Some Applications of Fourier Transform Infrared Spectroscopy to

17 Some Applications of Fourier Transform Infrared Spectroscopy to Pesticide Analysis STEPHEN R. LOWRY and CHARLES L. G R A Y 1

Downloaded by UNIV OF ARIZONA on June 4, 2017 | http://pubs.acs.org Publication Date: October 30, 1980 | doi: 10.1021/bk-1980-0136.ch017

Diamond Sharmock Corporation, P.O. Box 348, Painesville, OH 44077

F o u r i e r transform i n f r a r e d spectroscopy (FTIR) has provided support to a number of areas i n Diamond Shamrock's p e s t i c i d e program. Commercially a v a i l a b l e FTIR spectrometers o f f e r a number of advantages over d i s p e r s i v e instruments. Although some of the advantages are r e l a t e d to the a b i l i t y to perform computerized data m a n i p u l a t i o n s , the b a s i c design of the FTIR system does provide s u p e r i o r c a p a b i l i t i e s i n i n f r a r e d spectroscopy (jL) . A schematic diagram of a Michelson i n t e r f e r o m e t e r , the heart of the FTIR system, i s shown i n F i g u r e 1 (2) . The Michelson i n t e r f e r o m e t e r modulates each wavelength i n the i n f r a r e d r e g i o n at a d i f f e r e n t frequency i n the audio range. The modulated r a d i a t i o n passes through a sample chamber and i s measured by the d e t e c t o r . This s i g n a l , c a l l e d an i n t e r ferogram, i s converted by means of the F o u r i e r transform i n t o a s i n g l e beam spectrum. An interferogram and corresponding spectrum are shown i n F i g u r e 2. A normal i n f r a r e d spectrum i s obtained by r a t i o i n g the s i n g l e beam spectrum to a reference spectrum. The transmittance and absorbance s p e c t r a are shown i n Figure 3. Although t h i s seems to be an e l a b o r a t e method of obt a i n i n g a spectrum, s e v e r a l t h e o r e t i c a l advantages r e s u l t from r a p i d scanning FTIR. The f i r s t , F e l l g e t t ' s advantage, r e s u l t s from the f a c t that data from a l l frequencies are being measured by the detector simultaneously. This i s i n c o n t r a s t to d i s p e r s i v e instruments where only the small range of frequencies emerging from the e x i t s l i t at any one time are being measured. The second, Jacquinot's advantage, a r i s e s due to the increased energy throughput because no s l i t s are used i n the i n t e r f e r o meter. The t h i r d , Conne's advantage, r e s u l t s from the high degree of wavelength accuracy found i n most FTIR spectrometers due to the l a s e r reference system. This accuracy allows a l a r g e ^Current address:

N i c o l e t Instrument C o r p o r a t i o n 5225 Verona Road, Madison, Wisconsin

0-8412-0581-7/80/47-136-299$05.50/0 © 1980 American Chemical Society Harvey et al.; Pesticide Analytical Methodology ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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Moveable M i r r o r

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Diagram of a Michelson interferometer

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Interferogram (top) and resulting single beam spectrum after Fourier transformation (bottom,)

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


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PESTICIDE

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number of scans to be co-added, which reduces the s p e c t r a l n o i s e . These three advantages are the b a s i s of many of the improvements of FTIR over d i s p e r s i v e spectroscopy. Table I l i s t s some of the increased c a p a b i l i t i e s of FTIR. The most s i g n i f i c a n t improvement i s the a b i l i t y to o b t a i n an increased s i g n a l at a l l frequencies i n reduced time. This allows the a c q u i s i t i o n of good s p e c t r a i n s u b s t a n t i a l l y l e s s time, or the a c q u i s i t i o n of low s i g n a l s p e c t r a ( i . e . , micro samples) i n reasonable amounts of time.


Table

I

Advantages of FTIR Fundamental Advantages Multiplex Throughput Frequency Accuracy

F e l l g e t t ' s Advantage Jacquinot's Advantage Conne's Advantage Practical Applications Rapid Scan Opaque Samples Micro Sampling Spectral Subtraction

The remainder of t h i s paper w i l l d e s c r i b e s p e c i f i c a p p l i c a t i o n s of FTIR i n support of one of Diamond Shamrock's p e s t i c i d e programs. A l l the r e s u l t s described here were ob tained from s t u d i e s of a s i n g l e compound, thiofanox (Ρ), 3,3dimethyl-l-methylthio-2-butanone 0-[(methylamino)carbonyl]oxime. Thiofanox i s a potent systemic and contact carbamate i n s e c t i c i d e . The i n f r a r e d spectrum of thiofanox i s shown i n F i g u r e 4. The major peaks i n the spectrum are: the N-H s t r e t c h at 3380 cm , C-H s t r e t c h e s i n the 3000-2800 cm" r e g i o n , the carbonyl band at 1720 cm , the C=N at 1620 cm"" , CNH at 1500 cm" , and two other bands at 1235 cm"" and 950 cm" . -1

1

-1

1

1

1

1

Formulations The f i r s t a p p l i c a t i o n we would l i k e to d i s c u s s i n v o l v e s the a n a l y s i s of thiofanox formulated on a c l a y c a r r i e r . Two s p e c i f i c questions f o r which FTIR provided answers were: (1) are there i n t e r a c t i o n s between the compound and the c a r r i e r m a t e r i a l ? ; and (2) i s the a c t i v e i n g r e d i e n t completely removed by solvent extraction? The answer to these questions r e q u i r e d a comparison of s p e c t r a from the blank c a r r i e r and the formulated m a t e r i a l before and a f t e r e x t r a c t i o n . These s p e c t r a were obtained by thoroughly g r i n d i n g each sample, mixing with KBr, and p r e s s i n g


LOWRY

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Spectroscopy


17.


303

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i n t o a d i s c . One hundred 8192-point interferograms were coadded before a 16,384-point transform was performed. The spectrum of the f o r m u l a t i o n i s dominated by strong s i l i c a t e peaks from the c a r r i e r m a t e r i a l . F i g u r e 5 shows the spectrum of the f o r m u l a t i o n ( t o p ) , and the blank c a r r i e r (bottom). The only thiofanox peak which stands out c l e a r l y i s the carbonyl peak at 1720 cm" . Since a l l the s p e c t r a l data i s s t o r e d i n a d i g i t a l format, a p o i n t - b y - p o i n t weighed s u b t r a c t i o n can be performed between two s p e c t r a . The s u b t r a c t i o n of one spectrum from another i s based on the f o l l o w i n g equation: 1


Sample spectrum x FCS

- Reference spectrum x FCR

=0

(1)

where FCS and FCR are s c a l i n g f a c t o r s . G e n e r a l l y , FCS = 1 and FCR i s continuously v a r i e d u n t i l peaks disappear. On our instrument, a N i c o l e t Model 7199, t h i s process i s c a r r i e d out by v i s u a l examination on a scope d i s p l a y , which a u t o m a t i c a l l y d i s p l a y s the r e s u l t s of the s u b t r a c t i o n as FCR i s changed. The r e s u l t i n g d i f f e r e n c e spectrum w i l l then c o n t a i n only those peaks unique to e i t h e r of the two o r i g i n a l s p e c t r a . The spectrum obtained by s u b t r a c t i n g the blank c a r r i e r from the formulated m a t e r i a l i s shown i n F i g u r e 6. Considering the strong bands from the c a r r i e r which had to be s u b t r a c t e d , t h i s i s a very good spectrum. A comparison of t h i s with the spectrum of standard thiofanox i n KBr shows that most of the peaks have not changed during the f o r m u l a t i o n process. The only exception i s the N-H s t r e t c h at 3380 cm . T h i s peak i s completely missing i n the d i f f e r e n c e spectrum. The l o s s of t h i s peak suggests the N-H may be b i n d i n g to some of the groups on the c a r r i e r or to r e s i d u a l water. The procedure f o r determining the amount of a c t i v e i n g r e d i ent i n the formulated p e s t i c i d e i n v o l v e s a s o l v e n t e x t r a c t i o n followed by e i t h e r IR or HPLC a n a l y s i s . F i g u r e 7 shows a spectrum of the granules a f t e r e x t r a c t i o n , and the d i f f e r e n c e spectrum obtained by s u b t r a c t i n g the blank c a r r i e r from the e x t r a c t e d granules. I t can be seen that l i t t l e or no thiofanox remains on the granules a f t e r e x t r a c t i o n . The very small peak i n the 1700 cm"" r e g i o n may be due to t r a c e amounts of thiofanox, but the l e v e l i s below the accuracy l i m i t of the assay. A l s o , the negative peaks i n the d i f f e r e n c e spectrum i n d i c a t e that a component of the c a r r i e r i s removed by the e x t r a c t i o n process. These peaks appear to correspond to a carbonate s a l t , but an exact assignment has not been made. Although the FTIR i s not used r o u t i n e l y f o r the assay of thiofanox, i n cases where problems from i n t e r f e r e n c e s a r i s e or when d i s c r e p a n c i e s between r e s u l t s occur, the s u b t r a c t i o n capab i l i t i e s of FTIR can be used to o b t a i n q u a n t i t a t i v e i n f o r m a t i o n . F i g u r e 8 contains the spectrum of the e x t r a c t from the granules, a spectrum of the e x t r a c t i o n s o l v e n t , methylene c h l o r i d e , and the d i f f e r e n c e spectrum. The d i f f e r e n c e spectrum contains a -1

1


AND

GRAY

FTIR

Spectroscopy

305


LOWRY

Figure 5.

Spectra of a thiofanox formulation (top) and the blank carrier (bottom)


306

ANALYTICAL

METHODOLOGY


PESTICIDE

Figure 6.

Difference spectrum showing the thiofanox formulation with the carrier subtracted


LOWRY

A N D

GRAY

FTIR

Spectroscopy


17.

Figure 7. Spectra of a thiofanox formulation after solvent extraction (top) and the extracted formulation with the blank carrier subtracted (bottom)


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METHODOLOGY


308


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AND

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309

number of peaks which are f a r enough from the opaque regions of the solvent to e a s i l y permit a q u a n t i t a t i v e a n a l y s i s . F i g u r e 9 shows the spectrum of the e x t r a c t from the granules with the solvent already subtracted, a s i m i l a r spectrum of a standard thiofanox s o l u t i o n , and the r e s u l t i n g d i f f e r e n c e spectrum. Normally, the i n f r a r e d assay of thiofanox i s performed by comparing the i n t e n s i t y of the peak at 945 cm" of the standard and sample s o l u t i o n s . The amount of thiofanox i n the sample i s c a l c u l a t e d using the f o l l o w i n g equation: 1

Wt.

Sample = Wt.

Standard x

A


A

S

a

m

p

^

e

(2)

,

standard

where A i s the b a s e l i n e c o r r e c t e d absorbance f o r the sample and standard, r e s p e c t i v e l y , Using the s p e c t r a l s u b t r a c t i o n t e c h n i que would r e q u i r e that the i n t e n s i t y of the 945 cm" band i n the d i f f e r e n c e spectrum between the sample and standard go to zero. The equation would be: 1

A

sample

χ

FCS

- Α

, x FCS standard

s i n c e the s c a l i n g f a c t o r FCS

^

Λ

= 0

(3)

= 1, then

F C R

( 4 )

^standard or, based on the s p e c t r a l s u b t r a c t i o n , the weight of thiofanox the sample would be c a l c u l a t e d : Wt.

, = Wt. , , χ FCR sample standard

in

(5)

The advantage of the s u b t r a c t i o n method occurs when FCR i s d e t e r mined using the e n t i r e spectrum rather than a s i n g l e peak. This means that an i n t e r f e r e n c e at a s i n g l e peak w i l l not a f f e c t the a n a l y s i s to a s i g n i f i c a n t degree. Based on the s p e c t r a shown i n F i g u r e 9, the weight of e x t r a c t e d m a t e r i a l was 0.54 g. This represents an assay of 8.9% a c t i v e i n g r e d i e n t , compared to 9.0% thiofanox a p p l i e d during the f o r m u l a t i o n . This i s w e l l w i t h i n the accuracy f o r t h i s method. Gas

Evolution

Because of the known thermal i n s t a b i l i t y of carbamates at high temperatures, a method of i d e n t i f y i n g any v o l a t i l e species would be d e s i r a b l e . The r a p i d data a c q u i s i t i o n p o s s i b l e with FTIR would enable changes i n both c o n c e n t r a t i o n and composition to be monitored as a f u n c t i o n of time. A 10 cm gas c e l l was modified to enable the measurement of v o l a t i l e components from


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ANALYTICAL

M ET HO DO L O GY


310

Figure 9. Extract from formulation with solvent subtracted (top), thiofanox standard with solvent subtracted ( c e n t e r j , and difference between sample and standard with FCR = 1.376



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Spectroscopy

e i t h e r t e c h n i c a l thiofanox or formulated m a t e r i a l at v a r i o u s temperatures. A small opening was made i n the bottom of the c e l l , and a short neck attached. A sample i s placed i n a s m a l l sample cup which i s clamped to the neck of the c e l l . The e n t i r e c e l l i s then evacuated. A heating b l o c k i s placed around the cup, and the c e l l i s mounted i n the instrument. A f t e r a background spectrum i s a c q u i r e d , the c e l l i s heated. The temperature i s c o n t r o l l e d and monitored by means of a thermocouple attached to a thermal c o n t r o l u n i t . F i g u r e 10 shows the i n i t i a l spectrum of a sample of high q u a l i t y t e c h n i c a l m a t e r i a l at 100°C. The p o s i t i v e peaks i n the spectrum correspond to methyl isocyanate, which i n d i c a t e s the carbamate l i n k a g e i s being cleaved. The negative peaks i n the spectrum correspond to a decrease i n the l e v e l of water vapor and C0 o u t s i d e the c e l l as the instrument i s purged with n i t r o g e n . A spectrum obtained at 150°C i s shown i n F i g u r e 11. While s u b s t a n t i a l amounts of methyl i s o cyanate remain, numerous other peaks a l s o appear. These are due to the presence of isobutane, isobutene, and C0 . Experiments with lower q u a l i t y t e c h n i c a l m a t e r i a l and a formulation showed a very d i f f e r e n t behavior. When these samples were examined at 70°C, the major gaseous product was C0 . Although small amounts of methyl isocyanate were detected, i t appears that i m p u r i t i e s i n these samples are r e a c t i n g with thiofanox before the carbamate i s c l e a v e d . Because the C0 i s the dominant v o l a t i l e m a t e r i a l i n these s i t u a t i o n s , a method was developed to q u a n t i t a t e the e v o l u t i o n of C0 as a f u n c t i o n of time. This data should give the k i n e t i c r a t e f o r the thermal decomposition and provide a r a p i d s t a b i l i t y i n d i c a t i n g assay. F i g u r e 12 shows the C0 s p e c t r a from three commercially prepared standard gas mixtures. These s p e c t r a i n d i c a t e C0 can be e a s i l y detected down to 5 ppm by weight i n n i t r o g e n . The c a l i b r a t i o n curve based on these standards i s shown i n F i g u r e 13. The absorbance of the C0 band c l e a r l y f o l l o w s Beer's law i n t h i s r e g i o n . F i g u r e 14 shows a p l o t of C0 c o n c e n t r a t i o n as a f u n c t i o n of time f o r a t e c h n i c a l thiofanox sample at 70°C. T h i s graph i n d i c a t e s the e v o l u t i o n r a t e i s constant over the time p e r i o d of the experiment. The r a t e of decomposition can be c a l c u l a t e d from t h i s p l o t and i s 1.15 y£/g/min. 2

2

2

2

2

2

2

2

2

Micro Sampling With A p p l i c a t i o n s To M e t a b o l i t e

Studies

The f i n a l a p p l i c a t i o n of FTIR to be discussed i s the a n a l y s i s of microsamples. A major problem encountered i n the i n f r a r e d examination of microsamples i s the r e d u c t i o n i n energy reaching the d e t e c t o r (_3) . The s e n s i t i v i t y of FTIR proved b e n e f i c i a l i n work performed i n support of metabolite s t u d i e s of t h i o f a n o x . Samples were prepared by d i s s o l v i n g the compound i n a small amount of solvent (10-20 y £ ) , followed by withdrawing the s o l u t i o n i n t o a s y r i n g e . The t i p of the s y r i n g e was coated with a small amount of KBr, and the s o l u t i o n was slowly r e l e a s e d


312

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Volatile product obtained from heating high quality thiofanox to 100 C e

Volatile products obtained from heating high quality thiofanox to 150°C


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Calibration curve from CO standards z


PESTICIDE

ANALYTICAL

METHODOLOGY


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17.

LOWRY

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FTIR

Spectroscopy

315

i n t o the KBr as the solvent evaporated. The r e s u l t i n g powder was placed i n a micro d i e (1.5 mm diameter), and a p e l l e t prepared. F i g u r e 15 shows the spectrum of 10 yg of thiofanox prepared i n t h i s f a s h i o n . With d e t e c t i o n l i m i t s t h i s low, FTIR appears to be capable of d e t e c t i n g q u a n t i t i e s f r e q u e n t l y encountered i n TLC e x p e r i ments. To evaluate t h i s p o s s i b i l i t y , TLC p l a t e s were spotted with 10 yg and 20 yg q u a n t i t i e s of t h i o f a n o x . The p l a t e s were developed i n an acetone-hexane (1:1) s o l v e n t system, and the spots were detected under UV l i g h t or with i o d i n e vapors. I n i t i a l l y , the spot was scraped from the p l a t e and analyzed d i r e c t l y i n KBr. However, the s i l i c a g e l produced too much i n t e r f e r e n c e , and the thiofanox could not be detected. A second set of spots were scraped from the p l a t e , e x t r a c t e d with chloroform, and the s o l u t i o n was d r i e d on KBr using the procedure d e s c r i b e d above. F i g u r e 16 shows the e x t r a c t from a 20 yg spot. T h i s spectrum i s s t i l l dominated by the s i l i c a g e l peaks. By u t i l i z i n g the s u b t r a c t i o n c a p a b i l i t i e s of the FTIR system, the peaks from the s i l i c a g e l can be mathematically removed from the spectrum of the sample. F i g u r e 17 shows a spectrum of a blank spot from the TLC p l a t e , and the d i f f e r e n c e spectrum from these i s shown i n F i g u r e 18. This spectrum c l o s e l y matches the spectrum obtained from 10 yg of thiofanox shown e a r l i e r . Previous s t u d i e s at Diamond Shamrock have i n d i c a t e d that one degradation pathway f o r thiofanox i s shown i n F i g u r e 19 04). TLC experiments were performed on s e v e r a l of these m e t a b o l i t e s , and the spots were analyzed by FTIR. S e v e r a l of these s p e c t r a are shown i n F i g u r e 20. These r e s u l t s have demonstrated that FTIR can provide u s e f u l i n f o r m a t i o n f o r the i d e n t i f i c a t i o n of metabolites. Impurity

Study

With the e v e r - i n c r e a s i n g government r e g u l a t i o n s concerning p e s t i c i d e s , a s u b s t a n t i a l e f f o r t i s g e n e r a l l y r e q u i r e d to i d e n t i f y i m p u r i t i e s present i n a t e c h n i c a l product. Because thiofanox cannot be analyzed d i r e c t l y by the GC/MS method f r e q u e n t l y used i n t h i s type of study, p r e p a r a t i v e HPLC was used to l o c a t e new i m p u r i t i e s . As new i m p u r i t i e s were found, the f r a c t i o n s were f u r t h e r p u r i f i e d using a n a l y t i c a l columns. These f r a c t i o n s were then examined by FTIR, NMR, and mass spectrometry. Since the amount of sample i s o l a t e d using these columns i s extremely s m a l l , the s e n s i t i v i t y of FTIR proved extremely v a l u able i n p r o v i d i n g an o v e r a l l c h a r a c t e r i z a t i o n of s e v e r a l impurities. Conclusions Although

t h i s paper has d e s c r i b e d s e v e r a l a p p l i c a t i o n s of



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Spectrum of a blank TLC spot


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20bu

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Figure 18. Difference spectrum showing thiofanox TLC spot minus the blank


318

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N-0-C-NH-CH

ANALYTICAL

METHODOLOGY

3

(CH ) C-C 3

3

H -S-CH


2

3

NOH

N-O-C-NH-CH3 (CH ) C-C

(CH ) C-C; 3

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Some Applications of Fourier Transform Infrared Spectroscopy to

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