THE APPLICATION OF THE IODINE - AZIDE REACTION IN THIN-LAYER CHROMATOGRAPHY

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THE APPLICATION OF THE IODINE - AZIDE REACTION IN THIN-LAYER CHROMATOGRAPHY

STUDIES OF PESTICIDE PREPARATIONS

By

T. CSEHHATI* and F.OHSI

Department of Biochemistry and Food Chemistry, Technical University Budapest Received October 3, 1981

Presented by Prof. Dr. R. LASZTITY

Introduction

With chemicals bcing uscd in wider and wider areas for plant protection, toxicologieal and ecological aspects become increasingly important. The formulation analysis of pesticides cannot be limitcd nowadays to the selective determination of the active agent: the identification and quantitative determination of impurities is becoming equally significant. The process is of particular importance with phosphoric ester derivatives, since they are more ore less toxic to human beings. Gas chromatographic analysis (GC) of these active agents has been developed many years ago [1]. However, the technique could not supplant thin-layer chromatography (TLC) [2] for the following reasons:

a) thermally unstable phosphoric ester derivatives cannot be analyzed by GC;

b) if flame ionization detectors are used, one cannot state whether the peaks indicating impurities correspond to presumably toxic side products of the active agents, or to othcr, non-toxic components of the formulation;

c) a substantial part of the decomposition products formed in hydrolysis or oxidation proccsses are non-volatile. Consequently, GC methods can only be used when combined with additional processes to yield volatile products;

d) the TLC technique, using non-destructive, selective detection processes, allows to isolate, in a much simpler manner as ~ompared to traditional GC, volatile and non-volatile organic phosphoric ester derivatives in amounts in the order of milligrams for further studies of structure identification and toxic properties.

It is well known that P=S and C=S bonds have a catalytic effect on the reaction taking place between iodine and sodium azide [3]. This phenom- enon has long been in use for the detection by TLC of active agents of pesticides containing the above bonds [5, 6]. TLC determination of organic phosphoric ester derivatives are usually performed on silica gel layers [7-13], less

*

Research Institute for Plant Protection

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112 T. CSERHATl and F. ORS]

frequently on aluminium oxide [14<], Florisil [IS] or polyamide [16, 17] layers.

It is barely understandable why so little attention was pair to reverse-phase TLC separation, since R_Jvalues obtained by this technique characterize the lipophilic nature of the compound and are hence of b'Teat significance in optimum molecular design [18]. The objective of our study was to find the range of applicability of the iodine azide reaction in TLC analysis of phosphoric ester-based commercial pesticides, including the determination of both the active agent and the impurities.

Experimental

[vI aterials and apparatus

The constitutional formulae of the phosphoric el:iter derivatives studied are presented in Fig. 1.

For adsorption TLC, plates 20 X 20 cm, thickncss 0.25 mm were from Kieselgcl G (manufacturer: REANAL) prepared. For reverse-phase TLC, the plates were prepared by impregnation with a solution of paraffine oil in n- hexane (dimensions of the plates were similar to the above).

A Telechrom Video-densitometer (manufacturer: CHINOIN) was utilized for quantitative determination.

Methods

Stock solutions of the pesticides contaillillg the compounds listed in Fig' 1. in acetone were prepared, with concentrations of 2 mg aetiye agent per cm³ acetone. In adsorption TLC the solvent used was dichloromethane; ill reversc- phase TLC the solvent was a 1:1 or 3:1 mixture of methanol and water. The active agents were applied ill spots, ill anHlunts of 2 ... 40 pg. The plates were then dried ill air, sprayed with a freshly prepared 1.:1 mixture of 1. molll sodium azide and I molll iodins solutions, and subsequently, when - due to the eatalytie effeet of the P=S hond - the spoots beeame visible as white spots in the yellow background, the plates were sprayed 'with a 1

%

aqueous stareh solution. The area of the white spots ill the violet haekground was then measured instrumentally, and the equation hest describing the relationship area 1'S. amount of aetive agent was l:ieleeted. Sinee it is known that for homo- logue sequenees of related eompounds a relationship exists between their adsorptivity and lipophility, we attempted to calculate linear correlations hetween R_Jvalues measured in adsorption and reverse-phase TLC.

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o 0

CH30" N

CX)

^CH.J0,

~H0 00 ^~HsO, ¹

1 . cf

P-5 -cHi" ' ^iI ^N"

8

^• ^/11^P-5-CH-co~Hs ¹

15

^. ^/11^{P- 0- -} ^I'"

22

^• ^/^P-N^Jl 'C~ Yc:)i

CH:J 5 N CH.JO 5 CH-COOr H ~1-lsO 5 ~HsO S 11 '

2 ~ 5 0

Q ~s CH~

2 ^~I-\;O, ^~

^_

9 ~I-\;O,

^;:hSCH³

16)-N

^/oCH:J

23 .:1'-

^CH^r^CO-Nl-I-CH:J

P-o-~ . . . ,;-0 P-0\QJ. n '}-o-p CH:JO 5

• ~I-\;cr~ N-~ , ~Hso'~ Cl '

r

s " O C H : J '

g

©

^Oc;Hs ^CH:JO, ... C(}fr;] ...

o ^24,

^A'-5--GH²^-N-<m0 ^~

CH:JO ~ ~

3

, / p - o - p , ^SHsO,

^~-~Hs 10

. ^CH³^0"/P-5-CH&

~ 17

. ^C²^HsO,/P-5-C~-C-NH-~~ ^C2HsO,

~ g

r HsO 511 511 O-c;Hs CH:JO 511 I r \·L ~ ~

25

P-5-CH2-S-C²Hs ~

'"'2 cOO~Hs ^{"2 '}^'5 ^' ^/¹¹ ^tq

C2HsO S ...

CH:JO" ;CH, C₂HsO, CH30, CH:JO,

9

0

4

^P-o-c ^c-CH.J

11.

^P-S-CI-^L^5-C-(CH_J⁾³

18

P-0-fO\-N02

26

P-O-Cl-l-CH-S-CH '~

• /11 ' " /11 '';/ ' / 1 1 ~ • / \ '';/ 2 3 ...

CH~

^S N"'C,N C

2 HsO 5 CH₃0 5 CH3 CH3 0 5

~

NK;Hs)2

s.. ~

O-C C-OCH

!i:

12 ^Sf-\,o, 19

^CH³^0"

(§; 27

^CH30" ^\ ¹¹ ³ "<!

~

^c~-or^HS ^/p-^5-C~^Cl ^/p-O-⁰ ^-SCH^J P-S-CH2- N-N t:!

r-H 0 N-N l , . 11 "2 • 11 • / 11 ~

5

^"2⁵ "P-Q~ ^" ~I-\;O ⁵ ^CH³^{0 5} CH3 CH 30 S (')

, C

I-\O/~

^C!-i:J

~

2

~HsO ~CI

^{CH 0}

C~I

^{CH 0}

~

13

'P- 0- , C l

20

³^/'p-

°

⁰ ^Br

28

³ ^/'P1 -0 ·IQ\N0₂

~

CH 0 C H : J ' /11 " . 11 " \::!J 0

6

³>,-S-C~":'C ^-N/ C₂H_SO S Cl CH30 5 Cl CH30 S !!;]

· CH:JO

~

¹¹ ^'CHO ^;

o r: ^H

°

^rHO ^Cl ^r:^H

°

^III

CH3

14.,

^{L' 'S}^{'P-S _IQ\}

21.

^L ⁵^{'P -0}

iQ\-

^Cl

29

^'<⁵ "P-S-CH ~~ "<!

..c

^/11 'C2 /11 ):::!J /

2"Ji

7

_{• C}^~H5^0,^P,-^0-^He^C.^~^,,-CHt~) ^r:^"2^H⁵ ⁵ ^{CH 0 S}^{2 5} ^Cl ^r:^'<^{H 0}⁵ ^O~ ^I

2

Hs 0/ ~ 'N' ' '32

Fig. 1. The constitutional formulas of the thionphosphoric ester derivatives studied

...

W

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114 T. CSERHAT! and F, {jRS!

Results and discussion

The Rj values of the phosphoric ester derivatives studied are presented in Table 1.

Table 1

RI values thionphosphoric esters

No.

of compound

1 2 3 4 5 6 7 II 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

II III

I Reverse-phase, Reverse-phase.

Silica gel, methanol: water methanol: water

dicbloromethane 1 : 1 3 : 1

0.59 0.22 0.92 0.78 0.48 0.08 0.49 0.55 0.07 0.72 0.87 0.87 0.97 0.98 0.95 0.60 0.62 0.89 0.90 0.07 0.07 0.71 0.08 0.68 0.92 0.93 0.61 0.78 0.72

0.34 0.19 0.15 0.12 0.07 0.88 0.13 0.22 0.03 0.13 0.05

0.39 0.20 0.49

0.14 0.66

0.16 0.64

0.36 0.80

0.10 0.66

0.64 0.42 0.23 0.76 0.84

0.95 0.92

0.09 0.57

0.45 0.86 0.73 0.37

The following observations were made in the performation of the iodine- - azide reaction:

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IODINE-AZIDE REACTION IN THIN-LAYER CHROMATOGRAPHY 115

The compounds containing several higly electronaffine substituents (compounds 9, 13, 20 and 21) do not catalyze the reaction in our experimental conditions. By heating the plates, the spots can be made visible; however, the background also becomes much paler. Thus the I'elative error of instrumental measurement will increase, so that these compounds cannot reliably be deter- mined quantitatively by the iodine- azide reaction.

On the othcr hand, compounds with groups that will readily adsorb iodine (compounds 1, 2, 5, 7, 15 and 16), aftcl' spraying with thc iodine-azide solution, will develop brown spots instead of white spots in the yellow background. However, in contrast to the compounds of the previous group, this difficulty can be surmountcd by spraying the plates after to 100 oC, since at higher temperatures the extent of adsorption 'will decreasc, while the rate of the catalytic reaction will increase.

Thc developed plates prepared for calibratiun purposes to compounds 8 and 24 are presented in Fig. 2.

The visual detection limit of the cOIllpounds varies largely frOIll compound to cOIllpound, owing to the diffcrenees in thc substituents (0.5 ... 10 pg).

Since thc scnsitivity of thc Telechrom VidcodcllsitoIlleter is lower than that of the hUIllan cye, and the reproducibility of IlleasureIllent decreases dramatically below 10⁴digital signals (Fig. 3), it is expedient to work in the substance range of 10 to 40 f.Lg for quantitative determinations.

We attempted to approach the visibly non-linear relationship spot area vs. amount of substancc (Fig. 4) 'with logarithmic and log-log functions. The relationships calculated for cOIllpound 8 are as follows (y = amount of sub-

Fig. 2. Determination of thionphosphoric esters. Adsorption TLC, solvent CH2Cl_2•Compound 8 on plate Il, compound 24 on plate Ill. In both cases: 1-2 pg; 2-6 pg; 3,4,5-10 pg

6, 7, 8-20 pg; 9-30 pg; 10-40 pg; 11-50 pg

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116 T. CSERILiTI and F. OHS]

v

1.6

08

O'---,---,--i'>

Fig. 3. Dependence of the reproducibilityj'of videodensitomctric~arearIllcasureI1leIlts~(V) on area size (T)

33

17

I ~2

od

^, ^I

~

5 2']

Fig. 4. Relationship between the amount of substance applied (/Ig) and spot area (T). Curve 1 compound 8; curve 2 - compound 2'~

stance applied, {1g, x

=

digital value of area, n

=

8, r99%

=

0.8343, r99.9% =

= 0.9249):

{1g

=

-71.40

+

21.34 log x log {1g

=

1.20 0.59 log x log {1g = 0,86

+

^2.10·^10-5x

r

=

0.5751 r 0.8815 r

=

0,9697

The correlation between the logarithm of amount of substance applied and the area of the spot being linear at a reliability level of 99.9%, the above

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IODINE-AZIDE REACTION IN THIN·LAYER CHROMATOGRAPHY 117

calibration curve allows a quantitative determination of the substances with satisfactory accuracy.

The equations describing the relationship lipophility vs. adsorptivity (the Roman numerals indicate the solvent systems in Table I)

R jJ = 0.69-0.30 . Rfll R jJ = 0.71-0.03 . RjIlI

n = 17 n = 18

T = 0.2899

T = 0.0242

indicate that there is no correlation between adsorptivity and lipophility in the group of compounds studied.

The utilization of the method to detect impmities will be demonstrated by the following two examples. The separation of compounds 12 and 23, resp. and their decomposition products is presented in Figs 5 and Fig. 6. (In the experiments aiming to separate impurities, the solvents were changed as compared to the other runs.)

It may be seen from the figmes that the method is eminently suitable for the separation and detection of decomposition products containing the P=S bond, even at semi-preparative amounts of the substance.

IV

Fig. 5. Separatioll of compound 12 and its impurities. Reverse.phase TLC, methanol: water 4:1. 1 - 10 pg: 2-~O pg: 3-40 fIg: 4-60 ,ug: 5-80 fIg

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118 T. CSERHATI and F. ORSI

Fig. 6. Separation of compound 23 and its impurities. Adsorption TLC, benzene: acetone 9:1.

1-50 /lg; 2--100 /lg; 3-200 /lg; 4-300 /lg; 5-400 /lg

Summary

The analytical applicability:of the iodine-azide reaction catalyzed by the P=S bond was studied in thin-layer chromatographic detection and quantitative determination of thlon- phosphoric esters contained in 29 commercialipesticides. It was found that in the case of highly electronaffine Eubstituents the sensitivity of the reaction is very low and hence it is unsuit- able for quantitative determination. With substituents readily adsorbing iodine, it is expedient to carry out the reaction at elevated temperatures. The reaction was found to be suited for non-destructive quantitative determination of thionphosphoric esters and their decomposition products.

References

1. ZWEIG, G.: Analytical; Methods for Pesticides and iPlant Growth Regulators, V 01. VI, p.

191-231. New York-San Franciseo-London, Academic Press, 1974.

2. ZrVEIG, G.: Analytical I.Iethods for Pesticides and Plant Grov,"1;h Regulators, Vo!. VII, p.

3-89. New York-San Francisco-London, Academic Press, 1974.

3. JACIMIRSZKIJ: A kemia analizis kinetikus modszere (The Kinetic method of Chemical analysis). Budapest, Miiszaki Konyvkiado, 1966.

4. SVAB, J.: Biometriai modszerek a kutatasban (Biometric Methods in Research). Mezo gazdasagi Kiado, Budapest, 1973.

5. ZWEIG, G.: Handbook of Chromatography, Vo!. II, p. 151. USA, CRC Press, 1972.

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IODINE-AZIDE REACT1(jN IN THIN·LAYER CHROMATOGRAPHY 119

6. STAHL, E.: Diinnschichtchromatographie, p. 506. Berlin-Gottingen-Heidelberg, Springer Verlag, 1962.

7. ABBOTT, D. C.-BURBBIDGE, A. S.-TROMSON, J.-WEBB, K. S.: Analyst. 92, 171 (1967).

8. BRUKMAN, H.-WINTERLIN, W.: Bull. Environ. Contam. Toxicol. 1, 78 (1966).

9. MENDOZA, C. E.-WALEs, P. J.-BRAY, D. F.: Analyst. 93, 689 (1968).

10. FISCRER, R.-KLINGELHOFFER, W.: Pflanzenschutz Ber. 27, 165 (1961).

H. WALKER, K. C.-BEROZA, M.: JAOAC, 46, 250 (1963).

12. BAUMLER, J.-RrPPSTEIN, S.: Helv. Chim. Acta, 44, H62 (1961).

13. GUTR, J. A.: Pflanzenschutz Ber. 35, 138 (1967).

14. RAMASAMY, M. 0.: Analyst, 94, 1078 (1969).

15. HAMILTON, D. J.-SIMPSON, B. J.: J. Chromatogr. 39, 186 (1969).

16. NAGASAWA, K.-YOSHIDOME, H.: J. Chromatogr. 39, 282 (1969).

17. WANG, R. T.-CRou, S. S.: J. Chromatogr. 42,416 (1969).

18. ANDREW, J.-STUPER, W. E.-BRUGGER, P.: Computer Assisted Studies of Chemical Structure and Biological Function. New York, Wiley, 1979.

Dr. Tibor CSERBATI

Dr. Ferenc 6RSI

Research Institute for Plant Protection H-1521 Budapest

THE APPLICATION OF THE IODINE - AZIDE REACTION IN THIN-LAYER CHROMATOGRAPHY