Pesticide Analytical Methodology - ACS Publications - American

9 Recent Developments i n High-Performance

Thin-

-Layer Chromatography and Application to Pesticide Analysis H. E. HAUCK and E. AMADORI

Downloaded by UNIV LAVAL on July 12, 2016 | http://pubs.acs.org Publication Date: October 30, 1980 | doi: 10.1021/bk-1980-0136.ch009

E. Merck, D-6100 Darmstadt, Federal Republic of Germany

Summary The HPTLC pre-coated layers examined i n this paper, namely silica gel 60, RP, cellulose and silica gel 60 with concentrating zone, are new developments or modifications of existing TLC pre-coated layers offering new potential for pesticide analysis by thin-layer chromatography. A number of separations (of BHC isomers, of bromophos-ethyl and dimethoate. and of the active constituents of Trevespan 6038) were performed in order to provide a qualitative and quantitative comparison between TLC and HPTLC pre-coated plates, or between HPTLC plates coated with various sorbents, and i n order to demonstrate their use i n pesticide chemistry. I. Introduction S i l i c a gel 60, the most versatile and most frequently used TLC sorbent, was taken as a basis. The mean particle size of this sorbent was optimized; simultaneously, the particle size distribution was brought to within as narrow limits as possible (1-6). The sorbent material thus obtained was used to prepare HPTLC pre-coated plates silica gel 60, being followed subsequently by the development of other sorbents for processing into HPTLC pre-coated layers. The materials chosen were largely "reversed phase" sorbents, that is to say, chemically modified s i l i c a gels with a non-polar surface (7, 8), as well as microcrystalline cellulose (9). I n t h e c a s e o f HPTLC p r e - c o a t e d l a y e r s , i n p a r t i c u l a r , optimum u s e o f t h e a v a i l a b l e separating performance i s dependent on t h e compactness o f t h e 0-8412-05 81 -7/80/47-136-15 9$05.00/0 © 1980 American Chemical Society

Harvey et al.; Pesticide Analytical Methodology ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

160

PESTICIDE

ANALYTICAL

METHODOLOGY

s t a r t i n g s p o t s . These n a r r o w l y d e l i m i t e d s t a r t i n g spots are obtained w i t h the a i d of s u i t a b l e , r e l a t i v e l y i n v o l v e d a p p l i c a t i o n t e c h n i q u e s and by the a p p l i c a t i o n o f s m a l l v o l u m e s . I n o r d e r t o make better use o f t h e p o t e n t i a l performance o f HPTLC p r e - c o a t e d p l a t e s s i l i c a g e l 60, and to permit a p p l i c a t i o n of l a r g e r q u a n t i t i e s w i t h a r a p i d and n o n - p r o b l e m a t i c a l t e c h n i q u e , s p e c i a l HPTLC p r e - c o a t e d p l a t e s s i l i c a g e l 60 w i t h a s o - c a l l e d " c o n c e n t r a t i n g z o n e " were d e v e l o p e d (1J), 2 1 , 12).


2.

Experimental

2.1 R e a g e n t s and s o l v e n t s . solvenxs used i n t h i s work were from E. Merck, Darmstadt.

A l l the r e a g e n t s and of a n a l y t i c a l grade

2.2 A p p l i c a t i o n of the samples. The v o l u m e s of 0.75 p i were a p p l i e d as a dot u s i n g a p p r o p r i a t e glass c a p i l l a r i e s (E. Merck, Darmstadt). 2.5 Development. A l l the plates investigated here were each kept overnight at a r e l a t i v e h u m i d i t y o f 2 0 $ . The p l a t e s w e r e t h e n d e v e l o p e d t o h e i g h t s of 5 cm ( H P T L C ) a n d 7 cm ( T L C ) i n normal chambers, w i t h o u t chamber s a t u r a t i o n , u s i n g the appropriate solvent systems. 2.4 E v a l u a t i o n . The e v a l u a t i o n was c a r r i e d o u t b y r e f l e c t a n c e u s i n g a Z e i s s PMQ I I chromatogram spectrophotometer (Zeiss, Oberkochen), w i t h a d i r e c t l i n k o f the analogue o u t p u t t o an IBM 1800 p r o c e s s control computer. 5.

HPTLC p r e - c o a t e d

plates

s i l i c a

gel

60

O p t i m i z a t i o n o f t h e mean p a r t i c l e s i z e o f s i l i c a gel 60 and the consequent r e d u c t i o n of the p a r t i c l e size d i s t r i b u t i o n p r o d u c e d a number o f a d v a n t a g e s i n HPTLC p r e - c o a t e d p l a t e s s i l i c a g e l 60 p r e p a r e d from t h i s i m p r o v e d m a t e r i a l w h i c h t h e t r a d i t i o n a l TLC s i l i c a g e l 6 0 p r e - c o a t e d l a y e r s do n o t p o s s e s s . These a d v a n t a g e s a r e t o be f o u n d n o t o n l y i n t h e already m e n t i o n e d o p t i m i z e d mean p a r t i c l e d i a m e t e r and t h e narrower p a r t i c l e s i z e d i s t r i b u t i o n , but also i n the smoother and more homogeneous s u r f a c e o f the plates, which leads to an increase i n chromatographic performance. The i m p r o v e m e n t s made i n t h e HPTLC p r e - c o a t e d p l a t e s i l i c a g e l 60 as compared w i t h t h e TLC s i l i c a


9.

H A U C K

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HPTLC

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Pesticide

161

Analysis

g e l 60 p l a t e s meant t h a t HPTLC p r e - c o a t e d plates c o u l d a l s o be u s e d a d v a n t a g e o u s l y i n pesticide a n a l y s i s (13, 1£, ) · In this* paperHEhe advantages of HPTLC pre-coated p l a t e s s i l i c a g e l 60 ( E . M e r c k , D a r m s t a d t ) as compared w i t h TLC p r e - c o a t e d p l a t e s s i l i c a g e l 60 (E. Merck, Darmstadt), are presented i n the s e p a r a t i o n o f benzene h e x a c h l o r i d e (BHC) isomers. S e v e r a l p u b l i c a t i o n s (1,6, J J , 18) w e r e t a k e n a s a b a s i s f o r d e t e r m i n i n g the optimum c o n d i t i o n s for t h e s e p a r a t i o n a n d d e t e c t i o n o f BHC i s o m e r s , as regards both eluant and d e t e c t i o n mode. B o t h f o r TLC and f o r HPTLC p r e - c o a t e d plates s i l i c a g e l 60 the b e s t r e s u l t s were o b t a i n e d w i t h a solvent mixture c o n s i s t i n g of cyclohexane and c h l o r o f o r m i n a r a t i o o f 6 5 / 3 5 b y v o l u m e , w i t h a 5 cm m i g r a t i o n d i s t a n c e f o r H P T L C p l a t e s a n d a 7 cm m i g r a t i o n d i s t a n c e f o r t h e TLC p l a t e s . I n b o t h cases the p l a t e s were adjusted p r i o r to use to 20$ r e l a t i v e h u m i d i t y and were developed i n normal chambers w i t h o u t chamber s a t u r a t i o n . For b o t h types of plates, 0.75 μΐ q u a n t i t i e s of a t e s t m i x t u r e of α-, β-, γ-, δ - a n d 6-BHC w e r e a p p l i e d w i t h g l a s s capillaries ( E . M e r c k , D a r m s t a d t ) a t a c o n c e n t r a t i o n o f 10 m g / 10 m l d i s s o l v e d i n m e t h a n o l . Once s e p a r a t i o n h a d b e e n c o m p l e t e d , t h e b e s t v i s u a l i z a t i o n o f BHC i s o m e r s w a s a c h i e v e d by s p r a y i n g the p l a t e s w i t h 1$ o - t o l u i d i n e s o l u t i o n i n e t h a n o l f o l l o w e d b y 15 m i n u t e s i r r a d i a t i o n u n d e r UV l i g h t ( 2 5 4 n m ) . T h i s l e d t o the f o r m a t i o n of g r e y i s h - g r e e n spots on a greyish-brown background; even a f t e r 3 days the spots d i d not turn p a l e . The p l a t e s w e r e e v a l u a t e d w i t h a Z e i s s chromatogram spectrophotometer PMQ I I i n reflectance mode, w i t h the a n a l o g u e o u t p u t d i r e c t l y c o u p l e d to an IBM 1800 p r o c e s s computer. T a b l e I shows t h a t r e t e n t i o n o f t h e individual BHC i s o m e r s i s v i r t u a l l y i d e n t i c a l o n t h e T L C a n d HPTLC p r e - c o a t e d s i l i c a g e l 60 p l a t e s . The h R f v a l u e s o f t h e BHC i s o m e r s o n t h e H P T L C p l a t e s l i e only s l i g h t l y above t h o s e a c h i e v e d on the TLC pre-coated plates. Table II compares a number of a d d i t i o n a l c h r o m a t o g r a p h i c d a t a a c h i e v e d f o r BHC i s o m e r s e p a r a t i o n s o n TLC a n d HPTLC s i l i c a g e l 60 p l a t e s . These v a l u e s show t h a t t h e optimum s e p a r a t i o n c h a r a c t e r i s t i c s on HPTLC s i l i c a g e l 60 p l a t e s are a c h i e v e d a f t e r a m i g r a t i o n d i s t a n c e o f o n l y 5 cm, w h e r e a s on t h e TLC p l a t e s o p t i m u m s e p a r a t i o n i s a c h i e v e d a f t e r a m i g r a t i o n d i s t a n c e of 7 cm. T h i s means t h a t i n s p i t e o f a r o u g h l y 35$ s m a l l e r flow 1


for

5

1


162

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Table

METHODOLOGY

I . R e t e n t i o n data (hRf values) o f BHC i s o m e r s o n TLC and HPTLC p r e - c o a t e d plates s i l i c a gel 60; solvent system cyclohexane/chloroform 65/35 types of plates

Table

ANALYTICAL

:

hRf values of BHC isomers S

α

V

6

β

TLC s i l i c a gel 60

59,6

51,0

47,5

39,1

27,3

HPTLC s i l i c a gel 60

62,6

53,4

48,2

40,6

28,3

I L Chromatographic d a t a on the separation o f BHC i s o m e r s o n TLC a n d H P T L C p r e coated plates s i l i c a gel 60; solvent system: c y c l o h e x a n e / c h l o r o f o r m 65/35

types of plates

velocity coefficient κ C min/s ] 2

opt. migration distance C mm ]

development time Cs]

resolution R of s

α-and y-BHC

TLC silica gel 60

3,67

70

1540

1,34

HPTLC silica gel 60

2,38

50

1306

1,62


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Analysis

163

rate the required development t i m e f o r t h e HPTLC p l a t e i s a b o u t 240 s e c o n d s o r 15$ s h o r t e r t h a n on t h e TLC p l a t e . I n s p i t e o f t h i s s h o r t e r migration d i s t a n c e and development time, the chromatographic separation efficiency, e x p r e s s e d by the r e s o l u t i o n R b e t w e e n t h e two n e i g h b o u r i n g a - and γ-isomers o f BHC, i s about 21$ h i g h e r on the HPTLC p r e - c o a t e d plate s i l i c a g e l 60 t h a n on t h e c o r r e s p o n d i n g TLC pre-coated plate. As a means o f c o m p a r i n g t h e p o t e n t i a l for q u a n t i t a t i v e d e t e r m i n a t i o n s on t h e TLC and HPTLC pre-coated p l a t e s s i l i c a gel 60, the c a l i b r a t i o n c u r v e s o f 5-BHC w e r e recorded. I n F i g u r e 1 t h e p e a k h e i g h t s ( e x p r e s s e d i n mV, a f t e r e v a l u a t i o n by r e f l e c t a n c e u s i n g a Z e i s s PMQ II chromatogram spectrophotometer) are p l o t t e d against the a p p l i e d q u a n t i t y ( e x p r e s s e d i n ng) o f 5-BHC on t h e TLC and HPTLC s i l i c a g e l 60 p l a t e s . I t c a n be s e e n t h a t i n b o t h c a s e s a w e l l c o r r e l a t i n g c a l i b r a t i o n curve i n the shape of a straight line is obtained. It is also evident that f o r e q u a l a p p l i e d q u a n t i t i e s the peak h e i g h t s on the HPTLC p l a t e a r e s l i g h t l y more t h a n t w i c e as h i g h as on t h e TLC p l a t e . F o r t h i s r e a s o n and on a c c o u n t of the fact that the b a s e l i n e noise i s markedly l e s s on the HPTLC p l a t e e v a l u a t e d i n r e f l e c t a n c e mode a s a r e s u l t o f i t s more homogeneous and s m o o t h e r surface, t h e d e t e c t i o n l i m i t f o r 6-BHC u n d e r t h e given c o n d i t i o n s on the HPTLC p r e - c o a t e d p l a t e s i l i c a gel 6 0 i s c o n s i d e r a b l y l e s s a t 18 n g t h a n o n t h e TLC pre-coated p l a t e s i l i c a g e l 60 a t 40 n g .


g

4»

HPTLC

pre-coated

plates

RP

Now t h a t r e v e r s e d p h a s e c o l u m n p a c k i n g m a t e r i a l s h a v e been used s u c c e s s f u l l y i n column l i q u i d chromatography f o r a number of y e a r s , s u c c e s s has a l s o been achieved i n t h i n - l a y e r c h r o m a t o g r a p h y i n t h a t i t i s now p o s s i b l e to p r e p a r e HPTLC p r e - c o a t e d plates w i t h the same t y p e s o f s u r f a c e - m o d i f i e d s o r b e n t s and to use them f o r s e p a r a t i o n s (2» 8, 19, 20). The b a s i c s k e l e t o n o 7 t E e m o d i f i e d sorbents c o n s i s t s of a surface-active s i l i c a gel. Modification i s i n the form of a surface r e a c t i o n w i t h s p e c i a l l y active s i l a n e s at the s i l a n o l groups of the s i l i c a gel. Following e l i m i n a t i o n of these s i l a n o l groups, new s i l o x a n e g r o u p i n g s a r e f o r m e d , on w h i c h a l i p h a t i c h y d r o c a r b o n groups are c h e m i c a l l y bound by s i l i c o n carbon bonds to the s i l i c a g e l s k e l e t o n . Pre-coated plates w i t h these modified s i l i c a gels are designated:



164

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ANALYTICAL M E T H O D O L O G Y

Figure 1. Calibration curves for 8-BHC on a TLC pre-coated plate silica gel 60 (left) and on an HPTLC pre-coated plate silica gel 60 (right) (abscissae: applied quantity (ng); ordinates: peak height (mV))

Figure 2. Linear chromatograms for the separation of bromophos-ethyl and dimethoate on an HPTLC pre-coated plate silica gel 60 (left) and an HPTLC precoated plate RP-18 (right] (solvent system on silica gel 60: n-heptane/acetone 65/35; on RP-18: acetone/'water 80/20; application from left to right—(1st band) bromophosethyl; (2nd band) dimethoate; (3rd-5th band) mixture; migration distance: 5 cm; applied quantities: 0.75 /xL = 750 ng; detection: spraying with PdCl in ethanol heating to 120°C) 2



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HPTLC

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Pesticide

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HPTLC p r e - c o a t e d p l a t e R P - 2 , R P - 8 and R P - 1 8 . The f i g u r e f o l l o w i n g the RP a b b r e v i a t i o n r e f e r s to the c h a i n l e n g t h of the a l i p h a t i c hydrocarbon. One e x a m p l e f o r t h e u s e o f a n H P T L C pre-coated p l a t e w i t h a reversed phase m a t e r i a l i n the f i e l d of p e s t i c i d e a n a l y s i s i s the separation of bromophose t h y l and d i m e t h o a t e on a n HPTLC p r e - c o a t e d plate R P - 1 8 F 254 s (Ε. M e r c k , D a r m s t a d t ) . T h i s i s described and compared w i t h the s e p a r a t i o n of t h i s substance m i x t u r e on a n HPTLC p r e - c o a t e d p l a t e s i l i c a g e l 60 Ρ 254 (Ε. M e r c k , Darmstadt). The s o l v e n t s y s t e m most s u i t a b l e f o r separating b r o m o p h o s - e t h y l and dimethoate i s an 80/20 m i x t u r e of acetone/water f o r t h e HPTLC p r e - c o a t e d plate RP-18, and a 65/35 m i x t u r e of n-heptane/acetone i n the case o f t h e HPTLC p r e - c o a t e d plate s i l i c a gel 60. The o p t i m u m m i g r a t i o n d i s t a n c e w a s 5 cm i n b o t h cases. The a p p l i e d s u b s t a n c e q u a n t i t y was 0 . 7 5 p i o n b o t h plates, at a concentration of bromophos-ethyl and d i m e t h o a t e o f 10 mg/10 m l i n m e t h a n o l i n b o t h c a s e s . D e t e c t i o n o f s u b s t a n c e s p o t s was p e r f o r m e d by spraying w i t h a s o l u t i o n of 0.5 g palladium(ll)c h l o r i d e i n 100 m l o f e t h a n o l w i t h s u b s e q u e n t heating t o 120 C . The s p o t s a p p e a r i n g a r e b r o w n o n a g r e y p i n k b a c k g r o u n d . A Z e i s s PMQ I I chromatogram spectrophotometer c o u p l e d to an IBM 1800 process computer was u s e d f o r evaluations. F i g u r e 2 shows the s e p a r a t i o n o f bromophos-ethyl and d i m e t h o a t e on a n HPTLC p r e - c o a t e d plate s i l i c a g e l 60 ( l e f t ) and on a n HPTLC p r e - c o a t e d p l a t e RP-18 ( r i g h t ) . The a p p l i c a t i o n scheme was t h e same i n b o t h cases, namely from l e f t to r i g h t : 1st band : bromophos-ethyl 2nd band : dimethoate 3 r d - 5 t h band : m i x t u r e o f t h e two substances. The two c h r o m a t o g r a m s show t h a t i n c h a n g i n g f r o m the s i l i c a g e l 60 p l a t e t o t h e R P - 1 8 p l a t e t h e r e i s a r e v e r s a l of substance sequence. This i s also apparent i n T a b l e I I I w i t h t h e h R f v a l u e s o f t h e two sample substances. T a b l e IV compares a few c h r o m a t o g r a p h i c data r e l a t i n g to the s e p a r a t i o n of bromophos-ethyl and dimethoate on the HPTLC p r e - c o a t e d p l a t e s i l i c a g e l 60 and t h e HPTLC p r e - c o a t e d plate RP-18. These f i g u r e s show t h a t r e s o l u t i o n b e t w e e n b r o m o p h o s - e t h y l and d i m e t h o a t e on t h e HPTLC R P - 1 8 p l a t e i s r o u g h l y 60$ g r e a t e r t h a n on the s i l i c a g e l 60 p l a t e . T h o u g h t h e o p t i m u m m i g r a t i o n d i s t a n c e o f 5 cm i s t h e same i n b o t h c a s e s , t h e d e v e l o p m e n t time on the RP-18 p l a t e i s r o u g h l y t w i c e as l o n g as on the


166

PESTICIDE


Table

III

IV

METHODOLOGY

R e t e n t i o n d a t a ( h R f v a l u e s ) f o r "bromo p h o s - e t h y l and d i m e t h o a t e on a n HPTLC p r e - c o a t e d p l a t e s i l i c a g e l 60 (solvent s y s t e m : n - h e p t a n e / a c e t o n e 65/35) a n d o n an HPTLC p r e - c o a t e d p l a t e R P - 1 8 (solvent system: acetone/water 80/20) types of plates

Table

ANALYTICAL

hRf values of Bromophos-Ethyl

Dimethoate


70,7

30,7

HPTLC RP-18

27,3

79,8

Chromatographic data for the s e p a r a t i o n o f b r o m o p h o s - e t h y l and d i m e t h o a t e on HPTLC p r e - c o a t e d p l a t e s s i l i c a g e l 60 and HPTLC p r e - c o a t e d p l a t e s R P - 1 8

types of plates

velocity coefficient κ

opt. migration distance

development time

resolution R of Bromophos-Ethyl and Dimethoate s

C mm/s J

C mm ]

CsD

HPTLC s i l i c a Gel 60

3,73

50

814

3,43

HPTLC RP - 18

1,69

50

1841

5,53

2


9.

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s i l i c a g e l 60 p l a t e . T h i s i s a t t r i b u t e d t o t h e much lower velocity coefficient * of the solvent system used on the RP-18 p l a t e as compared to t h a t on the s i l i c a g e l 60 p l a t e . Quantitative i n - s i t u evaluation of the bromophose t h y l and dimethoate s e p a r a t i o n i n t h i s case of v i s u a l i z a t i o n has no r e a l v a l u e on a c c o u n t o f the r a t h e r non-homogeneous background c o l o u r a t i o n p r o d u c e d by s p r a y i n g w i t h the d e t e c t i o n r e a g e n t u s e d here. Therefore the r o u t i n e , q u a n t i t a t i v e in-situ e v a l u a t i o n of the u n s t a i n e d bromophos-ethyl and d i m e t h o a t e i n o u r l a b o r a t o r y i s c a r r i e d o u t b y UV measurement i n r e f l e c t a n c e mode. 5«

HPTLC p r e - c o a t e d

plates

cellulose

Apart from the sorbents already mentioned i n c o n n e c t i o n w i t h HPTLC p r e - c o a t e d l a y e r s , a m i c r o c r y s t a l l i n e c e l l u l o s e has a l s o been produced i n an average p a r t i c l e s i z e and a narrow p a r t i c l e size d i s t r i b u t i o n s u i t a b l e f o r HPTLC ( 9 ) . HPTLC p r e - c o a t e d p l a t e s c e l l u l o s e Ρ 254 s (Ε. M e r c k , D a r m s t a d t ) , composed of t h i s m i c r o c r y s t a l l i n e c e l l u l o s e , were used to separate Trevespan 6038. This i s a mixture of the substances i o x y n i l (3,5-diiodo-4-hydroxy-benzon i t r i l e ) , flurenol (9-hydroxyfluorenecarboxylic acid) a n d MCPA ( 2 - m e t h y l - 4 - c h l o r o p h e n o x y a c e t i c acid). For c o m p a r i s o n p u r p o s e s T r e v e s p a n 6038 was a l s o separated on HPTLC p r e - c o a t e d p l a t e s s i l i c a g e l 60 F 254 (Ε. Merck, D a r m s t a d t ) . The s o l v e n t s y s t e m m o s t s u i t a b l e f o r separating T r e v e s p a n on HPTLC p r e - c o a t e d p l a t e s cellulose c o n s i s t s of t e r t . b u t a n o l / w a t e r / 2 5 $ ammonia i n a r a t i o o f 80/20/1 by v o l u m e , w h i l s t f o r HPTLC p r e c o a t e d p l a t e s s i l i c a g e l 60 the b e s t m i x t u r e i s n-hexane/ethyl acetate/formic acid i n the ratio 4 0 / 6 0 / 0 , 5 . P r i o r t o t h e s e p a r a t i o n p r o p e r , t h e HPTLC pre-coated p l a t e s were adjusted to a r e l a t i v e h u m i d i t y of 20$. Q u a n t i t i e s of o.75 p i of a t e s t s o l u t i o n i n m e t h a n o l , composed as f o l l o w s , were a p p l i e d to the p l a t e s a n d m i g r a t e d o v e r 5 cm : Ioxynil : 0.6 pg F l u r e n o l : 0.8 pg MCPA : 2.8 pg. The p l a t e s , o n c e d e v e l o p e d , w e r e c h e c k e d v i s u a l l y u n d e r UV l i g h t a t 254 nm, w h i l e q u a l i t a t i v e a n d q u a n t i t a t i v e e v a l u a t i o n ensued w i t h the a i d of a Z e i s s c h r o m a t o g r a m s p e c t r o p h o t o m e t e r PMQ I I i n r e f l e c t a n c e mode a t a w a v e l e n g t h o f 2 8 0 n m . Owing to the d i f f e r i n g a c t i v i t i e s of the sorbents



168

PESTICIDE

ANALYTICAL

METHODOLOGY

used and the f a c t t h a t d i f f e r e n t solvent systems were employed f o r the d i f f e r e n t types of p l a t e s , the hRf values for the three c o n s t i t u e n t s of Trevespan 6038 differ q u i t e c o n s i d e r a b l y , as i s shown i n Table V. The s u b s t a n c e s e q u e n c e i s i d e n t i c a l i n b o t h cases, however. Table VI provides a f u r t h e r comparison of chromatographic data obtained for the s e p a r a t i o n of T r e v e s p a n 6038 on HPTLC p r e - c o a t e d p l a t e s s i l i c a gel 60 and c e l l u l o s e . A l t h o u g h t h e m i g r a t i o n d i s t a n c e was t h e same i n b o t h c a s e s , namely 5 cm, the development time for the HPTLC p r e - c o a t e d p l a t e s i l i c a g e l 60 i s considerably s h o r t e r t h a n f o r the HPTLC p r e - c o a t e d p l a t e cellulose on account of the h i g h e r flow r a t e . In both cases the three substance components were completely separated, though the R v a l u e s a c h i e v e d on the HPTLC s i l i c a gel p l a t e were considerably higher for t h i s p a r t i c u l a r s e p a r a t i o n t h a n t h e v a l u e s o b t a i n e d on t h e HPTLC cellulose plate. For q u a n t i t a t i v e determination of the individual Trevespan 6038 components, c a l i b r a t i o n curves were drawn up f o r t h e HPTLC p r e - c o a t e d p l a t e s i l i c a g e l 60 and f o r t h e HPTLC p r e - c o a t e d p l a t e cellulose. F i g u r e 3 shows t h e peak h e i g h t s f o r ioxynil, MCPA a n d f l u r e n o l ( e x p r e s s e d i n mV, a f t e r evaluation by r e f l e c t a n c e u s i n g a Z e i s s PMQ I I chromatogram spectrophotometer) plotted against the respective a p p l i e d q u a n t i t i e s ( e x p r e s s e d i n ng) for both the HPTLC s i l i c a g e l 60 p l a t e and t h e HPTLC c e l l u l o s e plate. Well c o r r e l a t i n g c a l i b r a t i o n curves i n form of s t r a i g h t l i n e s a r e o b t a i n e d o n b o t h t y p e s o f HPTLC p l a t e f o r a l l three sample substances i n the range of q u a n t i t i e s examined. Although the applied quantities a r e i d e n t i c a l , the peak h e i g h t s on t h e HPTLC p r e c o a t e d p l a t e c e l l u l o s e a r e somewhat h i g h e r t h a n t h o s e o b t a i n e d on t h e HPTLC p r e - c o a t e d p l a t e s i l i c a g e l 60. The d e t e c t i o n l i m i t s d e t e r m i n e d by t h e p e a k heights and by the b a s e l i n e n o i s e f o r the t h r e e substances examined are l i s t e d i n Table VII. T h e s e v a l u e s show t h a t f o r t h e separation performed the detection l i m i t s of a l l three sample s u b s t a n c e s on the HPTLC c e l l u l o s e p l a t e a r e somewhat l o w e r t h a n on t h e HPTLC s i l i c a g e l 60 p l a t e . 6.

A

HPTLC p r e - c o a t e d p l a t e s c o n c e n t r a t i n g zone further

new

development

s i l i c a

from

the

gel

60

field

with

of

HPTLC


9.

HAUCK

AND


Table

AMADORI

HPTLC

for Pesticide

V. R e t e n t i o n d a t a ( h R f v a l u e s ) for ioxynil, MCPA a n d f l u r e n o l o n H P T L C pre-coated p l a t e s s i l i c a g e l 60 ( s o l v e n t system: n-hexane/ethyl acetate/formic acid 40/60/0.5) and on HPTLC pre-coated plates cellulose (solvent system: tert. b u t a n o l / w a t e r / 2 5 $ ammonia 80/20/1) types of plates

Table

169

Analysis

hRf values of Ioxynil

MCPA

Flurenol


49,4

31,6

16,2

HPTLC cellulose

89,3

72,7

60,2

VI» C h r o m a t o g r a p h i c d a t a o f i o x y n i l , MCPA a n d pre-coated plates s i l HPTLC p r e - c o a t e d p l a t

types of plates

velocity coefficient κ C min/s 1 2

opt. migration distance Zf C mi ]

for the separation f l u r e n o l on HPTLC i c a g e l 60 and on es cellulose

development time Ce]

resolution R. of loxynil/MCPA

MCPA/Flurenol

HPTLC silica gel 60

3,67

50

858

2,45

2,24

HPTLC cellulose

0,67

50

4615

1,49

1,18



170

PESTICIDE

ANALYTICAL

M E T H O D O L O G Y

Figure 3. Calibration curves for ioxynil (*), MCPA (O), and flurenol (\Z\) on HPTLC pre-coated plates silica gel 60 (Mt) and on HPTLC pre-coated plates cellulose f right j (abscissae: applied quantity (ng); ordinales: peak height (mV))

Table

VII.

Detection l i m i t s ( i n ng) for i o x y n i l , MCPA a n d f l u r e n o l on HPTLC pre-coated plates s i l i c a gel 60 and on HPTLC pre coated plates cellulose types of plates

l i m i t of detection C ng ] Ioxynil

HPTLC s i l i c a gel 60 HPTLC c e l l u l o s e

MCPA

Flurenol

10

15

6

7

12

5



9.

HAUCK

AND

AMADORI

HPTLC

for Pesticide

Analysis

171

p r e - c o a t e d l a y e r s r e p r e s e n t s t h e HPTLC pre-coated p l a t e s i l i c a g e l 60 w i t h c o n c e n t r a t i n g zone. The s i z e o f t h e s p o t o f t h e s u b s t a n c e m i x t u r e a p p l i e d has a c o n s i d e r a b l e b e a r i n g on the separating performance a t t a i n a b l e on a t h i n - l a y e r p l a t e . The more compact the a p p l i c a t i o n of the s p o t o f substances t o be s e p a r a t e d , t h e more compact and t h u s t h e more favourable, t h e s p o t w i l l a l s o be a f t e r chromatography has t a k e n p l a c e . S u c h s m a l l s t a r t i n g s p o t s c a n be obtained w i t h the a i d of s u i t a b l e , sometimes laborious a p p l i c a t i o n techniques combined w i t h s m a l l a p p l i c a t i o n volumes. In order to improve the separating performance of HPTLC p r e - c o a t e d p l a t e s s i l i c a g e l 60 e v e n a t larger a p p l i e d v o l u m e s , a s may b e n e c e s s a r y a t l o w sample c o n c e n t r a t i o n s , and w i t h a r a p i d and s i m p l e technique of a p p l i c a t i o n , HPTLC p r e - c o a t e d p l a t e s s i l i c a g e l 60 w i t h s o - c a l l e d " c o n c e n t r a t i n g zones were developed (10, 11, 12). This type of plate c o n s i s t s of two d i s t i n c t Tâyer s e c t i o n s , namely the s e p a r a t i n g layer p r o p e r c o n s i s t i n g of s i l i c a g e l 60 and a c o n c e n t r a t i n g zone composed of an i n e r t , porous s i l i c o n dioxide. These two s o r b e n t m a t e r i a l s p a s s i n t o one a n o t h e r at a c l e a r l y d e f i n e d b o u n d a r y - l i n e i n s u c h a way t h a t the e l u a n t i s o f f e r e d no r e s i s t a n c e as i t p a s s e s through. What happens when a n HPTLC p r e - c o a t e d plate s i l i c a g e l 60 w i t h c o n c e n t r a t i n g zone i s u s e d i s as follows : the substance mixture i s a p p l i e d i n spots or s t r e a k s at any p o i n t and i n p a r t i c u l a r a t any height on the c o n c e n t r a t i n g zone and the s p o t s o r s t r e a k s are t h e n c a r r i e d by the e l u a n t to the b o r d e r between the two s o r b e n t m a t e r i a l s . A t t h i s b o u n d a r y - l i n e the substance spot i s automatically concentrated into a v e r y n a r r o w b a n d . Prom t h e b o u n d a r y - l i n e t h e n begins the chromatographic s e p a r a t i o n on the s i l i c a g e l 60 l a y e r , w i t h the c o n c e n t r a t i n g phase p a s s i n g over into the s e p a r a t i n g phase w i t h o u t i n t e r r u p t i o n . Improvements i n the separating performance a c h i e v e d w i t h HPTLC p r e - c o a t e d plates s i l i c a gel 60 w i t h c o n c e n t r a t i n g zone (E. Merck, Darmstadt) compared w i t h HPTLC p r e - c o a t e d p l a t e s s i l i c a g e l 60 w i t h o u t c o n c e n t r a t i n g zone, t a k i n g as an example the a p p l i c a t i o n of a r e l a t i v e l y l a r g e sample volume of 0 . 7 5 p i ( i n HPTLC sample volumes o f b e t w e e n 0.02 p i a n d 0 . 1 p i a r e common p r a c t i c e ! ) , i s demonstrated by the s e p a r a t i o n of Trevespan 6038, as already described. The s o l v e n t s y s t e m u s e d f o r b o t h t y p e s o f plates was n - h e x a n e / e t h y l a c e t a t e / f o r m i c acid i n a ratio of 4 0 / 6 0 / 0 . 5 b y v o l u m e . The c h r o m a t o g r a p h i c conditions, 1 1



172

PESTICIDE

ANALYTICAL

METHODOLOGY

a p p l i e d q u a n t i t i e s and sample c o n c e n t r a t i o n s are the same a s d e s c r i b e d u n d e r p o i n t 4 f o r t h e s e p a r a t i o n of T r e v e s p a n 6038 on HPTLC p r e - c o a t e d p l a t e s s i l i c a gel 60. By comparing the c h r o m a t o g r a p h i c d a t a g i v e n i n T a b l e V I I I i t c a n be s e e n t h a t f o r t h e optimum m i g r a t i o n d i s t a n c e o f 5 cm a n d w i t h a development t i m e s h o r t e n e d by 6 $ , t h e r e s o l u t i o n R on t h e HPTLC pre-coated p l a t e s i l i c a g e l 60 w i t h c o n c e n t r a t i n g zone i s on average about 26$ b e t t e r t h a n on the corresponding plate without concentrating zone. The c a l i b r a t i o n c u r v e s f o r t h e substances i o x y n i l , MCPA a n d f l u r e n o l o n t h e two H P T L C p r e c o a t e d p l a t e s s i l i c a g e l 6 0 , one w i t h and one w i t h o u t c o n c e n t r a t i n g zone, show, as i n F i g u r e 4, t h a t the peak h e i g h t s for i d e n t i c a l a p p l i e d q u a n t i t i e s are h i g h e r f o r a l l the substances examined on p l a t e s with a c o n c e n t r a t i n g zone t h a n on c o r r e s p o n d i n g plates without a concentrating zone. This i s a t t r i b u t e d to t h e a l r e a d y m e n t i o n e d more compact s p o t f o r m a t i o n on t h e HPTLC p r e - c o a t e d p l a t e s i l i c a g e l 60 w i t h concentrating zone. B e c a u s e o f t h e more h i g h l y c o n c e n t r a t e d spots o b t a i n e d on t h e HPTLC p r e - c o a t e d p l a t e s s i l i c a g e l 60 w i t h c o n c e n t r a t i n g zone, the d e t e c t i o n l i m i t s shown i n Table IX f o r t h i s type of p l a t e are lower t h a n i n t h e c a s e o f t h e HPTLC p r e - c o a t e d p l a t e s i l i c a g e l 60 without concentrating zone. The i m p r o v e m e n t s a c h i e v e d i n HPTLC pre-coated p l a t e s s i l i c a g e l 60 w i t h c o n c e n t r a t i n g zone as compared w i t h HPTLC p r e - c o a t e d p l a t e s s i l i c a g e l 60 a r e a l l t h e more p r o n o u n c e d t h e h i g h e r t h e a p p l i c a t i o n volume i s . There i s no p o i n t i n u s i n g HPTLC pre-coated p l a t e s w i t h o u t c o n c e n t r a t i n g zone at an a p p l i c a t i o n v o l u m e o f more t h a n 0 . 7 5 p i . However, on HPTLC p r e coated p l a t e s w i t h c o n c e n t r a t i n g zone i t i s possible to chromatograph even l a r g e r q u a n t i t i e s w i t h very good s e p a r a t i o n . A p a r t from these improvements i n separation performance, p a r t i c u l a r l y at large applied q u a n t i t i e s , the HPTLC p r e - c o a t e d p l a t e s s i l i c a g e l 60 w i t h c o n c e n t r a t i n g zone o f f e r the f o l l o w i n g additional advantages over corresponding pre-coated plates w i t h o u t the c o n c e n t r a t i n g zone : 1. F o l l o w i n g s p o t - w i s e o r s t r e a k - w i s e a p p l i c a t i o n o f a substance mixture, the spot or streak i s autom a t i c a l l y concentrated i n t o a compact starting l i n e d u r i n g the a c t u a l development process, separ a t i o n of t h i s s t a r t i n g l i n e then continuing without interruption. 2 . No s p e c i a l s k i l l s a r e r e q u i r e d d u r i n g a p p l i c a t i o n ,


9.

HAUCK

AND


Table

HPTLC

AMADORI

VIII,

for Pesticide

173

Analysis

Chromatographic data for the separation o f i o x y n i l , MCPA a n d f l u r e n o l o n HPTLC p r e - c o a t e d p l a t e s s i l i c a g e l 60 and on HPTLC p r e - c o a t e d p l a t e s s i l i c a g e l 60 w i t h c o n c e n t r a t i n g zone; solvent system: n-hexane/ethyl acetate/formic acid 4 0 / 6 0 / 0 . 5 ; m i g r a t i o n d i s t a n c e : 5 cm

types of plates

velocity coefficient κ

development time

resolution R

s

of

[ mm^/s ]

[s]


3,67

858

2,45

2,24

HPTLC s i l i c a gel 60 with cone, zone

3,86

806

3,27

2,64

Table

IX. D e t e c t i o n l i m i t s ( i n MCPA a n d f l u r e n o l o n p l a t e s s i l i c a g e l 60 coated plates s i l i c a trating zone types of plates

HPTLC s i l i c a gel 60 HPTLC s i l i c a gel 60 with concentrating zone

loxynil/MCPA

MCPA/Flurenol

ng) f o r ioxynil, HPTLC p r e - c o a t e d and on HPTLC p r e g e l 60 w i t h c o n c e n

limit of detection I ng ] Ioxynil

MCPA

Flurenol

10

15

6

7

13

5



174

PESTICIDE

ANALYTICAL

M E T H O D O L O G Y

Figure 4. Calibration curves for ioxynil (*), MCPA (O), and flurenol (£3) on HPTLC pre-coated plates silica gel 60 (left) and on HPTLC pre-coated plates silica gel 60 with concentrating zone fright) (abscissae: applied quantity (ng); ordinates: peak height (mV))


9.

3.


4.

5.

HAUCK

A N D AMADORI

HPTLC

for Pesticide

Analysis

175

in respect of geometry, p o s i t i o n i n g and spreading of the applied spots i n the concentrating zone. This simplification of application techniques leads i n i t s e l f to considerable saving of time. Apart from the concentrating e f f e c t , there i s also a certain "clean-up" process which takes place i n the c o n c e n t r a t i n g zone i n c e r t a i n cases; t h i s c a n r e p l a c e t h e e x t r a c t i o n o f substances t o be chromat o g r a p h e d , w h i c h may o t h e r w i s e have b e e n necessary. Possible decomposition or irreversible adsorption of substances on to the active s i l i c a g e l of the separating layer during drying of the applied spot i s a v o i d e d i n t h e i n e r t c o n c e n t r a t i n g zone. The s u b s t a n c e s come i n t o c o n t a c t w i t h t h e a c t i v e sorbent only after passing through the concentrat i n g zone and t h e n o n l y i n d i s s o l v e d form. I n t h i s respect, i t i s p o s s i b l e t o speak o f wet d o s i n g , as i n column l i q u i d chromatography. I t i s p o s s i b l e t o a p p l y sample substances by immersing t h e c o n c e n t r a t i n g zone o f t h e p l a t e into a d i l u t e sample s o l u t i o n . T h i s method c a n r e p l a c e a multiple, streak-wise application of very dilute sample s o l u t i o n s u s i n g s p e c i a l i s e d apparatus.

Literature Cited 1

Ripphahn, J.; Halpaap, H. J. Chromatogr., 1975, 112, 81. 2 Halpaap, H . ; Ripphahn, J. Kontakte (Merck), 1976, 3, 16. 3 Hezel, U . ; Blome, J.; Halpaap, H . ; Jänchen, D.; Kaiser, R. E.; Ripphahn, J. "Einführung i n die Hochleistungs-Dünnschicht-Chromatographie, HPDC"; Institute of Chromatography; Bad Dürkheim, 1976. 4 Zlatkis, Α . ; Kaiser, R. E . "HPTLC-High Performance Thin-Layer Chromatography"; J . Chrom. Library 9, Elsevier Publishing Company; Amsterdam-New York, 1977. 5 Halpaap, H . ; Ripphahn, J. Chromatographia, 1977, 10, (10), 613. 6 Halpaap. H . ; Ripphahn, J. Chromatographia, 1977, 10, (11), 643. 7 Kaiser, R. E.; Rieder, R. J. Chromatogr., 1977, 142, 411. 8 Halpaap, H . ; Ripphahn, J. Lecture, 13. Symposium on Advances i n Chromatography, 1978, St. Louis. 9 Hauck, H. E . Lecture, 2. Danube Symposium on Progress i n Chromatography, 1979, Carlsbad. 10 Halpaap, H . ; Krebs, K . - P . J. Chromatogr., 1977, 142, 823.


176

pesticide analytical methodology


11 G e r k e , Η . Ε . GIT F a c h z . L a b . , 1977, 2 1 , 685. 12 H a l p a a p , H . K o n t a k t e ( M e r c k ) . 1978, 1, 32. 13 J o r k , H . ; R o t h , B . J . C h r o m a t o g r . , 1977, 144, 39. 14 Davies, R. D.; Pretorius, V. J. Chromatogr., 1978, 155, 229. 15 D a v i e s , R. D. J . C h r o m a t o g r . , 1979, 170, 453. 16 G r a n g e r , C . ; Z w i l l i n g , J . P . Bull. Soc. Chim. F r a n c e , 1950, 873. 17 T h i e l e m a n n , H . Z . C h e m . , 1976, 16, 155. 18 T e w a r i , S. N . ; Sharma, I . C . J . C h r o m a t o g r . , 1977, 131, 275. 19 B r i n k m a n , U . A. T h . ; de V r i e s , G . J . HRC & C C , 1979, 2, 79. 20 B e c k e r , H.; E x n e r , J . ; B i n g l e r , T . J . C h r o m a t o g r . , 1979, 172, 420. Received March 3, 1980.


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