are dyed with Sulfur dyes. An
outstanding member of this family is Sulfur black. It dyes all cellulose
fibres, but particularly linen and jute, to a lustrous and deep black with
excellent wash and light fastness. Sulfur dyes are dyed from a dye
bath containing Sodium Sulfide and common or Glaubers Salt, and
are oxidized by airing or with some oxidizing agents (Sodium
Bichromate or Hydrogen Peroxide) in a fresh bath.

7. MORDANT DYES

Mordant dyes are so called because to apply them necessitates the use of
mordant. This group of dyes includes natural dyes: Logwood, Fustic and
Madder (now replaced by synthetic Alizarin) and a large group of synthetic
dyes with a widely differing constitution. The mordant dyes can be applied
to fibres by three different methods:
a. By mordanting the Fibre with a suitable metallic salt and then applying
the dye.
b. By dyeing the Fibre and subsequently after-treating it with a suitable
metallic salt so as to form an insoluble lake. This is the basis of 'after-
chrome' method used in particularly fast dyeing of black and brown color on
wool.
c. By the simultaneous application of the dye and mordant. In wool dyeing
as 'metachrome' or 'monochrome' process and is extensively used for dyeing
of brown and khaki colors.

USE: Since the dyes used in this process vary widely, not only by the
methods of application and dyeing of different fibres, reliable recipes and
instructions are to be followed carefully.

8. ACID DYES

These dyes comprise a large number of dyes used for the dyeing of wool,
silk and nylon. They vary considerably in their basic chemical structure, but

5
have one common feature - they dye from an acid dye bath. All acid dyes
can be grouped in three broad sub groups:

a. Level dyeing acid dyes. These dyes produce bright dyeing. The main
feature is their good leveling properties. They are dyed from a dye bath
containing strong acids (Sulfuric or Formic acid). These dyes exhibit low
wash and light fastness.

b. Acid milling dyes.

Selected because of their high and light fastness and are extensively
used for dyeing woolen fabrics that are subsequently milled. These
dyes require great care in application because uneven dyeing is
difficult or impossible to rectify. The dye bath requires the presence of
weak acid (acetic acid) or acid releasing salts (ammonium sulfate or
ammonium acetate) from which acid is liberated during dyeing.

c. Pre-metalized dyes
These dyes represent an extension of mordant dyes discussed in (7) above.
The metal component is being already incorporated in the dye during
manufacturing process. Very good light fastness even in pale shades
USE: The family of acid dyes is very large and diverse, varying
widely in their methods of dyeing, application and end use of the dyed
fabric. A choice of dyes should be made considering sometimes-
incompatible factors: - level dyeing, fastness, brightness and ease of
application. Care must be taken to use the appropriate method as
prescribed for a given dye. A number of acid dyes are also used to dye
nylon.

9. DISPERSE DYES

The introduction of a new regenerated cellulose acetate Fibre in 1920 led to

the necessity to develop an entirely new range of dyes. It was found that
cellulose acetate (or Celanese) Fibre had hardly any affinity for water-
soluble dyes. A new dyeing principle was introduced: dyeing with water
dispersed colored organic substances. These finely colored particles are

6
applied in aqueous dispersion to the acetate material and actually dissolved
in the fibres.

USE: Basically developed for dyeing of acetate fibres, Disperse dyes are
also used for dyeing of polyamide (Nylon) and acrylic (Orion & Acrylan)
fibres. With the addition of 'carriers' or swelling agents these dyes are also
used in dyeing of Polyester (Terylene, Tetron, Dacron, etc.)

10. OXIDATION DYES

These are not dyestuffs in the same sense as other soluble or disperse dyes,
but because of their exceptional fastness to light and washing are of great
importance. The most important member of this group is produced by
oxidation of aniline and is much used in dyeing of fur and leather goods.
USE: In addition to fur and leather dyeing, aniline black was almost
exclusively used to dye luster black umbrella fabric.

11. MINERAL AND PIGMENT DYES

Although it is preferred to use water soluble dyes in textile dyeing for two
reasons; ease of application and greater softness of the fabric, there are two
processes where pigment coloration is used:

a. Mineral khaki
Cotton army equipment where it is used because of its cheapness and
because it also renders fabric resistant to rotting and attack by insects in
damp conditions.

b. Synthetic resin printing

The introduction of heat setting synthetic resins has opened new fields in
textile printing. Mineral and organic pigments, as used in paint manufacture,
can now be applied to any fabric and rendered wash fast after heat treatment.
USE: Whilst the formation of mineral pigment on the fabric is used less and
less as a dyeing process, the use of pigment printing is continuously
increasing. The reason for this is the development of soft and flexible

7
 synthetic resins, available as binders (or adhesives) to secure the pigment to
the fabric without inducing a harsh or stiff finish.

MANUFACTURING OF AZO DYES

Azo colorant is manufactured by diazotization process. First aromatic amines transferred

into a diazonium compound. The reaction proceeds at low temperature and in the
presence of sodium nitrite and hydrochloric acid

HC1 & NaNo2/Diazotization

R—NH2 R—N=N]+ Cl'
[Diazonium Compound]

The diazonium compound reacts with coupling compound to form the actual dyestuff.

AZO DYE STUFF IN MARKET

According to the international survey in textile, more than 600 different auxiliary agents
involve in dyeing and printing. In addition to 8000 colorants azo dye stuff are named as
Acid, Basic, Mordant Direct, Disperse, and Solvent Or Food dyestuff.

Azo dye stuff, which can split off the toxic aryl amines. The list would be difficult to
keep up to date because new developments are frequently announced. Some dyestuff
manufacturer doesn’t want to disclose the chemical composition of their produc: because
they wish to keep them confidential.

According to acquire data base 1993 about 100 azo dye stuff, which are affected by the
prohibition of The German regulation on this class of dye. In the beginning of the
German
Regulation 20 aromatic amines were forbidden but now it is 24.

AZO DYES AND CARCINOGENICITY

Although both Natural and Synthetic chemicals may cause a Varity of effect at high
enough doges. The Effect most concern is Cancer. Chemical, which are known to cause
cancer, are called Carcinogenic and the process of cancer development is called
carcinigenicity. Azo dyes, which are splitting off toxic aryl amines, may lead cancer, due
to this reason azo dye stuff are known as carcinigenic. Some of the azo dyes have a
mutagenic character.

8
S.No Compound Name
1 o-Toluidine
2 p-Chloroaniline
3 p-Cresidine
4 4-Chloro-o-toluidine
5 2,4,5-Trimethyl amine
6 2,4-Toluendiamine
7 2-Napthylamine
8 2,4-Diaminoanisole
9 4-Aminobiphenyl
10 2-Amino-4-nitro toluene
11 Benzidine
12 4,4-Oxydianiline
13 4,5’-Diaminodiphenyl methane
14 o-aminoazotoluene
15 3,3 ’-Dimethylbenzidine
16 4,4-Thiodianiline
17 3,3 ’-dichlorobenzidine
18 3,3 ’-Dimethoxybenzidine
19 4,4 ’diamino-3 -3 ’ dimethyldiphenylmethane
20 4,4’-methylene-bis-(2-Chloraniline)

Above are the banned toxic arvl amines

9
 Analysis of Toxic aryl amines by Thin Laver Chromatography

Thin Layer Chromatography technique is oldest chromatographic technique.

Several methods were available for identification of toxic aryl amines by
TLC. This work was attempted to develop rapid, economical and reliable
test method to screen presence of toxic aryl amines by manufacturer and
user of synthetic dyes.

Apparatus: TLC developing chamber, TLC Plate 60 F254 (Supplied by

Emerck, India). UV-lamp.

Reagent: Methonal, Chloroform, Acetic acid, Ethyl Acetate, n-Hexane, 1-

Napthol and Potassium hydroxide.

Standard: 20 standard of Toxic aryl Amines from Sigma have been taken
for standardization.

Procedure:

30-mg/l standard solutions were prepared from each standard. 5 micro liter
of each standard was applied on the Thin Layer Chromatography plate using
micropipette. (Spot should be 2.5 mm above to bottom of the plate) .The
Thin Layer Chromatography plate was developed in two different mobile
phase i.e. ethyl acetate: n-hexane (9:1) and Chloroform: acetic acid (9:1)
until solvent front traveled up to 80 % of plate. Then plate was taken out and
allowed to dry and kept in nitration chamber. 1 % 1-napthol (spraying
reagent) was prepared previously and spread over Thin Layer
Chromatography plate. Observed the spot under UV-lamp and Response
factor *was calculated for individual standard of toxic aryl amines. Again the
mixture of 20 amines was analyzed using same procedure as individual and
Response factor was calculated (Table I and II) and compared with
individual. Separation of mixture of amines in Ethyl acetate: n-hexane
mobile phase was very good.

10
 TABLE-I

Mobile Phase -Chloroform: acetic acid

Name of the standard Toxic aryl Response Factor ( Rf)

amines

p-Chloramines 0.70

o-Toluidine 0.53

4,4-Diaminodiphenylmethane 0.37

2-Amino-4-nitrotoluene 0.78

3,3 -Dichlorobenzidine 0.74

4-Aminobiphenyl 0.69

Benzidine 0.47

2-Napthylamine 0.74

p-Cresidine 0.70

4,4-Oxydianiline 0.33

11
 TABLE-II

Mobile Phase -Ethyl acetate: n-hexane

Name of the standard Response Factor ( Rf) Response Factor (Rf)

Toxic aryl amines in individual In mixture

o-Toludine 0.90 0.89

p-Chloraniline 0.85 0.83

p-Cresidine 0.87 0.84

4-chloro-o-toluidine 0.81 0.82

2,4,5-Tri methyl amine 0.89 0.86

2,4-Tolyendiamine 0.43 0.42

2-Napthylamine 0.80 0.80

2,4-Diaminoanisole 0.41 0.40

4-aminobiphenyl 0.82 0.82

2-amino-4-nitrotoluene 0.93 0.92

12
Benzidine 0.62

4,4-oxydianiline 0.48 0.48

4,4-diaminodiphenyl 0.52 0.51

methane

o-aminoazotoluene 0.96 0.96

3,3-dimethylbenzidine 0.94 0.95

4,4-thiodianiline 0.65 0.65

3,3-dichlorobenzidine 0.89 0.88

3,3-dimethoxybenzidine 0.75 0.74

4,4-diamino-3,3- 0.60 0.60

dimethyldiphenylmetha
ne

4,4-methylene-bis-(2- 0.85 0.83

chloroaniline)

13
Analysis of Toxic aryl amines bv High Performance Thin Laver Chromatography
(HPTLO

High Performance Thin Layer Chromatography is an advance form of

TLC.From spotting to scanning is being done by machine. In this
experiment, CAMAG made HPTLC was taken for analysis.

Instrument: CAMAG HPTLC with automated multiple Developments.

Slit Dimension: 0.2*4mm

Mode: Scanning by absorption
Spectra: 200-400nm
Wavelength: 200nm (for 80% amines) and 230,280,360nm for others.

Standard & sample Preparation: Standard and sample was prepared in the
same manner as TLC

Method: Standard of 20 amines was spotted with the help of auto spotter
and developed using following gradient system. The minimum detection
limit of each amine was also determined. (Table-I, for gradient system and II
& III for detection limit)

Derivatization: After developing the TLC plate, the following method was
used for derivatization of plate.

1. Nox formation put 5ml beaker with about lgm sodium Nitrite in trough of
a twin trough chamber and added about 1ml acetic acid. After the reaction
has subsided, place the solvent free chromatograph plate in empty trough
and close the lid. After five min take it out and dry in steam of cold air.

2. After step one, spray N- (l-napthyl)-ethylenediammonium dichloride

(0.2% solution in 80:20 dichloromethane-methanol mixture) on
Chromatographic plate.

14
3. To neutralize excessive Nox ,dip the plate for I sec in ammonium
amidosulfonate solution (0.25gm in 70ml methanol dissolved and filled with
30ml dichloromethane) and dry in a hot air steam
4. To stabilize the chromatogram zone for at least one day, it is
recommended to dip the plate in a solution of n-hexane-paraffin oil (1:5)

TABLE!

Step n = 1 2 6 11

1 2 3 4
Bottle No.
40 30 20 10
Dichloromethane
40 30 20 10
Acetone
N_hexane 20 40 60 80

Drying Time 3 min after each run

Run Time 3 mm running distance increment

Wash Bottle 1 N Ammonia Solution

15
 TABLE-H

Name of the Response Limit of Colour

standard Toxic Factor (Rf) in detection (in ug)
aryl amines individual

o-Toludine 0.73 0.03 Reddish Brown

p-Chloraniline 0.70 0.02 Violet

p-Cresidine 0.70 0.02 Violet

4-chloro-o- 0.74 0.02 Yellow

toluidine

2,4,5-Tri methyl 0.68 0.03 Orange

amine

2,4- 0.28 0.2 Brown-Lila

Tolyendiamine

2-Napthylamine 0.75 0.075 Yellow

2,4- 0.26 0.075 Violet Brown

Diaminoanisole

4-aminobiphenyl 0.69 0.04 Violet

16
 2-amino-4- 0.78 0.02 Orang Violet
nitrotoluene

Benzidine 0.47 0.05 Smoke-Blue

4,4-oxydianiline 0.33 0.02 Violet

4,4- 0.37 0.02 Violet

diaminodiphenyl
methane

0- 0.81 0.02 Violet Brown

aminoazotoluene

3,3- 0.81 0.02 Grey

dimethylbenzidin
e

4,4-thiodianiline 0.65 0.65 Violet

3,3- 0.89 0.88 Brown -Lila

dichlorobenzidin
e

3,3- 0.75 0.74 Smoke-Blue

dimethoxybenzid
ine

17
4,4-diamino-3,3- 0.60 0.60 Orange-Brown
dimethyldipheny
lmethane

4,4-methylene- 0.85 0.83 Reddish -brown

bis-(2-
chloroaniline)

Table-Ill

Name of the Xmax (nm) Measurement Co lour

standard Toxic Wavelength
aryl amines (nm)

o-Toludine 225/280 230 Light-yellow

p-Chloraniline 235/290 230 Yellow-brown

p-Cresidine 280 280 Brown

4-chloro-o- 230/280 200/230 Yellow

toluidine

18
 2,4,5-Tri methyl 225/285 230 Light brown
amine

2,4- 210/290/365 360 Yellow-Brown

Tolyendiamine

2-Napthylamine 220/295 200 Yellow

2,4- 205/295 200 Deep Brown

Diaminoanisole

4-aminobiphenyl 270/360 200/280 Violet

2-amino-4- 0.78 0.02 Light Violet

nitrotoluene

Benzidine 280 280 Deep-Brwon

4,4-oxydianiline 240/290 200/230 Green-Violet

4,4- 240/285/390 230 Light-Brown

diaminodiphenyl
methane

0- 365/245 200/360 Reddish- Brown

aminoazotoluene

3,3-
dimethylbenzidine 280/205 280 Brown

19
4,4-thiodianiline 260 280 Brown

3,3- 280/210 280 Light-Brown

dichlorobenzidine

3,3- 300/210 280 Violet

dimethoxybenzid
ine

4,4-diamino-3,3- 240/285 200/230 Brown

dimethyldipheny
Imethane

4,4-methylene- 205/240/295 200 Light-Brown

bis-(2-
chloroaniline)

Thin Layer Chromatogram for Standard ( S1-S4), Leather sample

Extract (a) and fabric extract sample (c)

a a a S1 S2 S3 S4 b b b S1 S2 S3 S4 c c c

20
 Chromatogram of mixture of standard of the following Toxic aryl
amines
O-anisidine, Benzidine, p-chloraniline, 3,3’-dichlorobenzidine, 2-
napthvlamine. o-Toluidine, 4-Aminobiphenyl
tlethod Scan Calihration Spectrun Data End_ _ _ _ _ _ _ _ _ HELP

Havelength: 236 nn Cfif 3

Track: 3* noise level: 8,412flUj raw data file.: NIIRA29
1)4,82 S/H.'02020816 CfiflftG SOFIUflRE (c) 1995 SCANNERS: 820116

Track 3, Analysis c: STD MIXTURE

Peak 1 start ! max 1 end area
# ! Rf H ! Rf H r%i : Rf H F m
1. ! 0,06 0.4 : 0.08 98.0 5,89 ! 0.09 0.5 968.7 2,14
9 t 0.14, o.i : 0.18 126.7 7,60 1 0.22 1.7 3466.3 7,65
3 : 0,25 0.2 ! 0.29 251.8 15.12 ! 0.32 16.7 5305.4 11.71
4 : 0.32 i7.o : 0.37 90.6 5„44 ! 0.40 0.5 21.63.8 4..78
& ! 0.40 0,5 : 0.44 173.2 10.40 ! 0.4.6 101.7 3839.5 8 48
6 : 0.46 101.7 : 0.50 472.8 28.39 ! 0.55 58., 5 15350.6 33.89
7 ! 0.55 58.5 ! 0.59 282.3 16,95 ! 0,65 17.5 9346,8 20 63
8 : 0.71 o.i : 0.78 1,70 ..1 10.21 ! 0.82 1-7 4856.0 10,72
Total height = 1665.5 total area ~ 45297,1
21
 Chromatogram of Mixture of o-anisidine. Benzidine & p-chloroaniline

SPECTRUM :}1: HITRA23 ANCHRON INDIA 29/AUG/I997 18:56

Nethod Scan JflBHiBl. Calibration Spectrun Data End_ _ _ _ _ _ _ HELP

Ravelength: 236 nn CRH

Irach: 1? noise level: 0,412AUj raw data file: NIIRA29
D4J2 S/N:0202A0I6 CANAG SOFTUflRE (c) 1995 3: 028116

Track 1, Analysis a: BENZIDINE + O-DIAN 4 PCA

\ 1
Peak ! start max end area
# ! Rf H ! Rf H r%i Rf H ! 4 F r%i
1 1 0 14 6,4 ! n R '1 4300.1 44.44
0.1.9 214.9 54.69 0.23 .
Cj KJ
O 1
i 0.43 3.7 ‘ 0.48 114.6 29.18 0.53 2,0 : 3070.8 31.74
3 ! 0.78 5.3 ! 0.84 63 ,.4 16.13 0.89 2.6 : 2304.7 23.82
Total height = 392.9 total area = 9675.6

22
Spectrum of Mixture of o-anisidine. Benzidine & p-chloroaniline

Method Scan Integration Calibration jEBBSffilef [fata End_ _ _ _ _ _ _ _ _ HELP

1 3 a H 0,84 SJ 200
114,82 S/N:0202A016. CAMHG SOFTWARE (c) 1995 SCAMMER 3: 620116

23
Calibration curve of 3.3’-dimethoxvbenzidine

tnu

Spectra of standard 3,3’-dimethoxybenzidine and sample containing

33’-dim ethoxy benzidine

24
Spectra of standard Benzidine and sample containing Benzidine

tav]

2.5
 Analysis of Toxic arvl amines by High Pressure Liquid Chromatography with
Mass spectrometer

Instrumentation: Water HPLC/MS was taken for experiment

Sample Preparation: Same as Mentioned in TLC Method

The separation of 20 toxic aryl amines were optimized with a gradient of

ammoniumacetsolution pH=Ci8 Column.

Instrumental Condition: HPLC Column: Ci8 (5um*2.1* 150mm)

Gradient: 0.25 ml/min Flow
A-20 mm ammoniumacetatlsg.pH==4.2
B-Acetonitrile

Time (Min) Solvent A (%) Solvent B(%)

0 90 10
5 75 25
20 75 25
60 50 50
65 5 95
80 5 95

Injection Volume: lOul

Photo Array detector Condition: 200-400 nm

1 Spectra/Sec
1.2 nm Resolution

Mass Detector Parameter: Mass Range 60-300 Dalton

Scan Speed -1 Spectra/Sec
Nebulizer Temp- 85°C
Expansion Region Temp - 230°C
Helium Flow - 150 ml/min

26
 TABLE-1

Name of the standard Toxic Retention Time of Retention Time of

aryl amines Uv-Spectra (in Min) Mass Spectra (in Min)

o-Toludine
0.73 0.03
p-Chloraniline
0.70 0.02
p-Cresidine
0.70 0.02
4-chloro-o-toluidine
0.74 0.02
2,4,5-Tri methyl amine
0.68 0.03
2,4-Tolyendiamine
0.28 0.2
2-Napthylamine
0.75 0.075
2,4-Diaminoanisole
0.26 0.075
4-aminobiphenyl
0.69 0.04
2-amino-4-nitrotoluene
0.78 0.02

Benzidine
0.47 0.05

4,4-oxydianiline 0.33 0.02

4,4-diaminodiphenyl methane
0.37 0.02
o-aminoazotoluene
0.81 0.02
3,3 -dimethylbenzidine

27
 0.81 0.02
4,4-thiodianiline
0.65 0.65
3,3-dichlorobenzidine
0.89 0.88

3,3-dimethoxybenzidine 0.75 0.74

4,4-diamino-3,3- 0.60 0.60

dimethyldiphenylmethane

4,4-methylene-bis-(2- 0.85 0.83

chloroaniline)

Degradation of Azo dyes and releasing of Benzidine as a degradation

product

28
 Chromatogram at 280 and Total Ion Chromatogram

Chromatogramm at 280nm and TIC

Experiments on Percent recovery: To prove the accuracy of the test method,

percent recovery is the best method for checking. The percent recovery v/ere
done for all the Toxic aryl amines, in this method all the standard were
treated as sample, and result are given below

Toxic aryl amine Percent

Recovery
(In %)
2,4-Diaminoanisole 75
2,4-Diaminotoluol 54
4,4’-Oxydianiline 17
Benzidine 53
o-Toluidin 47
4,4’-Diaminodiphenylmethan
p-cresidin
p-Chloroanilin tlW
EsW-

yJ
29 i).-' ■
 3,3’ -Dimethoxybenzidine 31
3,3-Dimethylbenzidine 62
2,4,5-Trimethylaniline 49
4,4’-Thiodianilin 67
2-Napthylamin 47
3,3-Dimethyl,4,4’-diaminophenylmethan 83
4-Chloro-o-toluidin 48
4-Aminobiphenyl 50
3,3 ’-Dichlorobenzidine 48
4,4’-Methylen-bis-(2)-chloranilin 57

Result for HPLC with DAD Detector

Photodiode array Spectrum of standard Toxic aryl amines

4,4’-OxydianiIin O-Tolidine

mAU
i or
tl'r.U
250
200
IOC
150
SC
ioe
pf
50
0
00 225 250 " 2is ‘ ' 3&0 " 3&5 “ zEcTHis orri
200

_____ 200 225 250 ’ ' 275^' 300 ' 3^5 ' 350 ' 375 ' nrri ■

4,4’-Thiodianilin p-Cresidin
Tna i i i AU
200

150
100

■JJ
i0 200 7.25 "250 27s' 'HocT 325 350 " $5'
u nm
200 225 250 275 300 3^5 350 375
EEN BgtfZlDlKl
mAU :
V kmwkjttny/ ■.40
i 2n
i co-i
100
40 •20 /■
v(.:
20
:0 \
o ;
• I 30
i; >V. .
200 225 250 275 525 350 375 nrTV •'-'5 250 275 300' 325 350 375
..On-,
 imr.
175
150
125
100
75
50
25
0
260' ' 225 ‘ ' 260" ' 275 300 325~’ ' 3^0^ 375 nm
325 ' 3503?s‘nnj '

2-Napthylamine
4-Chlor-o-toluidin

m£U ^ i
i
'■ 250 :: ' •
200 '
i

325 550 375 nm

O-Aminoazotoulol 2-Amino-4-Nitroluol

! ' inAU
i'
! . 30
;; 40
20

-20 • ■
-40 I
300 325 350 3^5 niT 200 " 225 ' ‘ 250 " 2^5' ' 366 ‘ ' 3$5' ' 350^3)5 ’ nm

2,4-Diaminoanisol
p-Chloraniline

31
 mAU ;
mAU" 120 :
600 100 ■
500-i 80 i
400 1 i
so
300-J I i
! 40
200-j
| 20
j 100 j i 0 i
i o | 320 250 375 nro
2C0 22:
200 225 3CO 375 ni J!TV

4,4’Diaminodiphenylmethan 3,3’-DichIorbenzidin

•«t*

~mA'J 00

1'50
i 100 -j ',0 \ y \
50 -1
27; 225 ' 250^275 " 300 ‘ 325 ' 350 ^ 375^
0-i
200 225 260 325 350 375
•iiv nnoi

3,3-Dimethoxybenzidin 3,3’-Dimethylbenzidin

pinAlT
y) i:dj | 400
mAiO
J.V. I 300

CO j 200

1; 1C0
:■ 0
200 225 330 375
V

:oo 250’^275 300 325 350 375 nnr

3,3’"dimethyl-4,4,diammodiphenyImethane 4,4’-Metbylen-bis-(2)-chloraniline

32
 ;~fWU'r‘‘

150
I
125 :
■O' I 100 i
200 75 i
50 -]
100
25-!
0- 0-' i

200 225 J 250 '^215" 300' 3^5 ' ' 350 ' ’ 3?5^nrr I
f

2,4-Toluendiamin 2,4,5-TrimethyIaniIin

33
 HPLC Chromatogram of mixture of Standard-I
... . .. v.. t 24.11.98 12:15:00 3
^an-e SI
Anton

‘•/Quence :-'na C:\HPCHEM\1\SEQUENCE\2 4111.S

hoc. C : \HPCHEM\ 1 \METHODS\AMI 3 0 0.1 2
chcncjcd 22.01.98 12:45:41 by Anton
•.cetcnitril-Wasser

UAU1 b, Sig=24U,^ Ket=oo0,60^Z41 iT\uuT-03u1.u}

mAU.
i/0 ;
150 ' m
i °'3
<o
(6
lOO
18.133
15.716
90

/5 ! o
!■> 20.358

CO
50 ^ O)
•
•

25 ■ t
-
0 : ■ ■ -a-V
•<

o ' i 25
......... PMP1, Pressure

34
 HPLC Chromatogram of mixture of Standard-II

..jv.eti-.-n sJale 24.11.98 11:22:12

ample Same S2
.cq. Operator Anton
lilj V.

Sequence Fila : C:\HPCHEM\1\SEQUENCE\24111.E

;ethod : C:\HPCHEM\l\METHODS\AMI3 0 0i:: :■]
.ast changed : 22.01.98 12:45:41 by Anton
vceconi'cril -Sasser
“ DAD1 A, Sig=21it20'Ref=55(180“{2‘4T'rn002:020TX()"’
mAU' Oinj
175 2 t/llf ~rU
CO Utt
150
125
(S' i(JiD
o a
CD
o
in
o
CM

c0 s

30.923
▼
\

■
\ -------------------------- 1U-U---- 1 i—■ _________

0 5 10 15 20
DAU1 B, Sig=240i20”ReT=55U;80124TTTO)02;:020T7Dl~
.- 24.069

•wj
1/5
i r.n

125 <n
in
100- r-
75 <*i CO in
o
s c-g
50 r~-
CM
25
0 .. K._ _A._ X
-25
IB 15 20
PMP1 Pressure
bar.
175-
150
125
100

25
0
r.

o ’ y 10 15 20
PN1P i, How
ml'lnin
i • .
150
105
100
75
50
25
0
10 "iF 20

35
♦> Analysis of Toxic aryl amines by Gas Chromatography -Mass spectrometer

Sample Preparation; same as mentioned in earlier method

Standard: 5-ppm solution of each standard was taken for experiment. Sigma
& Aldrich Laboratory, USA, supplied Standrads

Instrument: Shimadzu make GC/MS (GC-17A, Mass QP5050)

Instrument and Chromatography Condition

Carrier Gas: Helium

Column: DB-5ms low Bleed (30meter, 25 mm ID, 25um Film

thickness)

Temperature
Progamme for
GC: 60°C -Holding Time 2 Minute
10°C/Minute to 220°C-Holding Time-2Minute
12°C/Minute to 260°C-Holding Time 1 minute
15°C/ Minute to 280°C- Minute to 280°C

Column Pressure: 100 kPa

Column Flow Rate: 1.6 ml.minute

Linear Velocity: 46.4

Total Flow Rate: 20 ml/minute

Mass Spectrometry Parameter

Interface Temperature: 300°C

Electron Impact Scan gains: 1.3kV
Scan Range: 50-350 Dalton
Scan Interval: 5second
Threshold Value: 1000

36
Solvent Cut Time: 3Minute
Acquisition Start Time: 5 Minute
Total Run Time: 30.67 minute
Injector Temperature: 250°C
Injection Volume: 1 ul
Injection Mode: Split less

Method of Detection Limit: lOOug/ml

GC Separation: The separation of all the standard aromatic amines were

done with Db-5ms low bleed column. Retention time has been shown in the
Table I & Chromatograph shown in Table II.

TABLE-I

Peak Compound Name Retention Time (in

No. Min.)
1 o-Toluidine 6.79
2 p-Chloroaniline 8.90
3 p-Cresidine 10.06
4 4-Chloro-o-toluidine 11.50
5 2,4,5-Trimethyl amine 11.57
6 2,4-Toluendiamine 11.80
7 2-Napthylamine 14015
8 2,4-Diaminoanisole 15.43
9 4-Aminobiphenyl 16.74
10 2-Amino-4-nitro toluene 17.83
11 Benzidine 21.18
12 4,4-Oxydianiline 21.26
13 4,5’-Diaminodiphenyl methane 21.38
14 o-aminoazotoluene 22.79
15 3,3 ’-Dimethylbenzidine 23.036
16 4,4-Thiodianiline 24.00
17 3,3 ’-dichiorobenzidine 25.11
18 3,3’ -Dimethoxybenzidine 25.025
19 4,4’diamino-3- 28.16
3 ’dimethyldiphenylmethane
20 4,4’-methylene-bis-(2-Chloraniline) 30.39

37
Characteristic ion (m/z) of Toxic aryl amines

Peak Compound Name Monitoring Ion

No.
1 o-Toluidine 106,107,77
2 p-Chloroaniline 127,65,129
3 p-Cresidine 122,137,94
4 4-Chloro-o-toluidine 106,77,141
5 2,4,5-Trimethyl amine 120,135,134
6 2,4-Toluendiamine 121,122,77
7 2-Napthylamine 143,115,116
8 2,4-Diaminoanisole 123,138,94
9 4-Aminobiphenyl 169,115
10 2-Amino-4-nitro toluene 77,152,116
11 Benzidine 184,92,156
12 4,4-Oxy dianiline 200,108
13 4,4’-Diaminodiphenyl methane 198,197
14 o-aminoazotoluene 106,225,77 i
15 3,3 ’-Dimethylbenzidine 212,106
16 4,4-Thiodianiline 216,184
17 3,3’ -dichlorobenzidine 252,254
18 3,3 ’-Dimethoxybenzidine 244,201
19 4,4’diamino-3- 226,211,225
3 ’dimethyldiphenylmethane
20 4,4’-methylene-bis-(2-Chloraniline) 231,266,195

38
 O- Tot.ui DtwE / 6-
C . H R O M A TO Grti2.Afv?

CHUOfcANtUNE / 8-90
ISJ
-

£_ c^esi Dins / io. o 6

j ~1 C

oj

^ i,_£Hl.ceo - G-TOLUlJ'«e/ n' F/1(. s:t

£
(2 T O T A L , i o n

_ 2,4,ff.-TR< MSrHYi-AKlUME/ T-
OF

~ Cr'—> 2, TotyEwDfAwi^e/ JI-8o

N APTHV*-A Ml « E / 1
^_£)

2, Lj- DOMINO P.MI.SOLE/

C H ^ O M A T o G if ^ A M ^
^ TAK bA R b

0IPMENYL- ) (6-74
4 _ ami no
vO
TOi-UtNE / IF-S3
' 2-Amino- NITW-
o
A R o m a t ic a m i n e s

i3> EN 2.1 JS I N E / 2 1• 1®
39

r r— OXV Dl Arvll UH e/ 21-2.6

NjJZ^, A-L,'- Dl MUlNO phenyl M£TH>VNE/2l-38
*7"—> Q_ AMIMQ A2-° TOOuEnB/ 22- 7?
j 21-3'- DIMETHYL- aEN3JONE/ j g,.gg

/" N V- THioDf amiuNE/ 2A.OU

"7, M-3, 3'_ DICHLOliOBL^N^v
,. 0/1 REM 21 •£>' AJ E/
,
2.S-/I
- ’ . „ 5(M£^rf8^^c/25-
c~ -H.

C U3
i,-V- W»«- 3'T-

L ^ ^.V-' mETHYLEmE-
KJ
o
fblS-C2-" CHt-CHifitW U *t£‘3 / 3>o-'2>c{

3
 ID Na»* Tin# H/Z Rr*a Cone.
1 Banzidin 37.251 181.15 1551628 8.983ppa
2 3 .3 '-D ia a th y lb a n z id in 39.8TT 212.28 869778 8.581ppa
TIC
181.88
212.88
TIC Chromatograph for sample, which shows presence of Benzedine & 3.3,-Di chlorobenzidine.
t List>
No SI Mol.Wgt. Hoi.For*./Compound Naas CftS No. Entry LIB*
t 8T 18*1C12Ht2N2
92-87-5 II
Banzidin
2 *48 169 C12H11N
92-87-1 9
f-Aainodiphonyl
3 38 127 C6HeClN 166-H7-8 26
p-Chloranilin
H 35 1*13 C10H9N 91-59-8 7
2-Naphtylaain
5 36 152 C7H8H2O2 99-55-8
2-ft«ino-*4-nitrotoluol
Library Naaa
(1) AftOH_AH.LIB
Spectra of Benzidine Matched in Library
Li»t>
o-Aainoazotoluol
33 152 C7H8N2O2 33-55-8 8
2-Aaino-4-nitrotoluol
34 141 C7H8C1N 35-63-2 4
4-Chlor-o-toluidin
33 184 C12Hf2N2 32-8T-5 11
Bonzidin
Spectra of 3,3 ’-DimethylBenzidine Matched in Library
Separation of Isomers

In the list of banned amines p-chloraniline and o-Toluidine are found in

isomers also. To check the presence of isomers of these amines, the
following method was developed.

p-Chloraniline isomers separation

Column for GC-Mass: DB-5ms

GC-Condition: Initial temp 40°c for 2 min.

Rate 1- 5°c / min to 90°c / min for 15 min.

Rate 2- 30°c / min to 280°c / min for 5 min.

Carrier Gas: Helium

Flow rate: 0.7 ml/min
Injector temp. : 250°c
Transferline temp. : 280°c
Injection Volume: 1 ul.

Standard preparation: 5-ppm mixture standard of o, m, p-chloraniline was

prepared in ethyl acetate.
Sample analysis: Detected sample has to be analyzed on this condition to
confirm the presence of respective amines.

o-Toluidine isomers

Column for GC-Mass: DB-5ms

GC-Condition: Initial temp 40°c for 2 min.

Rate 1- 3°c / min to 55°c / min for 15 min.

Rate 2- 30°c / min to 280°c / min for 5 min.

Carrier Gas: Helium

Flow rate: 0.7 ml/min
Injector temp.: 250°c
Transferline temp. : 280°c
Injection Volume: 1 ul.

43
Standard preparation: 5-ppm mixture standard of o, m5 p-chloraniline was
prepared in ethyl acetate.
Sample analysis: Detected sample has to be analyzed on this condition to
confirm the presence of respective amines.

❖ Extraction of sample in ethyl acetate in stead of tert-butvl methyl ether

Ethyl acetate has been tried for extraction of sample for azo dyes in stead of
t-butyl methyl ether, as it is very expensive solvent. It has been found that
ethyl acetate solvent was giving very good response. One experiment was
carried out using red color cotton fabric (dyed with Congo red, first reported
dye, which contains Benzidine). This sample was extracted using t-butyl
methyl ether and ethyl acetate and results were as follow.

Ethyl acetate extracted fabric t-butyl methyl ether extracted fabric

Benzidine-400ppm Benzidine-1 OOppm

❖ Conclusion: It has been concluded that the GC-Mass method using ethyl
acetate as a solvent for extraction is the best method for detection and
quantification of toxic aryl amines. TLC method can used only for
screening purpose> HPTLC method can be used for quantification as
well as
Identification, but cost is more than GC-Mass with El source. Now two
different detectors for HPLC i.e. DAD and Mass were taken in this
experiment, but its sensitivity is less comparing to GC-Mass and it is not
giving m/z value for analyzed compound. HPLC-Mass is providing m/z
spectra but cost is very high. Hence considering prices and features of these
instruments, it has been found that GC-Mass with El source is quite good for
this purpose.

44
❖ Analysis of Disperse Dye
Introduction: After the ban of Azo dyes, which are releasing toxic aryl
amines as a degradation product from synthetic dyes, the scientist have
discovered that some disperse dyes are allergenic and carcinogenic. At
present there is no legislation on the ban on disperse dye all over the world,
but some European countries are very concess about the carcinogenicity of
disperse dye on human health, hence they have made a draft on banning of
disperse dye and covered seventeen disperse dye in first stage. Disperse dye
is mainly used in dyeing of Polyester and sometimes for Nylon fiber. In the
literature survey, it has been found that there is no suitable method for
identification and Quantitation. In 1996,some work was done by Dr.Heinz-
Dieter Winkeler on identification of disperse dye from textile, but it was
limit for five disperse dye only.

The present work was done on the analysis of seventeen disperse dye by
TLC and HPLC with Diode array detector.

List of Disperse Dyes used for analysis

Serial Number Name of Disperse dye

1 Disperse Blue -1
2 Disperse Blue -3
3 Disperse Blue -7
4 Disperse Blue -26
5 Disperse Blue-35
6 Disperse Blue-102
7 Disperse Blue-106
8 Disperse Blue-124
9 Disperse Red-1
10 Disperse Red-11
11 Disperse Red-17
12 Disperse Yellow-1
13 Disperse Yellow-3
14 Disperse Yellow-9
15 Disperse Yellow-49
16 Disperse Orange-1
17 Disperse Orange-3
18 Disperse Orange-37/76

45
Structure of Some Disperse Dye:

Disperse Yellw-3

Disperse Orange-3

Disperse Red-1

46
Disperse Blue-1

47
Thin Laver Chromatography Spectra standard ban disperse dves
08
Analysis report of UV

Serial Number Name of Disperse dye A-max

1 Disperse Blue -1 640nm
2 Disperse Blue -3 640nm
3 Disperse Blue -7 650nm
4 Disperse Blue -26 625nm
5 Disperse Blue -35 625nm
6 Disperse Blue -102 625nm
7 Disperse Blue -106 600nm
8 Disperse Blue-124 600nm
9 Disperse Red-1 500nm
10 Disperse Red-11 525nm
11 Disperse Red-17 500nm
12 Disperse Yellow-1 360nm
13 Disperse Yellow-3 350nm
14 Disperse Yellow-9 370nm
15 Disperse Yellow-49 430nm
16 Disperse Orange-1 475nm
17 Disperse Orange-3 475nm
18 Disperse Orange-37/76 450nm

Experimental:

Instrumentation: Water makes HPLC (Model 515) with Diode Array

Dectector (Model 975) was used for analysis.

Chemical: Acetonitrle, Sodium Acetate

Instrumental Condition:

Column : C18 (25cm*4mm*5|um)

Flow Rate of Mobile Phase : lml/min
Column Temp. : 50°C
Detector : Diode array detector
Detection Range : 400-700 nm

49
Quantitation WaveLength : 450nm, 420nm, 570nm, 640nm
Injection Volume : 20 pi

Gradient Program:

Time (in min) Mobile Phase-A Mobile Phase-B

0 90 10
15 90 10
50 0 100
60 0 100
65 90 10
80 90 10

Standard Preparation:

1. 10-ppm solution of each standard was prepared for Individual HPLC

analysis and for TLC analysis.
2. Two sets of mixed working standard were prepared as follows.

Set-1

A. 20mg of Disperse Blue-1, Blue-35, Blue-106, Red-11, yellow-9 and

Orange-3 were dissolved into 100 ml volumetric flask with 50%
Acetonitrile.
B. 20mg of Disperse Red-1, Blue-124, Orange-37, Orange-1 were
dissolved into 100 ml volumetric flask with 50% Acetonitrile.
C. 10ml of the standard solution was pipetted into 100ml volumetric
flask and diluted with 50% aceonitrile
D. 10ml of the diluted standard solution was pipetted into 100ml
volumetric flask and diluted with 50% acetonitrile.

Set-2

A. 20mg of Disperse Blue-3, Blue-7, Yellow-1 and lOOmg of Blue-26 and

were dissolved into 100 ml volumetric flask with 50% Acetonitrile.

50
B 20mg of Disperse Red-17, Yellow-39, Yellow-49, Blue-102 were
dissolved into 100 ml volumetric flask with 50% Acetonitrile.
C. 10ml of the standard solution was pipetted into 100ml volumetric flask
and diluted with 50% aceonitrile ,
D. 10ml of the diluted standard solution was pipetted into 100ml volumemc
flask and diluted with 50% acetonitrile.

Chromatographic Separation
Separation of all the seventeen disperse^ dyes in standard using jsnidient
Acetonitrile/0.1 sodium acetate (Mobile P^ase
Acetonitrile/0.1 sodium acetate (Mobile Phase A-90: 10) gave c-ear
separation. The following showed separation.

Chromatograph-1
bo'a4 - 741 oi

^34-^3 97
L '_ * 2 ^3 r3 S 3

2 6 .1 2 6

|--4 1 .2 1 3
^ 4 3 .0 7 9
>I 3 6 .8 3 1
[-^ 29.691
^ 2 0 .8 3 7

j--33.125
cm

2 5 .0 4 8
6 .8 7 5

4 3 - 8 1 JU--
%
121847

I
I
I
I
<
jr-

I
i
!

20 40
nun

51
 Report Of Chromatograph -1

Disperse Dye Channel (Wave Retention Time Area

Length) ( min)
Blue-1 1 8.790 3050
2 8.818 9139
3 8.831 25772
Blue-1 1 18.124 4504
2 18.116 14990
3 18.116 52663
Blue-1 2 29.239 6365
3 29.239 22796 |

Chromatograph-2

Abs

52
Report Of Chromatograph -2

Disperse Dye Channel (Wave Retention Time Area

Length) (min)
Blue-1 1 8.790 3050
2 8.818 9139
3 8.831 25772
Blue-1 1 18.124 4504
2 18.116 14990
3 18.116 52663
Red-11 2 29.239 6365 !
3 29.239 22796 !
Yellow-9 1 16.218 49150 |
2 16.219 86678 !
Blue-35 3 20.834 11217 1
(peak-1) 4 20.834 15194 I
Blue-106 1 23.364 4960
3 23.392 84770
4 23.393 75528
Orange-3 1 26.138 188917
2 26.138 192953
4 26.126 4168
Yellow-3 1 26.910 28914
2 26.904 57670
Blue-1 3 29.684 16239
(Peak-3) 4 29.691 8369
Red-1 1 31.398 189129
2 31.398 97361
3 31.429 1202
4 31.397 56594
Blue-35 3 33.119 10041
(peak-2) 4 33.125 5108
Blue-124 1 35.516 5048
3 35.526 50822
4 35.526 70930
Orange-37 1 41.213 62358
2 41.214 67474
4 41.213 3406

53
 Blue-35 3 43.063 8394
(peak-3) 4 43.079 2775
Orange-1 1 43.812 89306
2 43.812 60477
4 43.812 7218

Chromatograph-3

.tAcs
i 3 Cftg 4 50iwi5 <TJ
03 O
-o* n 3 m
OtN r* OS <n r* m r- IQ m
o »n C1
CM CD
<Doon *M
1 03
os
1 os
os 03
3 1 .5

‘0 •
4FE \ CM CM as O
—A . A it m

3 Ch2 42Ohm 01
2 01p* 01ss s
OS
* o
of-4
m
in
OS in
1- 03<M
JLi <N
JU—i.
i
eJll^.8^72

-.--^.2-9.799
. 512

r-_ r^ « -.B 7 8
1 8 .5 7 8

4 6 .0 6 8
' 383$?§2S F 8 ’ 97 i9 .5 3 3
5 .1 ©

— 3 1 .6 7 7

•«:••v
4 9 .1 0 5
145.024
Z99
914

17

—"=40-f882
5 .1 9 8

- 1 8 .6 0 9
- 1 9 .9 9 3

_~--29.795
-7 .5 6 5

r -3 1 .6 7 3

4 9 .1 1 3
(>46.094
3 4 .4 1 5

20 40
nu.n

54
Report Of Chromatograph -3

Disperse Dye Channel (Wave Retention Time Area

Length) (min)
Blue-1 3 5.183 2440
(peak-3) 4 5.198 2288

Blue-7 1 7.537 1198

(peak-1) 3 7.564 16220
4 7.565 8896
Blue-7 3 11.182 8306
(peak-2) 4 11.200 2393
Blue-3 3 11.472 15848
(peak-2) 4 11.470 7793
Yellow-1 1 14.964 10051
2 14.994 24070
Blue-26 3 18.578 10161
(peak-1) 4 18.609 26287
Red-17 1 19.987 51043
2 19.983 28146
4 19.993 23949

Report Of Individual Chromatograph (Chromatograph shown)

Serial Number Name of Disperse dye Retention Time (min)

1 Disperse Blue -1 4.55,80818
2 Disperse Blue -3 8.264,21.977
3 Disperse Blue -7 No good separation
4 Disperse Blue -26 No good separation
5 Disperse Blue -35 No good separation
6 Disperse Blue -102 33.194,34.926,36.167
7 Disperse Blue -106 19.684
8 Disperse Blue -124 30.637
9 Disperse Red-1 26.353
10 Disperse Red-11 10.028

55
 11 Disperse Red-17 14.0184
12 Disperse Yellow-1 10.774
13 Disperse Yellow-3 22.357
14 Disperse Yellow-9 12.544
15 Disperse Yellow-39 19.366,24.173
16 Disperse Yellow-49 28.259
17 Disperse Orange-1 37.422
18 Disperse Orange-3 20.493
19 Disperse Orange-37/76 37.738

Sample Preparation:

0.5gm-textile material was taken and cut into small pieces and taken into
70ml-screw caped culture media tube.
There after 10 ml of acetonitrile added in tube and kept at 70°C for 15 min.
in ultrasonic bath. After cooling down, 10 ml of HPLC grade water added to
the sample solution then the sample solution was filtered and used for
TLC/HPLC Analysis

HPLC-DAD Chromatograph is attached here.

56
 Untitled Report
Reported by User: System Project Name: ANUJ

SAMPLE IN F 0 RM ATI ON

•ample Name: Dye Blue-1- 20 ppm Acquired By: System

•ample Type: Standard Date Acquired: 9/12/02 11:54:38 AM
'iat 1 Acq. Method Set; DTE MTH
ijection#: 1 Date Processed: 9/30/02 3:52:17 PM
ijection Volume: 20.00 ul Recessing Method: Default
!un Time:' 90.0 Minutes Channel Name: VWInChl
•ample Set Name: Roc. Chnl. Descr.: FCA 640.4 nm

Auto-Scaled Chromatogram

Peak Results
Name RT Area Height Amount Units

1 4.455 35569 2021

2 - 8.818 101231 3888

3 22.715 68989 1919

57 *

ort Method: Untitled Rinted 3:58:22 PM 9/30/02 Page: 1 of 1

 Untitled Report
wA Reported by User: System Project Name: ANUU

SAMPLE INFORMATION

Sample Name: Dye Blue-3- 20 ppm Acquired By: System

Sample Type: Standard Date Acquired: 9/12/0212:38:17 FM !
i/iai: 1 Acq, Method Set: DYE MTU
njection#: 3 Date Processed: 9/30/02 4:01:14 PM
njection Volume: 20.00 ul Processing Method: Default !
RunTime: 90.0 Mnutes Channel Name: VWIn Ch2 i
Sample Set Name: FToc. Chnl. Descr: PDA 639.3 nm

Auto-Scaled Chromatogram

Peak Results
Name RT Area Height Amount Unite

1 4.264 2838 258

2 8.483 15183 582

3 21.977 FB165 1657

irt Method: Untitled Printed 4:02:42 PM 9/30/02 Page: 1 of 1

 Untitled Report
TA Reported by Lteer: System Froject Name: ANUJ

SAMPLE INFORMATION

Sarrple Name: Dye Sue- 102 - 20ppm Acquired By: System

Sample Type: Uhknov/n Date Acquired: 9/16/02 2:04:54 PM
/tat 1 Acq. Method Set: DTE MTH
ijection#; 3 Date Rocessed: 9/30/02 4:24:52 PM
ijection Volume: 20.00 ul Rocessing Method: Default
?un Time: 90.0 Mnirtes Channel Name: VWh Ch1
ianpie Set Name: 3 Roc. Chnl. Descr.: PDA 640.1 nm

Auto-Scaled Chromatogram

Peak Results
Name RT Area Height Amount Units

1 13.397 43623 : 889

2 33.194 22136 751

3 34.926 26618 861

4 36.167 503915 16750

Msthod: Untitled Printed 4:25:22 PM 9/3Q/Q2 Page: 1 of 1

1 Reported by User: System Project Name: ANUJ
Untitled Report

SAMPLE INFORMATION

mple Name: Dye Blue-106 - 20 ppm Acquired By: System

irrple Type: Unknown Date Acquired: 9/16/02 3:16:54 PM
at 1 Acq. Method Set: DTE MTH
action#: 4 Date Rocessed: 9/30/02 4:28:09 PM
action Volume: 20.00 ul Processing Method: Default
nTime: 90.0 Mnutes Channel Name: VWIn Ch1
rrple Set Name: Proc. Chnl. Descr,: PDA 6417 nm

Auto-Scaled Chromatogram

Peak Results
Name RT Area Height Amount Units

1 19.684 546262 11184

Go
Method: Untitled R’inted 4:28:52 FM 9/30/02 Page: 1 of 1
 Untitled Report
A Reported by User: System Project Name: ANUU

SAMPLE INFORMATION

ample Name: Dye Blue-124 - 20 ppm Acquired By: System

ample Type: Unknown Date Acquired: 9/16/0212:51:13 PM
tat 1 Acq. Method Set: DTE MTU
jection#. 2 Date ftocessed: 9/30/02 4:20:08 PM
jectfon Volume: 20.00 ul Rocessing Method: Default
unTme: 90.0 Mnutes Chanrval Name: VWInChl
ample Set Name: Proc. Chni. Descr.: FDA 641.3 nm

Auto-Scaled Chromatogram

Peak Results
Name RT Area Height Amount Units

t 30.637 457324 15280

61
sort Method: Untitled Printed 4:20:34 PM 9/30/02 Page: 1 of 1
 Untitled Report
^ Reported by User: System Reject Name: ANUU

SAMPLE INFORMATION

ample Name: DYE STD Y-1-10 PPM Acquired By: System

ample Type: Unknown Date Acquired: 9/5/0212:32:27 PM
iat 1 Acq. Method Set: DYE MTH
jection#: 2 Date Rocessed: 9/30/0212:14:25 FM
jection Volume: 20.00 ul Rocessing Method: Default
unTime: 90.0 Mnutes Channel Name: VWh Ch1
ample Set Name: Roc. Chnl. Descr.: PDA 230.6 nm

Auto-Scaled Chromatogram

0.030

0.025

0.020

0.015-

0.010 h-

0.005- / \

0.000- ._J
A --

2.00 4.00 6.00 6.00 10.00 12.00 14.00 16.00 18.00 20.00
Minutes

Peak Results
Name RT Area Height Amount Units
1 10.774 183528 4897
___ i_______

oort Method: Untiled Printed 12:16:07 PM 9/30/02 Page: 1 of 1

 Untitled Report
a Reported by User: System Project Name: ANUJ

SAMPLE INFORMATION

airple Naira DYE STD Y-3- 10FPM Acquired By: System

ample Type: Unknown Date Acquired: 9/5/02 3:03:03 PM
at 1 Acq. Method Set: CYE_MTH
iection#: 3 Date Rocessed: 9/30/02 12:26:09 PM
ection Volume: 20.00 ul Rocessing Method: Default
jnTmne: 90.0 Mnutes Channel Name: VWIn Ch1
ample Set Name: Roc. Chnl. Descr.: PDA 253.4 nm

Auto-Scaled Chromatogram

Peak Results
Name RT Area Height Amount Units

1 22.357 271391 7491

QS>
t Method: Unfilled Printed 12:28:08 PM 9/30/02 Page: 1 of 1
 Untitled Report
Reported by U5er System Project Name; ANUU

SAMPLE INFORMATION

iatTpie Narre: DTE STD Y-910 PPM Acquired By: System

iampte Type: Unknown Date Acquired: 9/5/02 11:41:15 AM
fiat 1 Acq. Method Set: CYE MTH
ijection#: 1 Date Processed: 9/30/0212:33:06 FM
ijection Volume: 20.00 ul Rocessing Method: Default
fcjnTime: 90.0 Mnutes Channel Name: WVInChl
Sample Set Name: Roc. Chnl. Deser.: PDA 231.5 nm

Auto-Scaled Chromatogram

Peak Results
Name RT Area Height Amount Unite

1. 12.544 146048 4136

€4
sport Method: Untitled Printed 12:35:01 PM 9/30/02 Page: 1 of 1
 Untitled Report
Reported by User System Project Name: ANUJ

SAMPLE INFORMATION

Sample Name: DYESTDY-39-10 PPM Acquired By: System

Sample Type: Unknown Date Acquired: 9/5/02 210:18 PM
Viat 1 Acq, Method Set: DYE MTH
Injection#: 2 Date Rocessed: 9/30/0212:39:32 PM
hjection Volume: 20.00 ul Rocessing Method: Default
RunTime: 90.0 Mnutes Channel Name: VWin Ch2
Sample Set Name: Roc. Chnl. Descr.: FDA 231.2 nm

Auto-Scaled Chromatogram

Peak Results
Name RT Area Height Amount Units

i 19.366 122060 2601

2 24.173 106373 2723

G5~
ort Method: Uhtitied Fainted 12:49:23 PM 9/30/02 Page: 1 of 1
 Untitled Report
TA Reported by User: System Project Name: ANUL)

SAMPLE INFORMATION

Sample Name: DYESTDY-4910 FPM Acquired By: System

Sample Type: Unknown Date Acquired: 9/4/02 3:53:09 PM
i/iat 1 • Acq. Method Set: DTE MTU
Injection#: 3 Date R-ocessed: 9/30/G2 12:48:37 PM
Injection Volume: 20.00 ul Processing Method: Default
RunTime: 90.0 Mnutes Channel Name: VWh Ch1
Sample Set Name: Roc. Chnl. Descr.: PDA 457.0 nm

Auto-Scaled Chromatogram

Peak Results
Name RT Area Height Amount Units

1 28.259 502556 20326

£6
Ht Method: Untitled Printed 12:50:28 PM 9/30/02 Page: 1 of 1
 Untitled Report
Reported by User: System Project Name: ANUU

SAMPLE INFORMATION

Sample Name: dye red-120 ppm Acquired By: System

Sample Type: Unknown Date Acquired: 9/10/02 11:42:21 AM
/tat ,1 Acq. Method Set: DVE MTH •
njectton#: 2 Date Rocessed: 9/30/0212:54:42 PM
njection Volume: 20.00 ul Rocessing Method: Default
RunTime: 90.0 Mnutes Channel Name: VWInChl
Sample Set Name Roc. Chnl. Descr.: PDA 498.8 nm

Auto-Scaled Chromatogram

35.00 40.00 45.00 50.00

Minutes

Peak Results
Name RT Area Height Amount Units

1 26.353 2767133 80594

port Method: Untied Rimed 12:55:30 PM 9/30/02 Page: 1 of 1

1 Reported by User: System Reject Name: ANUJ
Untitled Report

SAMPLE INFORMATION

SfTpteName: dye red-11 20 ppm Acquired By: System

ample Type: Unknown Date Acquired: 9/10/02 2:12:28 PM
iat 1 Acq. Method Set: DYE MTH
lection#: i5 Date Rocessed: 9/30/021:00:36 PM
lection Volume: 20.00 ul Processing Method: Default
.in Tine: 90.0 Mnutes Channel Name: VWh Ch1
anrpte Set Name: ' Roc. Chnl. Descr.: FDA 257.8 nm

Auto-Scaled Chromatogram

0.010

0.008

0.006

0.004

0.002-

0.000

-0.002-
5.00 10.00 15.00 20.00 25.00 30.00
Minutes

Peak Results
Name RT Area Height Amount Units

1 10.028 321259 10250

G8
irt Method: Untitled Printed 1:03:37 PM 9/30/02 Page: 1 of 1
 Untitled Report
T* Reported ty User: System Project Name: ANLU

SAMPLE INFORMATION

SarrpleName: Dye red-17 20 ppm Acquired By: System

Sample Type: Standard Date Acquired: 9/11/0210:35:11 AM
Viat 1 Acq. Method Set: DTE MTH
Injection#. 1 Date R-ocessed: 9/30/021:02:54 PM
Injection Volume: 20.00 ul R-ocessing Method: Default
Run Tire: 80.0 Mnutes Channel Name: VWlnChl
Sample Set Name: Rroc. Chnl. Descr.: PDA 514.5 nm

Auto-Scaled Chromatogram

0.014 •*r

oo
0.012 'if

0.010

0.008

0.006

0.004

0.002-

o.ooo:

-0.002“

-0.004

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00

Minutes

Peak Results
Name RT Area Height Amount Units
1 14.814 728495 15832

6l
wrt Method: UnttJed R-inted 1:04:29 PM 9/30/02 Page- 1 of 1
 Untitled Report
A Reported by User:' System Roject Name: ANUJ

SAMPLE INFORMATION
Sample Name: Dye orange-1 20 ppm Acquired By: System
Sample Type: Standard Date Acquired: 9/11/0211:25:09 AM
i/iat 1 Acq, Method Set: DYE MTH
Injection#: 4 Date Rocessed: 9/30/021:11:17 PM
njectton Volume; 20,00 ul Rocessing Method: Default
RunTime: ’ 80.0 Mnutes Channel Name: VMnChl
Sample Set Name Roc. Chni. Descr.: FDA 466.1 nm

Auto-Scaled Chromatogram

Peak Results
Name RT Area ’ Height Amount Unite

1 37.422 961722 36596

__ _________________ JO_____________________
t Method: Untied Printed -i: 15:44 PM 9/30/02 Page: 1 of 1
 1*
____________________
Reported fay User: System Froject Name: ANUU
Untitled Report

SAMPLE INFORMATION

irrple Name: dye orange-3 20 ppm Acquired By: System

ample Type: Unknown Date Acquired: 9/10/022:51:01 FM
iat 1 Acq. Method Set: DTE MIH
jection#. 7 Date FTocessed: 9/30/021:08:11 PM
jecdon Volume: 20.00 ul Processing Method: Default
un Time: 90.0 Mnutes Channel Name: VWIn Ch1
ample Set Name: FToc. Chnl. Descr.: PCA 443.6 nm

Auto-Scated Chromatogram

Peak Results
Name RT Area Height Amount Units

1 20.493 1750851 37899

sport Method: Untitled Printed 1:14:32 PM 9/30/02 Page: 1 of 1

 Untitled Report
Reported by User: System Roject Name: ANUJ

SAMPLE INFORMATION

tanpieName: Dye orange-37 20 ppm Acquired By: System

larrpteType: Standard Date Acquired: 9/11/021254:35 FM
Tat 1 Acq. Method Set: DYE_MTH
tjection#: 5 Date Rocessed: 9/30/02 1:13:35 PM
ijection Volume: 20.00 ul Rocessing Method: Default
unTime: 80,0 Mnutes Channel Name: VWIn Ch1
ample Set Name: Roc. Chnl. Descr.: PDA 440.2 nm

Auto-Scaled Chromatogram

Peak Results
Name RT Area Height Amount Units

1 34.738 1155836 45648

thod: Unfitted Rrinted 1:16:47 FM 9/30/02 Page: 1 of 1