Pesticide Analytical Methodology - American Chemical Society

7 Fluorescence and Ultraviolet Absorbance of Pesticides and Naturally Occurring Chemicals in Agricultural Products After HPLC Separation on a Bonded-CN Polar Phase Downloaded by GEORGETOWN UNIV on August 19, 2015 | http://pubs.acs.org Publication Date: October 30, 1980 | doi: 10.1021/bk-1980-0136.ch007

ROBERT J. ARGAUER Analytical Chemistry Laboratory, Agricultural Environmental Quality Institute, Agricultural Research, Science and Education Administration, U.S. Department of Agriculture, Beltsville, MD 20705 Abstract

Some pesticides and many naturally occurring chemicals fluoresce sufficiently that direct monitoring of their natural fluorescence during HPLC is feasible. The fluorescence intensities of over thirty pesticides in hexane and methanol were measured at excitation wavelengths of both 254 nm and maximum absorbance. Carbaryl at 0.2 ppm was used as a model pesticide to contrast the relative merits of the fluorescence and absorbance modes for HPLC detection. Actual samples studied included rice, corn, green peas, potato, cucumber, lima beans, and orange. Pollen gathered by foraging honey bees proved the most challenging of the agricultural products studied because of the highly complex chromatograms obtained for methylene chloride extracts. Highly significant is the finding that the fluorescence efficiency of some pesticides varied dramatically with a change in polarity of the mobile phase.

Fluorescence spectrometry i s used i n both research and s u r v e i l l a n c e to help assure both the farmer and the consumer a continued bounty of high q u a l i t y a g r i c u l t u r a l products while p r e s e r v i n g the q u a l i t y of the environment. My o b j e c t i v e i s to discuss some of our current research and to share with you s e v e r a l i n t e r e s t i n g observations where we have used fluorescence as a monitor i n high performance l i q u i d chromatography. In 1970, we described i n a book the research p u b l i s h e d by a l a r g e number of s c i e n t i s t s from many d i s c i p l i n e s who are c o n t r i b u t i n g to the development of fluorescence as a u s e f u l and powerf u l a n a l y t i c a l t o o l (1). Subsequently, i n 1977, I published a chapter devoted s p e c i f i c a l l y to the use of fluorescence as a p r a c t i c a l technique f o r the a n a l y s i s of c e r t a i n p e s t i c i d e s ( 2 ) . F i g u r e 1 i s taken from that chapter and i l l u s t r a t e s what I consider to be the f i v e fundamental approaches that we

This chapter not subject to U.S. copyright. Published 1980 American Chemical Society In Pesticide Analytical Methodology; Harvey, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

Downloaded by GEORGETOWN UNIV on August 19, 2015 | http://pubs.acs.org Publication Date: October 30, 1980 | doi: 10.1021/bk-1980-0136.ch007

104

PESTICIDE

PEST or PLANT M A N A G E M E N T

ANALYTICAL

METHODOLOGY

CHEMICAL

on or in air, animal, formulation, plant, soil, water

S E P A R A T I O N by

DERIVATIVE

liquid/liquid partition, GC,LC,TLC,other

FORMED

FLUORESCENCE MEASURED CONFIRMATION

Figure 1. Fundamental approaches generally used in the analysis of pesticides (or other chemicals) byfluorescence(or some other physical property)

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


7.

ARGAUER

Fluorescence

and UV

Absorbance

of

Pesticides

105

researchers g e n e r a l l y use to q u a n t i t a t i v e l y measure chemicals i n a p a r t i c u l a r s u b s t r a t e . One of the approaches i s an i d e a l i z e d a n a l y t i c a l approach and i n c l u d e s j u s t two s t e p s — - e x t r a c t i o n ( i n c l u d i n g d i l u t i o n or c o n c e n t r a t i o n of the e x t r a c t ) followed by a d i r e c t determination of a p h y s i c a l property that i s s p e c i f i c f o r the p e s t i c i d e i n q u e s t i o n . Because numerous c o e x t r a c t i v e s u s u a l l y i n t e r f e r e w i t h t h i s i d e a l i z e d approach, you and I u s u a l l y r e s o r t to one of the remaining four approaches to minimize the e f f e c t s produced by the i n t e r f e r e n c e s . Indeed, one of the speakers i n t h i s symposium has d i s c u s s e d one of these approaches, i . e . the p r e p a r a t i o n and a n a l y s i s of f l u o r e s c e n t d e r i v a t i v e s . Oftentimes though, when forming d e r i v a t i v e s , i n t e r f e r i n g com pounds a l s o are formed. Co-extractants o r i g i n a l l y present as n o n - i n t e r f e r i n g compounds might indeed now become h i g h l y f l u o r e s cent d e r i v a t i z e d i n t e r f e r i n g compounds. The approach then must i n c l u d e a s e p a r a t i o n of the p e s t i c i d e from the i n t e r f e r i n g coe x t r a c t a n t s , e i t h e r b e f o r e or a f t e r formation of the d e r i v a t i v e , and p r i o r to d e t e c t i o n . For purposes of t h i s symposium, I have l i m i t e d myself to s t i l l another of these approaches. That approach i n c l u d e s e x t r a c t i o n , s e p a r a t i o n by HPLC, and d i r e c t measurement of the r e l a t i v e l y high n a t u r a l f l u o r e s c e n c e inherent i n the molecular s t r u c t u r e of the p e s t i c i d e s themselves. You w i l l f i n d that s o l v e n t p o l a r i t y and i n s t r u m e n t a l parameters are important v a r i a b l e s when I attempt to c o n t r a s t the chromatograms obtained i n the absorbance mode with those obtained i n the f l u o r e s c e n c e mode. Experimental Instrumentation A Perkin-Elmer MFF-2A Fluorescence Spectrophotometer was used to determine the e x c i t a t i o n and emission wavelengths r e quired f o r a c h i e v i n g maximum f l u o r e s c e n c e i n t e n s i t y f o r the p e s t i c i d e s s t u d i e d . The MPF-2A contained a 150 watt xenon a r c and an e x c i t a t i o n monochromator with a g r a t i n g blazed at 300 nm as the e x c i t a t i o n u n i t ; a Haraamatsu R 777 p h o t o m u l t i p l i e r tube ( s e n s i t i v i t y range: 185 - 850 nm) and an emission monochromator g r a t i n g b l a z e d at 300 nm as the emission d e t e c t i o n u n i t . A DuPont Model 848 L i q u i d Chromatograph was used f o r HPLC ( F i g u r e 2). The accessory i n j e c t i o n device i n c l u d e d a Rheodyne Model 70-10 s i x - p o r t sample i n j e c t i o n v a l v e f i t t e d with a 20 μ l i t e r sample loop. A Whatman HPLC column 4.6 mm χ 25 cm that con t a i n e d P a r t i s i l PXS 1025 PAC (a bonded cyano-amino p o l a r phase u n s p e c i f i e d by the manufacturer) was used with v a r i o u s mobile phases a t ambient temperature and a f l o w r a t e of 1.25 ml/minute. A dual beam 254 nm photometric absorbance d e t e c t o r w i t h a c e l l volume of 6.3 y l was used f o r absorbance measurement. The e x i t port of the photometric absorbance d e t e c t o r c e l l was connected i n s e r i e s to a 70 μ-liter flow c e l l i n an Aminco Fluoromonitor. The F l u o r o m o n i t o r s e x c i t a t i o n l i g h t source c o n s i s t e d of a 1



106

PESTICIDE

INJECTION I LOOP

L.C.

AMINCO 70

COLUMN

ANALYTICAL

6.3 M I C R O L I T E R FLOW CELL

METHODOLOGY

RECORD ABSORBANCE A T 254 N M

FLDORO

MICROLITER FLOW CELL RECORD FLUORESCENCE 1

Vi Γ

G.E. 4 W A T T S GERMICIDAL LAMP

Figure 2.

R C A 931 Β (S-4 SPECTRAL RESPONSE)

CORION C O R P . 253.7 N M INTERFERENCE FILTER TO

COLLECTOR

Schematic of a fluorescence monitor in series with an absorbance detector in HPLC


7.

Fluorescence

ARGAUER

and

UV

Absorbance

of

Pesticides

107

General E l e c t r i c 4 watt g e r m i c i d a l lamp and a Corion C o r p o r a t i o n 2537 i n t e r f e r e n c e f i l t e r (15% t r a n s m i s s i o n a t 2537). The F l u o r o m o n i t o r s emission d e t e c t i o n u n i t c o n s i s t e d of a RCA 931B (S-4 s p e c t r a l response) p h o t o m u l t i p l i e r tube and a Corning 7-51 g l a s s f i l t e r t h a t t r a n s m i t s l i g h t of wavelengths between 310 and 410 nm. 1


Determination of R e l a t i v e Fluorescence of P e s t i c i d e s We s e l e c t e d over s i x t y commercially important p e s t i c i d e s f o r t h i s study. Some of the p e s t i c i d e s were known to f l u o r e s c e . Others were s e l e c t e d on the b a s i s of most l i k e l y to f l u o r e s c e . The chemical d e s i g n a t i o n s of the p e s t i c i d e s mentioned i n the t e x t are l i s t e d i n Table 1. Standard s o l u t i o n s of the p e s t i c i d e s were prepared i n e t h y l a c e t a t e a t c o n c e n t r a t i o n s of 1 mg/ml. The standard s o l u t i o n s were d i l u t e d w i t h hexane or methanol to prepare s o l u t i o n s that contained 2 μg/ml p e s t i c i d e f o r the i n i t i a l f l u o r e s c e n c e measurements. E x c i t a t i o n and emission band widths on the s p e c t r o f l u o r o m e t e r were adjusted to 4 nm. A solu t i o n of q u i n i n e s u l f a t e , 1 μg/ml i n 0.1 Ν s u l f u r i c a c i d , was used as a r e f e r e n c e i n determining the r e l a t i v e f l u o r e s c e n c e i n t e n s i t y of the p e s t i c i d e s . The wavelengths f o r e x c i t a t i o n and emission that would g i v e the maximum f l u o r e s c e n c e i n t e n s i t y i n both hexane and methanol were obtained next by u s i n g 1 cm^ quartz fluorometer c e l l s . F i n a l l y the e x c i t a t i o n monochromator was s e t at 254 nm, and the f l u o r e s c e n c e i n t e n s i t y was again measured at wavelength of maximum emission i n both hexane and methanol. Determination of C a r b a r y l i n Food Products E x t r a c t i o n . One hundred grams of samples ( c o r n , orange, p o t a t o , r i c e , cucumbers, l i m a beans, or green beans) were blended f o r f i v e minutes w i t h 300 ml of methylene c h l o r i d e and 10 ml of 10% s u l f u r i c a c i d . The f i l t e r e d e x t r a c t was d r i e d over anhydrous sodium s u l f a t e , and a 150 ml a l i q u o t was concentrated on a Rinco evaporator to near dryness. The concentrate was d i s s o l v e d i n t o 10 ml of methylene c h l o r i d e . Pre-HPLC Column Chromatography. F i v e grams of 60-200 mesh s i l i c a g e l powder (4.7% weight l o s t on i g n i t i o n ) s u p p l i e d by J. T. Baker Chemical Company were poured i n t o a 10 mm i . d . g l a s s column. The concentrated e x t r a c t r e d i s s o l v e d i n 10 ml methylene c h l o r i d e was t r a n s f e r r e d to the column and allowed to soak i n t o the s i l i c a g e l . Then 65 ml of a d d i t i o n a l methylene c h l o r i d e were added to the column. The f i r s t 10 ml of the e l u a t e were d i s c a r d ed, and the next 50 ml c o l l e c t e d i n a 125 ml Erlenmeyer f l a s k . Chromatography by HPLC. The 50 ml f r a c t i o n c o l l e c t e d from the "dry-column" was concentrated to near dryness on a Rinco evaporator. Two ml of methylene c h l o r i d e was next added to the


In Pesticide Analytical Methodology; Harvey, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980. Niagara NIA-10242 Furadan

Nix-Scald; Stop-Scald

Azinphosmethyl IBA; Hormodin Naphthalophos

2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate

2,3-dihydro-3-hydroxy-2,2-dimethyl-7benzofuranyl methylcarbamate

2,3-dihydro-2,2-dimethyl-3-oxo-7-benzofuranyl methylcarbamate

0-(3-chloro-4-methyl-2-oxo-2H-l-benzopyran-7yl)(),()-diethyl phosphorothioate

2- (α-naphthoxy)-Ν,N-diethylpropionamide

biphenyl

diphenylamine

6-ethoxy-l,2-dihydro-2,2,4-trimethylquinoline

Ο,Ο-dimethyl S-[ ( 4 - o x o - l , 2 , 3 - b e n z o t r i a z i n 3(3H)-yl)methyl] phosphorodithioate

indole-3-butyric acid

N-hydroxynaphthalimide d i e t h y l phosphate

Carhofuran

3-Hydroxycarbo furan

3-0xocarbofuran

Coumaphos

Devrinol

Diphenyl

Diphenylamine

Ethoxyquin

Guthion

Indolebutyric acid

Maretin

or N-phenylbenzenamine

Sevin

1- naphthyl methylcarbamate

Carbaryl

Santoquin

DPA; S c a l d i p

Biphenyl

Napropamide

Co-Ral

Basagran

3-isopropyl-lH-2,1,3-benzothiadiazin-4(3H)one 2,2-dioxide

Other Designation

Bentazon

Designation

methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate

Chemical

Designations of P e s t i c i d e s Mentioned i n Text

Benomyl

Chemical

Benlate; Tersan 1991

Pesticide

Table 1.


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

1-naphthaleneacetic

1-Naphthaleneacetic

2- naphthol

1- n a p h t h y l t h i o u r e a

2- Naphthol

1-Naphthylthiourea

Pirimor Hoe 2873; Afugan Curamil APL-Luster; Mertect TBZ; Tecto; Tobaz

2- (dimethylamino)-5,6-dimethyl-4-pyrimidinyl dimethylcarbamate

ethyl 2-[(diethoxyphosphinothioyl)oxy]-5methyl p y r a z o l o [ 1 , 5 - a ] p y r i m i d i n e - 6 - c a r b o x y l a t e

2- ( 4 - t h i a z o l y l ) b e n z i m i d a z o l e

3- (a-acetonylbenzyl)-4-hydroxycoumarin

Pirimicarb

Pyrazophos

Thiabendazole

Warfarin

Coumaf ene; K y p f a r i n

Butacide

Piperonyl

a-[2-(2-butoxyethoxy)ethoxy]-4,5(methylenedioxy)-2-propyltoluene

Butoxide

Zolone Chipman RP-11974 Niagara NIA-9241

(),iO-diethyl S-[ (6-chloro-2-oxobenzoxazolin-3-

Ρhosalone

y l ) m e t h y l ] phosphorodithioate

Dowicide 1

[l,l -biphenyl]-2-ol

An tu

Fruitone

o-Phenylpheno1

f

1- naphthol

1- Naphthol

acid

1-naphthaleneacetamide

1-Naphthaleneacetamide

acid

Rootone; Amid-Thin W

naphthalene

Naphthalene

Bay 36205; F o r s t a n Oxythioquinox Quinomethionate

cyclic S-(6-methyl-2,3-quinoxalinediyl) dithiocarbonate

Morestan

Other D e s i g n a t i o n

Chemical Designation

Pesticide

(Continued)

Chemical Designations of P e s t i c i d e s Mentioned i n Text

Table 1.


110

PESTICIDE

concentrate. Since the HPLC was t i o n loop, the amount of e x t r a c t e q u i v a l e n t to 500 mg of s t a r t i n g f o r the a g r i c u l t u r a l products i n

ANALYTICAL

METHODOLOGY

f i t t e d with a 20 u - l i t e r i n j e c i n j e c t e d i n t o the HPLC was sample per i n j e c t i o n as given s e v e r a l of the f i g u r e s .


R e s u l t s and D i s c u s s i o n I n i t i a l Measurements. The data i n Table 2 f o r the f l u o r e s cence of p e s t i c i d e s i n hexane and methanol were obtained with a single-beam spectrofluorometer. No attempt was made to adjust these values f o r e i t h e r the i n t e n s i t y d i s t r i b u t i o n of the e x c i t a t i o n source or the r e l a t i v e s e n s i t i v i t y of the emission u n i t with wavelength. However, s e v e r a l observations can be drawn from t h i s data that can be u s e f u l when a p p l i e d to a HPLC fluorescence detector. 1. P e s t i c i d e - s o l v e n t i n t e r a c t i o n s a f f e c t the f l u o r e s c e n c e of the p e s t i c i d e s . 2. S e n s i t i v i t y and s e l e c t i v i t y would be enhanced when one i s operating i n the f l u o r e s c e n c e mode i f the l i g h t source s e l e c t e d f o r e x c i t a t i o n were to emit l i g h t concentrated s o l e l y i n a narrow band a t wavelengths that correspond with the wavelengths of maximum a b s o r p t i o n (maximum e x c i t a t i o n ) f o r the p a r t i c u l a r pesticide. 3. Tunable dye l a s e r s could provide both high i n t e n s i t y and some s e l e c t i v i t y when used as e x c i t a t i o n sources i n a f l u o r e s cence monitor. However, mercury lamps provide a convenient and inexpensive e x c i t a t i o n source with emitted l i g h t l a r g e l y concent r a t e d at a wavelength of 254 nm. Many p e s t i c i d e s i n Table 2 f l u o r e s c e s u f f i c i e n t l y when e x c i t a t e d at 254 nm (xenon arc) so that the strong 253.7 nm l i n e i n a mercury lamp can be advantageously used f o r e x c i t a t i o n i n a HPLC f l u o r e s c e n c e monitor. Indeed i f one were to a d j u s t the data i n Table 2 f o r the quantum d i s t r i b u t i o n of the xenon a r c source with wavelength, the v a l u e s t a b u l a t e d f o r the f l u o r e s c e n c e i n t e n s i t y with e x c i t a t i o n at 254 nm would i n c r e a s e r e l a t i v e to the values given f o r the f l u o r e s c e n c e e x c i t e d at wavelengths of maximum e x c i t a t i o n / absorption. P e s t i c i d e s Separated on a CN-Bonded P o l a r Phase. F i g u r e s 3 and 4 c o n t a i n the chromatograms obtained f o r s e v e r a l of the p e s t i c i d e s l i s t e d i n Table 2. Various mobile phases were used to f a c i l i t a t e s e p a r a t i o n on the CN-bonded p o l a r phase. Detect i o n i s shown i n both absorbance and f l u o r e s c e n c e modes. I t would appear that f l u o r e s c e n c e d e t e c t i o n i s more s e n s i t i v e f o r some p e s t i c i d e s , while absorbance d e t e c t i o n i s more s e n s i t i v e f o r o t h e r s . However, comparisons of one manufacturer s absorbance monitor with another manufacturer's f l u o r e s c e n c e monitor could be m i s l e a d i n g . (I w i l l r e f e r to t h i s under Instrumental Parameters.) Furthermore, the u l t i m a t e u s e f u l comparison i s obtained when p r a c t i c a l samples are chromatographed s i n c e these 1


ARGAUER

Fluorescence

and UV A bsorbance


METHYLENE CILIRIIE/CljII 99.5/1.5

•-PIENYLPIEHL

of Pesticides

METHYLENE CILIIflE 1N%

•-MENYLPIENIL

1-NAHTIIL

0

16

8

11

TIME (NMITES)

Figure 3. HPLC of several pesticides (100 ng) on a CN-bonded polar phase with absorbance and fluorescence detection (APM = attenuation setting of photomultiplier signal)


11


112

PESTICIDE

NAPHTHALENE

ANALYTICAL

METHODOLOGY

1-NAPITIYLTItllCEA

PIPERONYL IITIXIDE

BIPHENYL

lEVtlNIl MAIITAN

HEXANE 111%

HEXANE/METHYLENE CILIIIIE 51:51 METHYLENE Clllt»E/CI II M.75/1.25 3

MTKiruaiK

MKSUI

JVA. 11

I

II

I

11

21

TIME [MINUTES] Figure 4.

HPLC

of several pesticides (100 ng) on a CΝ-bonded polar phase with absorbance and fluorescence detection



7.

ARGAUER

Fluorescence

and

UV

Absorbance

of

Pesticides

113

samples g e n e r a l l y c o n t a i n many n a t u r a l l y o c c u r r i n g chemicals that may absorb and f l u o r e s c e and be considered as i n t e r f e r i n g coextractives. To i l l u s t r a t e the e f f e c t s of c o e x t r a c t i v e s , I f o r t i f i e d s e v e r a l a g r i c u l t u r a l products with c a r b a r y l at 0.2 ppm. Carb a r y l was chosen as a model p e s t i c i d e to i l l u s t r a t e the e f f e c t s of c o e x t r a c t i v e s because i t s f l u o r e s c e n c e i n t e n s i t y l i e s i n the lower-middle range r e l a t i v e to the other p e s t i c i d e s i n Table 2. In a d d i t i o n , we were f a m i l i a r w i t h i t s gas chromatographic and s p e c t r o f l u o r o m e t r i c p r o p e r t i e s (2). F i g u r e 5 contains the chromatograms obtained when c a r b a r y l was i n j e c t e d i n t o the HPLC at s e v e r a l instrument s e n s i t i v i t y s e t t i n g s . Amounts of c a r b a r y l i n j e c t e d were between 1 ug and 4 ng. The chromatograms show l i n e a r i t y i n response with the amount of c a r b a r y l i n j e c t e d i n both the absorbance and f l u o r e s c e n c e modes. P r a c t i c a l a g r i c u l t u r a l samples when analyzed f o r c a r b a r y l , however, r e q u i r e a p r e l i m i n a r y column cleanup before i n j e c t i o n i n t o the HPLC i n order to preserve the i n t e g r i t y of the HPLC Column. Function of Pre-HPLC Column. The schematic i n F i g u r e 6 f o r a HPLC chromatogram r e p r e s e n t a t i v e of e x t r a c t s of a g r i c u l t u r a l products i l l u s t r a t e s use of s i l i c a g e l adsorption chromatography f o r the pre-HPLC cleanup step. The schematic shows that (A) a l a r g e p a r t of the c o - e x t r a c t i v e s can be removed i n the f i r s t f r a c t i o n from the precolumn, (B) the p o l a r i t y of the mobile phase can be adjusted so the p e s t i c i d e e l u t e s i n pre-HPLC column f r a c t i o n Β where the e l u a t e can be c o l l e c t e d and concen t r a t e d f o r i n j e c t i o n i n t o the HPLC, while (C) more p o l a r com pounds that would otherwise appear during HPLC have been e l i m i nated by permanent adsorption on the pre-HPLC Column. P r a c t i c a l A g r i c u l t u r a l Samples. Chromatograms i n Figures 7, 8, and 9 r e f l e c t HPLC i n j e c t i o n s of v a r i o u s crop e x t r a c t s e q u i v a l e n t to 500 mg of crop and 100 ng of c a r b a r y l . These chroma tograms show that f o r c a r b a r y l a t the described operating con d i t i o n s the f l u o r e s c e n c e mode of d e t e c t i o n i s more s e n s i t i v e and s e l e c t i v e than i s the absorbance mode of d e t e c t i o n f o r many of these a g r i c u l t u r a l crops. There are s e v e r a l d i f f i c u l t i e s with these chromatograms, however, as the amount of organic coext r a c t a b l e s increase r e l a t i v e to the amount of c a r b a r y l present i n the crops. Indeed, the chromatograms i n F i g u r e 8 show that c a r b a r y l at 0.2 ppm i n orange would be d i f f i c u l t to measure given the e x t r a c t i o n , precolumn cleanup procedure, and the 500 mg crop i n j e c t i o n d e s c r i b e d . However d i f f i c u l t , a g r i c u l t u r a l samples such as orange and p o l l e n c o l l e c t e d by f o r a g i n g honey bees that contain a l a r g e amount of co-extracted i n t e r f e r e n c e s can s t i l l be analyzed by HPLC. Four m o d i f i c a t i o n s i n the described procedure can be attempted.



Benomyl 286 340(306) Bentazon 280 Carbaryl 280 Carhofuran 3-Hydroxycarhofuran 280 3-Ketocarbofuran not detected Coumaphos 320 Devrinol 293 260 Diphenyl Diphenylamine 290 360 Ethoxyquin 290 Guthion Indolebutyric a c i d 290 340(330) Maretin 360 Morestan 284 Naphthalene 1-Naphthaleneacetamide 280 290 1-Naphthaleneacet i c a c i d 1-Naphthol 295 2-Naphthol 285 325(332) 1-Naphthylthiourea o-Phenylphenol 290 282 Phosalone P i p e r o n y l Butoxide 290 380 335 305 330 440(420) 340 340 375(365) 380 322 336 324 340 355 425(370) 335(320) 310 320

-

300 450(370) 332 304 304

-

16 10 41 31 49

48 4 71 85 50 23 120 90 27 87 not detected 6 107 67 320 22 22 6 27 13 34 20 20 35 160 100 90 59 7 6 230 18 6 8 66 59

52 7 22 60 40

Intensity

(2)

-

0.1 15 23 8 10 not detected 17 0.1 0.3 3 3 3 7 17 0.6 7 0.3 1.6

0.5 0.6 3.7 4 2

-

3 5 48 9 5 2 16 7 1 8 5 5 7 27 0.5 80 0.4 2

0.2 1.8 8 3 2.5

at e x c i t a t i o n s e t a t 254 nm and and emission a t maximum i rn Hexane Methanol

Fluorescence

at maxima i n Hexane Methanol

Relative

(2 yg/ml) i n Hexane and Methanol

Wavelength (nm) a t Maximum^ Excitation Emission

Fluorescence of P e s t i c i d e s

Common/Chemical Name

Table 2.


In Pesticide Analytical Methodology; Harvey, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980. 80 1 183 8

18 21 9 0.3

28 1 12 0.7

50 μ - l i t e r s of 1 mg/ml stock s o l u t i o n s o f p e s t i c i d e i n e t h y l a c e t a t e d i l u t e d with hexane or methanol t o 25 ml and measured i n a Perkin-Elmer Model MPF-2A Fluorescence Spectrophotometer.

78.0 7.8 0.77 0.10 0.033

R e l a t i v e Fluorescence I n t e n s i t y

72 23 210 5

(2)

1.0 0.1 0.01 0.001 0.000

Concentration (yg/ml)

380(355) 420 340(332) 390(340)

at e x c i t a t i o n s e t a t 254 nm and emission a t maximum i n Hexane Methanol

The wavelengths are given f o r p e s t i c i d e i n methanol and a r e the same i n hexane except as i n d i c a t e d i n parentheses.

310 252 310 310

at maxima i n Hexane Methanol

R e l a t i v e Fluorescence I n t e n s i t y

(1)

Quinine S u l f a t e E x c i t a t i o n λ 350 Emission λ 450 i n 0.1N S u l f u r i c a c i d

Reference:

Pirlmicarb Pyrazophos Thiabendazole Warfarin

Wavelength (nm) a t Maximum * ' Excitation Bnission

(Continued)

Common/Chemical Name

Table 2.


PESTICIDE

Absorbance —

0.08

ANALYTICAL

METHODOLOGY

0.01

0.04

Fill Scale I.Oif Carbaryl

0.5ig Carbaryl tUn Carbaryl


J

! W « i Carbaryl

__A_

Figure 5. Fluorescence and absorbance responses for carbaryl during HPLC on a CΝ-bonded polar phase (mobile phase: methylene chloride/methanol 99.5/0.5)

WITHOUT SILICA G E L PRE COLUMN

FRACTION -A

FRACTION

ι if I

WITH SILICA G E L PRECOLUMN

i:

FRACTION

FRACTION _

β

—

TIME

>

i — c

>

Function of pre-HPLC column cleanup chromatography on HPLC chromatograms



Figure 7. Potato and rice extracts: comparison of fluorescence and absorbance detection modes after HPLC on a CN-bonded polar phase (0,2 ppm = 100 ng carbaryl/500 mg crop equivalent injected; mobile phase: methylene chloride/methanol 99.5/0.5)



Figure 8.

Corn and orange extracts: comparison of fluorescence and absorbance detection modes after HPLC on a CN-bonded polar phase


Η

> >

8 m

Ο

Η

ζΛ

00


α

10 0

10 0

5

10

LIMA BEANS (0.2PPM)

θ TIME (MINUTES)

LIMA BEANS (Control)

0

6REEN PEAS (Control)

10

O

11

GREEN PEAS (0.2PPM)

Extracts of cucumber, lima beans, and green peas: comparison of fluorescence and absorbance detection modes after HPLC on a CN-bonded polar phase

CUCUMBER (0.2PPM)

Figure 9.

CUCUMBER (Control)



120

PESTICIDE

ANALYTICAL

METHODOLOGY

1. A d d i t i o n a l pre-HPLC cleanup s t e p s , t h e i r s e l e c t i o n based on the chemistry of the p e s t i c i d e and i m p u r i t i e s , a r e introduced t o help achieve the r e q u i r e d s e p a r a t i o n . 2. The p o l a r i t y of the mobile phase i s reduced o r m o d i f i e d t o f a c i l i t a t e separation. 3. The s i z e of the HPLC column i s increased to y i e l d an increased number of t h e o r e t i c a l p l a t e s f o r an i n c r e a s e i n the power f o r s e p a r a t i o n . 4. The d e t e c t a b i l i t y l e v e l of the method i s s a c r i f i c e d simply by reducing the amount of crop e q u i v a l e n t i n j e c t e d i n t o t h e chromatography, thereby reducing t h e s i z e of the i n t e r f e r i n g peaks. The chromatograms we obtained f o r samples of p o l l e n a t a d e t e c t a b i l i t y l e v e l f o r c a r b a r y l of 5 ppm are g i v e n i n Figure 10 and i l l u s t r a t e our use of the f o u r t h m o d i f i c a t i o n . Whereas chromatograms obtained as background c o n t r o l s f o r many a g r i c u l t u r a l crops vary w i t h the n a t u r a l chemical composi t i o n and degree of r i p e n e s s , chromatograms f o r p o l l e n can become f u r t h e r complicated by the u n p r e d i c t a b l e f o r a g i n g h a b i t s of honey bees (Apis m e l l i f e r a L . ) , and the question of what c o n s t i t u t e s a v a l i d background c o n t r o l sample. The beauty o f absorbance and f l u o r e s c e n c e modes as c o m p l i mentary d e t e c t o r s f o r HPLC l i e s i n t h e i r n o n d e s t r u c t i v e nature. F r a c t i o n s e l u t i n g from these d e t e c t o r s are r e a d i l y a v a i l a b l e f o r c o n f i r m a t i o n by mass spectrometry. Decrease of Fluorescence S i g n a l by Unresolved Co-Extracta b l e s . The f l u o r e s c e n c e peak f o r c a r b a r y l i n l i m a beans i n the chromatogram i n F i g u r e 9 i s somewhat s m a l l e r than expected. Two mechanisms can account f o r the decrease i n the f l u o r e s c e n c e s i g n a l f o r c a r b a r y l . I p r e f e r the mechanism t h a t suggests t h a t the c o - e l u t a n t s are absorbing some o f t h e e x c i t i n g l i g h t , thereby decreasing the l i g h t quanta a v a i l a b l e f o r a b s o r p t i o n by the c a r b a r y l molecules. A second mechanism suggests that the c o e l u t a n t s can quench the f l u o r e s c e n c e by i n t e r a c t i n g w i t h the e x c i t e d s t a t e o f the c a r b a r y l molecule t o cause the absorbed energy t o be d i s s i p a t e d along a s o l u t e - s o l u t e i n t e r a c t i n g nonf l u o r e s c e n t pathway. V a r i a t i o n of Fluorescence S i g n a l w i t h P o l a r i t y of the Mobile Phase. A change i n p o l a r i t y of the mobile phase o f t e n a i d s the r e s o l u t i o n of the p e s t i c i d e from i n t e r f e r i n g c o - e l u t a n t s . How ever, the r e l a t i v e r e t e n t i o n area f o r s e v e r a l chromatographic peaks obtained i n the f l u o r e s c e n c e mode of d e t e c t i o n was found t o vary as the mobile phase i n the HPLC was changed. The data i n Table 3 show the e f f e c t s of common chromatographic s o l v e n t s on the f l u o r e s c e n c e o f two p e s t i c i d e s . For maretin and ο-phenylphenol, the r e l a t i v e f l u o r e s c e n c e i n t e n s i t y , as measured i n a s p e c t r o f l u o r o m e t e r , increased s u b s t a n t i a l l y as the p o l a r i t y of the s o l v e n t i n c r e a s e d . However, as shown f o r pyrazophos i n


Fluorescence

ARGAUER

and

UV

Absorbance


Pollen ( Control ]

X7

0

>

, - l

I

of

Pesticides

Pollen ( 5.0 P P M )

I

I

I

l

10 0 Time ( Minutes )

i

l

t

,

*

k

10

Figure 10. Extracts of pollen collected by foraging honey bees: comparison of fluorescence and absorbance detection modes after HPLC on a CN-bonded polar phase (0.1 μg carbaryl/20 mg pollen equivalent injected; mobile phase: methylene chloride/methanol 99.5/0.5)


PESTICIDE

TABLE

3,

FLUORESCENCE

OF

(2

COMMON

UG/ML)

IN

MARETIN

WGZML)

(2

ANALYTICAL

AND

CHROMATOGRAPHIC

0

/

II

C

METHODOLOGY

o-PHENYLPHENOL

SOLVENTS

2 %

\ OCoH

2 5 n

0 _


S O L V E N T ^

HEXANE

100%

HEXANE/METHYLENE

50:50

CHLORIDE

ΕΧλ

ΕΜλ

330 340

365 375

PHENYLPHENOL

EX λ

ΕΧλ

RELATIVE FLUORESCENCE INTENSITY

20 100

290 290

320 320

19 34

RELATIVE FLUORESCENCE INTENSITY

METHYLENE

CHLORIDE

340

375

230

290

320

41

340

375

240

290

335

120

340

375

320

290

335

230

100%

METHYLENE

CHLORIDE/

METHANOL

95:5

METHANOL

100%

(1)

1 MG/ML OF STOCK WITH S O L V E N T .

TABLE

SOLUTIONS

OF

PESTICIDES

IN

ETHYLACETATE

FLUORESCENCE OF PYRAZOPHOS (2 P G / M L ) COMMON C H R O M A T O G R A P H I C SOLVENTS.

4.

DILUTED

IN

0 C H 0C 2

5

0 C

2 5 H

PYRAZOPHOS

RELATIVE SQLYEHI HEXANE

FLUORESCENCE 100%

21.0

HEXANE/METHYLENE

50:50

CHLORIDE

11.0

METHYLENE

CHLORIDE

METHYLENE

CHLORIDE/ETHYLACETATE

95:5

METHYLENE

CHLORIDE/ETHYLACETATE

85:15

METHYLENE

CHLORιDE/METHANOL

100%

8.5 8.4 7.5

99.5:0.5

7.7

METHYLENE

CHLORIDE/METHANOL

99:1

7.3

METHYLENE

CHLORIDE/METHANOL

95:5

5.4

METHANOL

(1)

1

100%

MG/ML

1.0

STOCK

ETHYLACETATE (2)

(L2) INTENSITY

SOLUTION DILUTED

EXCITATION

WAVELENGTH

WAVELENGTH

AT

420

OF

WITH SET

PYRAZOPHOS

IN

SOLVENT. AT

254

NM;

EMISSION

NM.


7.

Fluorescence

ARGAUER

and UV Absorbance

of

Pesticides

123

Table 4, the r e l a t i v e f l u o r e s c e n c e i n t e n s i t y a l s o can decrease s u b s t a n t i a l l y as the p o l a r i t y of the s o l v e n t i n c r e a s e s . Next the absorbance f o r o-phenylphenol, maretin, and pyrazophos was compared i n both hexane and methanol. A Beckman DG-GT Spectrophotometer was used to o b t a i n the absorbance v a l u e s . Table 5.

Absorbance o f P e s t i c i d e s i n Hexane and Methanol


Pesticide

o-phenylphenol maretin pyrazophos

Concentrât i o n

20 yg/ml 20 μ g/ml 10 yg/ml

Wavelength (nm) 286 343 248

Absorbance in m hexane methanol 0.59 0.60 0.74 0.72 1.32 1.26

No a p p r e c i a b l e d i f f e r e n c e i n absorbance values was observed between s o l u t i o n s i n hexane o r methanol (Table 5 ) . We t h e r e f o r e a t t r i b u t e the change i n fluorescence to s o l u t e - s o l v e n t i n t e r a c t i o n s between the solvent and the p e s t i c i d e i n i t s e x c i t e d s t a t e . Several Instrumental Parameters. In a dynamic flow system such as HPLC, the s i g n a l generated by the f l u o r e s c e n c e monitor i s p r o p o r t i o n a l to s e v e r a l v a r i a b l e s i n a d d i t i o n to the two ( p e s t i c i d e c o n c e n t r a t i o n and s o l v e n t used as the mobile phase) already d i s c u s s e d . These v a r i a b l e s i n c l u d e : 1. Size of the flow c e l l . 2. The geometry or design of the d e t e c t o r , which a l s o i n c l u d e s s e l e c t i o n of o p t i c a l f i l t e r s and types of l i g h t d e t e c t o r s with t h e i r d i f f e r i n g r e l a t i v e responses to l i g h t of v a r i o u s wave lengths. 3. The i n t e n s i t y of the e x c i t a t i o n source. 4. The s p e c t r a l (absorbance and f l u o r e s c e n c e ) c h a r a c t e r i s t i c s of the s o l u t e ( p e s t i c i d e ) . The 70 u - l i t e r s i z e of the flow c e l l c o n t r i b u t e d to the broadening of the chromatographic peaks observed i n the f l u o r e s cence mode and could account f o r some l o s s i n s e l e c t i v i t y . The second v a r i a b l e , detector design, could account f o r the unex pected r a t i o i n peak heights (compared with data i n Table 2) obtained i n Figure 4 f o r diphenyl and naphthalene: the g l a s s envelop that houses the photomu11ip1er tube may absorb some of the f l u o r e s c e n c e of biphenyl (emission maximum 305 nm) while the f l u o r e s c e n c e of naphthalene (emission maximum 322 nm) passes through. V a r i a b l e s 3 and 4 a r e found i n the f a m i l i a r r e l a t i o n s h i p , i . e . , that f l u o r e s c e n c e i n d i l u t e s o l u t i o n s i s d i r e c t l y propor t i o n a l to the absorbance of the s o l u t e Αλ a t the wavelength s e l e c t e d f o r e x c i t a t i o n , times the quanta of l i g h t Ιολ a v a i l a b l e at the wavelength s e l e c t e d f o r e x c i t a t i o n , times the f l u o r e s c e n c e quantum e f f i c i e n c y φλ f o r the s o l u t e . Oftentimes the wavelengths a v a i l a b l e f o r maximum i l l u m i n a t i o n i n an e x c i t a t i o n source do not



124

PESTICIDE

ANALYTICAL

METHODOLOGY

WAVELENGTH (NANOMETERS) Analytical Chemistry

Figure 11.

Relative energy distribution of a xenon-arc source (3)

SHORT WAVE

LONG WAVE PHOSPHOR COATED MERCURY LAMP

MERCURY LAMP 254

313

366

207

WAVE LENGTH (NANOMETERS) Figure 12. Relative spectral distribution of a low-pressure mercury vapor lamp with visible light cut-off filter as measured through the emission monochromator of a spectrofluorometer (2 nm slit width). (Typical of lamps used to view fluorescence/ fluorescence quenching on thin-layer chromatographic plates.)



7.

ARGAUER

Fluorescence

and UV

Absorbance

of

Pesticides

125

correspond with wavelengths of maximum l i g h t a b s o r p t i o n f o r the solution. In F i g u r e s 11 and 12 the r e l a t i v e energy d i s t r i b u t i o n of a xenon arc source and two mercury lamps i s compared. The 254 nm mercury l i n e emitted by a g e r m i c i d a l mercury lamp was s e l e c t e d f o r e x c i t a t i o n i n our HPLC f l u o r e s c e n c e monitor. The product of 10254 (a r e l a t i v e l y high energy source f o r i r r a d i a t i o n ) and A254 (the absorbance of the p e s t i c i d e at 254 nm) was expected to produce a s u f f i c i e n t l y l a r g e f l u o r e s c e n c e f o r many of the p e s t i c i d e s . Furthermore any 254 nm e x c i t a t i o n r a d i a t i o n that i s s c a t t e r e d by the flow c e l l and r e f l e c t e d toward the d e t e c t o r i s f i l t e r e d out by the g l a s s envelope surrounding the RCA 931B p h o t o m u l t i p l i e r tube and redundantly by an a d d i t i o n a l g l a s s f i l t e r placed i n the entrance window to the d e t e c t o r . I t was a n t i c i p a t e d however that there would be some l o s s of s e l e c t i v i t y due to f l u o r e s c i n g c o - e l u t a n t s that absorb a t 254 nm. Conclusions Many p e s t i c i d e s f l u o r e s c e s u f f i c i e n t l y when e x c i t e d at 254 nm to permit the advantageous use of the intense 253.7 nm emission from a mercury l i g h t source f o r e x c i t a t i o n i n a HPLC f l u o r e s c e n c e monitor. A f l u o r e s c e n c e monitor can conveniently confirm and support data obtained with an absorbance d e t e c t o r . However, any comp a r i s o n of the r e l a t i v e s e n s i t i v i t y / s e l e c t i v i t y of the absorbance mode vs the f l u o r e s c e n c e mode depends on the spectrochemical nature of both the p e s t i c i d e i t s e l f , and the co-extracted coe l u t a n t s found i n the a g r i c u l t u r a l product e x t r a c t e d . A j u d i c i o u s s e l e c t i o n of mobile phase i s r e q u i r e d to optimize s e p a r a t i o n of the p e s t i c i d e and c o - e x t r a c t i v e s on a CN-bonded p o l a r s t a t i o n a r y phase. Chromatographic peak areas i n the f l u o r e s c e n c e mode can be a f f e c t e d by the choice of the mobile phase because p e s t i c i d e solvent i n t e r a c t i o n s a f f e c t the f l u o r e s c e n c e . We have i d e n t i f i e d t h i s unusual phenomena f o r both the p e s t i c i d e s themselves and f o r u n i d e n t i f i e d c o - e x t r a c t i v e s from s e v e r a l a g r i c u l t u r a l crops. Quenching of the f l u o r e s c e n c e emitted by p e s t i c i d e s can occur when c o - e l u t a n t s are present i n l a r g e amounts. Acknowledgments The author appreciates the a s s i s t a n c e of Ernest J . M i l e s , P h y s i c a l Science T e c h n i c i a n , of the A n a l y t i c a l Chemistry Laborat o r y , AEQI, AR, SEA, USDA, B e l t s v i l l e , Md. i n o b t a i n i n g the data for this contribution.


PESTICIDE ANALYTICAL METHODOLOGY

126 Literature Cited


1. White, C. Ε., Argauer, R. J., "Fluorescence Analysis, A Practical Approach," Marcel Dekker, New York, N.Y., 1970 (contains numerous literature references) 2. Robert J. Argauer, "Fluorescence Methods for Pesticides." Chapter 4 in Analytical Methods for Pesticides and Plant Growth Regulators, Volume IX, "Spectroscopic Methods of Analysis," Academic Press, Inc., New York, N.Y., 1977. (contains over 100 literature references to fluorescence of pesticides) 3. Argauer, R. J. and White, C. E. Anal. Chem. 1964, 36, 368. RECEIVED February 7,

1980.


Pesticide Analytical Methodology - American Chemical Society

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