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Detection, Separation and Determination of Some Pesticides
SUMMARY
THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF
Doctor of Philosophy IN
CHEMISTRY
BY
H A S E E B A H M A D K H A N
DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY
ALIGARH (INDIA)
1989
SUMMARY
Today a large number of pesticides are being used for destroy
ing, repelling or reducing a wide range of pests in agricul
ture and public health. In our country, India, agriculture
is still the main source of livelihood and the pesticides
are amongst the most useful tools available to man in order
to get the crops properly grown and fruitfully harvested.
Besides other pesticides such as insecticides, rodenticides
fungicides etc., usage of herbicides and plant growth regula
tors is certainly \Jnavoidable for better crop yield. It is
reported that India is loosing annually Rs. 6000 crores worth
of agricultural production, of which weeds alone cause about
one third of the loss.
Herbicides are the chemical weed killers that provide
a more effective and economical means of weed control than
mechanical methods and are largely used for killing weeds
in numerous crops. Plant growth regulators are also respon
sible for better crop yield as they control growth, initiate
flowering, cause blossoms to fall, set fruit, cause fruit
and leaves to fall, control initiation and termination of
dormancy and stimulate root development.
It is certain that the world-wide crisis of food has
been considerably removed with the help of pesticides. On
the other hand, usage of pesticides in agriculture has imposed
many direct and indirect effects on the environment. The
concentration of pesticides in environmental components such
as air, water, flora and fauna is increasing day by day due
to their repeated and indiscriminate use, their surface run
off, accidental spills, fire accidents in pesticide warehouses
etc. Hence to meet the world-wide problem of environmental
protection and pollution control, it is necessary to identify
and estimate these pollutants in our ecosystem and then appro
priate measures be taken for their removal.
Several non-instrumental and instrumental techniques
are being used for pesticide residues analysis. Since instru
mental techniques are costly and sophisticated and require
skilled operators, therefore, either they are not available
or have not been installed on many laboratories of the third
world. Hence there is a growing interest in developing new,
simple and inexpensive techniques for pesticide residue analy
sis. In this thesis efforts have been made to develop tech
niques for detecting, separating and determining some carboxy-
lic herbicides and plant growth regulators. The results
obtained are summarised in four chapters.
A sensitive and selective novel technique has been
developed for the detection and semiquantitative determination
of phenoxy herbicides. The herbicide is heated under reduced
pressure in presence of hydrated zinc sulphate to split off
formaldehyde. The vapours of formaldehyde bubble into the
reagent (one or two tiny crystals of chromotropic acid sodium
salt in 0.20 mL of concentrated sulphuric acid). The sensiti
vity and selectivity of the test has been improved by
(i) using ZnS0,.7H„0 as a hydrolysing agent in place of conc
entrated sulphuric acid (ii) designing of a new vacuum spot-
test apparatus (Figure 1) and (iii) utilization of preferen
tial solubilities of phenoxy herbicides in different solvents.
The detection of phenoxy herbicides by this test is not
affected by the presence of non-volatile coloured compounds
because they are left behind in the reaction theatre
(tube A). The test gives positive response for 2,4-D acid
(LV), 2,A-D ethyl ester (LV), 2,4-D sodium salt (LV), 4-chlo-
rophenoxyacetic acid (V), phenoxyacetic acid (V) and 2,4,5-T
(V) with lower limit of detection 5,4,4,15,5 and 15 |j,g respec
tively. Besides phenoxy herbicides it also responses to
dichlorovos (V), formothion (V), malathion (V), thiometon
(V), N-bromosuccinimide (LBr), p-dimethylaminobenzaldehyde
(V) and hexamine (V) with their lower limit of detection
38,19,32,30,375,70 and 0.5 pg respectively. As 2,4-D sodium
salt is insoluble in benzene, it gives no response and traces
of 2,4-D acid and 2,4-D ethyl ester can be detected in
2,4-D sodium salt matrix by the use of benzene. However,
2,4-D sodium salt can be detected by preparing the test solu
tions in ethanol, water etc. In benzene medium the test can
NOTE : Abbreviations used are L = light, V = violet and
Br = brown
TO \acuu5ter pump
Stopper
sample zinc sulphate
Note — All dimensions are in mm
Figure 1 Apparatus for micro analysis of herbicides in flora, soil ana water.
be applied for detection and semi-quantitative determination
of free acid (2,4-D) generated by the hydrolysis of 2,4-D
sodium salt or 2,4-D ethyl ester in inorganics rich matrices
such as soil and water. This test has also been used for
the detection of 2,4-D acid in leaves, soil and water with
lower limit of detection 100, 25 and 10 p,g respectively.
The results discussed above indicate successful applicability
of the newly designed apparatus for the preliminary detection
of pesticides based pollutants.
TLC is widely used for the qualitative analysis as
well as for the specific, sensitive, precise and rapid quanti
fication of a wide variety of compounds. Analytical potential
of various coatings has been studied by many workers. In
this laboratory silica gel G plates have been tested with
a number of solvent systems for the separation of several
plant carboxylic acids such as cinnamic, citric, indole-3-
acetic, maleic, malic, malonic, p-naphthaleneacetic, p-naphth-
oxyacetic, oxalic, phenoxyacetic, salicylic and tartaric
acids. Silica gel plates impregnated with inorganic salts,
zinc oxide, detergent and grease were also studied. In most
cases the acids remain at the point of application or give
bad tailing due to which separations are not feasible. Hence
search of new coating materials for TLC was badly needed.
The previous work showed that calcium sulphate is a good
material for separating carboxylic acids. Barium sulphate
is more insoluble than calcium sulphate; it is a non-toxic,
cheap and chemically inert material that can also be used
as an adsorbent. Now we have tested barium sulphate and the
results show its advantages as well as disadvantages. Barium
sulphate coatings of 0.25-1.00 mm thickness were found to
be full of cracks after drying at 110° for 1 hr and the coat
ing was separated from the plate on dipping it in the solvent
for development. Thus the coatings of barium sulphate alone
are not suitable for TLC. Coatings of admixtures of barium
sulphate and calcium sulphate are however very stable and
give good TLC separations. Most of the acids on mixed coatings
move as compact spots while on calcium sulphate alone about
30% of the acids tail. Therefore many separations can be succe
ssfully achieved on mixed coatings. For example P-naphthalene-
acetic acid (2-4) from citric acid (0.0), indole-3-acetic
acid (0.0), maleic acid (0.0), malonic acid (0.0), oxalic
acid (0.0) and tartaric acid (0.0) on BaSO^/CaSO^ (50:50)
coating in carbon tetrachloride; malonic acid (0.0), maleic
acid (0.0), citric acid (0.0), oxalic acid (0.0) and tartaric
acid (0.0) from cinnamic acid (3-6.), P-naphthaleneacetic
acid (0.62) and P-naphthoxyacetic acid (0.65) on BaS0,/CaS0,
(30:70) coating in chloroform; cinnamic acid (0.0), j3-naphtha-
leneacetic acid (0.0) and p-naphthoxyacetic acid (0.0) from
citric acid (1.0), maleic acid (1.0), malic acid (1.0),
malonic acid (1.0), oxalic acid (1.0) and tartaric acid (1.0)
on BaSO^/CaSO^ coatings (containing BaSO, 0,30,50,70,80
f values are given in parentheses
or 90%) in distilled water; phenoxyacetic acid (0,8) from
cinnamic acid (0.0), indole-3-acetic acid (0.0), P-naphthale-
neacetic acid (0,0) and ^-naphthoxyacetic acid (0.0) on
BaSO,/CaSO, (30:70) coating in distilled water; citric acid
(0.0), oxalic acid (0.0) and tartaric acid (0.0) from cinnamic
acid (1.0), indole-3-acetic acid (1.0), maleic acid (1.0),
malic acid (1.0) malonic acid (1.0), |3-naphthaleneacetic
acid (1.0), p-naphthoxyacetic acid (1.0), phenoxyacetic acid
(1.0) and salicylic acid (1.0) on either BaSO^/CaSO^ (50:50)
coating in ethyl acetate or on BaSO,/CaSO, (30:70) coating
in propanol. The synthetic auxin, p-naphthaleneacetic acid,
which is generally used for the control of preharvest fruit
drop has been quantitatively separated from oxalic acid.
The percentage error of the volumetric method used for the
quantification has been found in the range -6.43% to +1.90%.
The TLC on admixtures of BaS0,/CaS0, containing more than
50% BaSO, becomes a time consuming process since the ascension
time increases with an increase in percentage of barium
sulphate. The above work shows that layers of BaS0,/CaS0,
have good analytical potential for several plant growth
regulators and carboxylic acids of importance in citrus fruit
industry. These coatings may also be suitable for the analysis
of acid pesticides.
NOTE : R^ values are given in parentheses,
8
As discussed above, BaSO,/CaSO, is a good TLC coating
material and in simple solvent systems it can be used for
separating agro-carboxylie compounds in binary mixtures.
Now it has been found that when BaSO,/CaSO, coating is impreg
nated with coconut oil, test materials move as compact and
symmetrical spots that result in many quaternary and ternary
separations. For example, 4-chlorophenoxyacetic acid,
2,4,5-trichlorophenoxyacetic acid, p-naphthoxyacetic acid,
indole-3-acetic acid and indole-3-propionic a c i d tail 0-10
on a BaSO,/CaSO, (40:40) coating while their movement is
0-2, 0.0, 0.0, 0-4 and 0-2 respectively on a BaSO^/CaSO^
(40:40) coating impregnated with 15 mL of coconut oil in
distilled water. Chromatographic behaviour of 4-chlorophenoxy
acetic acid and indole-3-acetic acid shows the decreasing
size of their spots with the increasing concentration of
coconut oil, i.e. their movements are 0-10, 2-5, 2-4 and
0.16 and 0-10, 2-7, 0-7 and 0.35 on BaSO^/CaSO^ (40:40) coat
ings containing coconut oil, 0,5,10 and 15 mL respectively
in tap water. R^ values of 2,4-D and benzoic acid are 0.7 and
5-8 on BaSO,/CaSO, coating while they are 0-1 and 0.25 respec
tively on coating impregnated with 15 mL of coconut oil. The
ternary and quaternary separations achieved are shown in
Figures 2,3 and 4. These chromatograms prove that the system
is specific for the separation of trichloroacetic acid (TCA)
from complex mixtures because it has R^ value 1.0 on all
the coatings and solvents under study. TCA has been separated
c m
10
8
G
U
2
0-
10
o
1
12
o
10
o
1
0 3
10
O
1
0
7
Q
10
O
1
0 8
0
11 o
1
0
12
6*
11 O
b 5
0
11
1
0 7
0
11
1
0 6
0
Figure 2 Separation of some herbicides on coating B in DW 1 = B0A,3=CIA . 7 = cC-NTAA.8=p-NTAA; 10=PAA; 11 = TCA and 12 = 2 . ^ . 5 - 1 .
C m
1 0 -8-6-
u-2-0-
10
o
6
0 12
0
11
o
6
0 12
u
10
0
6
0 7
(/
11
o 6
0 7
y
c m
10-8 6 -
U-
2
0
(a) (b)
Figure 3 Separation of some herbicides (a) On coating B in DW and (b) on coating C in T W
6 = IPA, 7=oc-NTAA,8=/3-NTAAJO = PAA
11 = TCA and 12 = 2,^,5-7.
11
0 10
0 6
(? ^ 2 |]
11
Q 0 6
0 7
0
11
0 10
O 6
0 8
0
cm
10-8-
4-
2-0-
11 o 10
0 2
0
11
O 10
0
4
11 o 10 o
12
0
11
O 10
Cb 1 o
11
O 10
Q)
3
0
11
O 10
0
9
0
11
O
10
^
7
0
11
O
10
0
8
0
11 o 10
^
6 Q
Figure 4 Separation of some herbicides on coating D in DW
1 = B0A,2=CPAA, 3 = CIA, 4=2 ,4 -D , 6=IPA, 7=oc-NTAA, 8 = Q-NTAA^9 = NXAA,10 = PAA, 11=TCA and 12=2,4,5-1.
12
quantitatively from benzoic acid, 2,4,5-T, indole-3-propionic
acid and a-naphthaleneacetic acid. The lower limit of the
spectrophotometric method used is 400 jjig with a percent
error of -4.13 to -25.00% and coefficient of variation 0.00
to 5.26%. Therefore for analysis below 400 |ig of TCA another
spectrophotometric method must be used. These results show
that several carboxylic herbicides and plant growth regulators
which could not be separated on normal-phase layers of calcium
sulphate alone or admixtures of calcium sulphate and barium
sulphate, can be separated and determined by RP-TLC on BaSO,/
CaSO, layers impregnated with coconut oil. Hence these layers
seem to be very useful for the analysis of complex mixtures
of commonly used carboxylic herbicides.
The normal-phase layers of BaSO,/CaSO, have been found
to be useful for separating many plant carboxylic acids in
single solvent systems. But in case of chlorophenoxy herbici
des, the separation potential is limited due to tailing
nature of the herbicides in some of the solvents such as
benzene, carbon tetrachloride and chloroform. However, compact
spots with R^ value 1.0 were obtained in dioxane, ethyl
acetate and propanol and some binary separations were achieved
in distilled water. The utility of reversed-phase layers
of BaS0,/CaS0, impregnated with coconut oil for analysing
complex mixtures has already been reported. However, separa
tion potential of Bas0,/CaS0, coatings in mixed solvent
systems has not been tested so far. Now such an attempt has
13
been made and the results obtained are discussed here. The
following separations which are not possible on BaSO,/CaSO,
as well as on BaSO./CaSO, impregnated with coconut oil in
single solvent systems, could be achieved in mixed solvent
systems: benzoic acid from 4-chlorophenoxyacetic acid; indole-
3-acetic acid from benzoic acid, cinnamic acid, 4-chloropheno
xyacetic acid, 2,4-D, a-naphthaleneacetic acid, p-naphthalene-
acetic acid, p-naphthoxyacetic acid and 2,4,5-T; P-naphthale-
neacetic acid from a-naphthaleneacetic acid and p-naphthoxy-
acetic acid; indole-3-propionic acid from benzoic acid, cinna
mic acid and p-naphthoxyacetic acid. Many other separations
have also been achieved. Indole-3-acetic acid has been separa
ted quantitatively from benzoic acid, a-naphthaleneacetic
acid and 2,4,5-T in carbon tetrachloride-propanol (100:0.5)
solvent system. The coefficient of variation for four obser
vations is found in the range of 2.36-9.54%.
CONCLUSION
On the basis of above discussion it is concluded that the
techniques developed viz (i) vapour-phase spot-test for the
detection and semiquantitative determination of phenoxy herbi
cides, (ii) normal-phase TLC on BaSO./CaSO, in simple solvent
systems, (iii) reversed-phase TLC on BaSO,/CaSO, impregnated
with coconut oil in aqueous solvents and (iv) normal-phase
TLC on BaSO,/CaSO, in mixed solvent systems, can be used
successfully for the detection, separation and determination
14
of several carboxylic herbicides and plant growth regulators.
Being simple and inexpensive, these techniques may be extre
mely useful, especially at the places lacking the facility
of sophisticated instrumentation.
Detection, Separation and Determination of Some Pesticides
THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF
Doctor of Philosophy IN
CHEMISTRY
BY
HASEEB AHMAD KHAN
DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY
ALIQARH (INDIA)
1989
T3821
DIZPARTMENT OF APPLIED CHEMISTRY
Phone : Onicc : 6042
ZAKIR HUSAIN COLLtGC OF IINGG. 0 TECHNOLOGY
ALIOAKll MUSLIM UNIVLUSIIV AIJUAKH—202002 (INDIA)
Rcf. No. Dated. T>^tu^, /9^f
••'•^- ^..
CERTIFICATE
This is to certify that the thesis entitled,
'Detection, Separation and Determination of Some
Pesticides' submitted for the award of the degree
of Doctor of Philosophy in Chemistry, is a faithful
record of Llie bonafide research work carried out at
the Department of Applied Chemistry, Z.H. College
of Engineering & Technology, Aligarh Muslim University,
Aligarh, by Mr. Haseeb Ahmad Khan under my guidance
and supervision and that no part of it has been
submitted for any other degree or diploma.
' i v - < • ' **-(Dr. U.S. Rathore)
Reader
ACKNOWLEDGEMENTS
I feel pleasure to express my deep sense of gratitude to my
supervisor of research. Dr. H.S. Rathore, whose inspiring
guidance and continuous interest in my research work enabled
me to complete and present it in the form of this thesis.
I wish to thank Dr. Ishtiaq Ali, Reader, Department of
Applied Chemistry for his active cooperation and help from
time to time.
I place on record my sincere thanks to the Chairman,
Prof. K.T. Nasim and Ex-Chairman, Prof. A.U. Malik of the
Department of Applied Chemistry, for providing necessary
research facilities.
My colleagues, Dr.(Mrs) Sudha Gupta, Mr. S.K. Saxena,
Mr. Anup Kumar, Miss Tahira Begum and Miss Rachna Sharma,
rendered timely assistance and help whenever required for
which I am obliged to all of them. Financial support provided
by the University Grants Commission, New Delhi and the award
of a Senior Research Fellowship by the Council of Scientific
and Industrial Research, New Delhi,is gratefully acknowledged.
Last but not the least I find myself short of words
to express my indebtedness to my parents, elder brother,
Dr.(Mrs) Najma Takreemullah, Dr. Mujahid A. Khan, Dr. Asif A.
Khan and my friends, for their sustained inspiration and
encouragement.
]^/HM^ Haseeb Ahmad Khan
C O N T E N T S
LIST OF ABBREVIATIONS
LIST OF PUBLICATIONS
LIST OF TABLES
LIST OF FIGURES
ABSTRACT
CHAPTER 1 GENERAL INTRODUCTION
1.1 Introduction
1.2 Herbicides and Plant Growth Regulators
1.3 Analysis of Pesticides
1.3.1 Spot-Test Methods
1.3.2 Volumetric Methods
1.3.3 Biological Methods
1.3.4 Chromatographic Methods
1.3.4.1 Column Chromatography
1.3.4.2 Paper Chromatography
1.3.4.3 Liquid-Liquid Chromatography
1.3.4.4 Thin-Layer Chromatography
1.3.4.5 Gas Chromatography
1.3.4.6 High Performance Liquid-Chromatography
1.3.5 Spectrophotometric Methods
1.3.5.1 UV and Visible Spectrophotometry
1.3.5.2 Densitometry
1.3.5.3 Fluorimetry and Phosphorimetry
1.3.6 Miscellaneous Techniques
CHAPTER 2 A NOVEL METHOD FOR THE DETECTION AND SEMIQUANTITATIVE DETERMINATION OF TRACE LEVELS OF 2,4-D AND RELATED COMPOUNDS
2.1 Introduction
2.2 Experimental
(i)
(iii)
(iv)
(V)
(vi)
2
2
15
16
18
19
23
23
25
26
28
34
36
37
37
40
40
41
51
60
2.3 Results 65
2.4 Discussion 65
2.5 Conclusion 79
CHAPTER 3 CHARACTERIZATION OF BARIUM SULPHATE AS A TLC MATERIAL FOR THE SEPARATION OF PLANT CARBOXYLIC ACIDS
3.1 Introduction 82
3.2 Experimental 82
3.3 Results 87
3.4 Discussion 87
3.5 Conclusion 102
CHAPTER 4 QUANTITATIVE SEPARATION OF TRICHLOROACETIC ACID FROM SOME CARBOXYLIC HERBICIDES ON REVERSED PHASE LAYERS OP BaSO^/CaSO^ IMPREGNATED WITH COCONUT OIL
4.1 Introduction 105
4.2 Experimental 108
4.3 Results 111
4.4 Discussion 112
4.5 Conclusion 119
CHAPTER 5 NORMAL PHASE THIN-LAYER CHROMATOGRAPHY OF SOME HERBICIDES AND RELATED COMPOUNDS ON ADMIXTURES OF BARIUM SULPHATE AND CALCIUM SULPHATE IN MIXED SOLVENTS
5.1 Introduction 122
5.2 Experimental 122
5.3 Results 125
5.4 Discussion 125
5.5 Conclusion 132
LIST OF ABBREVIATIONS
AcOH
BOA
Bu
BuOAC
CA
CIA
CPAA
2,4-D
p-DAB
DW
Et
Et20
EtOAc
EtOH
lAA
I PA
MA
Me
MeCN
Me 2 CO
MeOH
MEA
MOA
a-NTAA
P-NTAA
Acetic acid
Benzoic acid
Butyl
Butyl acetate
Citric acid
Cinnamic acid
4-Chlorophenoxyacetic acid
2,4-Dichlorophenoxyacetic acid
p-Dimethylaminobenzaldehyde
Distilled water
Ethyl
Diethyl ether
Ethyl acetate
Ethanol
Indole-3-acetic acid
Indole-3-propionic acid
Malic acid
Methyl
Methyl cyanide
Acetone
Methanol
Maleic acid
Malonic acid
a-Naphthaleneacetic acid
p-Naphthaleneacetic acid
(i)
NXAA p-Naphthoxyacetic acid
OA Oxalic acid
PAA Phenoxyacetic acid
RW River water
SA Salicylic acid
2,4,5-T 2,4,5-Trichlorophenoxyacetic acid
TA Tartaric acid
TCA Trichloroacetic acid
TW Tap water
l±±}
LIST OF PUBLICATIONS
1. Characterization of barium sulphate as a TLC material
for the separation of plant carboxylic acids, Chromato-
graphia, 23(6), 432-434 (1987).
2. Quantitative chromatographic separation of trichloroacetic
acid from some carboxylic herbicides on BaSO. — CaSO.
coatings impregnated with coconut oil, J. Planar
Chromatogr,, 1(3), 252-254 (1988).
3. Plain thin-layer chromatography of some herbicides and
related compounds on admixture of barium sulphate and
calcium sulphate in mixed solvents, J. Liq. Chromatogr.,
11(15), 3171-3181 (1988).
4. A novel method for the detection and semiquantitative
determination of trace levels of 2,4-D and related
compounds. Water Res. (1989). In Press.
(ill)
LIST OF TABLES
Table 1.1 Some pesticides, their action and applications.
Table 2.1 Study of the test under different conditions.
Table 2.2 Effect of different organics and inorganics on the detection of 2,4-D acid.
Table 2.3 Detection of some formaldehyde producing compounds in different solvents at 180°.
Table 2.4 Detection and semiquantitative determination of 2,4-D acid in soil.
Table 2.5 Detection and semiquantitative determination of 2,4-D acid in vegetation.
Table 3.1 Coatings Studied.
Table 3.2 Separations achieved experimentally in carbon tetrachloride.
Table 3.3 Separations achieved experimentally in chloroform.
Table 3.4 Separations achieved experimentally in distilled water.
Table 3.5 Separations achieved experimentally in ethyl acetate.
Table 3.6 Separations achieved experimentally in propanol.
Table 3.7 Quantitative separation of p-naphthaleneacetic acid from oxalic acid on coatings B in ethyl acetate.
Table 4.1 Coatings studied.
Table 4.2 Quantitative separation and determination of TCA on coating B in distilled water.
Table 5.1 Separations achieved on BaSO./CaSO.(1:1) coatings in different solvent systems.
Table 5.2 Quantitative separation and determination of lAA using carbon tetrachloride-propanol (100:0.5) as solvent system.
(iv)
LIST OF FIGURES
Figure 2.1 Mechanism of colour reaction of formaldehyde with chromotropic acid in cone. H_SO..
Figure 2.2 Production of formaldehyde from 2,4-D in presence of either cone. H^SO. or hydrated sulphates of Zn and Mn.
Figure 2.3 Oxidative cleavage with molten benzoyl peroxide to give formaldehyde.
Figure 2.4 Splitting of methylene ethers of o-diphenols to give formaldehyde.
Figure 2.5 Production of formaldehyde from monochloro-acetic acid and glycolic acid with cone. H^SO..
Figure 2.6 Use of alkalihypohalogenite or chloramine T to split out formaldehyde from a-amino acids.
Figure 2.7 Apparatus for micro analysis of herbicides in flora, soil and water.
Figure 3.1 Chemical structures of plant growth regulators separated on admixtures of barium sulphate and calcium sulphate.
Figure 3.2 Plot of time of developing the plates in different solvents vs pei in (BaSO. + CaSO.) coatings. different solvents vs percentage of BaSO.
Figure 4.1 Chemical structures of herbicides and plant growth regulators separated on BaSO./CaSO. layers impregnated with coconut oil.
Figure 4.2 Separation of some herbicides on coating B in DW.
Figure 4.3 Separation of some herbicides (a) on coating B in DW and (b) on coating C in TW.
Figure 4.4 Separation of some herbicides on coating D in DW.
(v)
ABSTRACT
The pesticides are being commonly used for pest control in
agriculture, forestry and public health. In agriculture,
an increase in production is directly linked with a reduction
in losses due to adverse biological factors, such as insects,
weeds, fungi and nematodes, which are controlled effectively
by using pesticides. Indiscriminate and frequent use of
pesticides on the other hand, has many adverse effects on
the environment and their concentration is increasing day
by day in environmental components such as air, water, vegeta
tion, soil and marine animals. Hence it is most important
to estimate these pollutants in our ecosystem.
Many instrumental and non-instrumental methods are
being used for the analysis of pesticides. Since instrumental
methods are costly and sophisticated there has been a growing
need for developing simple and inexpensive methods that can
be used successfully in the laboratories of the third world.
The research work presented in this thesis is related to
the detection, separation and determination of some
carboxylic herbicides and plant growth regulators.
In chapter 1 an attempt has been made to describe
various types of pesticides, their applications and recently
reported methods of their analysis. In chapter 2 a sensitive
and selective vapour-phase spot-test for the detection and
semiquantitative determination of phenoxy herbicides is
(vi)
discussed. This technique has also been used successfully
for the detection of traces of 2,4-D acid, 2,4-D ethyl ester
and 2,4-D sodium salt in formulations, leaves, soil and water.
In the next three chapters, thin-layer chromatography of
some herbicides, plant growth regulators and carboxylic acids
of importance in fruit growing industry has been described.
Since thin layers of silica gel for TLC analysis of
carboxylic herbicides were found to be of limited use, search
for new coating materials was badly needed. In chapter 3
the merits and demerits of BaSO./CaSO. admixtures as a TLC
coating material is reported. The synthetic auxin, p-naphtha-
leneacetic acid has been quantitatively separated from oxalic
acid on these coatings. For analysing complex mixtures, RP-
TLC on BaSO./CaSO. coatings impregnated with coconut oil
has been found to be more useful as discussed in chapter 4.
Trichloroacetic acid (TCA) has been separated from benzoic
acid, 2,4,5-T, indole-3-propionic acid and a-naphthaleneacetic
acid on reversed- phase layers and determined spectrophoto-
metrically. Chapter 5 describes the utility of mixed solvent
systems. Many important separations that are not possible
either by NP-TLC or RP-TLC in single solvents, can be achieved
successfully by NP-TLC in mixed solvent systems. This chapter
also reports the quantitative separation of indole-3-acetic
acid (lAA) from benzoic acid, 2,4,5-T and a-naphthaleneacetic
acid.
(vii)
CHAPTER 1
GENERAL INTRODUCTION
1.1 INTRODUCTION
At present nearly 1000 inorganic and organic compounds in
the form of about 10,000 formulations are used for destroy
ing, repelling or reducing a wide range of pests in agricul
ture and public health (1). In India agriculture is still
the main source of livelihood and the pesticides are amongst
the most useful tools available to man in order to get the
crops properly grown and fruitfully harvested.
The pesticides applied to kill or control insects,
nematodes, mites, molluscs, rats, fungi and weeds are known
as insecticides, nematicides, acaricides, molluscicides,
rodenticides, fungicides and herbicides respectively. Some
of the pesticides, their action and applications (2,3) are
summarized in Table 1.1.
1.2 HERBICIDES AND PLANT GROWTH REGULATORS
Herbicides are the chemicals used for killing weeds. They
have largely replaced (2) mechanical methods of weed control
in the past AO years. Herbicides provide a more effective
and economical means of weed control than mechanical methods
such as cultivation, hoeing and hand pulling. It would have
been impossible to mechanise fully the production of cotton,
sugar beets, grains, potatoes, corn etc. without using pesti
cides. There are other locations such as industrial sites,
roadsides, ditch banks, irrigation canals, fence lines,
recreational area, railroad embankments and power lines
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14
where herbicides are used extensively. Herbicides are classed
as selective when they are used to kill weeds without harming
the crop and as nonselective when the purpose is to kill
all vegetation. Both types of herbicides can be applied
to weed foliage or to soil containing weed seeds and seed
lings, depending on the mode of action. Each herbicide
affects the plant either by contact or translocation. Contact
herbicides kill the plant parts to which the chemical is
applied and are most effective against annuals. Complete
coverage is essential in weed control with contact herbi
cides. Translocated herbicides are absorbed either by roots
or above ground parts of plants and then moved within the
plant system to distant tissues. Their greatest advantage
is to control established perennials. For translocation
materials an uniform application is needed and complete
coverage is not required.
Plant growth regulators are the chemicals used to
alter the growth of plants, blossoms, or fruits and are
considered pesticides (2). They are also referred to as
plant regulating substances, growth regulants, plant hormones
and plant regulators. Hormones are the natural substances
produced by the plants that control growth, initiate flower
ing, cause blossoms to fall, set fruit, cause fruit and
leaves to fall, control initiation and termination of dorman
cy and stimulate root development.
Amongst different pesticides, carboxylic compounds
15
(Table 1.1), are largely used as plant growth regulators
as well as herbicides for better yield in a number of crops
and for killing total vegetation. Indole-3-acetic acid (lAA)
is a commonly occuring natural auxin while indolepropionic
acid and indolebutyric acid occur in a few species. Synthetic
auxins such as naphthaleneacetic acid, indolebutyric acid
and ^-chlorophenoxyace tic acid have been found to be more
effective than natural auxins (A). 2,4-D; 2,4,5-T and MCPA
are the best known auxin type herbicides used selectively
to kill most dicotyledonous (broad-leaf) plants in adequate
concentrations while they are comparatively ineffective
on most monocotyledenous plants.
1.3 ANALYSIS OF PESTICIDES
Beyond any doubt the world-wide crisis of food has been
considerably removed with the help of pesticides. On the
other hand, usage of pesticides in agriculture has imposed
many direct and indirect undesirable effects on the environ
ment i.e. chemical plant protection process is a profit-
induced poisoning of the environment.
The concentration of pesticides in environmental
components such as air, water, flora and fauna is increasing
day by day due to their repeated and indiscriminate use,
their accidental spills, land run-off, fires in pesticide
warehouses etc. Hence to meet the worldwide problem of envi
ronmental protection and pollution control, it is necessary
to identify and measure these pollutants in our ecosystem
16
and then proper steps be taken for their removal.
Several instrumental and non-instrumental methods
have been used for the detection, separation and determina
tion of pesticide residues in different environmental
matrices. The recently reported papers in this area are
summarized below :
1.3.1 SPOT-TEST METHODS
Spot tests are extremely useful for the preliminary charac
terization of a test material. These tests are simple,
sensitive, selective and rapid and can be applied directly
(solution phase) or indirectly (vapour phase). Therefore
different characteristics of the test materials and proper
set of conditions have been utilized in order to bring the
sensitivity, selectivity and specificity of the particular
test to a maximum.
A simple and inexpensive spot test (5) for the detec
tion of organic pollutants in water has been developed.
Citric acid impregnated paper in the presence of acetic
anhydride has been used as the reagent. The limits of identi
fication for the pesticides: amitrole, azobenzene, bavistin
and calixin have been found to be 0.04, 40.00, 2.00 and
3.20 yg respectively. A new specific, colorimetric spot
test (6) for the detection of malathion residues in water
has been developed. Activated charcoal is used to recover
and concentrate malathion from water samples. Ethanolic
malathion solution -is hydrolysed with potassium hydroxide
17
to give potassium fumarate which gives a red colour on heat
ing with acetic anhydride. The lower limit of detection is found
to be 1 mg/L.
A new technique, pressure capillary spot-test (7) has
been developed for the detection and determination of pollutants
including plant growth regulators lAA and gallic acid with their
lower limit of detection as 0.1 and 50 \ig respectively. A capi
llary containing cotton plug impregnated with p-dimethylamino-
benzaldehyde and trichloroacetic acid is used for colour develo
pment. This technique has been used for the semiquantitative
determination of lAA in wheat shoots. The above technique has
also been utilized (8) for the detection of trace levels of
organophosphorus insecticides, such as malathion, formothion,
thiometon, dichlorvos, methyl parathion, dimethoate and phos-
phamidon in water, soil and citrus leaves. The lower limit of
detection for the above pesticides in water has been found to
be 5,25,25,8,5,30 and 9 mg/L respectively. A fluorescence spot-
test (9) for the selective detection of traces of TCA in soil
and water samples has been developed. The test is based on the
formation of salicylaldazine of yellow-greenish fluorescence
from TCA. The test has been found to be useful for detecting
persistency of TCA in soil.
Two specific spray reagents for the detection of
carbaryl by thin-layer chromatography have been reported
(10). They are 1% m/v CuCl^ followed by 0.1% ammonium meta
18
vanadate and 0.5% m/v potassium hexacyanoferrate (III) in
0.5% m/v NaOH. The former reagent has relatively higher
sensitivity for the breakdown product of carbaryl, 1-naphthol^
whereas the later has the same sensitivity for both carbaryl
and 1-naphthol (1 jig).
1.3.2 VOLUMETRIC METHODS
Volumetric methods involve use of a chemical reaction between
substance to be determined (analyte) and another substance
used as titrant. Being simple and inexpensive it is also
used in analysing pesticides especially at the places where
sophisticated instruments are not available.
A volumetric method (11) for the determination of
malathion in emulsifiable concentrates has been developed.
Alkali cleavage of malathion gives 0,0-dimethyIphosphorodi-
thioic acid that forms a complex with Bi (III). The complex
is extracted with chloroform and the bismuth left uncomplexed
is titrated with the sodium salt of EDTA. Any free
0,0-dimethyIphosphorodithioic acid, that may be present,
interferes with the determination. This is rectified by
the complex formation without hydrolysing the sample. For
the determination of malathion one more simple, sensitive
and safe volumetric method has been reported (12). In this
method potassium permanganate in alkaline medium has been
used as an oxidizing agent. The lower limit of determination
has been found to be 0.1 mg of malathion. Quantitative
separation (13) of j3-naphthaleneacetic acid from oxalic
19
acid has been carried out. The acids are separated on TLC
plates coated with BaSO,/CaSO, (30:70) in ethyl acetate
developer. p-Naphthaleneacetic acid is eluted with 25 mL of
hot 2-propanol from the material scratched off the plate
while oxalic acid is rejinoved by 25 mL of hot distilled water.
Both the solutions are made up to 50 mL in 50% 2-propanol
by adding water or propanol and then titrating with standard
alkali and phenolphthalein indicator. A fast method (14)
based on argentometric titration has been developed for the
determination of TCA directly on the farms or in mobile
labs. An aliquot of the sample solution containing NaTCA
is treated with a 5-10 fold amount of 30% NaOH and the
mixture is heated for 2 hr using a reflux condenser. After
cooling dil HNO„ and AgNOo are added to the solution and
the excess is titrated with ammonium thiocyanate in presence
of ferroammonium aluminosilicates until a stable pink-
cinnamonic colour develops. A modified micro-Kjeldahl disti
llation method (15) has been developed for the determination
of secondary carbamate pesticides in technical materials
and in formulations. Methylamine, released on alkaline
hydrolysis of the sample, is steam distilled and absorbed
in boric acid solution which is titrated with HCl using
bromocresol green as indicator. The limit of detection is
found to be 0.02 m mole of active ingredient.
1.3.3 BIOLOGICAL METHODS
The toxically significant residues of pesticides of certain
classes which cause inhibition of the enzyme cholinesterase
20
have been analysed by biological methods. As it is possible
to apply these methods without clean-up, they are suitable
as screening tests but are non-specific. The techniques
based on enzyme - inhibition are simple, inexpensive, ultra
sensitive and rapid (16). These include enzyme inhibition
electrodes, enzymatic-chromatography and enzymatic-col-
oriraetry.
Guilbault and Kramer (17) have prepared a column
of immobilized horse serum cholinesterase in a flow system
and developed an assay of organophosphorus pesticides. The
fluorescence due to hydrolysis of a fluorescein or resorufin
ester by the cholinesterase is monitored. When the enzyme
is inhibited, no hydrolysis occurs and the fluorescence
is no longer produced. As little as 1 ppb of parathion,
systox or malathion can be assayed. A system for the
detection of pesticides (in ppb range) in air and water has
been described by Goodson et al. (18,19). The cholinesterase
is adsorbed on aluminium hydroxide gel during the precipita
tion of the aluminium hydroxide. The adsorbed enzyme is
suspended in a starch slurry and applied to a polyurethane
foam. For water assay a separate line applies sample to
the pad. In a second cycle the air and substrate buffer
solutions are passed through, and the presence of inhibitors
is noted by a change in the potential of the system. A device
is marked by Midwest Research on this system, called CAM
(Cholinesterase Antagonist Monitor), and is commercially
available for air and water monitoring.
21
An enzyme immunoassay for the determination of
atrazine in water and soil has been described by Bushway
et al. (20). An atrazine antiserum is prepared by derivati-
zing atrazine at the 2-chloro position and covalently conju
gating it to bovine gamma globulin using a modified
carbodiimide crosslinking procedure. The coefficients of
variation for atrazine in water samples at atrazine concen
tration of 4-50 ppb are reported to be 10.0 - 4.1% and for
atrazine in soil samples at 4-40 ppm are 16.3-8.4%.
El Yamani et al. (21) have developed automated systems based
on the use of a butyr ylcholinesterase electrode for the
detection of organophosphorus and carbamate pesticides at
very low (ppb) concentrations. This technique has been
utilized for the routine monitoring of river water pollution.
Schwedt et al. (22) have used reactivatable enzyme electrode
with acetylcholinesterase for the differential detection
of insecticides in trace range. Acetylcholinesterase is
immobilized in a matrix of bovine albumin by crosslinking
with glutaraldehyde directing at the surface area of pH
electrode. A reversible inhibition by methyl carbamate
insecticides is detectable from 0.1 mg/L (aldicarb) and
irreversible inhibition is detectable by phosphoric acid
ester insecticides from 50 Mg/L (dichlorvos) . After oxidation
by bromine water phosphoric acid thioester insecticides
(parathion ethyl) are detectable from 2.5 |ig/L. A reactiva
tion of the enzyme electrode by means of pyridine-2-aldoxi-
22
raethyliodide as well as differential detection of the three
insecticide groups in water and on fruits and vegetables
have also been demonstrated. An enzyme immunoassay (EIA)
has been developed (23) for the insecticide endosulfan and
its degradation products. The EIA is based on antibodies
raised against the diol of endosulfan by immunizing rabbits
with a keyhole limpet hemocyanin (KLH) endosulfandiol conju
gate. With this method, endosulfan can be detected in aqueous
solutions at a level of 3 ppb without any sample extraction
procedure. The measuring range has been found to be between
3 and AOO ng/mL,
TLC coupled with enzyme inhibition (16) has been
used for the sensitive and rapid detection of paraoxon.
In this method, solutions of 0.5 mg/mL horse serum cholines-
terase in either 50 mM sodium phosphate buffer (pH 7.7)
or 10 mM sodium tetraborate buffer (pH 9.2), or 1:10 diluted
human pool plasma in the phosphate buffer have been used.
For the colour development the spray reagents used are 1.5
mM Ellman's reagent [5,5'- dithio bis-(2-nitrobenzoic acid),
pure], and 7 mM butyrylthiocholine in phosphate buffer or
15 mg of indoxyl acetate in ethanol (ImL) and borate buffer
(10 mL) . The detection limit is found to be 0.3 ng. Bhaskar
(24) has developed a simple enzymatic method for field TLC
detection and determination of fenitrothion as fenitrooxon
in water with pig liver acetone powder as enzyme source.
It is also reported that pig liver acetone powder is more
advantageous than raw liver sources, owing to its easy
23
procurement and instant use. For colour development,
solutions of 1-naphthyl acetate in acetone followed by
p-nitrobenzenediazonium fluoroborate in acetone are sprayed.
The white spots appeared on the orange red background. The
lower limit of detection is found to be 1 ng. Determination
of parathion residues and its metabolite paraoxon in crops
has been carried out by TLC - enzyme inhibition method (25).
Unpolished rice (25 g) is ground, extracted with Me^CO and
the extract (10-20 p,L) is subjected to TLC, using C^H,,-
EtOAc (3:1) as solvent system. After heating at 50° for
10 min, the plate is sprayed with an enzyme extract from
mouse liver followed by the substrate containing p- naphthyl
ethyl ester. Then the spots are scanned for the quantitative
determination of parathion residues and its metabolites.
Recoveries are found to be 70.2 - 109.1% for paraoxon and
91.79 - 105.4% for parathion and the lower limit of detection
as 10 g.
Bhaskar and Kumar (26) have developed colorimetric
method for the determination of dimethoate and omethoate.
Pig liver acetone powder is used as enzyme source and
p-nitrobenzenediazonium fluoroborate as the chromogenic
reagent. By this method 1-10 jig of dimethoate and 50-1000
ng of omethoate can be estimated.
1.3.4 CHROMATOGRAPHIC METHODS
1.3.4.1 Column Chromatography (CC)
Different types of stationary phases are used in CC depending
24
upon the mode of separations, by adsorption, ion-exchange,
partition etc. CC is commonly applied for separation and
preconcentration of pesticides.
Ion exchangers (27) have been used for the preconcen
tration of herbicides in natural water samples. The anionite
AV-17-8, a highly alkaline monofunctional polymer containing
fixed ion-exchanging groups N Me™ in CI form, is highly
effective in concentrating TCA , a,a- dichloropropionate,
2,3,6-trichlorobenzoate, 2-methoxy-3,6-dichlorobenzoate and
2,4-D from model solutions and rice paddy effluents.
Molecular sorption on cationites KU-2-8 and KU-23 are used
for concentrating N-containing nonelectrolytes, 3,4-dichloro-
propanide and yolan (S-ethy1-N-hexamethyleneiminothiocarba-
mate). Ion exchange column (28) has been used for preconcen
tration of 2,4,5-TP and other acidic herbicides from plant
extracts. The plant material is extracted with aqueous basic
buffer, the extract is loaded on a strong anion exchanger
column to extract the acidic compounds and to elute the
neutral and cationic substances, thus attaining on-column
trace enrichment. By mobile phase selection (pH change),
elution from the column is possible. Kanatharana (29) has
studied three cation exchange columns for the determination
of paraquat residues in soil. An improved silica gel cleanup
method (30) has been developed for organophosphorus pesti
cides. This method involves a 3.5 g silica gel column with
1% acetic acid wash to condition the column prior to the
addition of the sample. Percentage recovery (and standard
25
deviation) of phorate and disulfoton are 96(5-6) and 98(1.0)
respectively. Recoveries range from 92-101% for carbopheno-
thion, chlorpyrifOS, DEF, diazinon, disulfoton, Et parathion,
fenthion, leptophos, malathion, phorate and temephos. Hoke
et al.(31) have used Baker-IOSPE system for concentrating
herbicides such as 2,4-D; 2,4,5-T and silvex on a Baker
C,Q high capacity solid phase column. Atrazine; siraazine;
2,4-D; silvex and 2, 4,5-T have been extracted (32) from
natural water samples using solid phase extraction on dispo
sable C, o columns. The adsorption behaviour (33) of carboxy-
i o
lie herbicides and plant growth regulators such as phenoxy-
acetic acid, TCA, benzoic acid, cinnamic acid, lAA,
j3-naphthaleneacetic acid and P-naphthoxyacetic acid on alumi
nium oxide (active and neutral) has been studied and the
possible use of aluminium oxide in filter systems for remo
ving these compounds from water is suggested. Columns packed
with (34) activated granular charcoal, or cheapest and
unconventional adsorbent, flyash can also be used for remo
ving above carbxylic pollutants. The retention capacity
of several carboxylic herbicides and plant growth regulators
on polyurethane foam has also been studied (35). The foam
plugs have been found to be useful for the separation of
|3-«aphthy lace tic acid from acetic, benzoic, citric and trich
loroacetic acids.
1.3.4.2 Paper Chromatography (PC)
This is the simplest method of detection and separation.
It has a reasonable degree of sensitivity and selectivity
26
for chlorinated organophosphorus insecticides and phenoxy
acid herbicides. The greater sensitivity and concurrent
determination possible with gas chromatography has led
to PC being used now mostly for confirmation of the relati
vely non-specific gas chromatography compounds. Thin layer
chromatography has replaced PC in pesticide residues analy
sis because of its increased resolution and shorter develop
ment time .
Pain and Pal (36) have separated lAA from honey
by PC in propanol-ammonium hydroxide-water (10:1:1) or
ethanol (70%). Ehrlich's reagent has been used as the loca
tion reagent. Rathore et al. have separated certain carboxy-
lic herbicides and plant growth regulators besides other
carboxylic acids on papers impregnated with calcium sulphate
and calcium carbonate (37) and hydroxides of aluminium
and cadmium (38) in simple solvent systems.
1.3.4.3 Liquid-Liquid Chromatography
since the basic principle of liquid-liquid chromatography
and liquid-liquid extraction is very similar both of
the techniques are discussed under this heading. In the
first technique the separation depends on the sufficiently
different partition coefficients of the compounds to be
separated, in the selected solvent systems. The later tech
nique involves a solvent system consisting of two iramissible
solvents, a solute tends to partition preferentially into
one solvent rather than the other. The utility of these
27
techniques is in separating and preconcentrating the pesti
cides prior to their estimation.
Hamann and Kettrup (39) have used liquid-liquid
extraction with CH„C1« for the preconcentration of phenoxy
acid herbicides in water samples. Eleven triazine herbicides
(40) have been extracted from natural waters using CH„C1„.
Acetonitrile, hexane partitioning (41) has been used for
the extraction of methoprene from poultry manure. Oxamyl
residues (42) have been determined in potato tubers after
their extraction and liquid partitioning. Crown ethers
and their derivatives have been used (43,44) successfully
for the separation of several metal ions. The separation
mechanism depends upon the size of metal ions, so it is
very useful for the separation and purification of alkali
metals. Crown ethers typically contain central hydrophobic
cavity with diameter ranging from 1.2 - 6.0 A°. The cavity
is ringed with electronegative binding polyether oxygen
atoms which in turn are surrounded by a collar of CH„
group forming a flexible frame work exhibiting hydrophobic
behaviour. Hence it seems that they may be of greater
importance especially for the separation and concentration
28
of small size metabolites which are formed due to degradation
of pesticides. It is wellknown that most of the organophos-
phorus and carbamate pesticides are degradable and in some
cases the metabolites are more toxic than the parent pesti
cides. Therefore their separation and estimation is important
and use ofextraction with crown ethers can be made successfully.
1.3,4.4 Thin-Layer Chromatography (TLC)
TLC has grown rapidly in the recent years and is now widely
accepted as a quick and efficient technique for the detection,
separation and determination of very small quantities of
a majority of pesticides. TLC is lacking in the precise
specificity of gas-liquid chromatography but it is more
precise and sensitive than PC. Although most advances in
the pesticide analysis have taken place during the past
few years in the field of GC and HPLC, TLC has retained
the status as a valid and simple method of qualitative and
quantitative analyses of pesticide residues and their metabo
lites. It appears that many papers on this subject originate
from researches in less developed countries, which is plausi
ble due to the lack of sophisticated instrumentation and
29
the shortage of repair facilities in these countries. In
order to achieve better results, TLC can be performed by
many ways. For pesticides analysis TLC has been used by
the following procedures.
1.3.4.4.1 Normal-phase thin-layer chromatography
(NP-TLC)
This is the simplest type of TLC in which a solvent system
is allowed to develop the normal phase plates. This mode
of development suffices for many applications, and it is
commonly used for pesticides analysis.
Several carboxylic acids and some carboxylic herbi
cides and plant growth regulators have been separated (45)
by TLC on glass plates coated with calcium sulphate plus
activated charcoal, p-DAB, flyash, silica gel, starch, cal
cium nitrate and cupric sulphate. This technique may also
be suitable for other acid pesticides. TCA herbicide has
been separated (46) from plant growth regulators lAA,
p-naphthaleneacetic acid, p-naphthoxyacetic acid and from
other organic acids on TLC plates coated with calcium sulph
ate containing ammonium molybdate, alumina, calcium carbonate,
cupric sulphate, ferric chloride, magnesium sulphate, phthalic
anhydride, titanium oxide and zinc oxide. Determination
30
of herbicide residues from a group of derivatives of aromatic
acids in plant material has been carried out (47). Wheat,
rye, rice, wheat flour, corn flour, semolina, barley groats,
oat flakes, potatoes and carrots are extracted at pH 1.2
(H„SO,) with ethanol and re-extracted with ethyl ether,
water and chloroform for purification. TLC on silica gel
using Cc^Hr-C^H, ,-AcOH (5:12:2) as the solvent system gives
the best separation of herbicides from co-extracted impuri
ties, whereas C^H, ,-AcOH (7:3) system gives the best separa
tion of dichlorprop from dicamba. The spots are visualized
under UV after spraying silver nitrate solution. Sherma (48)
has used quantitative TLC for the determination of organoch-
lorine pesticides in water samples. The pesticides are
extracted from water using a C-18 solid phase extraction
cartridge instead of conventional liquid-liquid extraction.
The concentrated pesticides are separated on silica gel
layers using EtOAc, C^H^^ - C^Hg (1:1) and C ^ H ^ - Et20(l:l)
as eluents, followed by detection with ammoniacal silver
nitrate solution and quantification by densitometric scanning.
Recoveries of methoxychlor and p,p'-DDT have been found to
be > 90% at fortification concentrations of 2.5 and 0.25
ppb respectively.
1.3.4.4.2 Reversed-phase thin-layer chromatography
(RP-TLC)
Recent introduction of chemically bonded reversed phase
TLC plates has made RP-TLC an important new tool. RP-TLC
expands the scope of separations by TLC to include compounds
traditionally not separable by NP-TLC because of their high
31
polarity. RP-TLC involves the use of a nonpolar, usually
hydrocarbonaceous stationary phase and a relatively more
polar mobile phase.
Mixtures of (49) eptam with pebulate or cycloate,
thiobencarb with molinate, pebulate or vernolate and pebulate
with vernolate have been separated on silufol UV-254 impreg
nated with vaseline oil using Me„CO-H„0 (6:4) as solvent
system. Detection thresholds have been found in the range
of 0.01-0.02 mg/L. Quantitative separation (50) of TCA from
some carboxylic herbicides has been made on BaSO,/CaSO,
coatings impregnated with coconut oil. Many ternary and
quaternary separations have been achieved using aqueous
solvents. The lower limit of determination of TCA has been
found to be 400 [jg.
Separation and detection (51) of phenoxyacetic herbi
cides such as 4-chlorophenoxyacetic acid; 2 , 4-D;phenoxyacetic
acid and 2,4,5-T and plant growth regulators such as benzoic
acid, cinnamic acid, gallic acid,IAA,IPA, a-naphthaleneacetic
acid, p-naphthaleneacetic acid and ^-naphthoxyacetic acid
have been carried out by ion-pair reversed-phaseTLC on cal
cium sulphate coatings impregnated with an ion pair reagent
cetrimide and different oils such as olive, paraffin and
silicone using distilled water as a solvent and bromophenol
blue as a detector. Limits of detection have been found
to be 20-60 jig.
32
1.3.4.4.3 Two-dimensional thin-layer chromatography
(2-D-TLC)
In this developing technique, there is the application of
normal, multiple or stepwise development in two dimensions.
2-D-TLC has been used (52) for the detection, separation
and determination of several compounds including pesticides
and other environmental pollutants.
Kavetskii et al. (53) have used 2-D-TLC on silufol
for the analysis of 2,4-D; 2,4'-DDT; 4,4'-DDT; 4,4-DDD;
4,4'-DDE; a-HCH and y~HCH in water, soil and plants. Initially
polar pesticides are separated using CHC1„-Et^0 (5:2) and
then the nonpolar ones are separated using C^H,,-Me^CO (20:1).
Detection thresholds for 2,4-D are found to be 0.002 mg/L
in water and 0.1 mg/kg in the soil and plants. Jost and
Herbert (54) have studied the application of 2-D-TLC of amino
acids, bile acids, pesticides and sex hormones on precoated
plates of CNF„,c; • 2-D-TLC on calcium sulphate coated glass
plates has been applied (55) for the separation of herbicides,
fungicides and other organic acids occuring in fruit juices,
using common solvents such as chloroform, carbon tetrachlo
ride, ethyl acetate, benzene and distilled water.
1.3.4.4.4 Sequential thin-layer chromatography
(S-TLC)
When a mixture contains compounds that differ considerably
in polarity, a single development with one mobile phase
may not provide the desired separation. However, if the plate
33
is developed successively with different mobile phases t'lat
have different selectivities or strengths, a separation
will probably be forthcoming.
Riebel and Rellich (56) have analysed methamidophos
in potato tubers and foliage on silufol plates using C,H, ,-
Me2C0(l:l) followed by CHCl„-Me2CO-MeOH(1 : 1 : 1 ) solvent
systems. The spots have been visualized by spraying a dilute
swine liver homogenate and then with alcoholic p-naphthol
acetate. Separation and identification (57) of carboxylic
herbicidal substances such as 2,4-D; 2,A,5-T and TCA and
plant growth regulators such as benzoic acid, cinnamic acid,
lAA, R-naphthaleneacetic acid, p-naphthoxyacetic acid and
phenoxyacetic acid have been made by S-TLC on calcium sul
phate layers with acetone, benzene, carbon tetrachloride,
chloroform, ethyl acetate, dioxane and propanol as solvents
and broraophenol blue as detector. A mixture of eight herbi
cides (58) has been analysed by S-TLC on silica gel Merck
no. 5721 plates. The plates are first developed in C^H,,-
Et^0(3:l) solvent system, dried in an air stream for one
hour, and then developed again using C^H, ,-Me„CO-AcOH
(35:25:0.05) solvent system,
1.3.4.4.5 Multiple development thin-layer chromato
graphy (M-TLC)
If a single development of a plate indicates a partial sepa
ration, the plate is allowed to dry and then reinserted
into the developing chamber for redevelopment. This may
be repeated any number of times until a satisfactory
34
separation is achieved.
Triazine herbicides (59) have been separated and
determined on silica gel GF254 plates using C,H^,-BuOAc
(60:40) as a solvent system. When the solvent front reaches
6.5 cm from the starting line, the plate is removed and
dried and then allowed to develop to 12 cm. Good separations
have been achieved for six triazines.
1.3.4.4.6 Preparative thin-layer chromatography (PLC)
PLC may be defined as the thin layer chromatography of rela
tively large amounts of materials in order to prepare and
isolate quantities of separated substances for further work
such as additional chromatography, IR analysis, melting
point determination, or synthesis. The layer thickness range
for PLC is 0.5-10 mm but the commonly used thicknesses are
1.0 mm and 2.0 mm.
Layers (0.75 mm) of admixture of barium sulphate
and calcium sulphate have been used (13) for the separation
of carboxylic herbicides, plant growth regulators and carbo-
xylic acids of importance in citrus fruit industry. Layers
(1.0 ram) of the above admixture have been found to be more
useful for the quantitative separation of p-naphthaleneacetic
acid (9.31 mg) from oxalic acid (3.15 mg).
1.3.4.5 Gas Chromatography (GC)
GC is the most versatile and sensitive method for pesticide
residues analysis.
35
Noble (60) has reported a method based on packed
column and capillary gas liquid chromatography for the iden
tification of phenoxy ester herbicides. The ester ification
method with acetyl chloride gives a mean yield of 98% with
a range of phenoxy acids. This method can be used to produce
standards for the specific quantitative analysis of esters
in formulations. GC coupled with mass spectrometry has been
used (61) for the analysis of phenoxy propionic acid deriva
tives possessing herbicidal activity. Fluazifop-Bu,haloxyfop-
ethoxyethyl and quizalofop-Et as well as their metabolites
are first converted to their corresponding methyl esters
and then separated on several gas chromatographic columns
(e.g. OV-1, OV-17, OV-25). Herbicidal carboxylic acids (62)
have been analysed by ECD gas chromatography using columns
filled with (3% OV-225 on 80/100 gas chrom Q ) . The enbloc
derivatization (ester form) is made by direct ingestion
of a mixture of herbicides and pentafluorobenzy 1 bromide
in acetone. This procedure is fast and particularly conven
ient for low level (e.g. 0.04 ng in 2,4-D and 2,4,5-T) screen
ing, identification and analysis of highly acidic chloro-
benzoic, chlorophenoxyalkanoic and arylacetic acids in the
presence of less acidic carboxylic acids and phenols. Penta-
fluorobenzyl and methyl esterification methods(63) for seve
ral common acid herbicides (dalapon, MCPA, MCPP, dicamba,
dichlorprop, dinoseb, silvex, 2,4-D, 2,4-DB and 2,4,5-T)
have been evaluated and compared. GC/electron capture
detection limits for two of seven pentafluorobenzy1 esters
are better than the corresponding methyl esters and
36
comparable for the other five herbicide esters. The linear
dynamic concentration range is generally wider for the methyl
esters. The choice between methyl or pentafuorobenzyl esters
is contingent on the relative importance of detection limits
and linear dynamic range.
1.3.4.6 High-Performance-Liquid Chromatography (HPLC)
HPLC continues to find a useful place as an analytical tool
for the separation and determination of pesticide residues.
HPLC is preferable for analysing thermally unstable pesticides
that can not be analysed directly by GC due to their tendency
to break down on GC columns. It is also reported (64) that
the additional steps, taken for the preparation of thermally
stable derivatives for GC analysis, make the procedure time
consuming and concomitantly increase the chances of pesticide
loss .
Gyorffi and Kandenczki (65) have used capillary fused
silica columns for higher sensitivities and better resolution
of sixteen pesticide residues including lindane, parathion,
captan, endosulfan and fenarimol in citrus. A simple HPLC
method (66) for the determination of carbaryl deposited after
aerial application has been reported. The carbaryl is collec
ted on a filter paper, washed with acetone and the solution
is concentrated and chromatographed on a 5|im-0DS column using
MeCN-H20 as eluent and monitored at 267 nm. Carbaryl has
2 been detected in the range of 35-3500p,g/m within 200 m from
the boundry of aerial application.
37
Detection of organophosphorus pesticide residues
(67) has been carried out by reversed-phase HPLC using
Finepak SIL C,„ packed columns. The eluent used is MeOH-
H„0 (65:35) and the detection is made with the help of UV
detector at 254 nm. Sensitivity of the detection by this
method is inferior to those by GC and TLC methods. Davy
and Francis (68) have used RP-HPLC for the analysis of
enantiomers of fluazifop and other phenoxypropionic acids
such as mecoprop, dichlorprop and fenoprop. L-prolyl-n-
octylamide - Ni(II) and acetonitrile-H^O (50:50) have been
used as the mobile phases. Derivatization, combined with
RP-HPLC (69) has enabled simultaneous determination of multi
ple plant growth regulators in seedlings and developing
seeds of rice. a— Bromo-2'-acetonaphthone has been used
as a derivatization agent for the common carboxylic func
tional groups among abscisic acid, gibberellins and lAA.
1.3.5 SPECTROPHOTOMETRIC METHODS
Spectrophotometry of pesticide residues does not achieve
the sensitivity of TLC and GC techniques. It may not be
able to distinguish between the parent compound, metabolites
and hydrolysis products but can be used with chromatography
as a confirmatory technique.
1.3.5.1 UV and Visible Spectrophotometry
Tsitovich et al.(27) have determined several herbicides
in natural water samples spectrophotometrically. For
38
preconcentration of herbicides ion-exchange methods have
been used. 2,4-D is eluted with 2M NaCl and determined
by using butyIrhodamine and a green filter. Dalapon is deter
mined by UV spectrophotometry at 200-203 nm. TCA is eluted
with 4M NaCl and determined at 510 nm using pyridine. Yolan
(S-ethy1-N-hexaraethyleneiminothiocarbamate) is eluted with
NaCl in H^O-EtOH(1:1)and determined at 206 nm. Determination
of (70) the herbicides, chlortoluron, metobromuron,monoli-
nuron, linuron, chlorbromuron, metribuzin, atrazine, terbu-
tryn, metolachlor and ioxynil has been carried out. The
herbicides are extracted from soil and determined by UV
and visible spectrophotometry. The detection limit has been
found to be 0.1 |j,g/raL. Sastry and Vijaya (71) have reported
four methods using different chromogenic reagents for the
determination of fenitrothion and methyl parathion. The
first method is based on the reaction of the reduced pesti
cides with metol and 10, at pH 3 and measurement of absor-
bance at 520 nm. The second method is based on the oxidative
coupling of the reduced compounds with thymol and measurement
of absorbance at 600 nm. The remaining two methods are based
on the diazo coupling of the reduced amine with phloroglu-
cinol and chromotropic acid and measurement of absorbance
at 410 and 480 nm respectively. Seshaiah and Mowli (72)
have described a simple and sensitive spectrophotometric
method for the determination of malathion with methylene
blue. The malathion is decomposed with alkali and the resul
tant dimethyldithiophosphate is extracted with methylene
blue in chloroform. The absorbance of the organic layer
39
is measured at 652.1 nm. A new, simple, rapid and sensitive
method (73) for the determination of organophosphorus pesti
cides such as parathion, malathion, quinolphos and monocroto-
phos in plant materials has been reported. The method invol
ves the formation of 12-molybdophosphoric acid from orthoph-
osphate and excess of raolybdate in acid solution followed
by the reduction with succinyldihydrazide to give molybdenum
blue. The dye extracted in butanol shows absorption at 780nra.
This method is free from the interference of Mo, As and
Cu. The molar absorptivity and Sandell's sensitivity are
4 3 -1 -1 3.75x10 dm mol cm and 0.0008 p,g respectively.
Rangaswamy et al.(74) have developed a colorimetric
method for the determination of carbendazim (MBC), benorayl
and their degradative product, 2-aminobenzimidazole (2-AB).
Alkaline hydrolysis of MBC with 6.5 N NaOH yields methanol
and 2-AB. Methanol on oxidation gives formaldehyde which
is coupled with the chromotropic acid reagent and then deter
mined. By this method as little as 1.5 p,g of methanol can
be estimated. 2-AB on alkaline iodination forms reddish
brown N-iodo-2-AB with an absorption maximum at 400 nm and
serves as a measure of MBC with a lower limit of determina
tion as 3 ^g. Benomyl is hydrolysed with 0.1 N HCl in a
water bath for 15-30 min to cleave the butylaminocarbony 1
group. The resultant MBC is alkali hydrolysed as above.
The linear relationship between the absorbance at 580 nm
and the concentrations of methanol and MBC are valid upto
25 and 150 mg/5 mL of reaction mixture respectively. In
40
the case of 2-AB, this relationship at 400 nm is obeyed
upto 40 |j,g. Recoveries of 87-100% have been achieved.
1.3.5.2 Densitometry
Herbicide residues (75) have been analysed by using spectro-
densitometry coupled with TLC. The diffuse reflection elec
tronic spectra of herbicides of different groups have been
obtained in the UV region and the optimal wavelengths for
scanning in routine analysis have been determined. The subs
tances studied are characterized by the intense band in
the 220-280 nm region, the place of which and quantity of
resolution depend on the substituents, present in the
chromophore group. Spectrodensitometry coupled with S-TLC
has been used (58) for the separation and determination
of diphenyl ether group herbicides including nitrofen, oxyfl-
uorfen, bifenox, fluorodifen, acifluorfen, femesafen, chloro-
xuron and diphenoxuron.
1.3.5.3 Fluorimetry and Phosphorimetry
A sensitive fluorimetric method (76) has been developed
for the determination of indole-derivative plant growth
regulators (lAA, IBA and IPA) of the auxin group. The method
is based on the reaction of auxins with o-phthalaldehyde
in concentrated sulphuric acid. Yuan and Cao (77) have deter
mined DDVP and related compounds by fluorescence method.
These compounds react with o-dianisidine in the presence
of sodium perborate to form fluorescent products which can
be measured by fluorimetry.
41
Trautwein and Guyon (78) have determined the pesticides
2,4-D, p-naphthoxyacetic acid and silvex by low-temp phospho-
rimetry. Excitation and emission wavelengths are 284 and
480 nm respectively for 2,4-D, 320 and 500 nm respectively
for P-naphthoxyacetic acid and 290 and 464 nm respectively
for silvex. The limit of detection for the above pesticides
is found to be 0.10 mg/L.
1.3.6 MISCELLANEOUS TECHNIQUES
Some important techniques not described above are discussed
here.
Tsunoda and Kishi (79) have used mass spectroscopy for
the analysis of N-methylcarbamate pesticides. They have
recorded electron impact (EI) and chemical ionization mass
spectra of fifteen N-methylcarbamates and S-methyl-N-hydroxy-
thioacetamide. These compounds give intense peaks of [M-57 ]
on EI mass spectra. Positive-ion fast-atom bombardment [FAB]
mass spectra (80) of the monosodium and disodium salts of
metabolites of azinphos-Me, azinphos-Et, demeton-S-Me and
omethoate have been obtained with detection limits of 3-5 (jg.
Identification of (81) alkyldipyridilium herbicides
(paraquat and diquat) has been made by using IR spectra of
their insoluble salts prepared by adding H_PtCl,, HAuCl,,
picric acid or Reinecke salt to 2 mg/mL aqueous solutions
of these herbicides. The minimum limits for precipitation of
the salts are found to be 10-100 [ig/mL paraquat or diquat.
42
Paraquat chloroplatinate is more soluble than diquat chloro-
platinate whereas both chloroaurates are insoluble to the
same extent. This differential solubility enables fraction
ation from their mixtures.
The utility of (82) surface-enhanced Raman scattering
(SERS) spectrometry for the determination of trace levels
of various chlorinated pesticides including chlordan, carbo-
phenothion, bromophos, chloropyrifos-Me, dichloran, linuron
and 1-hydroxychlordene has been described. The silver coated
substrates consisting of submicron spheres on solid surfaces
have been used as SERS-active media.
Polarography has been applied to the detection and
estimation of several pesticides. Quantitative polarography
is based on the measurement of the diffusion current, i.e.
height of the polarographic wave. A differential pulse
polarographic method (83) using the dropping mercury
electrode has been reported for the determination of herbi
cides, atrazine, prometrine and simazine. The limit of detec-
_ Q
tion is found to be 8x10 M, corresponding to about
15 |_ig/L. Goicolea et al. have determined (84) chloridazon
in soil by differential pulse polarography. The measurements
have been carried out in 0.1 M NaClO, and Britton - Robinson 4
buffer (pH 1.81) by using a Hg-drop electrode. A reduction
wave is obtained at -0.9V. Effects of drop time, modulation
amplitude, scanning rate, Hg pressure, temperature and pH
have been evaluated. The acid-base dissociation constants
are reported to be 3.2 (pK ) and 6.0 (pK„). The limit of
43
detection is found to be-~'0,166 ppm, Fenitrothion and methyl
parathion have been determined (85) by polarography in an
ethanolic solution at pH 7.0 using a Sorensen's buffer solu
tion as supporting electrolyte and 0.5% gelatin as suppressor
at 25°. The method is applicable even in the presence of
malathion which foils other analysing techniques. The minimum
concentrations for the polarograms recorded are 0,104 mg
and 0.166 mg and the half-wave potentials, -0.95 V and -1.0 V
for fenitrothion and methyl parathion respectively.
Radioanalytical methods have also been used for pesti
cide residues analysis. Kiigemagi et al. (86) have deter
mined oxamyl residues in peppermint hay and oil by radio-
isotope-dilution technique, Kannangara et al. (87) have
analysed 2,4-D by radioimmunoassay.
On the basis of the above discussion, it is clear
that several non-instrumental and instrumental techniques
are being used for pesticide residues analysis. Since instru
mental techniques are costly and sophisticated and require
skilled operators either they are not available or have
not been installed in most of the laboratories of the third
world. Therefore there is a growing interest in developing
new, simple and inexpensive techniques for pesticide residues
analysis,
In the present work, efforts have been made to develop
techniques for detecting, separating and determining some
carboxylic herbicides and plant growth regulators. In
44
chapter 2 a vapour phase spot-test for the detection and
semiquantitative determination of 2,4-D and related compounds
is discussed. TLC on admixtures of BaSO, and CaSO, using
simple solvents is described in chapter 3. The research
work on RP-TLC and NP-TLC is presented in chapters 4 and
5 respectively.
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49
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CHAPTER 2
A NOVEL METHOD FOR THE DETECTION AND SEMIQUANTITATIVE DETERMINATION OF 2,4-D AND RELATED COMPOUNDS
2.1 INTRODUCTION
In continuation to the previous work, carried out in this
laboratory (1-5), for developing new, simple, non-instrumental
and inexpensive methods of detection, separation and determi
nation of herbicides present in environmental samples one
more procedure is described.
In Feigl's book (6) a reagent containing chromotropic
acid (1,8-dihydroxynaphthalene-3,6-disulphonic acid) in con
centrated sulphuric acid is reported for the detection of
traces (0.14 [j,g) of formaldehyde in presence of fructose,
saccharose, furfural, arabinose, lactose and glucose. The
test is highly sensitive in concentrated sulphuric acid.
The chemistry of this colour reaction is not known with cer
tainty. It is probable that phenolic chromotropic acid conden
ses with formaldehyde followed by oxidation to a p-quinoidal
compound of violet colour (Figure 2.1). This reaction can
also be used for detecting compounds such as phenoxyacetic
acid and its halogen derivatives and monochloroacetic acid
which split out formaldehyde when hydrolysed or pyrolysed
(Figures 2 .2 and 2.5).
In this colour reaction sulphuric acid acts as a
dehydrating agent, oxidant and water donor. Cellulose and
hydrated sulphates of zinc and manganese, which lose water
when heated above 150° act in the same way as concentrated
sulphuric acid. However, their analytical utility has not
been fully explored.
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58
Freed (7) has remarked that when the test is applied
directly, there is interference by compounds which split
off formaldehyde when heated with concentrated sulphuric
acid. However, the test becomes quite selective for phenoxy-
acetic acid and its halogen derivatives if their solubilities
in benzene are utilised. The interfering compounds can thus
be taken out of the reaction theatre, including those which
caramelize or char when heated with concentrated sulphuric
acid and so impair the recognition of the colour reaction.
The test can be made even more selective by the vapour
phase detection in which formaldehyde split out is detected
in the vapour phase by contact with chromotropic acid and
concentrated sulphuric acid. However, the sensitivity is then
only about one tenth as great (8).
The survey of the literature (2) shows that the sensi
tivity of the vapour phase detection can be improved by per
forming the test under vacuum. The use of differential solubi
lity of the test material can be made in enhancing the selec
tivity by performing the test in different solvents. The
proper selection of conditions as well as the reagent for
splitting out formaldehyde from a particular functional group
can also contribute to the selectivity of the test because
a wide range of compounds split out formaldehyde under diff
erent set of conditions and reagent. For example (-OCH„ and
>N-CH„ groups): anisole, antipyrine, methyl red, methyl orange
etc. give formaldehyde through oxidative cleavage with molten
benzoyl peroxide at 120° (Figure 2.3). Methylene ethers of
59
o-diphenols (C^H.< >CH„ type): apiole, berberine, narcotine D 4 Q Z
etc. give formaldehyde when heated alone or along with concen
trated sulphuric acid at 170-180° (Figure 2.4). In the case
of dry heating, the hydrolysis is brought about by the water
split out of the test material. It acts as superheated steam
at the site of its production and accomplishes hydrolysis
which can not be brought about by boiling water. Also when
the material is heated with concentrated sulphuric acid,
it is the water in the acid which as quasi superheated steam
is responsible for the hydrolysis. Glycolic acid and mono-
chloroacetic acid yield formaldehyde when warmed with concen
trated sulphuric acid at 100° and 150-180° respectively
(Figure 2.5). Glycolic acid on dehydration directly gives
formaldehyde whereas monochloroacetic acid is first hydrolysed
into glycolic acid which gives formaldehyde. a-Amino acids
give aldehydes on treating with alkali hypohalogenite. Glycine
splits out formaldehyde when treated with chloramine T or
nitrous acid at 100° (Figure 2.6). By the action of sodium
hypochlorite, glycine is oxidatively decarboxylated and deami-
nated to produce formaldehyde. Chloramine T gives best results
and functions as a hypochlorite donor because of its hydroly
sis. Choline hydrochloride produces formaldehyde and acetal-
dehyde when heated with MnS0,.4H„0 or MnS0,,H„0 at 180°.
4 2 4 2
Phenoxyacetic acid and its halogen derivatives give formal
dehyde on warming for a short time with concentrated sulphuric
acid at 150° (Figure 2.2).
Therefore in view of the above facts, now an attempt
60
is made to raise selectivity as well as sensitivity of the
test by the use of (i) solid ZnS 0, . 7H„0 for splitting out
formaldehyde from phenoxy herbicides, (ii) preferential solu
bility in different solvents, and (iii) newly designed appar
atus in which the test tube is connected with another ' U'
shaped side tube containing the reagent between two bulbs
and vacuum is created to bubble the traces of formaldehyde
into the reagent. The newly designed apparatus is used succe
ssfully to detect traces of 2,4-D and related compounds in
soil, vegetation and water samples. The results obtained
are discussed in this chapter.
2.2 EXPERIMENTAL
2.2.1 APPARATUS AND MATERIALS
Glass tube (Figure 2.7), vaccustier pump 17 cm vacuum (Atlan
tis Applications Engineering Pvt. Ltd., India), temperature
controlled heating mantle (Sunvic, U.K.), hot air drier,
electric oven, centrifugal machine (Baird & Tatlock Ltd.,
England), graduated micropipettes , porcelain tile with grooves ,
petri dishes (8.0 cm diameter) etc. were used.
Benzene (Glaxo, India), chromotropic acid sodium
salt (CDH, India), 2-propanol (Merck, India), sulphuric acid
(BDH, India), ZnS0^.7H20 (SD Fine Chem., India), 2,A-D ethyl
ester 34% EC (Unique Farmaid Pvt. Ltd., India), 2,4-D sodium
salt 80% wettable powder (Mascot Agrochemicals Pvt.Ltd.,India)
dichlorovos 76% EC (Pesticides India Ltd.), formothion 25%
EC ( Sandoz India Ltd.), malathion 50% EC (Cyanamid India
Ltd.), phosphamidon 85% EC (Hindustan Ciba-Geigy Ltd., India),
TO vacuuster pump
Stopper
Sample zinc sulphate
Note — All dimensions are in mm
Figure 2.7 Apparatus for micro analysis of herbicides in flora, soil ana water.
61
62
thiometon 25% EC (Sandoz India Ltd.). p-dimethylaminobenzal-
dehyde, 2,4-D acid and other herbicides were from Sigma,
USA. All other reagents used were of analytical grade.
2.2.2 SOLUTIONS
All the solutions (1%) were prepared in benzene or otherwise
as stated. If the compounds were insoluble in benzene their
saturated solutions were used.
2.2.3 COLOURING REAGENT
A mixture of one or two tiny crystals of chromotropic acid
sodium salt and 0.20 mL of concentrated sulphuric acid in
'U' tube was used as the reagent.
2.2.4 ENVIRONMENTAL SAMPLES
(i) Leaves
(ii) Soil (fertile)
: leaves of Duranta plumieri of family verbenaceae of the commonly grown hedge plant were used.
: sand 32.68%, silt 57.00%, clay 10.06%, CEL 16.00 mg/lOOg, organic matter 0.61% and pH 7.75.
(iii) Soil (nonfertile) : sand 50.00%, silt 27.60%, clay 22.40%, CEL 17.30 mg/lOOg, organic matter 0.19% and pH 8.33.
(iv) Water (river)
(v) Water (tap )
dissolved oxygen 8.2-8.7 ppm, total hardness 122-141 ppm, total dissolved solids 124-175 ppm, total alkalinity 174.0-189.4 ppm, chloride 9.2-9.9 ppm, sulphate 6.5-6.7 ppm, phosphate 0.42-0.47 ppm, BOD 5.20-5.61 ppm, COD 6.5-6.7 ppm and pH 8.70-8.80(9).
total alkalinity as CaCO„ 261-268 ppm, total hardness 135-144 ppm, chloride 13-14 ppm, sulphate 14.0-18.5 ppm, fluoride 0.99-1.00 ppm, nitrate-nitrogen 2.1-2.5 ppm, cadmium 1.25-1.50 ppm, copper 4.50-5.00 ppm,
63
chromium 2.0-2.5 ppm, manganese ISIS ppm, nickel 8.5-10.0 ppm, lead 0.7-0.8 ppm, zinc 132-139 ppm, electr-, ical conductivity 132.2-134.5 jj ohms
cm"- and pH 7.5-7.6 (10).
2.2.5 GENERAL PROCEDURE
Solution (1 drop) of the test material was taken in tube
A, evaporated to dryness, cooled to room temperature (37±2°),
35 mg of ZnS0,.7H„0 was added into it and triturated with
a glass rod. The reagent was taken in 'U' tube through the
end B. Tube A was stoppered and tube B was connected to the
suction pump through a rubber tube. About 2 cm of the lower
portion of the apparatus (Figure 2.7) was dipped into the
oil bath at a desired temperature for 2 min. The colour so
developed was carefully observed. Then the coloured solution
was poured into a groove of the tile in order to visualize
the colour for longer time and to use the intensity of the
colour for semi-quantitative analysis.
The lower limit of identification was determined by
starting with known volumes of standard solutions of the
compound under examination.
2.2.5.1 Detection of 2,4-D Acid in Leaves
A small portion of leaves (500 mg) was taken in different
petri dishes, sprayed with a known solution of 2,4-D acid,
the system was covered and left for 24 hr at room temperature
(37+2°). The leaves so sprayed were transferred into a separa
ting funnel and treated with 5 mL of propanol. The aliquot
64
was centrifuged, the clear solution was evaporated to dryness,
the solid left was treated with water (1.5 mL), the insoluble
portion was removed by filtration and then 2,4-D acid was
detected in the aqueous solution by the above procedure.
2.2.5.2 Detection of 2,4-D Acid in Soil
A small portion (5g) of non-fertile or fertile soil was taken
in different petri dishes and 1.25 mL of 2,4-D acid solution
(0.2%) in benzene was applied dropwise with continuous shaking.
After 36 hr 2,4-D acid was detected in the soil by the follow
ing procedure. 2,4-D acid was eluted by shaking a definite
portion of the soil with 2 mL of propanol in a test tube
and then the undissolved material was removed by centrifug-
ation. The clear supernatant aliquot was transferred to
another test tube, concentrated on water bath till the volume
was 0.20 mL, transferred into the tube A of the apparatus
and then 2,4-D acid was detected as above.
2.2.5.3 Detection of 2,4-D Acid in Water
Known solutions of 2,4-D acid in distilled water (DW) or
river water (RW) or tap water (TW) were prepared and the
lower limit of detection was determined as above.
2.2.5.4 Detection of 2,4-D Ethyl Ester in Formulation
Known solutions of 2,4-D ethyl ester (formulation) were pre
pared in benzene, ethanol and ether while stable suspensions
of 2,4-D ethyl ester (formulation) of known concentration
were prepared in TW and the lower limit of detection was
65
determined as above.
2.2.5.5 Detection of 2,4-D Sodium Salt in Formulation
Known solutions of 2,4-D sodium salt (formulation) were pre
pared in ethanol and TW and the lower limit of detection
was determined as above. 2,4-D sodium salt was insoluble
in benzene and ether and its stable suspension was also not
possible in these solvents.
2.3 RESULTS
Results of detection of phenoxyacetic acid under different
conditions such as temperature, time of heating, different
salts and salt concentration are given in Table 2.1. Data
of detection of substances containing different functional
groups and detection of 2,4-D acid in presence of a wide
variety of substances are recorded in Table 2.2. Results
of detection of 2,4-D ethyl ester and 2,4-D sodium salt in
formulations and some formaldehyde generating compounds disso
lved in different solvents are recorded in Table 2.3. Results
of detection of 2,4-D acid in different environmental samples
are recorded in Tables 2.4 and 2.5. The lower limit of detec
tion of 2,4-D acid in distilled water, river water and tap
water is found to be 10 |ig. The diagram of the apparatus
used is given in Figure 2.7.
2.4 DISCUSSION
The facts described in introduction show that the colour
reaction under study has good analytical potential. However,
TABLE 2.1 STUDY OF THE TEST UNDER DIFFERENT CONDITIONS
Effect of the
following
Amount of phenoxyacetic
acid (P-g) Temperature
(°) Colour
1. Salts 36.00 160
2. Sulphuric acid (mL) in reagent
0.00 180
LV (CaS0^.2H20),
Lv (NH^)2Fe(S0^)2.6H20)
LV (Na2S202.5H20),
V (CuS0^.5H20),
V (MgS0^.7H20),
VIV (KHSO^),
DV (MnS0^.H20),
DV (ZnS0^.7H20).
LBr (0.1), NC (0.2).
3. Temperature (°)
18.00 LV (150), V (160),
DV (170), DV (180),
DV (190), BrDV (200)
4. Time (min) 0.00
0.00
18.00
160 NC (1), NC (2), NC (3),
VlBl (4), VlBl (5).
180 NC (1), NC (2), NC (3),
LBl (4), LBl (5).
160 LV (1), DV (2), DV (3),
DV (4), DV (5).
TABLE 2.1 (Continued)
67
18.00 180 V (1), DV (2), DV (3),
DV (4), DV (5).
5. ZnSO,.7H„0 4 2 (mg)
18.00 160 V (17.5), DV (35),
LV (70).
NOTE : Name of the salt (1), volume of concentrated sulphuric
acid (2), temperature (3), time (4) and amount of
ZnSO, .7H„0 (5) are given in parentheses.
Br = brown, Bl = black, D = dark, L = light, NC = no
colour, V = violet and VI = very light.
TABLE 2.2 EFFECT OF DIFFERENT ORGANICS AND INORGANICS ON THE DETECTION OF
2,4-D ACID
Colour (Lt in |ig)
160° 180° Name of Compounds Compound Compound Compound Compound
alone +50 |.tg of alone +50|j,g of 2,-4-0 acid 2,4-D acid
Aldehydes
Acetaldehyde (S)
Benzaldehyde (s)
p-Dimethylaminoben-zaldehyde (S)
Formaldehyde (SP)
Paraldehyde(S)
Vanillin (S)
Amides & imides
Acetamide (I) NC V
Acrylamide (I) NC V
Benzamide (I) NC V
N-Bromosuccinimide (S) LBr (940) DV
Phthalimide (I) NC DV
Amines
Aniline (S )
Aniline HCl (I)
Diethanol amine (I)
Diethyl amine (S)
Diphenyl amine (S)
Hexaraine (S)
Bl
VlBr
V (120)
DV
Bl
NC
Bl
BrV
V
DV
Bl
DV
Bl
LBr
V (70)
DV
Bl
NC
Bl
DBr
DV
DV
Bl
DV
NC
NC
NC
LBr
NC
(375)
DV
DV
DV
BrV
DV
LY
NC
NC
LY
NC
LV (0, .5)
DV
V
DV
DV
DV
LY
NC
NC
LY
NC
LV (0, .5)
DV
DV
DV
DV
DV
69
TABLE 2.2 (Continued)
Triethyl amine (I) VlBr
Trimethyl amine (I) LG(P)
Anhydrides
Acetic anhydride (S) NC
Phthalic anhydride (I) NC
Carbohydrates
Arabinose (I)
Dextrin (I )
Starch (I)
Sucrose (I)
Carboxylic acids
Acetic (S)
cis-Aconitic (I)
trans-Aconitic (I)
Adipic (I)
L-Alanine(I )
L-Arginine (I)
Ascorbic (I )
L-Aspartic (I )
Barbituric (I)
Benzoic (S)
4-Chlorophenoxy-acetic (S )
Cinnamic (S)
Citric (I)
2,4-D acid (S)
Formic (I )
VlBr
DV
LV
LV
VlBr
G(P)
NC
NC
Br
DV
V
V
NC
NC
NC
NC
DV
DV
DV
DV
NC
NC
NC
NC
DV
DV
DV
DV
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
V (18)
NC
NC
V (18)
NC
DV
DV
DV
DV
DV
DV
DV
DV
DV
DV
DV
DV
DV
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
V (15)
NC
NC
V (9)
NC
DV
DV
DV
DV
DV
DV
DV
DV
DV
DV
DV
DV
DV
TABLE 2.2 (Continued)
70
Fumaric (I)
Gallic (I)
Glycine (I)
Indole-3-acetic (I)
Indole-3-propionic (I)
DL-Isocitric (I)
Itaconic (I)
a - K e t o g l u t a r i c ( I )
Male ic ( I )
L-Mal ic ( I )
a - N a p h t h a l e n e -a c e t i c (S)
j 3 - N a p h t h a l e n e -a c e t i c (S)
p - N a p h t h o x y a c e t i c (SP)
O x a l i c ( I )
Phenoxyacetic (S)
D(-) Quinic (I)
Salicylic (I)
Tartaric (I)
2-Thiobarbituric (I)
Trichloroacetic (S)
2,4,5-T (S)
Esters
Ethyl acetate (S)
Hydrocarbons
Benzene (S)
Hexane (S) i
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
DV
DV
DV
DV
DV
DV
DV
DV
DV
DV
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
DV
DV
DV
DV
DV
DV
DV
DV
DV
DV
NC
NC
NC
NC
DV
DV
DV
NC
NC
NC
NC
V (10)
NC
NC
NC
NC
NC
V (18)
V
BrV
DV
DV
DV
DV
DV
DV
NC
NC
NC
V (5)
NC
NC
NC
NC
NC
V (15)
V
BrV
DV
DV
DV
DV
DV
DV
NC
NC
NC
DV
DV
DV
TABLE 2.2 (Continued)
71
Kerosene (S)
Paraffin liquid (S)
Ketones
Acetone (S)
Acetophenone (S)
Methyl ethyl ketone (S)
Phenols
4-Chlorophenol (S)
o-Nitrophenol (S)
Phenol (S)
Pyrogallol (I)
Resorcinol (I)
Ureas
Urea (I)
Thiourea (I)
Ureide
Uric acid (I)
Inorganics
Ammonia (I)
Ammonium chloride (I)
Bleaching powder (I)
Calcium carbonate (I)
Calcium chloride (I)
Calcium oxide (I)
Ferrous sulphate (I)
Magnesium sulphate (I)
VIG (P)
NC
VIV
VIV
LG(P)
NC
LV
LV
NC
NC
NC
NC
NC
NC
DV
DV
DV
DV
DV
DV
NC
NC
NC
NC
NC
NC
DV
DV
DV
NC
NC
NC
NC
NC
BrV
DV
DV
DV
BrV
, NC
NC
NC
NC
NC
BrV
DV
DV
DV
BrV
DV
DV
DV
NC
NC
NC
NC
NC
NC
NC
NC
DV
DV
DV
DV
DV
DV
DV
DV
NC
NC
NC
NC
NC
NC
NC
NC
DV
DV
DV
DV
DV
DV
DV
DV
72
NC
NC
NC
NC
NC
NC
NC
DV
DV
DV
DV
DV
DV
DV
NC
NC
NC
NC
NC
NC
NC
DV
DV
DV
DV
DV
DV
DV
TABLE 2.2 (Continued)
Potassium dihydrogen phosphate (I) NC DV NC DV
Potassium permanganate (I)
Sodium bicarbonate (I) NC
Sodium carbonate (I)
Sodium hydroxide (I)
Sodium nitrate (I)
Sodium nitrite (I)
Sulphur (I)
Pesticides
Bavistin (I)
2,4-D ethyl ester (S) LV (A)
2,4-D sodium salt (I) NC
Dichlorvos (S)
Formothion (S)
Malathion (S)
Phosphamidon (S)
Thiometon (S)
Miscellaneous
Chloramine-T (I)
Detergent (I)
Methyl orange (SP)
Methyl red (SP)
Soap (I)
Note : Abbreviations used are:G = grey, P = pinkish, Lt = lower limit of detection, (I) = insoluble, (S) = soluble, and (SP) = sparingly soluble. See footnote of Table 2.1 for other abbreviations.
NC
LV (A)
NC
V (57)
V (19)
V (64)
NC
V (30)
DV
DV
DV
DV
DV
VIV
DV
NC
LV (4)
NC
V (38)
V (19)
V (32)
NC
V (30)
DV
DV
DV
DV
DV
V
DV
NC
NC
NC
NC
NC
DV
DV
DV
DV
DV
NC
NC
NC
NC
NC
DV
DV
DV
DV
DV
TABLE 2.3 DETECTION OF SOME FORMALDEHYDE PRODUCING COMPOUNDS IN DIFFERENT SOLVENTS AT 180°
Compound Solvent used Colour Lower limit of detection ( \^g)
2,4-D acid
2 , 4-D ethyl ester
2,4-D sodium salt
Benzene
Ethanol
Ether
TW
Benzene
Ethanol
Ether
TW
Ethanol
TW
LV
LV
LV
LV
LV
LV
LV
LV
LV
LV
5.0
5.0
5.0
5.0
4.0
4.0
4.0
4.0
4.0
4.0
Glycine
Hexamine
TW NC
Methyl orange
Methyl red
Benzene
Ethanol
TW
Ethanol
Ethanol
LV
LV
LV
NC
NC
0.5
0.5
0.5
-
NOTE : See experimental section and footnotes of Table 2.1
for abbreviations .
TABLE 2.A DETECTION AND SEMIQUANTITATIVE DETERMINATION OF 2,4-D ACID
IN SOIL
Colour Amount of Amount of soil (mg) 2,4-D (]ig) Nonfertile soil Fertile soil
50 25 LV LV
100 50
150 75 DV DV
NOTE : See footnote of Table 2.1 for abbreviations
TABLE 2.5 DETECTION AND SEMIQUANTITATIVE DETERMINATION OF 2,4-D ACID IN
VEGETATION
Amount of Amount of Colour leaves (mg) 2,4-D (lig )
500 25 NC
500 50 VIV
500 100 LV
NOTE : See footnote of Table 2.1 for abbreviations
76
the following aspects of the test had not been thoroughly
studied (i) the effect of different hydrolysing agents in
place of concentrated sulphuric acid, (ii) designing of new
vacuum spot test apparatus, (iii) preferential solubilities
of phenoxy herbicides in different solvents and (iv) applica
tion of the test for non-instrumental analysis of herbicides
residues for developing countries.
Table 2.1 shows that out of the eight hydrolysing
agents under study only two e.g. MnSO, .H„0 and ZnSO, .7H^0
give dark violet colour for phenoxyacetic acid. The colour
developed by the use of ZnSO,.7H„0 was found to be more inte
nse than that by MnSO,.H^O. Therefore ZnS0,.7H„0 is more
suitable for the newly developed technique. The test gives
no response when concentrated sulphuric acid is used in place
of above salts. It seems that in this case formaldehyde is
not free to move from reaction theatre to the reagent even
under reduced pressure. It may be due to the hydrogen bonding
or high solubility of formaldehyde in sulphuric acid.
An intense colour was produced when 35 mg of ZnSO,.7H^0
were taken while a larger amount fades the colour (Table
2.1). It may be due to the retention of formaldehyde with
the reaction mixture. Phenoxyacetic acid (18 jjg) and
ZnS0,.7H«0 (35 mg) give light violet colour at 150°, violet
at 160°, dark violet from 170-190° and brownish dark violet
at 200°. The appearance of dark violet colour is better than
that of brownish dark violet. Therefore any temperature in the
range of 170-190° is more suitable for performing the test.
'3Q2-/ 77
•'•*•• ..- , r .- '"
When the reagent (chromotropic acid in sulphuric acid)
was heated for 3 min at 160° and 180° there was no change
in colour while reagent turned black when heated for more
than 4 min. the reagent gives dark violet colour in 2 min
for 18 jjg of phenoxyacetic acid at 160° as well as 180°.
Hence it is clear that the heating for 2-4 min is sufficient.
As stated in the introduction that formaldehyde can be
detected in the vapour phase but the sensitivity decreases.
In the newly developed apparatus this drawback has partially
been overcome by the application of vacuum e.g. the lower
limit of detection of phenoxyacetic acid was found to be
10 ng and 75 ng in vacuum and without vacuum respectively.
The test under study gives positive response for benzene
soluble phenoxy herbicides such as 2,4-D acid, 2,4-D ethyl
ester, 4-chlorophenoxyacetic acid, phenoxyacetic acid and
2,4,5-T except 2,4-D sodium salt which is insolube in benzene
(Table 2.2). Besides phenoxy herbicides it also gives response
to dichlorvos (V), forraothion (V), malathion (V), thiome-
ton (V); N-bromosuccinimide (LBr), p-dimethylaminobenzal-
dehyde (V) and hexamine (V). N-Bromosuccinimide gives light
brown (LBr) colour and it may be detected selectively but
its sensitivity is very poor (375 [j, g) . The reaction is sensi
tive enough for phenoxy herbicides and it is highly sensitive
for hexamine (0.5 [ig). The intensity of the violet colour
produced for 2,4-D acid was found to be proportional to its
concentration and the reaction could be used for visual semi
quantitative determination of 2,4-D acid (10-100 fig) in water
78
samples. It is also clear from Table 2.2 that non-volatile
coloured compounds such as methyl orange and methyl red do
not affect the detection of 2,4-D acid because they are left
behind in the reaction theatre (tube A ) .
Data recorded in Table 2.3 shows the utility of prefe
rential solubility and formaldehyde producing reagent in
order to raise the selectivity of the spot-test under study.
For example 2,4-D acid and 2,4-D ethyl ester give positive
response when their solutions are prepared in benzene or
ethanol or ether or TW. Their lower limit of detection is
also the same in all the solvents used i.e. 5 }j,g of 2,4-Dacid
and 4 ng of 2,4-D ethyl ester. It shows that the sensitivity
of the test is independent of the solvents used. As 2,4-D
sodium salt is insoluble in benzene, it gives no response
and traces of 2,4-D acid as well as 2,4-D ethyl ester can
be detected in 2,4-D sodium salt matrix by the use of benzene.
However, 2,4-D sodium salt can be detected by making the
test solutions in ethanol, TW etc. and it can also be deter
mined in the presence of 2,4-D acid or 2,4-D ethyl ester
by the use of two solvents i.e.benzene for 2,4-D acid/2,4-D
ethyl ester and TW for total phenoxy herbicides. Hence it
is obvious that in benzene medium the test can be applied
for the detection and semi-quantitative determination of
free acid (2,4-D) generated by the hydrolysis of 2,4-D sodium
salt or 2,4-D ethyl ester in inorganics rich matrices such
as soil and water. The sensitivity and selectivity of the
test can also be enhanced by the use of specific reagent
79
for the splitting out of formaldehyde i.e. hydrated zinc
sulphate can split out formaldehyde from phenoxy herbicides,
some organophosphorus insecticides, N-bromosuccinimide,
p-dimethylaminobenzaldehyde, hexamine etc. while it gives
no formaldehyde from glycine, methyl orange, methyl red
etc. The interference due to non-volatile substances which
are soluble in benzene can be checked by the use of newly
designed apparatus which leaves non-volatiles in reaction
chamber. The results show that this technique is good for
the selective detection and determination of some pollutants.
Tables 2.4 and 2.5 show that the test can be used
for the detection of 2,4-D acid in leaves and soils success
fully. The sensitivity of the test is in the following
sequence : water > soils > leaves i.e. the lower limit
of detection in leaves, soil and water is 100, 25 and
10 jig respectively. Lower sensitivity of the test in soils
than that in water may be due to strong adsorption of
2,4-D acid on soils (11). However, the sensitivity is lowest
in leaves, it may be due to strong absorption (11) as well
as inefficient method of extraction. A simple extraction
procedure was used in order to show the basic importance
of the method. However, recovery of 2,4-D acid can be raised
by the use of a reported extraction method (12).
2.5 CONCLUSION
The results discussed above indicate successful applicabi
lity of the newly designed apparatus for the preliminary
80
detection of pesticides based pollutants in different
environmental samples. It may prove very helpful in places
where sophisticated instruments are either not available
or have not been installed so far.
REFERENCES
1. Qureshi.M.; Rathore.H.S. and Sulaiman,A.M. (1982) Water Research, j_6 , 435.
2. Rathore,H.S.; Gupta,S. and Khan,H.A. (1986) Anal. Letts., 12, 1545.
3. Rathore,H.S.; Ali,I. and Khan,H.A. (1988) J. Planar Chromatogr. , 2_, 252.
4. Rathore,H.S. ; Ali.I. and Kumar,A. (1989) Intern. J. Environ. Anal. Chem., 2^, 199.
5. Rathore,H.S.; Ali,I.; Sharma,S.R. and Saxena,S.K. (1988) Intern. J. Environ. Anal. Chem., 3_3, 209.
6. Feigl,F. and Anger,V. (1966) Spot Tests in Organic Analysis, Elsevier Scientific Publishing Co., 7th Ed., pp. 19,165,356,434,461,466,501.
7. Freed,V.H. (1948) Science, 1 ^ , 98.
8 Feigl,F. and Anger,V. (1972) Spot Tests in Inorganic Analysis, Elsevier Publishing Co., 6th Ed., pp.53-54.
9. Khan,M.A. (1982) Physico chemical studies on industrial wastes and their effects on soil, plants and river waters. M.Phil. Thesis (Chemistry), Aligarh Muslim University, Aligarh, India, p. 58.
10. Raziuddin (1986) Studies on industrial wastes and their effects on rivers, soils and underground waters. Ph.D. Thesis (Chemistry), Aligarh Muslim University, Aligarh, India, pp. 190-191.
11. Klingman,G.C. and Ashton.F.M. (1975) Weed Science: Principles and Practices, John Wiley & Sons, Inc., pp. 57,79,215, 220.
12. Chau,A.S.Y. and Afghan,B.K. (1982) Analysis of Pesticides in Water, CRC Press, Inc., Florida, Vol. II, pp. 216-218.
CHAPTER 3
CHARACTERIZATION OF BARIUM SULPHATE AS A TLC MATERIAL FOR THE SEPARATION OF PLANT CARBOXYLIC ACIDS
3.1 INTRODUCTION
TLC is widely used (1) for the qualitative analysis as well
as for the specific, sensitive, accurate, precise and rapid
quantification of a wide variety of compounds. Analytical
potential of numerous coatings has been studied. The previous
work (2-5) shows that calcium sulphate coated glass plates
can be used to separate organic acids occuring in plant
tissues. Calcium sulphate coatings (6) impregnated with
alumina, charcoal, flyash, silica gel and various reagents
can be used for the analysis of carboxylic herbicides. Barium
sulphate is more insoluble than calcium sulphate (7); it
is a non-toxic, cheap and chemically inert material that
can also be used as an adsorbent. Barium sulphate has been
used in TLC for the separation of sulpha drugs (8) and pharm
aceuticals (9). As barium sulphate is more inert than calcium
sulphate, it may have different chromatographic characteri
stics. This chapter reports the chromatographic studies of
some plant growth regulators listed in figure 3.1 and some
carboxylic acids of importance to the fruit growing industry,
on coatings of barium sulphate alone and its mixture with
calcium sulphate.
3.2 EXPERIMENTAL
3.2.1 APPARATUS AND MATERIALS
Stahl apparatus with universal applicator (adjustable thick
ness of the applied layers from 0.25-2.0 mm), glass plates
(20x4 cm), glass jars (25x5 cm), microburette (5 mL), conical
-CHrCHCOOH
Cinnomic acid
Ou - C H 2 - C - O H II 0
I ndo le -3 -ace t i c acid
p - Nophthaleneacetic acid p-Naphthoxyacet ic acid
< ^ 0 - C H 2 - C - C H ? - C - O H
Phenoxyacetic acid
Figure 3.1 Chemical s t ructures of plant growth regulators separated on admixtures of barium sulphate and calcium sulphate.
83
84
flask (100 mL), measuring flask (100 mL), lambda pipette
(0.1 mL), vaccupet, temperature controlled electric oven
and magnetic stirrer with hot plate etc. were used.
Barium sulphate (GSC, India), calcium sulphate dihy-
drate, carbon tetrachloride, chloroform, ethyl acetate,
2-propanol, sodium hydroxide and phenolphthalein were from
Merck, India; bromophenol blue and the carboxylic acids were
from Sigma, USA. All other reagents used were of analytical
grade .
3.2.2. PREPARATION OF SOLUTIONS
Solutions (1%) of plant growth regulators and other carboxy
lic acids were prepared in ethanol and distilled water (DW)
respectively. For quantitative separation studies solutions
of p-naphthaleneacetic acid (0.1 N) and oxalic acid (0.1 N)
were prepared in propanol and DW respectively. Solutions
of sodium hydroxide and phenolphthalein (1%) were prepared
in DW and propanol respectively.
3.2.3 PREPARATION OF PLATES
A slurry of various coatings in distilled water was applied
to the glass plates with applicator to give a film of 0.75 mm
(or 1.00 mm for quantitative determination) thickness.
Layers of 0.25 mm thickness were found to be suitable for
the separation of small amounts while thick layers (0.75 mm)
were suitable for large amounts. The plates were first
allowed to dry at room temperature and then in an oven at
110° for 1 hr . The composition of the coatings is shown
TABLE 3.1 COATINGS STUDIED
Coating Barium sulphate Calcium sulphate Distilled water (g) (g) (mL)
A - 30 70
B 30 70 130
C 50 50 130
D 70 30 130
E 80 20 130
F 90 10 130
G 30 - 60
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98
30 50 70 80 90
% of BaSO/i In (Baso^ • casOii)Coatings
Figure 3-2 Plot of time of developing the plates in
different solvents vs percentage of Ba SOA
In ( BaS04 * CasO/; ) Coatinfis.
Distilled water Ethyl acetate C hioroform
•—-• Carbon tetrachloride Ethanol Propanol
99
86
in Table 3.1.
3.2.4 SPOTTING OF TEST SOLUTIONS
Test solutions were spotted onto the plates with a fine capi
llary. The plates were kept at 30° (room temperature) for
15 min to remove the solvent and then developed.
3.2.5 VISUALIZATION OF CHROMATOGRAMS
The compounds under study were visualized on TLC plates by
spraying 0.1% ethanolic alkaline bromophenol blue solution.
3.2.6 MEASUREMENT OF R^ VALUES
For tailing, the front limit (RI) and the rear limit (RT)
were measured while for compact spots R^ values were calcula
ted by the following relation :
Distance travelled by substance (cm) R^ =
Distance travelled by solvent front (10 cm)
3.2.7 QUANTITATIVE SEPARATION OF p-NAPHTHALENEACETIC ACID FROM OXALIC ACID
A known volume of both the acids was spotted on plates of
coating B. The plate was developed with ethyl acetate and
the acids were located with the reagent. The acids were sepa
rated again on a fresh plate and the previously indicated
portions of the coating were removed with a spatula. R-Naphth-
aleneacetic acid was eluted with 25 mL of hot propanol while
the oxalic acid was removed by 25 mL of hot distilled water.
Both the solutions were made up to 50 mL by adding propanol
87
or water or 'both, in order to make water+propanol (1:1).
3.2.8 DETERMINATION OF ACIDS
The following procedure reported by T.S.Ma (10) was used
for the determination of acids. A known volume of an acid
solution (0.1 N) was taken in a 100 mL conical flask,
50 mL of propanol (50% v/v with DW) were added. The contents
were boiled on a hot plate and then titrated with standard
aqueous sodium hydroxide solution (0.0084 N) using phenol-
phthalein as indicator.
3.3 RESULTS
Some important separations achieved on different coatings
in common solvents are summarized in Tables 3.2-3.6. The
results of quantitative separation of j3-naphthaleneacetic
acid from oxalic acid are recorded in Table 3.7. Plots of
time taken by the solvent in ascending 10 cm of plate versus
composition of the coatings'are given in Figure 3.2.
3.4 DISCUSSION
In this laboratory silica gel G plates have been tested with
a number of solvent systems for the separation of the acids
under study. Silica gel plates impregnated with inorganic
salts, zinc oxide, detergent and grease have also been studied.
In most cases the acids remain at the point of application
or give bad tailing due to which separations are not feasible.
The previous work (2-6) showed that calcium sulphate is a
better material for separating acids. Now we have tested
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96
100
barium sulphate and the results show its advantages and dis
advantages .
It is clear from Table 3.3 that chloroform is an exce
llent solvent for analytically difficult separations on
BaSO,/CaSO, coatings. The following is the order of separation
potential of the solvents : chloroform > ethyl acetate >
DW > propanol > carbon tetrachloride. Mixed BaSO,/CaSO, coat
ings can also be used to separate plant growth regulators
such as cinnamic, indole-3-acetic , R-naphthaleneacetic ,
p-naphthoxyacetic and phenoxyacetic acids from other organic
acids such as citric, maleic , malic, malonic, oxalic, sali
cylic and tartaric acids which are generally present in leaves
and fruits (Tables 3.2 to 3.6).
It is known (11) that the growth, production and marke
ting of citrus fruits depend upon the concentration of organic
acids. Total acidity along with concentration of sugars is
an important criteria of fruit maturity in oranges and grape
fruits. BaSO,/CaSO, coatings seem to be very useful for the
analysis of organic acids present in fruits. For example
citric, malic, malonic, oxalic and tartaric acids etc. are
present in orange juice, grape juice, lemon juice, lime juice
and apple juice and their separations can be achieved on
BaSO,/CaSO, coatings using chloroform, ethyl acetate or pro
panol as developers. The synthetic auxin, p-naphthaleneacetic
acid, which is generally used for the control of preharvest
fruit drop (12) can be quantitatively separated from oxalic
acid (Table 3.7).
101
Barium sulphate coatings of thickness 0.25-1.00 mm
were found to be full of cracks after drying at 110° for
1 hr and the coating was separated from the plate on dipping
it in the solvent for development. Thus the coatings of barium
sulphate alone are not suitable for TLC. Coatings of mixtures
of barium sulphate and calcium sulphate are however very
stable and give good TLC separations. Most of the acids on
mixed coatings (B to F) move as compact spots while on calcium
sulphate alone (A) about 30% of the acids tail. Therefore
many separations can be successfully achieved on mixed coat
ings. The absence of tailing on barium sulphate may be due
to its lower solubility product (1.08x10 ) than that of
-4 calcium sulphate (1.96x10 ) in water at 25°.
Figure 3.2 shows the time taken by the solvent to
ascend a 10 cm length of mixed coated plates. The ascension
time increases with the increasing percentage of barium sul
phate and becomes a time consuming process for amounts grea
ter than 50%. It has been observed that the mobility of acids
increases with the increasing solubility of acids in a parti
cular solvent i.e. cinnamic acid is more soluble in ethanol
than in distilled water and its Rj. value is 1.0 in ethanol
and 0.0 in water. Malonic, tartaric and salicylic acids
behave similarly. The R , value of tartaric, citric, oxalic
and malonic acids is 1.0 while the solubility (13,14) of
barium tartrate (0.0279 g/100 mL) , barium citrate (0.0572 g/
100 mL), barium oxalate (0.0760 g/100 mL) and barium malonate
(0.2120 g/100 mL) is very low in water. R„ values of cinnamic,
102
malic and slicylic acids are 0.00, 0.78 and 0.50 respectively
while the solubilities of barium cinnamate (0.7260 g/100 mL),
barium malate (0.8830 g/100 mL) and barium salicylate
(28.6500 g/100 mL) are higher than those of the salts men
tioned above. Similar is the trend of R^ values and solubi
lities of calcium salts of these acids in water. These obser
vations show that there is no precipitation of the acids
as their barium or calcium salts on the coating. It is a
simple partition chromatography between the adsorbent and
the solvent.
3.5 CONCLUSION
The above work shows that layers of BaS0,/CaS0, have good
analytical potential for plant carboxylic acids. These coat
ings may be suitable for the analysis of acid pesticides.
REFERENCES
1. Sherma,J. (1986) Anal. Chem., 5^, 69R.
2. Rathore,H.S.; Sharma.S.K. and Kumari.K. (1981) Anal. Letts. , 1_4, 1327 .
3. Rathore,H.S.; Kumari,K. and Garg.M. (1983) J. Liq. Chroma-togr . , 6, 973.
4. Rathore,H.S.; Kumari.K. and Agarwal.M- (1985) J. Liq.
Chromatogr., 8, 1299.
5. Rathore,H.S. and Kuraari.K. (1982) Anal. Letts., j_5 , 873.
6. Ahmad,S.R.; Ali,I.; Rathore,H.S. and Gupta,S. (198A) J. Liq. Chromatogr., 7_, 1321.
7. Mellor,J.W. (1961) A Comprehensive Treatise on Inorganic and Theoretical Chemistry, Longmans, William Clowes and Sons Limited, Vol. Ill, pp. 777-790.
103
8. Sanghavi.N.M. and Khatib,S.I. (1982) Indian Drugs, 1_9_, 319.
9. Kamath,P.R, and Sanghavi,N.M. (1985) Indian Drugs, ^ , 336.
10. Ma,T.S. (1969) The Chemistry of Carboxylic Acids and Esters, Wilog, London, p. 871.
11. Vandercook,C.E. (1977) Citrus Science and Technology, Ch. 5, p. 208.
12. Bidwell,R.G.S. (1974) Plant Physiology, Macmillan Publishing Co., Inc., New York, p. 674.
13. Seidell (1940) Solubilities of Inorganic and Metal Organic Compounds, D. Van Nostrand Co. Inc., New York, 3rd Ed. Vol. I, pp. 130-140.
14. Stephen,H. and Stephen,!. (1963) Solubilities of Inorganic and Organic Compounds, Pergamon Press, London, Vol.1, part I, pp. 56-71,
CHAPTER 4
QUANTITATIVE SEPARATION OF TRICHLOROACETIC ACID FROM SOME CARBOXYLIC HERBICIDES ON REVERSED PHASE LAYERS OF BaSO,/CaSO, IMPREGNATED WITH COCONUT OIL
4.1 INTRODUCTION
Trichloroacetic acid (TCA) is an effective soil sterilant
that is widely used for perennial weed and grass control
on non-crop land. It is used selectively to control seedling
grasses and some broadleaf weeds in sugar beets and sugarcane.
TCA is used either alone or in a mixture with other herbici
des (e.g. Eptam, Nortron, Hexilure, 2,4-D, propazine, and
Dalapon) for weed control in numerous crops such as sugar
beet (1,2), fodder beet (3), winter oil seed rape (4) and
carrot (5). Among the phenoxy acid herbicides, 2,4-D and
2,4,5-T are more effective and are largely used in commonly
grown crops such as wheat, barley, oats, corn, paddy, and
sugarcane. The LD^^ (rat) of TCA; 2,4-D; 2,4,5-T and a-naph-
thaleneacetic acid are 5000 mg/kg, 370 mg/kg, 500 mg/kg
and 1000 mg/kg respectively. These values show that the
mammalian toxicity of the herbicides under study is not
of primary importance. However, as they tend to accumulate,
their concentration in soil, plants, water and marine animals
increases day by day. Hence there is a growing interest in
new methods of herbicides analysis.
It has been shown in chapter 3 that BaSO./CaSO, coat
ings have a high separation potential for carboxylic acids
(6). Paper impregnated with olive oil, paraffin, grease,
vaseline, etc. has been used for the separation of fatty
acids (7). However, the analytical potential of BaSO./CaSO,
coatings impregnated with fatty oils for separating carboxylic
herbicides has not been studied so far. Therefore in
CI I
CI — C—COOH I
CI
COOH
Trichloroacetic acid Benzoic acid
CH=CH-C-OH II 0
Cinnamic acid
N^ • C H 2 - C - O H II 0
N H
I n d o l e - 3 - acetic acid
•CHo-CHo—C-OH II 0
C H 2 - C - O H • II
0
N H
Indole-3-propionic acid oC-Naphthaleneacetic acid
( Continued )
106
p-Naphthaleneacetic acid p-Naphthoxyocetic acid
< ^ 0 - C H 2 - C - 0 H C I - \ \ A>-0 -CH2-C-0H
PhenoxyacetiC acid A-Chlorophenoxyocetic acid
CI
Cl-<\ / ) - 0 - C H 2 - C - 0 H
CI
C l n \ / ) - 0 - C H 2 - C - 0 H
Cl
2,4- Dichlorophenoxy acetic acid 2,4,5 — Trichlorophenoxyacetic acid
Figure 4.1 Chemical structures of herbicides and plant growth regulators separated on BaS04 /CaS04 layers impregnated with coconut oil .
107
108
continuation of the previous work carried out in our labora
tory (6,8,9), now the chromatographic behaviour of the herbi
cides and plant growth regulators (see Figure 4.1 for chemical
structures) on BaSO./CaSO, coatings impregnated with coconut
oil in distilled water and tap water is examined.
4.2 EXPERIMENTAL
4.2.1 APPARATUS AND MATERIALS
Stahl apparatus with a universal applicator, glass plates
(20x3 cm), glass jars (25x5 cm), Bausch and Lomb spectro-
nic-20 spectrophotometer, water bath and temperature contro
lled electric oven were used.
Bromophenol blue, herbicides and plant growth regula
tors (Sigma, U.S.A.), coconut oil (Tata, India) and TCA (CDH,
India) were used. All other reagents used were of analytical
grade.
4.2.2 PREPARATION OF PLATES
A slurry of various coatings in distilled water was applied
to the glass plates with the applicator to give a film of
0.50 mm thickness. The plates were first allowed to dry at
room temperature and then in an oven at 80° for half an hour.
4.2.3 PREPARATION OF REAGENT FOR TCA DETERMINATION
The reagent is prepared by mixing 40 mL of distilled water
and 0.5 mL of NaOH (15%) in 100 mL of pyridine.
TABLE 4.1 COATINGS STUDIED
Coating Barium sulphate Calcium sulphate Coconut oil Distilled water (8) (g) (mL) (mL)
40 40 0 100
40 40 5 100
40 40 10 100
D 40 40 15 100
110
4.2.4 SPOTTING OF TEST SOLUTIONS
Test solutions (1% ethanolic) were spotted onto the plates
with a fine capillary. For determination purposes varying
amounts such as 10 p.L to 50 |iL of TCA (2% aqueous solution)
were applied. The spots were dried with the help of a hot
air blower and then the plates were developed.
4.2.5 VISUALIZATION OF CHROMATOGRAMS
The herbicides and the plant growth regulators were visualized
on the plates by spraying an ethanolic alkaline solution
of bromophenol blue (0.1%).
4.2.6 MEASUREMENT OF R VALUES
For tailing, the front limit (RI) and the rear limit (RT)
were measured while for compact spots R^ values were calcula
ted by the procedure given in chapter 3.
4.2.7 QUANTITATIVE SEPARATION OF TCA
Known volumes of herbicides were applied to the plates coated
with B. The plates were developed with distilled water. Pre
viously indicated portions of the coatings were scratched
with a spatula. TCA was eluted from the coating material
with 5 mL of distilled water and determined spectrophotomet-
rically by the following procedure (10). An aliquot was trans
ferred to a conical flask containing 5 mL of 100% NaOH and
10 mL of acetone. The flask was agitated for 5 min and then
4 mL of the colouring reagent was added. The flask was heated
I l l
on a water bath at 70° for 7 min. It was cooled and then
1 mL of boiled water was added. The coloured fraction was
separated with a separating funnel and its absorbance was
recorded at 520 nm against the reagent blank.
4.3 RESULTS
Some ternary and quaternary separations achieved on different
coatings are shown in Figures 4.2, 4.3 and 4.4 , and are summa
rized below :
(i) Ternary separations on coating B using distilled water
as solvent :
PAA (0.7) - BOA (0.4) - 2,4,5-T (0-1) or CIA (0-2) or
a-NTAA (0-1) or p-NTAA (0-1)
TCA (1.0) - BOA (0.4) - 2,4,5-T (0-1) or CIA (0-2) or
a-NTAA (0-1) or p-NTAA (0-1)
PAA (0.7) - IPA (0.3) - 2,4,5-T (0-1) or a-NTAA (0-1)
TCA (1.0) - IPA (0.3) - 2,4,5-T (0-1) or a-NTAA (0-1)
(ii) Quaternary separations on coating C using tap water
as solvent :
TCA (1.0) - PAA (0.65) - IPA (0.3) - 2,4,5-T (0.1) or
a-NTAA (0.07)
or p-NTAA (0-1)
(iii) Ternary separations on coating D using distilled water
as solvent :
TCA (1.0) - PAA (0.5) - CPAA (0-2) or 2,4-D (0-1) or
2,4,5-T (0.0) or BOA (0.25)
112
or CIA ( 0 - 1 ) or NXAA ( 0 - 1 ) or
a-NTAA ( 0 - 1 ) or p-NTAA ( 0 . 0 )
or IPA ( 0 . 1 5 )
NOTE : Rj. values are given in parentheses.
Results of quantitative separation of TCA are recorded
in Table 4.2. Coefficient of variation (C.V.) for three
sets of results was calculated by the following expressions :
2 2
C.V. =
n-1
O xlOO
1
Where a = standard deviation, x, x„ = measured values, |i = average value and n = number of sets.
Abbreviations used are BOA = benzoic acid, CPAA = 4-chlorophenoxyacetic acid, CIA = cinnamic acid, 2,4-D 2,4-dichlorophenoxyacetic acid, lAA = indole-3-acetic acid, IPA = indole-3-propionic acid, a-NTAA = a-naphthaleneacetic acid, p-NTAA = p-naphthaleneacetic acid, NXAA = ^-naphthoxy-acetic acid, PAA = phenoxyacetic acid, TCA = trichloroacetic acid and 2,4,5-T = 2,4,5-trichlorophenoxyacetic acid.
4.4 DISCUSSION
Despite the preeminence of HPLC for carboxylic pesticides
residue determination, TLC continues to be researched and
used for qualitative and quantitative analyses under circums
tances where sophisticated apparatus is not available (11).
There have been many TLC studies of herbicides, mainly on
10-
8-
6-
u-
2-
0-
10
o
1
Q 12 o
10
1
0-3
10
O
1
D
7
Q
10
O
1
0 8
0
11 o
1
0
12
11 o
b 3
0
11
C7
1
0 7
11
o 1
0 8
0
Figure U-l Separation of some herbicides on coating B in DW 1 = BOA, 3= CIA . 7 = °C-NTAA.8=j3-NTAA, 10=PAA, 11 = TCA and 12 = 2.^.5-T.
113
c m
1 0 -8 -6^ u-2-0-
'
10
o
6
0 12
0
11
o
6
0 12
D
10
0
6
0 7
(7
11
o
6
0 7
(;j
c m
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4-2-0-
11
0 10
0 6
0 ^ 2 0
11
Q 0 6
« 7
0
11
0 10
o 6
0 8
t'
( a ) ( b )
Figure 4-3 Separation of some, herbicides
(a) On coating B in DW and (b) on coating C in T W
6 = IPA,7=cC-NTAA,8=j9-NTAAJ0 = PAA
11 = TCA and 12 = 2,4,5-1.
114
cm
10-8-6-
2-0-
11
o 10
0 2
0
11
O 10
0
11 o 10
o
12
o
11
o 10 C5 1
o
11
o 10
o 3
0
11
o 10
0 9
0
11
o 10
0
7
0
11 c
10
0
8
0
11
o 10
0 6 O
Figure 4-4 Separation of some herbicides on coating D in DW
1 = B0A,2-CPAA, 3 = CIA, 4=2 ,4 -D , 6 = IPA, 7=oc-NTAA, 8=p-NTAA^9=NXAA,10 = PAA, 11=TCA and 12=2,4,5-1.
115
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117
silufol or silica gel plates and with complex solvent systems.
For example chlorophenoxy herbicides (12) have been separated
on TLC plates made of silufol or a 2:3 mixture of silica
gel and kieselguhr by using cyclohexane-benzene-HOAc-liq.
paraffin and cyclohexane-benzene-Me„CO as the eluents. Tria-
zines and chlorophenoxy herbicides (2,4-D and 2,4,5-T) have
been determined (13) on preadsorbed silica gel layers impre
gnated with AgNO„ in solvent systems CHCl„-Me„CO for tria-
zines and hexane-AcOH-Et„0 for phenoxy herbicides. Triazine
herbicides (14) have been separated and determined on silufol
with C^H^CHo-MeoCO, EtOAc-CHCl„ or CHCl„-Me„CO and on silica 6 5 3 2 3 3 2
gel GF 254 with hexane-BuOAc as solvent systems.
Our previous work shows that BaSO,/CaSO, coatings plus
simple solvent systems such as chloroform, carbon tetrachlo
ride, propanol and water can be used for separating agro-
carboxylic compounds. However, in general, unsymmetrical
spots were found and this system could only be used for
separating simple mixtures in the form of binary separations,
Now it has been found that when BaSO,/CasO, coating is impre
gnated with coconut oil, test materials move as compact
and symmetrical spots that result in many quaternary and
ternary separations. For example, 4-chlorophenoxyacetic
acid, 2,4,5-trichlorophenoxyacetic acid, p-naphthoxyacetic
acid, indole-3-acetic acid and indole-3-propionic acid tail
0-10 on a BaSO^/CaSO, coating (A) while their movement is
0-2, 0.0, 0,0, 0-4, and 0-2 respectively on a BaSO^/CaSO^
coating impregnated with 15 mL of coconut oil (coating D)
118
in distilled water. Chromatographic behaviour of 4-chloro-
phenoxyacetic acid and indole-3-acetic acid shows the decrea
sing size of their spots with the increasing concentration
of coconut oil, i.e. their movements are 0-10, 2-5, 2-4
and 0.16 and 0-10, 2-7, 0-7 and 0.35 on coatings A,B,C and
D respectively in tap water. R^ values of 2,4-D and benzoic
acid are 0.7 and 5-8 on coating A while they are 0-1 and
0.25 respectively on coating D.
The chr omatograms shown in Figures 4.2, 4.3 and 4.4
prove that the system is specific for the separation of
TCA from complex mixtures because it has R^ value 1.0 on
all the coatings and solvents under study. Table 4.2 shows
that TCA can be separated from benzoic acid, 2,4,5-T, indole-
3-propionic acid and a-naphthaleneacetic acid in amounts
ranging from 400 |jg to 1000 |4g. The recommended preplant
application of TCA is 10-40 kg/ha three months before seeding
or transplanting in sugar beet (1,2), carrot (5) and fruit
tree nurseries (16). Hence it is clear that TLC coupled
with spectrophotometry can be applied for the separation
and determination of TCA. The lower limit of the spectro-
photometric method used is 400 ^g. Therefore for analysis
below 400 pg of TCA another spectrophotometric method must
be used .
It is also clear that comparatively costlier organic
solvents such as chloroform, carbon tetrachloride, acetone,
ethyl acetate and ethanol are not very useful on these
119
coatings while many ternary and quaternary separations can
be achieved in distilled water and tap water.
The ascension times for water over 10 cm of plates
coated with A, B, C and D are 1, 1, 2 and 3 hr, respectively,
showing that development time increases with the increasing
concentration of coconut oil.
4.5 CONCLUSION
The above results show that several carboxylic herbicides
and plant growth regulators which cannot be separated on
normal phase layers of calcium sulphate alone or admixtures
of calcium sulphate and barium sulphate, can be separated
and determined by RP-TLC on BaSO./CaSO layers impregnated
with coconut oil. Hence these layers seem to be very useful
for the analysis of complex mixtures of commonly used
carboxylic herbicides.
REFERENCES
1. Kvasov,V.A. and Kurakov, V. I. (1986) Agrokhimiya, j_]_, 95,
2. Kvasov.V.A. and Sergeev.V.M. (1984) Khim. Sel'sk Khoz., ig, 40.
3. Borona.V.P.; Prokopenko,L .S . and Zaverukha,L.M. (1987) Zasheh. Rast., 2 , 34.
4. Ward,J.T. and Turner,E.W. (1985) Proc. Bro. Crop Prot. Cong. Weeds, 1_, 223.
5 . M e r e z h i n s k i i , Y . G . ; S e m e n o v , A . G . and L u k ' y a n c h e n k o , A . S . ( 1 9 8 6 ) Khim. S e l ' s k K h o z . , 3_, 6 2 .
6 . R a t h o r e . H . S . and K h a n , H , A . ( 1 9 8 7 ) C h r o m a t o g r a p h i a , 2 3 , 4 3 2 .
120
7. Zweig.G. and Sherma,J. (1976) Handbook of Chromatography, Vol. I, CRC Press, Ohio, USA, p. 369.
8. Rathore,H. S. ; Kumari,K. and Agarwal,M. (1985) J, Liq. Chromatogr . , 8, 1299.
9. Rathore,H.S. ; Mi,I.; Ahmad,S.R. and Gupta,S, (1984) J. Liq. Chromatogr., 1_, 1321.
10. Snell,F.D. and Snell,C.T. (1953) Colorimetric Methods of Analysis, 3rd Ed., Vol. Ill, D.Van Nostrand Company Inc., New York, p. 336.
11. Sherma,J. (1987) Anal. Chem., _5i, 18R.
12. Hermenan,H. and Grahl,K. (1984) Acta Hydrochim.
Hydrobiol. , 12^, 685.
13. Sherma,J. (1986) J.Liq. Chromatogr., 9, 3433.
14. Kuthan,A. (1983) Veda Vysk. Praxi, 1±, 55.
15. Wiemer,B. (1985) Acta Hydrochim. Hydrobiol., V^_, 521.
16. Martynenko.A.I. (1985) Subtrop. Kul' t. , 5 , 117.
CHAPTER 5
NORMAL PHASE THIN-LAYER CHROMATOGRAPHY OF SOME HERBICIDES AND RELATED COMPOUNDS ON ADMIXTURES OF BARIUM SULPHATE AND CALCIUM SULPHATE IN MIXED SOLVENTS
5.1 INTRODUCTION
Calcium sulphate has been used for the separation of carboxy-
lic herbicides by normal-phase (1), ion-pair reversed-phase
(2), sequential (3) and two-dimensional (4) thin-layer
chromatography. Normal-phase thin-layer chromatography on
admixtures of calcium sulphate and barium sulphate (5) has
been used for the separation of plant carboxylic acids in
single solvent systems (Chapter 3). The utility of reversed-
phase TLC on irapr.egnated BaSO,/CasO, layers has already
been discussed in chapter 4, which shows that the separation
potential of BaSO./CaSO, coatings can be enhanced by impreg
nating the coating with coconut oil.
Literature shows that a number of papers have been
devoted to separate carboxylic herbicides by TLC on
silufol (7,8) or a 2:3 mixture of silica gel and kieselguhr
(7), and preadsorbed silica gel layers impregnated with
AgNOo (9) in mixed solvent systems. However, separation
potential of BaSO./CaSO, coatings in mixed solvent systems
has not been tested so far.
Therefore, in continuation to previous work, now
an attempt has been made to investigate the separation poten
tial of BaSO./CaSO, coatings in mixed solvent systems. The
results obtained are discussed in this chapter.
5.2 EXPERIMENTAL
5.2.1 APPARATUS AND MATERIALS
Stahl apparatus with a universal applicator, glass plates
123
(20x3 cm), glass jars (25x5 cm), Bausch and Lomb spectro-
nic-20 spectrophotometer, centrifugal machine (Baird and
Tatlock Ltd., England), magnetic stirrer (Sunvic, U.K.)i
glass coated magnetic bars (approx. length 0.8 cm), tempera
ture controlled electric oven (Tempo, India), lambda pipette,
10 mL conical flask etc. were used.
Ferric chloride anhydrous (Ranbaxy, India); methanol
(Glaxo, India); perchloric acid (Merck, India) and bromo-
phenol blue, herbicides and plant growth regulators were
from Sigma, USA. All other reagents used were of analytical
grade.
5.2.2 PREPARATION OF PLATES
A slurry containing barium sulphate (50 g), calcium sulphate
(50 g) and distilled water (130 mL) was applied to the glass
plates with an applicator to give a film of 0.50 mm thickness.
The plates were first allowed to dry at room temperature
and then in an oven at 110° for one hr.
5.2.3 PREPARATION OF REAGENT FOR IND0LE-3-ACETIC ACID (lAA) DETERMINATION
The reagent is prepared by mixing 1 mL of 0.5 M ferric chlo
ride solution in 50 mL of 35% (v/v) perchloric acid.
5.2.4 SPOTTING OF TEST SOLUTIONS
Test solutions (1% ethanolic) were spotted onto the plates
with a fine capillary. For determination purpose 100 ng of
124
lAA (10 |j,L of 1% methanolic solution) and varying amounts
of benzoic acid, a-naphthaleneacetic acid and 2,4,5-T such
as 50 p,g to 100 |ig (5 [iL - 10 (J. L of 1% methanolic solution )
were applied. The spots were dried with the help of a hot
air blower and then the plates were developed.
5.2.5 VISUALIZATION OF CHROMATOGRAMS
The herbicides and plant growth regulators were visualized
on the plates as yellow spots on blue background, by spraying
an ethanolic alkaline solution of bromophenol blue (0.1%).
5.2.6 MEASUREMENT OF R^ VALUES
For tailing, the front limit (RI) and the rear limit (RT)
were measured while for compact spots R- values were calcula
ted by the procedure given in chapter 3,
5.2.7 QUANTITATIVE SEPARATION OF lAA
Known volumes of a standard solution of lAA were applied
on TLC plates. The plates were developed with carbon tetra-
chloride-propanol (100:0.5) solvent system. Previously indica
ted portions of the coating were scratched with a spatula
and collected in a conical flask of 10 mL capacity, 2.5 mL
of methanol were added into it, the mixture was stirred for
5 min, and then transferred to the centrifuge tube. The coni
cal flask was washed with 0.5 mL of methanol and the washings
were transferred to the same centrifuge tube. The solid por
tion was removed by centrifugation and lAA was determined
125
in clear solution by the following method (10). The freshly
prepared reagent (2 mL) was rapidly added dropwise to the
centrifugate with continuous agitation and the system was
allowed to stand in dark for one hr for colour development.
Finally the absorbance was measured at 510 nm against a blank
containing methanol (3 mL) and the reagent (2 mL),
5.3 RESULTS
The separations achieved on BaSO,/CaSO, coatings using mixed
solvent systems are recorded in Table 5.1. Results of quanti
tative separations of indole-3-acetic acid from benzoic acid,
«-naphthaleneacetic acid and 2,4,5-T are given in Table 5.2.
To calculate analytical parameters, the expressions
given in the result section of chapter 4 were used.
5.4 DISCUSSION
Previous publications (1-5) from this laboratory show that
calcium sulphate and barium sulphate are good TLC materials.
Thin layers of barium sulphate are not as good as those of
calcium sulphate alone. Admixtures of barium sulphate and
calcium sulphate give uniform, smooth and stable layers and
have very good separation potential for carboxylic acids.
The time of development (5) of TLC plates increases
with the increasing percentage of barium sulphate in the
admixtures (Figure 3.2) and the admixture containing barium
sulphate/calcium sulphate (1:1, w/w) is most suitable for
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thin-layer chromatographic studies. As discussed in chap
ter 3, the above admixture can be used for separating plant
carboxylic acids such as citric, maleic, malic, malonic,
oxalic, salicylic and tartaric acids and some plant growth
regulators such as cinnamic acid, indole-3-acetic acid,
n-naphthaleneacetic acid, p-naphthoxyacetic acid and phenoxy-
acetic acid in single solvent systems. But in case of chloro-
phenoxy herbicides, the separation potential is limited due
to tailing nature of the herbicides in some of the solvents
such as benzene, carbon tetrachloride and chloroform. However,
compact spots with R, value 1.0 were obtained in dioxane,
ethyl acetate and propanol and some binary separations were
obtained in distilled water.
A number of ternary and quaternary separations were
achieved on reversed-phase coatings of BaSO,/CaSO, (1:1)
impregnated with coconut oil (6) in water while organic sol
vents were found to be ineffective. The separation potential
increases with an increase in the percentage of coconut oil
used for impregnation. Unfortunately the time of development
also increases with increasing percentage of coconut oil.
Table 5.1 shows that thin layers of BaSO./CaSO, can 4 4
be used for several binary separations of carboxylic herbi
cides and related compounds using mixed solvent systems.
The following separations, which were not possible on BaSO,/
CaSO, as well as on BaSO./CaSO, impregnated with coconut
oil in single solvent systems, could be achieved in mixed
132
solvent systems : benzoic acid from 4-chlorophenoxyacetic
acid; indole-3-acetic acid from benzoic acid, cinnamic acid,
4-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid,
a-naphthaleneacetic acid, p-naphthaleneacetic acid, p-naphth-
oxyacetic acid and 2,4,5-trichlorophenoxyacetic acid; p-naph-
thaleneacetic acid from a-naphthaleneacetic acid and ^-naphth-
oxyacetic acid; indole-3-propionic acid from benzoic acid,
cinnamic acid and ^-naphthoxyacetic acid (Table 5.1).
Data recorded in Table 5.2 show that indole-3-acetic
acid can be separated quantitatively from benzoic acid,
a-naphthaleneacetic acid and 2,4,5-trichlorophenoxyacetic
acid in carbon tetrachloride-propanol (100:0.5) solvent system.
5.5 CONCLUSION
Admixture of barium sulphate/calcium sulphate (1:1) is a
good thin-layer chromatographic material. It can be used
for some binary separations of carboxylic herbicides and
related compounds in water as developer. The above admixture
impregnated with coconut oil can be used for ternary and
quaternary separations using water as developer. Many binary
separations which are not possible on normal-phase layers
using single solvent systems as well as on reversed-phase
layers, can be achieved on normal-phase layers of the same
admixture using mixed solvent systems.
133
REFERENCES
1. Rathore,H.S.; Ali,I.; Ahmad,S.R. and Gupta,S. (1984) J, Liq. Chroraatogr., 1_, 1321.
2. Rathore,H.S.; Mi,I.; Sharma.S.R. and Saxena.S.K, (1988) Intern. J. Environ. Anal. Chem., 3_3, 209.
3. Rathore.H.S. and Saxena.S.K. (1987) J. Liq. Chromatogr., 10, 3623.
4. Rathore,H.S. , and Gupta,S. (1987) J. Liq. Chromatogr., iO, 3659.
5. Rathore,H.S. and Khan,H.A (1987) Chroraatographia, 23, 432.
6. Rathore.H.S. ,• Ali,I. and Khan,H.A. (1988) J. Planar Chromatogr .,• 1, 252.
7. Hermenan,H. and Grahl.K. (1984) Acta Hydrochim. Hydrobiol., j_2, 685.
8. Kavetskii,V.N.; Bublik,L.I. and Fuzik,G.V. (1987) Zh. Anal. Khim. , 4J_, 1302.
9. Sherma,J. (1986)j.Liq.Chromatogr., £, 3433.
10. Mahadevan,A. and Sridhar.R. (1986) Methods in Physiological Plant Pathology, 3rd Ed., Sivakami Publications, p. 270.
Characterization of Barium Sulphate as a TLC Material for the Separation of Plant Carboxylic Acids
H.S.RathoreVH.A.Khan
Chemistry Section, Z .H. College of Engineering and Technology, Aligarh Muslim University, Aligarh 202 001, India
Key Words
Thin layer chromatography
Barium sulphate/calcium sulphate
Carboxylic acids
Citrus fruit
Summary
A mixture of barium sulphate and calcium sulphate has
been shown to be a good TLC material for the detection
and the quantif ication of a variety of carboxylic acids
known to be plant growth regulators and carboxylic
acids of importance in the citrus frui t industry.
lnt''oduction
TLC is used for the qualitative and quantitative analysis of
a wide variety of compounds (11 and the potential of
numerous coatings has been studied. Our previous work
(2, 3, 4, 5, 6] showed that glass plates coated wi th calcium
sulphate can be used to separate organic acids occurring in
plant tissues. Barium sulphate is more insoluble than cal
cium sulphate (7 | ; it is a non-toxic, cheap and chemically
inert material that can also be used as an adsorbent. Barium
sulphate has been used in TLC for the separation of sulpha
drugs (8) and pharmaceuticals | 9 | . This paper reports the
separation of some carboxylic acids of importance to the
fruit growing industry on coatings of barium sulphate alone
and mixed wi th calcium sulphate.
The solutions used were prepared by the procedure report
ed previously | 6 | .
Plates were prepared as follows — A slurry of the various
coatings in distilled water was applied to the glass plates
wi th the applicator to give a f i lm thickness of 0.75 mm (or
1.00 mm for quantitative determinations). Layers of 0.25
mm were found to be suitable for the separation of small
amounts while thick layers (0.75 mm) were suitable for
large amounts. The plates were first allowed to dry at room
temperature and then in an oven at 110 ''C for 1 hour. The
composit ion of the coatings is shown in Table I.
Test solutions were spotted onto the plates wi th a fine ca
pillary. The plates were kept at 30 °C (room temperature)
for 15 minutes to remove solvent and then developed.
For tailing, the front l imit (Rl) and the rear l imit (RT)
were measured while for compact spots Rf values were cal
culated in the usual way.
Quantitative separation of /^-naphtha/eneacetic acid from
oxalic acid
A known volume of both acids was spotted on plates coat
ed wi th coating B. The plate was developed wi th ethyl ace
tate and the acids were located wi th 1 % ethanolic alkaline
bromophenol blue. The acids were separated again on a
fresh plate and the previously indicated portions of the
coating were removed wi th a spatula. /3-naphthaleneacetic
acid was eluted wi th 25 ml of hot 2-propanol from the
material scratched off the plate while the oxalic acid was
removed by 25 ml of hot distilled water. Both the solutions
were made up to 50 ml in 50 % 2-propanol by adding water
or propanol and then t i trat ing wi th standard alkali and phe-
nolphthalein indicator.
Experimental
A Stahl apparatus wi th a universal applicator made in India
was used to coat glass plates (20 x 4 cm). The plates were
developed in glass jars (25 x 5 cm).
Barium sulphate was from GSC, India. Calcium sulphate
dihydrate, carbon tetrachloride, chloroform, ethyl acetate,
2-propanol, sodium hydroxide and phenolphthalein were
from Merck, India. Bromophenol blue and the carboxylic
acids were f rom Sigma, U.S.A. A l l other reagents were of
analytical grade.
Results
Most important separations achieved on different coatings
in common solvents are: NTAA (2 -4 ) from CA (0), lAA
(0), MEA (0), MOA (0), OA (0) and TA (0) on coating C in
carbon tetrachloride; CIA (3 -8 ) from CA (0), MEA (0),
MOA (0), OA (0) and TA (0) on coating A in chloroform;
MOA (0), MEA (0), CA (0), OA (0) and TA (0) from CIA
( 3 - 6 ) , f\JTAA (0.62) and N X A A (0.65) on coating B in
ch loroform; CIA (0), l A A (0), N T A A (0) and N X A A (0)
f rom CA (I), MEA (I), MA (I), MOA (I), OA (I), PAA (I),
432 Clirnrnnloqr.-iphu) Vol ?3. Ni i . 6, June 1987 Oriq i i i ; l l5
0009-5893/87/6 0432-03 $ 0 3 00/0 © 1987 Fnedr. Vieweg & Sohn Verlagsaesellschaft inhH
Table 1. Coat
Coating
A
B C D E F
G
Ba
ngs studied
rium sulphate
(g)
-. 30
50
70
80 90
30
Calcium su
(gi
30
70
bO
30
20
10
Iphate D:st lied V
(ml)
/O
130
130
130
1 30
130
GO
StPr
SA (I) and TA (I) on coatings A, B, C, D, E and F in
distilled water; CA (0), OA (0) and TA (0) f rom CIA (I),
lAA (I), MOA (I), NTAA (I), N X A A | l ) , PAA (I) and SA
(I) on coatings A, B and C in ethyl acetate; CA (0), OA (0)
and TA (0) f rom CIA (I), lAA (I), MEA (I). MA (I), MOA
(I), NTAA (I), NXAA (I), PAA (I) and SA (I) on coating B
in propanol.
Rf values are given in parenthesis. Abbreviations used are
CIA = cinnamic acid, CA = citric acid, l A A = indole-3-
acetic acid, MEA = maleic acid, MA = malic acid, MOA
= malonic acid, NTAA = |3-naphthaleneacetic acid, N X A A
= j3-naphthoxyacetic acid, OA = oxalic acid, PAA = phen-
oxyacetic acid, SA = salicyclic acid and TA = tartaric acid.
Quantitative results for the separation of ^-naphthalene-
acetic acid f rom oxalic acid are shown in Table I I . Plots of
the t ime taken by the solvent to ascent 10 cm against the
composition of the coating is shown in Fig. 1.
% of BaSO, in
50 70 80 90
BaSO^ + CaSO^) Coatings
Fig. 1
Plot of time of developing tfie plates in different solvents vs percent
age of BaS04 in (BaS04 ^ CaS04) Coatings.
• • Distilled water • • Carbon tetrachloride
Ethyl acetate x x Ethanol A A Chloroform • • Propanol
Discussion
In this laboratory silica gel G plates have been tested wi th
a number of solvent systems for the separation of the acids
under study. Silica gel plates impregnated wi th inorganic
salts, zinc oxide, detergent and grease have also been stud
ied. In most cases the acids remain at the point of applica
t ion or give bad tailing so that separation is not feasible.
Our previous work showed that calcium sulphate is a better
material for separating acids. Now we have tested barium
sulphate and the results show its advantages and disadvant
ages.
Chloroform is an excellent solvent for analytically di f f icul t
separations on BaS04/CaS04 coatings. The fol lowing is the
order of separation potential of the solvents: chloroform
> ethyl acetate > distilled water > propanol > carbon
tetrachloride. Mixed BaS04/CaS04 coatings can also be
used to separate plant growth regulators such as cinnamic,
indole-3-acetic, /3-naphthaleneacetic, j3-naphthoxyacetic and
phenoxyacetic acids f rom salicylic and tartaric. It is known
[ 1 0 | that the growth, production and marketing of citrus
f ru i t depend upon the concentration of organic acids. Total
acidity along wi th concentration of sugars is an important
criterion of frui t maturi ty in oranges and grape fruits.
BaS04/CaS04 coatings seem to be very useful for the ana
lysis of organic acids present in frui t . For example citric,
malic, malonic, oxalic and tartaric acids etc. are present in
many frui t juices and their separations can be achieved on
Table I I . Quantitative Separation of (Jnaphthaleneacetic Acid from Oxalic Acid on Coatings B in Ethyl Acetate
Amount loaded (mg)
9.310 9.310 9.310 9.310
(J-naphthaleneacetic acid
Amount found (mg)
9.433 8.711 9.110 9.117
Percentage error
- 1 32 - 6,43 - 2.14
- 1 42
Amount loaded (mgl
3.160
3 150 3.150
3.150
Oxalic nrid
Amount found
(ri iq)
3.090 3.177
3.090 3.067
Percentage error
- 1.90
+ 0.85
- 1.90
- 2.63
Chromatographia Vol. 23, No. 6, June 1987 Origmals 433
BaS04/CaS04 coatings wi th chloroform, ethyl acetate or
propanol. The synthetic auxin, /3-naphthaleneacetic acid,
which is generally used for the control of preharvest f ru i t
drop [ l l ] , can be quantitatively separated from acids such
as oxalic acid (Table II) .
Barium sulphate coatings of thickness 0.25—1 mm were
found to be ful l of cracks after drying at 110 °C for 1 hour
and are not suitable for TLC. Coatings ot mixtures of bari
um sulphate and calcium sulphate are however very stable
and give good TLC separations. Most of the acids on mixed
coatings (8 to F) move as compact spots while on calcium
sulphate alone (A) about 30 % of the acids tail so that more
separations can be successfully achieved on mixed coatings.
The absence of tailing on barium sulphate may be due to
its lower solubil i ty product (1.08 x 1 0 ^ ' ° ) than calcium
sulphate (1.96 x 10"'*) in water at 25 °C.
Fig. 1 shows that the time taken by the solvent to ascend a
10 cm length of mixed coated plates. The time required
increases wi th the percentage of barium sulphate and be
comes a time consuming process for amounts greater than
50 %. It has been observed that the mobi l i ty of acids in
creases wi th the increasing solubil ity of acids in a particular
solvent i.e. cinnamic acid is more soluble in ethanol than in
distilled water and has an Rf value of 1 in ethanol and 0 in
water. Malonic, tartaric and salicylic acids behave similarly.
The Rf value of tartaric, citric, oxalic and malonic acids is
1 while the solubil ity [12, 13] of barium tartrate (0.0279 g/
100 ml), barium citrate (0.0572 g/100 ml) , barium oxalate
(0.076 g/100 ml) and barium malonate (0.212 g/100 ml) is
very low in water. Rf values of cinnamic, malic and sali
cylic acids are 0, 0.78 and 0.50 respectively while the solu
bilities of barium cinnamate (0.726 g/100 ml), barium ma-
late (0.883 g/100 ml) and barium salicylate (28.65 g/100
ml) are higher than that of the salts mentioned above.
These observations show that there is no precipitation of
the acids as their barium or calcium salts on the coating. It
is a simple part i t ion chromatography between the adsorb
ent and the solvent.
References
M l Joseph Sherma, Anal. Chem., 58 (1986) 69 R. | 2 | H.S. Rathore, S.K.Sharma. K. Kumari, Anal Chem., 14 (1981)
1327 | 3 | H. S. Rathore, K. Kumari, M. Garg, J. Liq. Chromatogr., 6
(1983) 973. | 4 | H. S. Rathore, K. Kumari, M. Agarwal, J. Liq. Chromatogr., 8
(1985) 1299. | 5 | H.S.Rathore, K.Kumari. Anal. Letts.. 15 (1982) 873. 16) S.R.Ahmad, I.AIi, H.S. Rathore, S.Gupta, J. Liq. Chromatogr.,
7 (19841 1321. | 7 | J. W. Mellor, in A comprehensive Treatise on Inorganic and
Theoretical Chemistry, Longmans, Vo l . I l l , Wiliam Clowes and Sons Limited, London, 1961, pp. 7 7 7 - 7 9 0 .
| 8 | N.M.Sanghavi, S.I.Khatib, Indian Drugs, 19 (1982) 319. | 9 | P.R.Kamath, N.M.Sanghavi. Indian Drugs, 22 (19851 336.
| 1 0 | Carl E. Vandercook. in Citrus Science and Technology, 1977, Ch 5, p. 208.
1111 R. G. S. Bidwell. in Plant Physiology, Macmillan Publishing Co., Inc., New York, 1974.
\^7\ Seidell, in Solubilities of Inorganic and Metal Organic Compounds, 3rd Ed, Vol . I, D. Van Nostrand Company Inc., New York, 1940, pp. 130 -140 .
|13) H.Stephen, T.Stephen (Editors), in Solubilities of Inorganic and Organic Compounds, Vol . I, Pergamon Press, London, 1963, pp. 5 6 - 7 1 .
Received: March 2, 1987 Revised manuscript received: Apri l 6, 1987 Accepted: Apri l 16, 1987
434 Chromatographia Vol . 23, No. 6, June 1987 Originals
Short Communications
Quantitative Chromatographic Separation of Trichloroacetic Acid from Some Carboxylic Herbicides on BaS04-CaS04 Coatings Impregnated with Coconut Oil
H. S. Ralhore*. I. All, and H. A. Khan
Key words:
TLC Trichloroacetic acid Carboxylic herbicides Impregnated BaS04-CaS04 as stationary phase
1 Introduction
Trichloroacetic acid (TCA) is used alone or in admixture other herbicides (e.g. Eptam, Nortron, Hexllure, 2,4-D, Propaiine, and Delapon) for weed control in numerous crops such as sugar beet (1,2), fodder beet [3], winter oil seed rape [4], and carrot (5). Among the phenoxy acid herbicides, 2,4,-D and 2,4,5-T are more effective and are largely used in commonly grown crops such as wheat, barley, oats, com, paddy, and sugar cane. The LD50 (rat) of TCA; 2,4-D; 2,4,5-T and a-naphthaleneacetic acid are 5000 mg/kg, 370 mg/kg, 500 mg/Vg, and 1000 mg/l<g respectively. These values show that the mammalian toxicity of the herbicides under study is not of primary importance. However, as they tend to accumulate, their concentration in soil, plants, water, and marine animals increases day by day. Thus there is a growing interest in new methods of herbicide analysis.
We have previously shown [6] that BaSOj-CaSO^ coatings have a high separation potential for carboxylic acids. Paper impregnated with olive oil, paraffin, grease, vaseline, efc. has been used for the separation of fatty acids [7]. In continuation of our previous work [6, 8, 9], we now examine the chromatographic behavior of the herbicides on BaS04-CaS04 coatings impregnated with coconut oil in distilled water and tap water.
2 Experimental
Stahl apparatus with a universal applicator, glass plates (20 x 3 cm), glass jars (25 x 5 cm), Bausch and Lomb spectronic-20 spectrophotometer, water bath, and temperature controlled electric oven.
Bromophenol blue, herbicides, and plant growth regulators were from Sigma, USA. Coconut oil and TCA were from Tata and CDH, India respectively. All other reagents used were of analytical grade.
A slurry containing BaSO< (40 g), CaS04 (40 g), coconut oil (0, 5, 10 and 15 ml for coatings A, B, C, and D respectively) and distilled water (100 ml) was applied to the glass plates with the applicator to give a film thickness of 0.50 mm. The plates were first allowed to dry at room temperature and then in an oven at SOX for half an hour.
The reagent is prepared by mixing 40 ml of distilled water and 0.5 ml of 15% NaOH in 100 ml of pyridine.
The tap water used contained dissolved oxygen (8 ppm), temporary hardness (135-144 ppm), and permanent hardness (350 ppm). Its conductivity was 132.25-134.56 p o h m ' c m ' and pH 7.5-7.6.
Test solutions (1 % ethanolic) were spotted onto the plates with a fine capillary. For determination purposes varying amounts such as 10 pi to 50 pi of TCA (2% aqueous solution) were applied. The spots were dried with the help of a hot air blower and then the plates were developed.
For tailing spots, the front limit (Rl) and the rear limit (RT) were measured while for compact spots fl, values were calculated in the usual way |9].
The herbicides on the plates were visualized by spraying with an ethanolic alkaline solution of bromophenol blue (0.1%).
Known volumes of herbicides were applied to plates coated with B. Ttie plates were developed with distilled water. Previously indicated portions of the coating were scratched with a spatula. TCA was eluted from the coating material with 5 ml of distilled water and determined spectrophotometrically as follows [10]. An aliquot was transfen^ed to a conical flask containing 5 ml of 100% NaOH and 10 ml of acetone. The flask was agitated for 5 min and then 4 ml of the coloring reagent was added. The flask was heated on a water bath at 70 X for 7 min. It was cooled and then 1 ml of boiled water was added. The colored fraction was separated with a separating funnel and its absorbance was recorded at 520 nm against the reagent blanl<.
3 Results
Some ternary and quaternary separations achieved on different coatings are shown in Figure 1, 2. and 3. Results of quantitative separations of TCA are recorded in Table 1. Percentage error and coefficient of variation for three sets of results were calculated by the fonnula given in our previous publication [17].
H. S. Rathore, I. All, and H. A. Khan. Department of Applied Chemistry, Z. H. College of Engineering and Technology, Aligarh Muslim University, Aligarti 202002, India.
Abbreviations used: (1) BOA = benzoic acid, (2) CPAA • 4-chlorophenoxyace-tic acid. (3) CIA - cinnamic acid. (4) 2,4-D - 2.4-dichlorophenoxyacetic acid, (5) lAA - indole-3-acetic acid, (6) IPA - indolepropionic acid, (7) a-NTAA - a-naphlhaleneacetic acid, (8) p-NTAA - p-naphthaleneacetic acid, (9) NXAA - p-naphthoxyacetic acid, (10) PAA - phenoxyacetic acid, (11) TCA - trichloroacetic acid and (12) 2.4.5-T - 2,4,5-trichlorophenoxyacetic acid.
252 Journal 0I Planar Chromatography C' 1988 Dr. Allted Huelliig Publishers
Short Communications
Table 1
Quantitative separation and determination of TCA on coating B In distilled water.
Mixture separated TCA(1)-BOA(0.4)-2,4,5-T{0-1) Amount of TCA applied (^g)
200 400 600 800
1000
Amount of TCA found (vig)
Nil 300 600 750
1000
TCA(1HPA(0.3>-2,4,5-T(0-1) Amount of TCA applied (vig)
200 400 600 800
1000
Amount of TCA found (pg)
Nil 300 550 750 900
TCA(1HPA(0.3Hi-NTAA (0-1) Amount of TCA applied (ug)
200 400 600 800
1000
Amount of TCA found (pg)
Nil
300 550 800 950
% error
--25 - 5.53 - 4.13 - 5
Coefficient of variation (C.V.)
— 0.00 5.09 3.76 5.26
Rf values are given in parenttieses. The concentration of different acids loaded on the plate is BOA - 20 ug. 2.4.5-T - 20 ug. IPA - 20 ug and Q-NTAA - 20 ug.
10
8
6
4
2
0
10 o
1
Q 12
o
10
O
1
0 3
P
10
o
1
D
7
a
10
o
1
0 8
0
11 O
1
0
12
o
11
o
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0
11
1
0 7
9
11
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0
10 8 G i.
2 0-
11
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^
2
0
11
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^
11
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O
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11
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o
11
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3
0
11
o 10
u 9
0
11
o 10
^
7
0
11 c ?
10 0
0
11
o 10
61
6 O
Figure 1
Separation o( some herbicides on coating B in DW. 1 • BOA, 3 • CIA, 7 • a-NTAA, 8 - p-NTAA, 10 - PAA, 11 - TCA and 12 - 2,4,5-T.
Figure 3
Separation of some herbicides on coating D In DW. 1 - BOA, 2 - CPAA, 3 • CIA, 4 - 2,4-D, 6 - IPA, 7 » o-NTAA, 8 - p-NTAA, 9 - NXAA, 10 - PAA, 11 • TCA and 12 - 2,4,5-T.
Cm
1 0 -
8 -
fi-1
4-2-0-
10
o
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0 12
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o 6
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(a) (b) Figure 2
Separation of some herbicides, (a) On coating B in DW and (b) on coating C In tap water. 6 - IPA, 7 - a-NTAA, 8 - p-NTAA, 10 - PAA, 11 - TCA and 12 - 2,4,5-T.
4 Discussion
Despite tlie preeminence of HPLC for carboxylic pesticide residue determination, TLC continues to be researctied and used for qualitative and quantitative analyses under circumstances wtiere sophisticated apparatus is not available [11]. Ttiere have been many TLC studies of herbicides, mainly on silufol or silica gel plates and with complex solvent system, e.g.:
Chlorophenoxy herbicides [12] have been separated on TLC plates made of silufol or a 2:3 mixture of silica gel and kieselguhr by using cyclohexane-benzene-HOAc-liq. paraffin and cyclohexane-ben-zene-MejCO as the eluents. Triazines and chlorophenoxy herbicides [13] (2,4-D and 2.4,5-T) have been determined on preadsorbent silica gel layers Impregnated with AgNOj in solvent systems CHCIa-MejCO for triazines and hexane-AcOH-EtjO for phenoxy herbicides. Triazine herbicides [14] have been separated and determined on silufol with toluene-acetone, ETOAc-CHCI,, or CHCl3-fv1e2CO and on silica gel GF 254 with hexane-Bu acetate as solvent systems. Chromatographic determination [15] of 2,4-0 and its salts has been carried out on silufol plates with Bu^O, BuOAc, amyl acetate or BujO-BuOAc as solvent systems. Organic acids [9] have been separated by TLC using CaSOj plates. This technique may be suitable for acid pesticides.
Our previous work [6] showed that BaSO^-CaSOj coatings plus simple solvent systems such as chloroform, carbon tetrachloride, ethanol, and water can be used for separating agro-carboxylic compounds. However, in general, bad tailing was observed and this system could not be used for separating complex mixtures. Now it has been found that when a BaSO^-CaSOj coating is impregnated with coconut oil, test materials move as compact spots that result in many quaternary and ternary separations, for example, chlorophenoxyace-fic acid, 2,4,5-T, p-naphthoxyacetic acid, indole-3-acetic acid, and
Journal of Planar Chromatography VOL I.SEPTEMBER 1988 253
Short Communications
indolepropionic acid tail 0-10 on a BaSO^-CaSO, coating (A) while their movement is 0-2, 0, 0, 0-4, and 0-2 respectively on a BaSO,-CaS04 coating impregnated with 15 ml of coconut oil (coating D) in distilled water. Chromatographic behavior of chlorophenoxyacetic acid and indole-3-acetic acid shows the decreasing size of their spot with the increasing concentration of coconut oil. i.e. their movements are as 0-10, 2-5, 2-4, and 0.16 and 0-10, 2-7, 0-7, and 0.35 on coatings A,B,C, and D respectively in tap water. R, values of 2,4-D and benzoic acid are 0.7 and 5-8 on coating A while they are 0-1 and 0.25 respectively on coating D.
These data show the compactness of the spot as well as retardation of their movement that suggest the possibility of quaternary separations and the analysis of complex mixtures. The results obtained (Figures 1-3) show that the system is specific for the separation of TCA from complex mixtures because it has Ri value 1 on all the coatings and solvents under study. Table 1 shows that TCA can be separated from benzoic acid, 2,4,5-T, indolepropionic acid, and a-naphthaleneacetic acid in amounts ranging from 400 pg to 1000 pg-The recommended preplant application of TCA is 10-40 kg/ha 3 months before seeding or transplanting in sugar beet (1,2), can-ot [5], and fruit tree nurseries (16). Hence it is clear that TLC coupled with spectrophotometry can be applied for the separation and determination of TCA. The lower limit of the spectrophotometric method used Is 400 \ig. Therefore for analysis below 400 pg of TCA another spectrophotometric method must be used.
It is also clear that comparatively costly organic solvents such as chloroform, carbon tetrachloride, acetone, ethyl acetate, and ethanol are not very useful on these coatings while many ternary and quaternary separations can be achieved in distilled water and tap water.
The ascension times for water over 10 cm of plates coated with A, B. C and D are 1,1,2, and 3 h, respectively, showing that development time Increases with the increasing concentration of coconut oil.
The above results show that BaSO^-CaSO^ impregnated with coconut oil is an excellent TLC material for the detection, separation, and determination of commonly used carboxylic herbicides in water.
Acknowledgment
Financial assistance by the University Grants Commission, New Delhi to one of us (HAK) is gratelully acknowledged.
References
(1) V. A. Kvasov and V. I. Kurakov, Agrokhlmiya 11 (1986) 95-98.
(2) V. A. Kvasov and V. M. Sergeev, Khim. Selsk Khoz. 10 (1984) 40-41.
[3] V. P. Borona. L. S Prokopenko. and L. M. Zaverukha, Zasheh. Past. 2
(1987) 34-36.
[4) J. T. Ward and E. W. Turner. Proc. Bro. Crop. Prot, Cong. Weeds 1 (1985) 223-230.
(5) Yu. G. Merezhinskii. A. G. Semenov, and A. S. Luk'yar\chenko. Khim.
Selsk. Khoz. 3 (1986) 62.
[6] H. S. Ralhore and H. A. Khan. Chromatographia 23 (1987)432-434.
[7J G. Zweig and J. Sherma, in Handbook of Chromatography, third printing, Vol. 1st. CRC Press 18901, Ohio 44128. USA (1976) 369.
|8J H. S. Ralhore. K. Kuman and M. Agarwal. J. Liq. Chromatogr. 8 (1985) 1299-1317.
19) H. S. Ralhore. I. Ali. S. R. Ahmad, and S. Gupla. J. Liq. Chromatogr. 7 (1984) 1321-1340.
[10] f, 0. Sf7e// and C. T. Snell. in Colorimetric Methods of Analysis, III Ed., Vol. 3rd.. D. Van Nostrand Company Inc., New York (1953) 336.
(11] J. Sherma. Anal. Ghem. 59 (1987) 18R-31R,
(12) H. Hermenan and K. Grahl. Acta Hydrochim. Hydrobiol., 12 (1984) 685-687.
(13) J. Sherma. J. Liq. Chromatogr. 9 (1986) 3433-3438.
(14) A. Kuthan, Veda Vysk. Praxi 71 (1983) 55.
115) B. Wterr\er. Acta. Hydrochim. Hydrobiol. 13 (1985) 527-529.
(16) A I. Martynenko. Subtrop. Kult. 5 (1985) 117-118.
|17) H. S Ralhore. S. Gupta, and H. A. Khar}, J. Ind. Poll. Cont. 2 (1986) 17-23.
fvis received: February 5, 1988 Accepted by SN: May 2. 1988
254 VOL. I.SEPTEMBER 1988 Journal of Planar Chromatography
JOURNAL OF LIQUID CHROMATOCRAPHV, 11(15). 3I7I-3UI (I98S)
PLAIN THIN-LAYER CHROMATOGRAPHY OF SOME HERBICIDES AND RELATED
COMPOUNDS ON ADMIXTURE OF BARIUM SULFATE AND CALCIUM SULPHATE IN MIXED SOLVENTS
M.S. R A T H O R E ' AND H. A. KHAN Dcparintcnt of Applied Chcnusiry
7..U. CulU'KC of Etii^inccriiit; and Technology Altgarh Muslim Uniwrsiiy
Aligarh - :02002 India
ABSTRACT
Soae c a r t o x y t l c h e r t l c l d e s and plant frowth r e ( u l a t o r a •uob aa benzoic a c i d , '•-chlorophenoxraoFtlc ao ld , c lnnaa lc ao ld , 2 ,N-0, i D d o l e - 3 - a e 8 t l o nc ld , indolaproplonlc a c i d , 06 -naphtha-l e n e a c e t l c aold, /I - c a p h t h a l e n e e c e t l c ac id , fi -nanhthoxyaoetlo a c i d , phenoiyacet lo a o l d , TCA and 2,' i ,5-T hare bean separated on BaSO.-CaSO. ( I l l ) o o a t l a c a In alxed aolTent ajratens.
Q u a n t l t a t l r e l eparat lona of l n d o l e - 3 - a o e t i o aold (100 m ) fro» 3O-IOO ^c of benzoic a c i d , c<. -napbtbaleneacet lc acid and Z,"*,?-! hare been oarr led out auocaaaful ly .
INTBODUCTION
Calclua sulpbate vaa used for tbe separation of
c a r b o i y l l c herb ic ides by p l a i n (p-TLC ) , l o n - p a l r reverse -phase
(IP-RP TLC ) , s equent ia l (S-TLC)' and two-dimensional (JD-TLC)
t h i n - l a y e r ehroMatocmphles . P-TLC on adalxture of CaSO. and
( " p y r u i h l O I'lKN hy Marvel D f l k c t . \nc
3172 RATHORE AND KUAN
BaSO. vaa ««ed for the separat ion o f a ouaber of oartooxTllo
aclda such a« oinnaalc, c i t r i c , lndola-3-«cet le , Balalo, Batlo,
•alonlo, (i-naphtbaleneaoetlo, P-aapbthoxr"ce'tic, oxalic,
pbenozyaoetlo, sa l lor l l c and tartaric in i l o c l e aolTent Byateas.
It vaa reported In oar preTlons paper that the aeparatlon
potential of Ba50.-CaS0. ooatlnc* oan be enhanced by lapre(Datlnf
the ooatlnc vltb coconat o i l .
Literature showi that a naaber of papers were deroted 7 8 to separate cartoxyllc herbicides by TLC on sl lnfol ' or a
213 alztare of s i l i c a (el and Kieselcahr , and preadsorbent 9
s i l i ca gel layers Ivprri^aated vlth A^NO.. In alxed solTent
systeas. Hoverer separation potential of Ba90.-CaS0, ooatlnfs
In Mixed solTeat systeas baa not been tested so far.
Therefore, In continuation to our previous irork, now
such an ntteapt hns bern aade. The resul ts obtained are
discussed In this paper.
KIPKBIMSOTAL
Apparatus
Stabl aoparatus vlth a anlTersal applicator, c^oss
plates (20 x 3 CB), glass Jars (25 x 5 en), Bausch and Lomb
spectronlc-20 spectrophotoaeter, centrlfajal aachlne (Balrd 4
Tatlock Ltd. (Bogland), eagnetlc s t irrer (SunTlc,U.iC. ] , glass
coated nagnetlc bars (spprox.length 0.8 ca) , teaperature
controlled e lectr ic oren (Teapo,India), laabda pipette, conical
flask 10 al etc. vere used.
HERBICIDES AND R E L A T E D COMPOUNDS 3173
Cbealoal*
Ferr ic c h l o r i d e anhydroua (KaabaxTiIndia); •atbanol
(Glaxo,India ) ; perch lor i c a c i d (Merck,India) and broaopbenol
b l a a , b e r b l c l d e * and plant t r o v t h regalatora were froa S lgaa ,
n .S .A . All o ther reacenta aaed were of a n a l r t l o a l grade.
Freparatlon of F l a t e t
A i l u r r r contalnln^^ BaSO. (50 g ) , CaSO. (50 j ) and
d l a t l l l e d water (13^ • ! ) '*'* applied to tbe t i e ' s p l a t e s with
the a p p l i c a t o r to g l r e a f l l a th lcknese of 0 .50 mm. The
p l a t e s were f l r e t a l lowed to dry at rooa teaperature and then
In an OT^D at 110 C for one hour.
Preparation of Beagent for I n d o l e - 3 - " c e t l c acid (lAA)
Determination
The reagent Is prepared by a lx lng 1 • ! of 0.5M f e r r i c
ch lor ide s o l u t i o n In 50 al of 35% ( T / T ) perch lor i c a c i d .
Spott ing of Test S o l u t i o n s and H Values
Test s o l u t i o n s (1% e t h a n o l i c ) were spotted onto the
p l a t e s with a f ine c a p i l l a r y . For d e t e m l n a t l o n purpose
100 fig of lAA (10 pi of 1< ae thano l l c s o l u t i o n ) and rarylng
aaounts of benzoic a c i d , oC -naphthaleneacet lc acid and 2, ' i ,5-T
8uch as 50 HE to 100 fig (5 p i - 1 0 pi of 1% nethanol Ic s o l u t i o n s )
were a p p l i e d . The spota were dried with the help of a hot a i r
blower and then the p l a t e s were developed.
For t a i l l o g , the front H a l t (HI) and the rear H a l t
(RT ) were aeasured while for co»naot spots R. ra lues were
c a l c u l n t e d In the usual way .
3174 RATHORE AND KHAN
I d o n t l f l o a t i o n Kethod
The berb lo lde* and grovtb r e r a l a t o r s on p l a t e s vere
r l a u a l i z e d by •prarlag e t b a n o l i o a l k a l l n a a o l a t l o n ot
broBopbenol blue (O. l j t ) .
Qaant l tat iT« Separat ion of lAA
rnoiro Toltuaaa of i tandard aolut lon of UUL vere appl ied
on TLC p l a t e a . Tbe p la tea vera developed In oarboo t e t r a -
chlorlde-prDpanol ( 1 0 0 ( 0 . 5 ) s o l r e n t syateai. Prer lous ly
Indicated port lona of tba c o a t l n i vere acratcbed v l t b a
•patnla and c o l l e c t e d la a con ica l f lask (10 • ! c a p a c i t y ) ,
2»5 • ! o f aetbanol vere added Into I t , the Blxture vaa s t i r r e d
for 7 a l n , and then trans ferred ID tbe c e n t r l f u c e tube. Tbe
conical f laak vaa vasbed v l t b 0 .5 al of aetbanol and tbe vaablngs
vere t rans ferred Into tbe ssne oen tr l fuce tube . The s o l i d
portion vaa reaoTed by c e n t r l f u c a t l o n and UUL vaa detens lned 10
In c l e a r s o l u t i o n by the fo l lov ln( ( aetbod , Tbe f resh ly
prepared r e a i e n t (2 • ! ) v s s added Into tbe c e n t r l f n g a t e
dropvlse but rapidly v l t b contlQuoua a g i t a t i o n and tbe s y s t e a
vas placed In dark for one hour for co lour deve lopaent . F i n a l l y
tbe absorbance vas aeasured at 51* na aga ins t a blank c o n t a l n l n c
aetbanol (3 a l ) and reagent (2 a l ) .
CSPLT3
Tbe separations acblered on BaSO.-CaSO. coatings aslng
alxed aolTent aysteas are recorded In Table 1. Results of
quantltatlTe aaparatlona of IJLA froa benzoic acid, o6 -naphtba-
leneacetlc acid and 2,'i,5-T are glron In Table 2.
HERBICIDES AND RELATED COMPOUNDS 3175
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3176 RATHORE AND KHAN
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• 04
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oo ^ o • o
i r \ TH
* o o
o r~ TH
o • O
o ITl VO
*
o ir> VO • »
l f \ N ITi
*
»rv r-VO • r
o i n CO •r
o ITv
r» • r
o ITl
r-•V
v\ r-VO
•* 8 0^ • r
r-
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o
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OD
O i n
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o
n • r
o «r\
O
i n
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o
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o
at
<J3
-r o
• r
GO 00 00
o o o
00 O VO * m *
* O VO
* * * o o o
s O in 8 ?
o
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o
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o o o o 8 o o
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c
e
HERBICIDES AND RELATED COMPOUNDS 3)79
To o a l c u l a t a • o a l r t l e a l p a ra a e t t r t the f o l l o « l n t
r « l « t l o n a vara uaad
a- •
C.T . -
0 - 1
cr a 100
whare<r - atandard d e r l a t l o n ; x^.x , . . • aeaaarad ra luaa ,
p • »r»rag« r a l u e , n - nupbar of aata and C.T. » o o e f f l o l e n t
Of r a r l a t l o n .
DiaCtJSaiOM
1-5 Prarloua publ ioat lona f r o * tbta laboratory aboH
that o a l o i u a aulphate and barlaa sulphate are food TLC aiater-
l a l a . Thin layera of barluji aalpbata are not aa good aa that
o f oa lo luB aalphata a l o a a . Ad«ixtarea of barlua aulphate and
a a l o l a a aulphate R I Y * ualform, aaootb and Btabl« l a y e r a and
haTe Tery food feparat lon potent ia l for oarboxyl lo ao lda .
The t l » e of derelopBent^ of Tl<C p l a t e Increeaea v l t b
th« Inaraaalnc percentaje o f bar lu* sulphate In the adalxtutea
and the aflalxture oonta ln lnf barlua a u l o h a t e - o a l c i o a sulphate
( l J l , * / w ) l a »0Bt s u i t a b l e l o r t h i n - l a y e r chro«atojraphlc s t u
d i e s . The aboTe adalxtore can be aaed for separat lnc
oarboxy l l c h e r b i c i d e s In s l n ( l a s o l v e n t ayateaa. The
aeparat lon p o t e n t i a l Is H a l t e d due to t a i l i n g nature of the
herb lc ldea in aoae of the so l ren ta anch aa benzene, carbon
t e t r a c h l o r i d e and c h l o r o f o r a . HoveTer, compact spo t s with
Rj ra lue 1 were obtained In dloxan, e thyl a o e t a t e and propanol
and soae binary separat lone vera obtained In <H«t l l l ed v a t e r .
3180 RATHORE ^SD KHAN
A nuBber o f t e r t l a r r and qaatemarr aeparvtlona vere
aehlered on Ba50.-Ca90. ( I l l ) o o a t l n f a iBprecnatad v l t h 6
ooeonut o i l In water v t i l l e orxanlo io lTenta vera foand to be
I n e f t e o t l T a . The aaparatlon t>otantlal Isoreaeea v l t h l a e r a a s -
Ing percentace of oooonat o i l aaed for l a p r e c n a t l o n . Cafor-
tnnata ly the t l a e of deTelopaent a lao Inereaaea v l t h l»eraaalDx
peroentafe of cooonat o i l .
Table 1 ahova BaS0^-Ca90^ oan be naed for aereral
b inary aeparatlona o f oarfaozTllc herble ldea aad r e l a t e d
ooBpounda aalnf a lxed aolTent ayateaa. The f o l l o v l n f f.rpara-
t l o a a , vhloh are not poaalble on BaSO.-CaSO. as wel l at »o
BaSO.-CaSO. laprafnatad with ooconnt o i l In a ln^le ao lrra t
ayateas , oan be achlered In alxad aolrant ayataaal BOA froa
CfAA} lAA fro« BOA, CIA, CFAA, 2,'i-D, oc-WTAA, p-NTAA. SIAA
and 2,*,5-T5 fi -NTAA fro» < -NTAA and HIAA; IPA froa KU, CIA
and HIAA ( tab le l ) .
Data reoorded In Table 2 abow that lAA oan be
aeparated q a a n t l t a t l r e l r froa BOA, f<- .4fTAA and 2,%,5-T la
oarbon te traohlor lde-propanol ( 1 0 0 » 0 . 5 ) .
COHCLD3I0H
Adalzture of BaSO.-CaSO. ( i J l ) l a a good t h l n - l r r p r
ohroaatograpblo m a t e r i a l . I t can be used for aoae blncrr
separat ions of c a r b o z r l l c herb le ldea and r e l a t e d coapou- ls
In water aa dere loper . The abore adalr tare i»pregnat»-d v l t h
oooonnt o i l oan be used for ternary and quaternary a r p c n t l o n s
ualnx water as dere loper . Many binary aeparatlona whtti are
not poaa lb le In a l n g l e aolrant ayateag aa well aa on l i q i m c -
HERBICIDES AND RELATED COMPOUNDS 3181
nat*d o o a t l n g i , eaa %• aolil«T«d on tb« • « • • ad«lxtar« by
• I z e d aolTent a y s t v a s .
AcnovuDnmrrr
The aathora are thaokfo l t o Prof. K.T.Naaia, Chairman,
DapartMeat of Appllad Chaair try , I . H . C o l l a c e of K n f c A Taoh. ,
A l l carb Kaalla U n l r e r a i t y , Al l fBrh for p r o r l d l n f reaaarob
f a e i l l t l e a . On« of tba aathora (HAJC) l a a lao thankful to
CoanoLl of S o l e n t l f t o end Indnatr la l Beaaartib, Mrw Dalhl for
the award of a aenlor raaaarob f e l l o v a h l p t o h i * .
Bf fBNCKS
1. Bj i tbora .B.S . ; A l l , I . 2 Abaad,3.B. and G a p t a . S . , J .L lq . C h r o M t o f r . , J. (199%) 1321-15*0.
2. t a t h o r a . H . S . { A l l , I . ; Sbaraa .S .B. and Saxena.S.K. , I n t a r o . J . B n n r o n . A n a l . C h e - . , ^1 ( 1 9 8 8 ) 2 0 9 - 2 1 7 .
1. t a t h o r a , S . S . and Saxcaa ,S .K. , JU. lq .ChroBatocr . , £0 (1987) 3623-3636.
h. t a t b o r a . B . S . and O a p t a . S . , J .L lq .Cbroaatogr . , j ^ (1987) 3659-5671.
5. Bat taor« , l . 3 . and Khan,H.A., Chraaatofraphia, tj (1987) *32-A3*.
6 . IUtbor«,H.S.S A l l , I . and IKan.B.A. Jenmal of Planar Cbro«*t«(rapb7->iodani TLO (1988) In praaa.
7. Bamanan.H. and Gr«hl ,X. , Aeta Brdroeb la .Brdroblo l . , ^ {19M) 685-687.
8 . C a T e t i k l l . T . I . : B a b l l k . L . I . and PnElk.G.V., Zb.Anal. Tbin. *2 (19»7) 1302-13M.
9 . 3 h e r » a , J , , J . t l q . C h r o n a t o t r . , £ (1986) >H33-3'«38.
10. Mabsderan.A. and S r l d b a r . B . , Natboda In P b y a l o l o i l e a l P lant Patbolocr , 3rd Kd. , S lrakanl Pnbl loat iona (1986) 270.
\NR—MS 68(j
liiili-r «r> \ n l IKl. N i ' 0. r r l^<" """ f"^-' Pruned in Grcil Ilril.ii" All t i th l \ rc-ti.;i. ' (.'(<p\ nchl
(I(1JM.'M K9 ?3 on -r 0(10 ' PS'J ffr^'amon Press pic
A NOVEL METHOD FOR THE DETECTION AND SEMIQUANTITATIVE DETERMINATION OF TRACE
LEVELS OF 2,4-D AND RELATED COMPOUNDS
H. S. RATHOKK* and H. A. KUAN
Dcpurlmcnl of Applied C"lH'n)i'-lr>. Z. H Collccc of Enj:inccrinp j n d Tcchnnlogy. Aliearh Muslim L'ni \crsi l \ . A l i w r h : o ; 0 0 : . Indi:i
(Fir.ii retellcJ Aticu'l I9SS: aiccpli-it m rnisti/ form Fvhnniry I9S9I
Abslract—A sensitive and sclcciixc novel Icchniquc has been developed for llie deicclion and seniiquanli-(alive dclcrminalion of phcnow herbicides The herbicide is healed in presence of li>dialed zinc sulphale to split ofl' formaldchvdc The pressure is reduced in order to bubble the formaldehyde \apours into ihc rcagcnl (one or l»o lin\ crsslals of chromotropic acid sodium sail in 0 2(1 ml ol concenlraled sulphuric acid). This technique has been used successfully for the detection of traces of 1.^V> acid. 2.J-D ethyl csler and 2A-D sodium salt in formulations, leaves, soil and water
Key Mords—vacuum spot-test. ^.J-D. flora, formulations, soil, water
INTHODtniON
In coniinuaiion to our previous work (QiircSlii et al. 19fe2. kathorc 1/«(.. 198(1, lysKa.h. I9S9) of developing new. simple, non-insirumcnial and inexpensive n'.c'ihods of dtiection. separation and dcierminaiion of herbicides present in environmental samples. no« one more procedure is described.
In Feigl's book (Feigl and Anger. 1966) a reagent is reported containing chromotropic acid in concentrated sulphuric acid for the detection of traces (O.I4;/e) of formaldehyde in presence of fructose, saccarosc. furfural, arabinose, lactose and glucose. The lest is highly sensitive in concentrated sulphuric acid. It can also be used for detecting compounds such as phenoxyacelic acid and its halogen derivatives and monochloroacetic acid which split out formaldehyde when hydrolysed or pvrolyscd The chemistry of this colour reaction is not knovsn with certainty. It is probable that phenolic chromotropic acid condenses with formaldehyde followed by oxidation to a /j-quinoidal compound of violet colour:
SOjll SOjH
so,II so,n In this reaction sulphuric acid acts as dehydrating
aueiu. oxidant, and water donor Cellulose and
*To whom correspondence should be addressed
hydralcd sulphates of 7inc and niancanese which lose water when heated above 150 C act in the same hshior\ 3i co;icLn;ralc(l sulphuric acid. However, their analytical utility has not been fully explored.
Freed (19-iis') has remarked that when the test is applied directly, there is interference by compounds which split off formaldehyde when healed with concentrated sulphuric acid However, the lest becomes quite selective for phenoxyacelic acid and its halogen derivatives if use is made of their solubility in benzene. The interfering compounds can thus be taken out of the reaction theatre, including those which carameli/e or char when heated with concentrated sulphuric acid and so impair the recognition of the colour reaction
The test can be made even more selective by the vapour phase deicciion in which the fonnaldchyde split out is delected in the vapour phase by contact with chromotropic-acid and concentrated sulphuric acid. However, the sensilivity is then only about one-tenth as great (Fcicl and .Anger. 1972).
The survey of the literature (Ralhore rl al.. 1986) shows that the sensitivity of the vapour phase detection can be improved b\ performing the test under vacuum The use of dilTcreniial solubility of the lest material can be made in enhancing the selectivity by perfonninc the test in different solvents. The proper selection of conditions as well as ihc reagent for spliUmc out foniialdehyde from a parlicular functional group can also conlribuie to the sclectiviiy of the test because a wide range of compounds split out fc>rnialdchvde under di/Iercnl set of conditions and reagent for example ( - O t l l ; and >N—C\\, group): :;nisolc. anlipvnne. methvl red. methyl orancc cic cive foniialdehvde ihrouuh oxidative
H S RATtfoRr and U A K H A N
cleavage w i th m o l l c n bcn7.a>l perox ide at 120 C.
Methy lene ethers o f o -d iphcno ls
' 0 ^ (C^
( \ /
C l l : t y p e )
apiole. berbcr inc. narcot ine etc. c i \ c f o m i a l d e h \ d c
when heated alone or a long w i t h concent ra ted sul
phur ic acid at 170-180 C. g lycol ic acid and
monoch loroacc t ic acid yield fo rma ldehyde v.hen
warmed w i t h concentrated su lphur ic acid at 100 and
150-180 C. respectively. i - . -Xmino acids give alde
hydes on t reat ing w i t h a lka l i hypoha logen i te . G lyc ine
splits out fo rma ldehyde when treated w i t h ch lo r -
aminc -T or n i u o u s acid at 100 C. Cho l i ne h y d r o
ch lor ide produces fo rma ldehyde and aceta ldchydc
when heated w i t h M n S O , 4 H . O o r M n S O , H -O at
180 C. Phenoxyacetic acid and its halogen der ivat ives
give fo rmaldehyde on br ie f w a r m i n g v^iih concen
trated su lphur ic acid at 150 C.
Therefore, m view o f the above (acts, an a l t cmp t
is now made to raise, seieciivi iv as wel l as sensi i ivuy
o f the test by the use o f (i) sol id ZnSO^ " H O for
sp l i t t ing out fo rma ldehyde f r o m phcnoxv herbic ides,
(i i) preferent ia l so lub i l i ty in di f ferent solvents, and
( i i i ) newly designed apparatus in wh ich the test tube
is conn i c t i d vviih anuMie' •l '" '-^hap•,•d •^ide tube
con ta in ing the reagent between two bu lbs and
vacuum is appl ied to bubble the traces o f f o rma lde
hyde in to the reagent. The newly designed appara tus
is used successfully to detect traces o f 2 .4- t ) and
related compounds in soi l , vegetat ion and water
samples. The results ob ta ined are discussed in this
paper.
F.\[•F.HIM^.^T^t.
Apparatus and maicriah
A glass tube (Fig I), vaccuslicr pump P c m vacuum (Atlantis Applications Engineering Pvl. L id . India), icm-
To v o r u j s l f pump
Stopper
Fig 1 .Apparatus for micro analvsis of hcrhKidcs in flora, soil and water .Ml dimensions arc in mm
pcralurc controlled healing manllc (Sunvic. England), hot air drier, clecinc oven, ccnirifugal machine (Haird & Tallock L id . rii>;land). gradualcd micr(i pipclles. porcelain lilc with grooves and Peiri dishes (8cm dia) eic were used.
Ben7ene (Cilaxo. India), chromolropic acid sodium sail (CDM. India). 2-propanol (Merck. India), sulphuric acid ( B D I L India). ZnSO, 711.-0 (SD Fine Chem., India), 2.4-D cthvl esier . '4% EC (Unique Farmaid Pvl Ltd. India). 2,4-D sodium sail SO°c W P (Mascot .Agrochemicals Pvl. Ltd. India), dichlorovos 76% EC (Pesiicidcs India Ltd), formolhion 1S'\ EC (Sando? India Lid), malalhion 50°'o EC (Cvanamid India Lid), phosphamidon 85% EC (Hindusian Ciha Geigv L id . India), ihioniclon 25°''o EC (Sando? India Lid), /i-dinielhylaniinohcn/aldehvdc. 2.4-D acid and iMher herbicides v^ere from Sigma. U.S.A. Al l other reagents used were of analytical grade.
Sdltniiiny.
All the solulions (l"'o) vverc prepared in bcn7ene unlil olhcrv^ise slated. I f ihe compounds were insoluble in benzene, ihcir saluraled solulions were used.
C(i/iiiini:i: rcaccril
A mixiure of one or |vio linv crvslals of chromolropic acid sodium -.all .ind (I 20 ml of concenlraled sulphuric acid in .1 " l " lube vias used as reagcnl
E'li !tininnii!iil \aifipli-\
LI lit f^ Leaves of ihe Ptiratiui phrnicri of family vcrbcn-.iccie of ihe commonly grown hedge planl were used,
Sinl U<riil(\. The ferlile soil was composed of sand ' 2 ^ v " „ Mil S7(K)%, cb-, ! ! " " " . CEL KiO'Jn-.L If!!), organic mailer 0.61 and pH 7 75
Soil (nun-fi-riilc). The non-ferlile soil was composed of sand 50.(K)%. sill 27.60%,. clay 22,40"/„. CEL n . t O m g 10(1 g. organic mailer 0 | y% and p l l 8.1.1.
II (Her {riiIT). The river waler was composed of 8.2 8.7°o dissolved oxygen ppni. 122 141 ppm total hardness. 124 175 ppm lotal dissolved solids, 174-1894 ppm total alkalinity. 9.2 9.9 ppm chloride. 6.5-6.7 ppm sulphate. 0.42- 0 47 ppm phosphate. 5.2 5 61 ppm BOD. 6.5-6.7 ppm COD and pH 8 70 8.80 (Khan. I9S2I.
Haii-r {lap) The lap waler was composed of 261-26(1 ppm lotal alkalinity as CaCO,. 1.15 144 ppm lotal hardness as CaCO,. 1.1-14 ppm chloride. 14-18 5 ppm sul-phale. 099 -1 ppm fluoride. 2.1 2.5 ppm nilrale-nitrogcn, 1.25 I 50 ppm cadmium. 4 50 5 ppm copper. 2-2.50ppm chromium. I 5 - I 6 p p m manganese. 8.50 lOppm nickel. 070 0SOppm lead. I.12-I.19ppm zinc. 1.12.25-114 56 ; iohm ' cm ' electrical conductivity and pH 7.50-7.60 (Raziuddin. 1986).
General pnncdiirc
A solution (1 drop) of the test malerial was laken in lube .•\. evaporaicd lo dryness, cooled lo room lempcralurc (.1'' 1 2 C l . .15 mg of Zi iSOj 7M.O was added into il and Inluraied wiih a glass rod The reagenl was laken in a " U " lube ihrough end B Tube ,A was sloppcrcd and lube B was connected to the suction pump through a rubber tube. .•\boui 2 cm of the lower portion of the apparatus (Fig 1) was dipped into the oil bath al a desired temperature for 2 mm The colour so developed was noted Then the coloured solution was poured inlo a grove of the tile in order lo visualize Ihc colour for a longer linic and lo use Ihe intensity of the colour for semi-quanlilalive analysis.
The lower limit of idenlificalion was determined by sl.i i l ing wiih kn<^wn volumes t^f st.indard solutions of a compi-^und concerned,
O f h i i i i M i !>(• 2 . 4 - D m i l / m / r u i i v
•\ vniall portion of leaves (.S(KI mg) was taken in difTerenl Pclri dishes, sprayed wi ih a definite conccniralion of 2.4-D acid, ihc svstcm was covered and left for 24 h al room
Dclemiin.i l ion of Iracc ICNCIS of 2.4-D
Tabic I Siuetv of ihe lest under diffctcni condiimns
Si n o . Cffcci of ihc foIlr»y.?n^'
Arm-uni of
phcnoxN Jcclic aod ifi^) TcmpciJiurc < r C) Cv lour
SJIIS
2 Sulphuric acid {nil( \n reagcnl 3 Tcmpcraiurc ( Cl
^ Time (mini
^nSO, 7M.O (mfl
v. no
0 00 i s m )
(UKI
OIKI
n ixi
IS (Kl
IWI LV (CaSO, :H.Ol. LV ((NH, | ;Fc(SOJ: fiH.Ol. l \ (Na.S-O, 5i).C)|. V (CuSO, 5H.OI. V (MfSO, 7H.Oi. VIV (KHSOJ. DV (MnSO, li-Ol, DV (ZnSO, 7H.0)
IKO LBr (0 11. N C (0 21
l \ (I.Vn. \ ' (1601. DV (1701. DV (1801.
D\' (IW). lirDV Cimi IWI NC (II NC Cl NC (.1|. VIHI (41. VIHI (51 Kso NC (II. NC Cl. NC (.•'I. LBI (4). LBI (.M. !(•(' L\ 111. DV C). D\ {}). DV (41. DV (.<;) IMl V (II. DV Cl. DV (M. DV (4|. U\ (SI IWI \ 117 ,M. DV (IM. L\' (701.
Name of ihc ?uilt (1 | . \oIumc of conccniraied sulphuric jcid (2l. icn^pcraiuic C l. nmr (41 and amouni of ZnS04-7H.O (5i are given in parcnlhesei. Br =• brown. Bl - hIacV.. D = Jjrl.. L = lighi. NC = no coloui V - violei and VI = vcrv lichl
lempcralurc (. 7 ± 2 C) The leaves so spravtd were Irans-fcrred inio a separating funnel and Ueaied with 5 ml of propanol . The ahquol was ccntrifugcd. the clear solulion was evaporated to dryness, the solid left was treated with water (1.5 ml), the insoluhlc port ion was removed h> filtration and then 2.4-D acid was delected in the aqueous solulion hy the above procedure
Dcicciinn of ^.-l-D IHUI in MHI
A small portion (5g) of non-fertile or fertile soils was taken in diflerenl glass Petri dishes and I 2,*- ml of 2.J-I) acid solulion (0.7°/ol in bcn/cne was applied dropwisc with cont inuous shaking ..^fle^ .^hh. 2.4-D acid wa^ delected i r the soil by the following pioccduie 1.4-0 jcid wjs cluted by shaking a definite port ion of the soil with 2 ml of propanol in a test tube and then the undis<«i|vcd material was removed by ccntrifugation The cleat supe rna tam aliquot was transferred to another test tube, concentrated on waler bath till the volume was 0 2(1 ml, transferred into lube A of (he appara tus and then 2.4-D acid was detected as above
Dcli'dion <)/ J.'i-D IHHI in uiiur
Known solutions of 2.4-D acid in distilled water ( D W | or river waler | R \ \ ) or tap water (T\V) were prepared and the lower limit of detection was determined as above
Dclcclicn of 2.4-D cllivl cidr in furniiihiiuin
Known solutions of 2.4-D cihvl ester (formulj i ionj were prepared in benzene, ethanol and elhcr while stable suspen
sions of 2.4-D ethyl ester (foriiiulalioni of known concentration were prepared in TW and the lower limit of detection was determined as above
Dfit'f tinn ol J.-i-P Mulnin) sail in iinnnilaiion
Known solution- of 2.4-D sodium sail (formulation) were prepared in elhanol and TW and Ihc lower limil of deleclion wa<; determined as above 2.4-D sodium sail was insoluble in benzene and eiher and its slable suspension was also not possible in ihcse solvents
RESLl.T?:
R e s u l t s ol dc t cc iu i i i o f p l i c n o v y a c c t i c ac id in dilTcr-
cn t c o n d i t i o n s s u c h as t e m p e r a t u r e , t i m e o f h e a l i n g .
dilTcrcnl sa i l s a n d s a h c o n c e n t r a t i o n a r c g iven in
T a b l e 1. D a i a of d c i e c t i o n of s u b s l a n c e s c o n t a i n i n g
dilTcrcnl r u n c l i o n a l g r o u p ; ; -dnd d e l e c l i o n o f 2 . 4 - D
ac id in p r e s e n c e o f w i d e va r i c iy o f s u b s t a n c e s a r e
r e c o r d e d in T a b l e 2 R e s u l t s o f d c i e c t i o n of 2 . 4 - D
e t h y l es te r a n d 2 . 4 - 0 s o d i u m salt in f o r m u l a t i o n s a n d
Siomc f o r m a l d e h y d e g e n e r a t i n g c o m p o u n d s d i s so lved
in different s o l v e n t s a r e r e c o r d e d in T a b i c "S. R e s u l t s
/ i f d e l e c l i o n o f 2 . 4 - D a c i d in d i / f c r cn ! e n v i r o n m e n t a l
s a m p l e s a r c r e c o r d e d in T a b i c 4. T h e lower limil o f
d e l e c l i o n o f 2 . 4 - D acid in d is t i l led vvalcr. r iver w a t e r
Tabic ^ GITcct ol dillcrcnl orcanu^ and inorcanics on Ihr dtlr^lion ol ZA-\'> acid
Name of comp*>undv
AUilndci
Acelaldchvdc (Si Ben/aldchvdc iSl
p-Dimethvl.iminohen/aldchvdc (Si Formaldchvdc (SPi Paraldehvde (Si \ an i l lm (Si Anuiit s tinj tnut'h\
A.cctamide ( l l .Acrvlamidc ( I I Bcnraniide ( l l .N.'-Rrom(»NUccinimidc Piliclamide i l l
i S i
C ompoun alone
B l
VIH,
\ ' ( i :<n DV
Bl
N C
NC
N C
N C LBr ('J4ri
XC
IfJi
( d
1
C
( <*nipo
Mi„i : .4 -D
Bl
H i V
\ DV
HI
D \
\ \ \ D \
n\
l.l.HH l l
and -
: of
acid
I m \i^\
COnipouni
alone
B l
1 Bi
V ( '( I I D \
HI
NC
NC
NC
N C
1 M M -
NC
ISO C
Compound *
j Mi/ ip of : .4 .D acid
B l
D B i
D V
D \
Bl
D \
D \
DV
DV
"si HrV D V
H S R A T H O R I and H A K H A S
Table 2—Kontmucd
Name of compounds
Colour (L{ in ^g t
160 C ISOC
C ompi^und -* Compound + C(»mp<>und -^f'/'P c* (on ipound .''('/Jf of
alone 2.J-D j c i d alone 2.4-D acid
Aniline (S) Anilmc MCI (I> Diclhanol amine (! l Dielh>l amine (S( Diphenvl amine (S) Hcxaminc (S) Tnclhyl amine (It Trimeihyl amine (I(
Anh\dru{c\
Acelic anhydride (S) Phlhalic anhydride Carhoh\iiraiv% Arabmose (I) Dcxlnn ( I ) Starch ( I ) Sucrose 0) Curhvxxlu utui\ Acetic (SI t M-Aconrdc (I) (rt j fn-Aconil ic {\\ Adipic ( l ( I -Manine (1) 1 vAriiinmc ( h Ascorbic {\\ l.-ANpariic (!( Biirbiiuric ( f l Hcn/*MC (S| 4-C hlorophcnoxyacftic (S) Cinnamic '*^* Ci inc (I) : .4 .D acid (S) Formic (11
Fumaric 0) Gallic (I) Glycine ( I ) lndolc-3-acelic (I) Indolc-3-propionic ( I ) DL-Uocitric ( I ) haconic (.fj 3-Kctoglutaric s^X} Malcic (I) I.-Malrc ( I I j-Naphlhalencacctic (Si /^-NaphlhalcncacTlic (S) /f-Naphthox\acetic (SP) Oxalic (I) PhcnoKyaclic (Si l>-( —) Quinic (I) Salfcyclic (I) Tartaric (I) 2-Thiobjrbuunc \\^
Trichloroacetic (S) 2.4.5T (S) Uric (I)
Ethyl .icetaie <S\ HxdriHUrhims
Benzene fS) Hexanc (S) Kerosene (S) Paraffin l iguid (S) Kt'(imv\ Acetone (S) AceKiphcnone (Si Meihyl ethyl Veione (Si Phcnoh
4-Chlorophcnol (Si o-Nitrophenol (S| Phenol(S) Pyrogallol (II Rcsorcinol (II
L I NC NC
\\ NC 1 V ( ( K i \ I B r L G l P l
NC NC
NC NC NC NC
NC NC NC NC NC NC NC NC NC NC \ ( IX l
NC NC V d K l NC NC NC NC NC NC NC NC D \ NC NC NC NC NC NC V ( IO ) NC NC NC NC NC \ ( I M NC
D \ V DV
D \ DV
VlUr DV
LV LV
DV
D \ D \ D \ '
D \ DV DV
D \ D \ D \ DV
D \ D \ D \
D \ DV
DV
D \ DV DV DV DV DV DV
NC DV
D \ V
\' BrV DV
DV DV DV DV DV
DV
LV NC NC LV NC LV ((I M VIHr C. (P)
NC NC
NC NC NC NC
NC NC NC NC NC NC NC NC NC NC V ( ; ' . NC NC V O r
NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC V ( M NC NC NC NC NC \ ( I M NC
DV DV DV DV D\ '
Br DV
\' V
DV D \ DV DV
DV
DV DV
in-DV
D \ DV
D \ D \ DV
D\ ' DV
DV
D \ ' DV DV
D \ DV
DM DV DV DV DV V V BrV DV
u\ DV
D \ D \ D\ '
DV
NC DV
NC NC \ 1 G ( P | NC
NC NC NC
NC NC NC NC NC
DV DV VIV M \
DV D \ D \
BrV D\-D \ ' DV BrV
NC D\
NC NC L G l P l NC
NC NC NC
NC NC NC NC NC
DV
V)\ LV LV
DV
D% D \
BrV DV
D\ DV BrV
(iinlinucil
Dclcrmin.'ilion of trace levels of 2.4-D
Tjhic . rontinucii
Name of tomrKiunds
Colour (LI in fjg)
160 c i snc
Compound 4 C()mpourid + Compound .Vi/ifof Compound 5(>/jpof
alorvr 2A-0 and alone 2A-D acjd
( r f i j ^
I rcj (1) Thicurcj III /(tdret/Mtt <
^n)monrJ Ammonium chfondc iU RIciichmp powder ( I I
Cul t ium larbonaic l l ) C j k i u m (.hUinJc ( I I Cdk ium t u i d t (1) Ferrous sulphate l i j Mj j ;ncsium sulphj ic (IJ PiXjssium dihvJri 'pcn phosphj ic ( l l PoijNMum pcrmjncan;nt ( l l Sodium b ic j rK>nj ie ( I ) Sodium carK.»njtc ( l l S\^dium hydroxide ( l l Sodium n i i r j ie i l l Sodium nilr i le ( l l Sulphur ( l l Pi\ti, i,l,\ H.nlslin i l l :•> D . - l ln l Olcr (SI ; J- l ) s.-dium -.jl l ( I I D i ih lo ro ios ( S I rt>rm<iihion (Si M.iUihion iS l Phosphamid(>n (Si Thi.a,,, S. Afi>,.//u.i.,u« t ' h I o r a m i n f T (I) r ic lc rcrn l ( l l Mclhv l orance (SPl M(Mli\l i r d iSPl Soap ( l l
NC NC
NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC
NC 1 \ (^1 NC V (?7| \ ( N l \ (<J| N(
\ ( . ' ( i |
NC NC NC NC NC
DV DV
D V DV DV DV D V DV DV
D\ D V DV DV DV DV
D \ D \ DV
DV
in-D \ DV DV VIV
D \
D \ ' DV DV DV DV
NC NC
K C NC NC NC NC NC NC N C NC NC NC NC N ( NC NC NC
NC l.V (41
NC V (-(SI V ( N ) V (.V| NC V (3(11
NC NC NC NC NC
DV DV
DV DV
DV DV DV
D \ ' DN D \ D \ DV DV D \ '
n\ n\ D\
n\'
D \
DV DV
I ) \ DV
\-l ) \
D\ DV DV DV
D \
AbbrcMaiions used G. prey. P. pinlish sparinpiv sttluble All other dhhrc\
: l-i. lower limii ofdeteciion: (II iations arc defined in Table I.
Insoluble: (Si. soluble, and (SP|.
and lap waicr is found lo be 10/tg. The diagram of of the icsl had noi been ihoroughly suidicd (i) (he (he apparatus used is given in Fig. 1.
DISCI .SSION
The facts described in the Introduction show that the colour reaction under study has good
ciTcct of din'erent hvdrolysing agents in place of concentrated sulphuric acid, (ii) designing of new vacuum spot test apparatus, (iii) prcrerential solubilities of phcnoxy herbicides in difTcrcnt solvents and (i\) application of the test for non-instrumental analysis of herbicides residues for dc\cloping
analytical potential However, the following aspects countries.
Table . Dttcvtion of some forntaldehsde produsmc comp^^unds m diflcreni soUent^ at ISO C
C oiTip*>und
: . - l -D acid
: - l D eihsl esicr
Z -i-O sodium -Jll
CiUtine Metamine
Melhs 1 orance Melhvl red
Solxeni used
Benzene Ethanol Elher TW Ben/ene t i thanol Elher TV. Clhanol TW T\V
Ben/ene Clhanol T \ \ t l h a n o l Ethano!
co lour
LV
l.V LV I V LV t V LV LV t \ LV
NC LV LV L \ NC NC
Lower l imit of de teclion (t/p)
^ s
<. 5 J 4 4 4 4 4
— Oi (1 5 (1?
_ -
AbhrcMJiions drc dtfhnrd in T.tblc I and in Expcnmcnial section
H S. RATHORI and H A KUAN
Tjbic •*. Dclcclion and s<rm;quuniildi)\c dcicrniin;njon of 2,J-D acid in soil jnd vcpct;ition
Sorl Lcaxr*
C oli^u: Amount o( AmounI of
•rf>il ^4 -1 ) N o n fcriilc rciitlc (mp) | ; jp) Mill soi)
Ahbrc\ijlion*. art defined in Tabic I
Amount of Amounl of ic.ncs ZA'O imp) tHp) Colour
50 100 150
25
50 75
LV V
DV
LV
\ DV
5(KI 500 5(K1
25 50
100
NC VIV LV
Tabic 1 shows ihat out of the eight hydrolysing agents under study only two, e.g.. MnSOjH;0 and ZnS0j-7H-0. give dark violet colour for phenoxy-acctic acid. The colour developed by the use of ZnSOj• 7H;0 was found to be more intense than that by MnSO, H:0. Therefore. ZnSO^-TH-O is a suitable salt for the newly developed technique. The test gives no response when concentrated sulphuric acid was used in place of aho\c salts. It seems that in this case formaldehyde is not free to move. c\en under reduced pressure, from reaction theatre to the reagent. It may be due to the hydrogen Kindmg or high soluhilily of formaldehyde in sulphuric acid.
An intense colour vvas obtained when .'."img of ZnSOj• 7H-0 were taken while the amount more than .•; :ng fades the colour (Table I). It may be due to the retention of formaldehyde with the reaction mixture. Phcno.xyacciicacid (I8/ig)and ZnSOj-TH-O (. .'i mg) gives a light violet colour at 150 C. siolet at 160 C. dark violet from 170-190 Cand brownish dark violet at 200 C. The appearance of the dark violet colour is better than the brownish dark violet. Therefore, any temperature in the range 170-190 C is suitable for performing the test.
When the reagent (chroniotropic acid in sulphuric acid) was heated for 3 min at 160 and 180 C. there is no change in colour while reagent turned black when heated for more than 4 min. The reagent gives a dark violet colour in 2 min for 18/ig ofphenoxyacciic acid at 160 as well as 180 C. Hence, it is clear that a brief heating of 2-4 min will suflice.
As staled in the Introduction, formaldehyde can be detected in the \apour phase but the sensitivity decreases. In the newly developed apparatus this draw back has partially been overcome b\ the application of vacuum, e.g.. the lower limit of detection of phenoxyacetic acid was found to be 10 and 75//g in vacuum and without vacuum respectively.
The test under study gives a positive response for benzene soluble phenoxy herbicides such as 2.4-D acid. 2.4-D ethyl ester. 4-chlorophcnoxyacciic acid, phenoxyacetic acid and 2.4.5-T except 2.4-D sodium salt which is insoluble in ben/enc (Table 2) Besides phenoxy herbicides it also gives response to dichloro-vos (V). formothion (\'). malaihion ( \ ) . thiometon (V); N-bromosuccinimide (LBr). /7-dimeihylamino-benzaldehyde (V) and hexamine (V). N-Bromosuc-cinimide gives a light brown (LBrl colour and it may
be detected selectively but has poor sensitivity (.•'75/(g). The reaction is sensitive enough for phenoxy herbicides and it is highly .sensitive for hexamine (0.5/ig). The intensity of the violet colour produced for 2.4-D acid was found to be proportional to its concentration and the reaction can be used for visual scmiquaniilaiivc dcierminalion of 2.4-D acid (!0-100;jg) in water samples. It is also clear from Tabic 2 that non-volatile coloured compounds such a*, mcihyl orange and methyl red do not alfecl the deicction of 2.4-D acid because they arc left behind in the reaction theatre (tube A).
Data recorded in Table ,' shows the uliliiy of preferential solubility and formaldehyde producing reagent in order to raise '.lie sc'ecii.i;'. v.fili.- \l^,.l-i.•vl under suidx. ('or example. 2.4-D acid and 2.4-D ethyl ester give positive response when their solutions were prepared in benzene, clhanol. ether or TW Their lower limit of detection is also the same in all the solvents used. i.e.. 5/ig o( 2.4-D acid and 4/(c of 2.4-D ethyl ester. It shows that the .sensitivity of the test is independent of the solvents used. As 2.4-D sodium salt is insoluble in benzene it gives no response and traces of 2.4-D acid as well as 2.4-D ethyl ester can be detected in 2.4-D sodium sail matrix by the u.se of benzene. However. 2.4-D sodium salt can be detected by making the test solutions in ethanol. TW etc.. and it can also be determined in the presence of 2.4-D acid or 2.4-D ethyl ester by the use of two solvents, i.e.. benzene for 2.4-D acid 2.4-D ethyl ester and TW for total phenoxy herbicides. Hence, it is obvious thai in ihc benzene medium, the test can he applied for the dcicciion and semiquaniiuiiive dc-lerniinaiion of free acid (2.4-D) ccneraicd b\ the hydrolysis of 2.4-D sodium salt or 2.4-D ethvl ester in inorganics rich matrixes such as soil and water The sensiiiviiy and selectivity of the test can also be raised by the use of specific reagent for splitting out formaldchvde. i.e.. hydrated zinc sulphate can split oul formaldehyde from phenoxy herbicides, some organophosphorous insecticides. N-bromosuccini-midc./J-diniethylaminobenzaidchvde. hexamine etc . while it gives no formaldehyde from glycine, mcihyl orange, methyl red etc. The interference due to non-volatile substances which arc soluble in benzene can be checked by ihc use of newly designed apparatus which leaves non-volatilcs in reaction chamber The results show that this technique is good for
Dclcrminiilion of Iracc levels of 2.4-1)
(he selective dcteciion and determination of some
pollutants.
Table 4 shows that the test can be used successfully
for the detection o r2 .4 -D acid in lea\cs and soil. The
sensitivity of the test is in the followinu sequence;
water > soil > leaves, i.e.. the lower limit of detection
in leaves, soil and v\atcr is 100. 25 and 10 (ic respect
ively. Lower sensitivity of the test in soils than thai
in water may be due to strong adsorption of 2.4-D
acid on soils (Klincman and Ashton. 1975). However,
the sensitivity is lowest in leaves, it ma\ be due
to strong absorption (Klingman and Ashlon. 1975)
as well as inefficient method extraction. A simple
extraction procedure was used in order to show the
basic importance of the method. However, recovery
of 2.4-D acid can be raised by the use of a reported
extraction method (Chau and .Afghan. 1982). Further
work on the use of this method for quanti tat ions of
phenoxy herbicides in real samples is in progress.
C O N C I . I S I O N
The results discussed a b o \ e prove the applicabilitv
of the newly developed apparatusRir the prelimmarv
detection of pcslicides-based pollutants in the
different cnviroimicntal samples, ' t iiity p u n e vcr".
helpful in places where sophisticated instruments are
either not available or have not been installed so far
.•ltknii»U{/i;tnniirs — Financial assistance eranlcd hv the Council of Scientific and Industrial Research. New Delhi to one of us (HAK) is gralefullv acknowledged
RF.tKRF.VCF-S
Chau A S ^' and Afghan B K (1982) Aiwlvm of Av//</,/,i m Wuur. Vol 11. pp 216 218. CRC Press. FJ
Fcicl F and .Anger \ ' (jydfi) Spin TcMs in Orf^ainc Analysis. 7"lhedn. pp 19. 165. .1.''6, 4.U. 461. 4h6. .SOI. Elsevier, js'ew York
Feigl F and Anger V (1972) Spot Tesix in Innrgaiuc Allahsn. 6lh cdn. pp 5. 54. Elsevier. Ncv. York.
Freed V IF (1948) Sucue 107. 98 Khan M .\ (19S2) Physico chemical studies on industrial
wastes and their cITccis on soil, plants and river waters NFPhi! thesis (Cheniislrvi. Aligarh Muslim University. Aligarh. India
Klingman G C. and Ashlon F. M. (1975) H'ccJ Science: Primiplcs and Praclicc.^. pp. 57. 79. 215. 220. Wiley. New York.
Qureshi M . Ralhore H. S. and Sulaiman A. M (1982) A new sens.-livc and simple spol icsl for ihc dclcclion of icrliarv amme pollutants in waler Hi;;. Res. 16,4.15-440.
Ralhore H S . Ali I. and Khan H. A. (l9X8a) Quantitative chromatographic separation of Irichloroacelic acid from some cartiowlic herbicides on BaSO, CaSO^ coatings imprepnalcd wii)i coconut oil. J Phinur Chromaui. Mod. TLC I, 2.';2 2.';4
Rathorc 11 S . .Ali 1 and Kumar A (1980) Capillars spot losi for ihe detection of irace levels oforpanophosphorus inscclicidcs in walcr. soil and vcgclalion Ini J emir. .Aiiiih I Cluni In press
Ralhore H S. Gupta S and Khan H. A (1986) Pressure t.ipill.iiv spot test for dcteciion of pollulanls in crops, vegetation and environment. .Unihi Leu 19, 1545 If-M)
Ralhore IF S. Ali I . Sh^irma S R and Saxcn:; S CQS.Sb; Scparaiion and dcleciion of some phenoxy herbicides and plant growth regulators by ion-pair reversed phase Ihin-laver chromalographv. Ini J emir. Anahl Cheni 33, 209 217.
Ra7iuddm (1986) Studies on industrial wastes and their ellccls on rivers, soils and underground waters. Ph D thesis (Chemislrv) Aligarh Muslim University. .Aligarh. India
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