Development of an Analytical Method for Benzidine-Based Dyes

2 Development of an Analytical Method for Benzidine-Based Dyes

Downloaded by UNIV OF ARIZONA on January 7, 2013 | http://pubs.acs.org Publication Date: April 2, 1981 | doi: 10.1021/bk-1981-0149.ch002

EUGENE R. KENNEDY and MARTHA J. SEYMOUR National Institute for Occupational Safety and Health, Robert A. Taft Laboratories, 4676 Columbia Parkway, Cincinnati, OH 45226 Benzidine has been an important dye industry intermediate since 1890 (J_,2). Over 200 dyes based on benzidine are listed in the Colour Index or are in commercial use. Although the potential of benzidine to cause bladder cancer has been well documented (3^,5), i t was originally believed that when chemically incorporated into a dye, the carcinogenic hazard was removed (6). Recent evidence (7.,8,9.) has shown that benzidine-based dyes can be reduced to benzidine in living systems and eliminated by the usual benzidine metabolic pathways. Because of this fact, the National Institute for Occupational Safety and Health has recommended that certain benzidine-based dyes be recognized and handled as carcinogens (9). The only published method available for the determination of personal exposure to benzidine-based dyes utilized the analysis of urine for benzidine and benzidine metabolites ,_H). This method does not allow for quantitation of a daily exposure, since benzidine and its metabolites have been found in the urine of hamsters fed a benzidine-based dye up to 168 hours after a single dosing (J2.) · A method for the determination of personal exposure to azo dyes and diazonium salts has been developed (^3), but it is not specific enough to determine an exposure to a benzidine-based dye. In the development of a sampling and analytical method for benzidine-based dyes, the most important feature was the verification of the benzidine moiety in the dye molecule. Since reduction of some of these dyes in vivo was known to release benzidine, a human carcinogen, the determination of benzidine released by chemical reduction is the most logical approach. Specificity for a particular dye was not reasonable due to the large number of benzidine-based dyes and the possibility of dye substitution. The method should provide quantitative collection and recovery at the microgram level and be free This chapter not subject to U.S. copyright. Published 1981 American Chemical Society

In Chemical Hazards in the Workplace; Choudhary, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

C H E M I C A L HAZARDS IN

22

THE

WORKPLACE

from i n t e r f e r e n c e s , e s p e c i a l l y those a r i s i n g from benzidine congeners, such as o - t o l i d i n e ( 3 , 3 - d i m e t h y l b e n z i d i n e ) and o - d i a n i s i d i n e (3,3 -dimethyoxybenzidine). Various c o n d i t i o n s for r e d u c t i o n of the azo l i n k a g e s i n these dyes were known (14), so cleavage of the benzidine moiety was p o s s i b l e . A l s o , c o n d i t i o n s f o r the a n a l y s i s of aromatic amines were w e l l e s t a b l i s h e d (Jj2,i5,16.,ΥΠ . P r e l i m i n a r y work i n our l a b o r a t o r y had shown t h a t benzidine and i t s congeners could be separated by high pressure l i q u i d chromatograph (HPLC). Based on t h i s reasoning, a method f o r the determination of benzidine i n benzidine-based dyes was developed. f

f


Experimental Apparatus. The HPLC system used i n t h i s study, assembled from modular components manufactured by Waters A s s o c i a t e s , c o n s i s t e d of two Model 6000A pumps, a Model 660 s o l v e n t programmer, a Model 440 u l t r a v i o l e t d e t e c t o r w i t h a 280-nm f i l t e r and a Waters Model 710A I n t e l l i g e n t Sample Processor. Data were recorded on a S o l t e c Model B281 dual channel s t r i p chart recorder and i n t e g r a t e d by a Hewlett-Packard Model 3354A Laboratory Automation System. The columns used f o r the analyses were a Waters A s s o c i a t e s y-Bondapak C ^ and a Waters Model RCM100 R a d i a l Compression Module w i t h a R a d i a l Pak A c a r t r i d g e . The mobile phase was 60% methanol (Burdick and Jackson) and 40Î of an aqueous phosphate b u f f e r . This phosphate b u f f e r was prepared by d i s s o l v i n g 3.390 g (0.025 mole) o f KH P04 ( F i s h e r S c i e n t i f i c Corp.) and 3.530 g (0.025 mole) of Na HP04 (Matheson Coleman B e l l ) i n 1 L of water (J_8). A helium purge was maintained i n the s o l v e n t r e s e r v o i r s to e l i m i n a t e d i s s o l v e d a i r . The columns were maintained at ambient temperature and run at a 2-mL/min flow r a t e . R e s u l t i n g pressure at t h i s flow r a t e was 500-1900 p s i for the R a d i a l Compression Module and 3000 p s i f o r the μ-Bondapak column. A precolumn f i l t e r could not be used because the e x t r a dead volume reduced r e s o l u t i o n of r e d u c t i o n products. M i l l i p o r e 37 mm Mitex (Teflon) f i l t e r s (5.0 ym) w i t h backup pads and t h r e e - p i e c e c a s s e t t e s were used f o r the spiked f i l t e r s t u d i e s . A Doerr Model 0272X vacuum pump w i t h a 10-port sampling manifold and 1-L/min c r i t i c a l o r i f i c e ( M i l l i p o r e Corporation) was used to p u l l a i r through the dye-spiked f i l t e r c a s s e t t e s f o r the s t a b i l i t y s t u d i e s . Two methods of exposing the c a s s e t t e s to h u m i d i f i e d a i r were employed. For the 28-day storage study, a i r was p u l l e d d i r e c t l y through midget impingers f i l l e d w i t h a saturated sodium c h l o r i d e s o l u t i o n . For the 7-day storage study, a i r was blown through a l a r g e saturated sodium c h l o r i d e - f i l l e d impinger and then s u p p l i e d to a 10-port manifold to which the c a s s e t t e s were attached. A i r was p u l l e d through the c a s s e t t e s 2

2


K E N N E D Y A N D SEYMOUR

2.

Benzidine-Based

Dyes


u s i n g the p r e v i o u s l y described vacuum pump. With t h i s system the c a s s e t t e s were always maintained a t atmospheric pressure. R e l a t i v e humidity generated by e i t h e r o f these techniques was approximately 75% (19)* V i s i b l e - s p e c t r u m s t u d i e s of the course of the dye r e d u c t i o n were performed on a Beckmann Model 25 U l t r a v i o l e t - V i s i b l e Spectrophotometer scanning the r e g i o n o f 750-350 nm. Reagents. The dyes used i n t h i s study (see F i g u r e 1) were obtained from the f o l l o w i n g sources: Congo Red (Colour Index (C.I.) D i r e c t Red 28, C.I. No. 22120) A. D. Mackay I n c . ; D i r e c t Black GX ( C . I . D i r e c t Black 38, C.I. No. 30235), D i r e c t F. Blue 2B 250% ( C . I . D i r e c t Blue 6, C.I. No. 22610), D i r e c t Brown BRL 200$ ( C . I . D i r e c t Brown 95, C.I. No. 30145) F a b r i c o l o r Inc.; Evans Blue ( C . I . D i r e c t Blue 53, C.I. No. 23860), Benzo Azurine G ( C . I . D i r e c t Blue 8, C.I. No. 24140) P f a l t z and Bauer I n c . Benzidine was obtained from Sigma Chemical Co., o - t o l i d i n e from F i s h e r S c i e n t i f i c Corp. and 3 , 3 - d i m e t h o x y b e n z i d i n e ( o - d i a n i s i d i n e ) from Eastman Kodak Company. A n i l i n e , £-aminophenol, £-phenylenediamine and £-nitroaniline used i n the i n t e r f e r e n c e study were obtained from Chem S e r v i c e I n c . The phosphate b u f f e r s o l u t i o n used f o r r e d u c t i o n o f the dyes was prepared by d i s s o l v i n g 1.179 g (0.0087 mole) o f KH P04 and 4.300 g (0.0303 mole) o f Na HP0ij i n water to make 100 mL o f s o l u t i o n . For the r e d u c t i o n o f the dyes, a s o l u t i o n was prepared which contained 10 mg o f sodium h y d r o s u l f i t e ( F i s h e r S c i e n t i f i c ) i n 1 mL o f the above b u f f e r s o l u t i o n (0.087 Μ KH P0i| - 0.303 M Na HP0i|). This sodium h y d r o s u l f i t e c o n t a i n i n g s o l u t i o n was prepared immediately before a d d i t i o n to the desorbed dye to prevent decomposition o f the sodium h y d r o s u l f i t e . A l l s o l u t i o n s , i n c l u d i n g the HPLC aqueous phase, were f i l t e r e d through a 0.22-ym c e l l u l o s e e s t e r membrane f i l t e r before use t o prevent plugging o f the HPLC system d u r i n g a n a l y s i s . f

2

2

2

2

Procedure. A primary standard s o l u t i o n o f benzidine i n methanol was prepared. Standards o f lower c o n c e n t r a t i o n s were prepared by d i l u t i o n o f the primary standard. T y p i c a l l i q u i d chromatograph c a l i b r a t i o n curves were l i n e a r i n the r e g i o n from 0.38 t o 30.6 ug/mL. The standards were s t a b l e f o r s e v e r a l months when stored i n the dark. Aqueous s o l u t i o n s o f known dye-formulation c o n c e n t r a t i o n were prepared. F i l t e r samples f o r the s t a b i l i t y and r e p e a t a b i l i t y s t u d i e s were prepared by s p i k i n g the f i l t e r w i t h a known volume o f the dye s o l u t i o n u s i n g f i x e d volume p i p e t t e s or volumetric s y r i n g e s . The f i l t e r s were then d r i e d i n a d e s s i c a t o r f i l l e d w i t h anhydrous calcium s u l f a t e and phosphorous pentoxide. The d e s s i c a t o r provided more r a p i d and uniform d r y i n g o f the


CHEMICAL HAZARDS IN T H E W O R K P L A C E

CHEMICAL STRUCTURE AND NAME

COLOUR INDEX N*

C.I. DIRECT RED 28 NH

NH

2

(§φ-Ν=Ν

S0 Na

3

3


C.I. DIRECT

BLUE

6

NH OH

OHNH

2

N

No0 S

=

-

N

^

^

N

S0 No

3

3

C.I. DIRECT N a 0

22 120

Ί

S0 No

j è ^

2

N=N φ ^ |

2

= ^ è l ^ Na0 S SOjNa

2 2 6 10

N

3

BROWN 95

2 v

0—Cu—a

C

ΗΟ-^-Ν = Ν - ^ - 0 - Ν = - Ν τ Λ ρ Ν = Ν-Ο H O ^ S0 Na

30145

M

3

C.I. DIRECT

BLUE

53

NH pH 3 W^T-N = N - p ^ ^ N

OH N H =N^WS0 Na

2

N o 0

2

S

S0 Na

L

3

C.I. DIRECT

n

3 BLUE

^ J ^ V c O ,

3

23860

S0 Na 3

8 OCH,

< ^ N - N - & ^ N OH Figure 1.

3

-

j°3

N-Çb

N Q

2 4 , 4 0

OH

Structures and names of dyes used in this study



2.

KENNEDY AND

SEYMOUR

Benzidine-Based

Dyes

f i l t e r s than a i r d r y i n g . The f i l t e r s were then stored at room temperature i n a f i l t e r s h i p p i n g case u n t i l r e d u c t i o n and analysis. The spiked f i l t e r s were placed i n a 50-mL beaker w i t h the spiked side up and 1 mL of water was added. The beaker was shaken so that a l l of the f i l t e r area had been washed by the water. One mL o f the r e d u c t i o n - b u f f e r s o l u t i o n without the sodium h y d r o s u l f i t e was added to the f i l t e r and water. The beaker was shaken a second time. The f i l t e r was then turned over (spiked side down) and the beaker placed i n an u l t r a s o n i c bath f o r 15 minutes. At the end o f t h i s p e r i o d , the s o l u t i o n i n the beaker was c o l o r e d . A 1-mL a l i q u o t of t h i s s o l u t i o n was t r a n s f e r r e d to a 4-mL v i a l . One mL o f a f r e s h l y prepared s o l u t i o n of 100-mg sodium h y d r o s u l f i t e i n 10-mL phosphate r e d u c t i o n b u f f e r was then added to the v i a l . The v i a l was then capped and shaken s e v e r a l times during the course of an hour. During t h i s time, the o r i g i n a l c o l o r of the s o l u t i o n disappeared or changed to a d i f f e r e n t c o l o r , depending on the dye present. This s o l u t i o n was then i n j e c t e d i n t o the l i q u i d chromatograph. A 10-yL a l i q u o t was used, g i v i n g a measurement l i m i t of 0 . 3 8 ng benzidine/yL. The a n a l y t i c a l r e p r o d u c i b i l i t y at t h i s l i m i t was 10% c o e f f i c i e n t of v a r i a t i o n (CV). 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 work on the r e d u c t i o n r e a c t i o n i n v o l v e d a study of the completeness o f the r e a c t i o n and v e r i f i c a t i o n of the r e d u c t i o n product, benzidine. The presence of benzidine i n the reduced dye sample was confirmed by gas chromatographic/ mass spectrometric a n a l y s i s . In order to determine the completeness o f the dye-reduction r e a c t i o n , the r e d u c t i o n o f C.I. D i r e c t B l a c k 3 8 , C.I. D i r e c t Brown 95 and C.I. D i r e c t Blue 6 were s t u d i e d i n d i v i d u a l l y i n the v i s i b l e spectrum. A b a s e l i n e was recorded using the phosphate r e d u c t i o n b u f f e r i n both c e l l s . Subsequent a d d i t i o n s o f known amounts of dye and scanning allowed absorption maxima and molar a b s o r p t i v i t y to be determined. Then 3 mg o f sodium h y d r o s u l f i t e was added to the d y e - c o n t a i n i n g c e l l . The c o n c e n t r a t i o n of dye remaining a f t e r r e d u c t i o n was c a l c u l a t e d using Beer's Law. The remaining dye v a r i e d from 0 to 6% o f the o r i g i n a l amount of dye added (Table I ) . Reduction was complete w i t h i n 3 0 minutes. In the i n i t i a l phases of the a n a l y t i c a l method development a Waters A s s o c i a t e s C-j3 y-Bondapak column was used. With c e r t a i n dyes, such as C.I. D i r e c t Black 3 8 , a n i l i n e i s used as a t e r m i n a l group (Figure 1 ) . This column caused the benzidine peak of the reduced dye to obscure a peak due to a n i l i n e . I f these two compounds were not r e s o l v e d , the method could not d i f f e r e n t i a t e between benzidine-based and a n i l i n e - b a s e d dyes. This problem was removed by use of the Waters R a d i a l Compression Module Model RCM 1 0 0 w i t h a R a d i a l Pak A c a r t r i d g e .


25

26

C H E M I C A L HAZARDS IN

Table I V i s i b l e Spectrum S t u d i e s o f Benzidine-Based


C.I. D i r e c t Blue 6 C.I. D i r e c t Brown 95 C.I. D i r e c t Black 38

WORKPLACE

Dyes

Concentration (ug/mL) Before After Reduction Reduction

Molar Absorptivity

Dye

THE

26.5

9.1

0.1

23.8

18.1

1.1

19.0

15.9

0.0

This system r e s o l v e d the a n i l i n e peak ( r e t e n t i o n time ( r t ) = 2.67 min) from the benzidine peak ( r t = 2.27 min) as can be seen i n Figure 2. Other p o t e n t i a l i n t e r f e r e n c e s were s e l e c t e d for study by l o o k i n g at the expected fragments from the r e d u c t i o n of v a r i o u s dyes. Reduced dye samples were spiked w i t h a n i l i n e ( r t = 2.67 min), £-aminophenol ( r t = 1.97 min), £-phenylenediamine ( r t = 1.93 min) and £-nitroaniline ( r t = 3.16 min). None o f these m a t e r i a l s i n t e r f e r e d w i t h the d e t e c t i o n of the benzidine peak. To determine i f other types o f dyes might i n t e r f e r e w i t h the a n a l y s i s , two s e t s of f i l t e r s were spiked a t low and high l e v e l s s e p a r a t e l y w i t h C . I . D i r e c t Red 28 (13.7 yg and 137 yg), C . I . D i r e c t Blue 53 f o r m u l a t i o n ( o - t o l i d i n e - b a s e d ) (21.2 yg and 212 yg) and C . I . D i r e c t Blue 8 f o r m u l a t i o n ( o - d i a n i s i d i n e - b a s e d ) ( 2 3 - 3 yg and 233 y g ) . R e s u l t s from the analyses showed there were no i n t e r f e r e n c e s from the other dyes present ( F i g u r e 3 and Table I I ) . The c o e f f i c i e n t of v a r i a t i o n f o r the average a n a l y t i c a l method recovery ( C V ^ r ) has been defined by the f o l l o w i n g equation (20) to account f o r the propagation of e r r o r r e s u l t i n g from the d i v i s i o n of the amount of benzidine found on the f i l t e r by the amount i n the l i q u i d sample: CVAMR = L ( C V ) 2 L

where:

+

(GV )2)]1/2 F

C V l = c o e f f i c i e n t of v a r i a t i o n f o r the l i q u i d samples. C V = c o e f f i c i e n t of v a r i a t i o n f o r the f i l t e r samples. F

Three of the dyes ( C . I . D i r e c t Blue 8, C.I. D i r e c t Black 38 and C.I. D i r e c t Brown 95) had been analyzed f o r r e s i d u a l benzidine content during previous work ( 21_). None of the dyes contained s u f f i c i e n t q u a n t i t i e s of r e s i d u a l benzidine t o r e q u i r e a c o r r e c t i o n to be made to the t o t a l amount of benzidine found i n the reduced dye samples.


2.


Benzidine-Based

27

Dyes


-BENZIDINE

0.002 ABSORBANCE UNIT

Figure 2. Chromatogram of a 10-μΣ injection of reduction products of C.I. Direct Black 38 at the 124.0- g level showing resolution of benzidine (reten tion time = 2.27 min) and aniline (reten tion time = 2.67 min). Chromatographic conditions: Radial Pak A; 2 mL/min; 60% methanol/40% phosphate buffer; ambient temperature. h

INJECT 0

1 2 3 4 TIME , (min )

-BENZIDINE

•0.002 ABSORBANCE UNIT

DIANISIDINE ^

/(KTOLIDINE

JU INJECT 0 1 2 3 4 5 6 TIME , (min )

Figure 3. Chromatogram of a 10-μΣ injection of reduction products of a mix ture of C.I. Direct Red 28 (28.8 μg), C.I. Direct Blue 53 (28.6 μg) and C.I. Direct Blue 8 (34.4 μg) showing resolution of benzidine (retention time = 2.33 min), o-dianisidine (retention time = 4.02 min), and o-tolidine (retention time = 4.27 min). Chromatographic conditions as in Figure 2.


C H E M I C A L HAZARDS IN T H E W O R K P L A C E


28

Since no c h e m i c a l l y pure dyes (greater than 95$ p u r i t y ) were a v a i l a b l e , recovery s t u d i e s were done by reducing and a n a l y z i n g a l i q u i d sample o f the dye along w i t h the spiked f i l t e r . These l i q u i d samples were prepared by adding a q u a n t i t y o f dye s o l u t i o n (2-200 yL) equivalent t o one-half the amount present on the spiked f i l t e r t o 1 mL o f the r e d u c t i o n b u f f e r s o l u t i o n . To t h i s s o l u t i o n 10 mg o f sodium h y d r o s u l f i t e i n 1 mL o f the r e d u c t i o n b u f f e r was added. The sample was then analyzed i n the same manner as the spiked f i l t e r sample. The amount o f dye contained i n the f i l t e r and l i q u i d spiked samples was the same, but concentration d i d vary. However, volume v a r i a t i o n s became g r e a t e r than 9% (200 yL a d d i t i o n to 2 mL o f s o l u t i o n ) on only the most concentrated samples. Since t o t a l sample amount was the same f o r f i l t e r and l i q u i d samples, any d i f f e r e n c e s i n sample amounts would be due t o recovery l o s s e s . Table I I Recovery o f Free Benzidine from F i l t e r Samples Containing C.I. D i r e c t Red 28, C.I. D i r e c t Blue 53, and C.I. D i r e c t Blue 8 Loading o f C.I. D i r e c t Red 28 (yg)

Average Recovery ( $ )

Coefficient of V a r i a t i o n ($)

98.0 106.0

13.7 136.7 a

a

4.3

2.6

Averages o f s i x samples.

Based on the analyses o f these l i q u i d samples an approximation o f amount o f the b e n z i d i n e - c o n t a i n i n g dye compound i n a p a r t i c u l a r dye f o r m u l a t i o n could be made u s i n g molecular weight c a l c u l a t i o n s . For 30 samples o f C.I. D i r e c t Red 28 f o r m u l a t i o n (molecular weight (M ) = 696) a t the 15-yg l e v e l , the b e n z i d i n e - c o n t a i n i n g compound was found t o compose 91.2% o f the f o r m u l a t i o n (CV = 1.1$). With the other dyes the f o l l o w i n g compositions were found: C.I. D i r e c t Blue 6 f o r m u l a t i o n ( M = 932), 30.7$ benzidinec o n t a i n i n g compound (23 samples a t the 48.8 - 52.0-yg l e v e l ) CV = 8.5$; C I . D i r e c t Black 38 f o r m u l a t i o n ( M = 781), 41.6$ b e n z i d i n e - c o n t a i n i n g compound (18 samples a t the 14.92-yg l e v e l ) CV = 8.5$; C.I. D i r e c t Brown 95 f o r m u l a t i o n (M = 759), 39.4$ b e n z i d i n e - c o n t a i n i n g compound (24 samples at the 30.8-yg l e v e l ) CV = 16.7$. An examination o f the side-by-side analyses o f l i q u i d and f i l t e r samples should be able t o detect any day-to-day v a r i a t i o n s i n analyses. When the raw data from the l i q u i d samples was s t u d i e d , there appeared t o be such a day-to-day v a r i a t i o n i n the r e s u l t s . However, a n a l y s i s o f variance r

r

r

r



2.


Benzidine-Based

Dyes

i n d i c a t e d no v a r i a t i o n a t the 0.05 l e v e l of s i g n i f i c a n c e . To f u r t h e r i n v e s t i g a t e any p o s s i b l e sources o f v a r i a t i o n , the method was subjected to a ruggedness t e s t as described by Youden ( 2 2 ) , u s i n g benzidine c o n c e n t r a t i o n as the response v a r i a b l e . The HPLC mobile phase b u f f e r and r e d u c t i o n b u f f e r were prepared w i t h 10% a d d i t i o n a l disodium hydrogen phosphate and 1 0 $ a d d i t i o n a l potassium dihydrogen phosphate f o r the high l e v e l s i n the t e s t . Sodium h y d r o s u l f i t e c o n c e n t r a t i o n i n the r e d u c t i o n b u f f e r was a l s o increased by 1 0 $ . The mobile phase makeup was changed t o 3 5 $ b u f f e r / 6 5 $ methanol f o r the high l e v e l . Flow r a t e was increased to 2 . 2 mL. An o l d and new column were used to evaluate column backpressure. Ten yL o f sample s o l u t i o n were removed before a d d i t i o n o f the reductant s o l u t i o n to simulate volume v a r i a t i o n caused by use of out o f c a l i b r a t i o n p i p e t t e s . The major c o n t r i b u t i o n s to the v a r i a t i o n r e s u l t e d from HPLC b u f f e r c o n c e n t r a t i o n , column backpressure and mobile phase composition. According t o instrument s p e c i f i c a t i o n s , mobile phase makeup was c o n t r o l l e d to w i t h i n + 2 $ o f set p o i n t so t h a t any v a r i a t i o n should have been randomized during the t e s t i n g . During l a t e r work i t was found that ambient temperature v a r i a t i o n s (+ 5 ° C) caused changes i n volumes d e l i v e r e d by the pumping system. This volume change amounted to as much as 0 . 2 mL/min. This problem was solved by premixing the mobile phase. Column backpressure could a l s o have been a c o n t r i b u t i n g f a c t o r t o the v a r i a t i o n i n volume d e l i v e r y s i n c e s o l v e n t c o m p r e s s i b i l i t y i s a f f e c t e d g r e a t l y by temperature at lower pressures ( 2 3 ) . Based on t h i s ruggedness t e s t , HPLC mobile-phase b u f f e r c o n c e n t r a t i o n could be a major source of v a r i a t i o n . To minimize t h i s v a r i a t i o n i n the method, care should be used when p r e p a r i n g t h i s b u f f e r . The a n a l y s i s method was evaluated w i t h f o u r d i f f e r e n t benzidine-based dye f o r m u l a t i o n s ( F i g u r e 1 ) . The r e s u l t s of a r e p e a t a b i l i t y study u t i l i z i n g spiked samples are shown i n Table I I I . S p i k i n g l e v e l s were a r b i t r a r i l y chosen but do r e f l e c t the lower l i m i t o f the method. A l l c o e f f i c i e n t s of v a r i a t i o n passed the B a r t l e t t s t e s t (24) f o r homogenity at the 1 $ s i g n i f i c a n c e l e v e l , except C.I. D i r e c t Black 3 8 . With the lowest l e v e l of C.I. D i r e c t Black 3 8 excluded, the c o e f f i c i e n t s of v a r i a t i o n f o r the remaining two l e v e l s were homogeneous. The pooled a n a l y t i c a l c o e f f i c i e n t s of v a r i a t i o n (CV-j) f o r each dye are as f o l l o w s : C.I. D i r e c t Red 2 8 , 0.055; C.I. D i r e c t Blue 6 , 0 . 0 8 3 ; C I . D i r e c t Brown 9 5 , 0 . 1 0 8 ; C.I. D i r e c t B l a c k 3 8 , 0 . 0 5 8 ( 2 levels only). To i n v e s t i g a t e the storage s t a b i l i t y of these dyes, a 28-day storage study was undertaken. F i l t e r s were spiked and d r i e d by the procedure d e s c r i b e d i n the experimental s e c t i o n . S i x t y l i t e r s o f h u m i d i f i e d a i r ( 7 5 $ r e l a t i v e humidity) were drawn through each spiked f i l t e r before storage, u s i n g the p r e v i o u s l y d e s c r i b e d vacuum pump system. During the f


30

C H E M I C A L HAZARDS IN T H E W O R K P L A C E

Table I I I Recovery o f Benzidine from Benzidine-Based Dye F i l t e r Samples A f t e r One-Day Storage

Dye C.I. D i r e c t Red 28


C.I. D i r e c t Blue 6 C.I. D i r e c t Brown 95 C.I. D i r e c t Black 38

a

Loading o f Dye (ug) 13.7 27.3 273.4 15.0 30.0 300.0 12.1 24.3 242.6 6.2 12.4 124.0

Based on s i x samples.

Average Recovery(%) 94.4 101.0 109.0 100.0 98.9 108.0 78.5 96.4 110.p 78.1 91.6 102.0

a

D

Coefficient of V a r i a t i o n (%) 7.2 5.6 2.5 6.6 11.7 4.8 9.2 14.8 6.6 16.6 7.9 2.2

^Based on f i v e samples.

h u m i d i f i c a t i o n step u t i l i z i n g the midget impingers, s e v e r a l o f the samples were a c i d e n t a l l y wetted w i t h the sodium c h l o r i d e s o l u t i o n . This was not n o t i c e d u n t i l the samples were prepared f o r a n a l y s i s . These contaminated samples were not included i n the data a n a l y s i s . A l s o , o c c a s i o n a l plugging o f an impinger w i t h s a l t was noted. This r e s u l t e d i n a p a r t i a l vacuum being created i n the c a s s e t t e . When the impinger was unplugged, the rush o f a i r might p o s s i b l y have dislodged a p o r t i o n o f the dye from the f i l t e r causing greater v a r i a t i o n i n the a n a l y s i s . When s e v e r a l o f the c a s s e t t e s were opened for a n a l y s i s , the spiked dye spot seemed to be l y i n g loose on the f i l t e r . These samples a l s o were not i n c l u d e d i n the data a n a l y s i s . A pump m a l f u n c t i o n i n the HPLC system i n v a l i d a t e d the day-14 analyses f o r C.I. D i r e c t Blue 6, C.I. D i r e c t Brown 95 and C.I. D i r e c t Black 38. The remaining data from the storage study i s shown i n Table IV. The v a r i a b i l i t y o f the r e c o v e r i e s was subjected to a n a l y s i s o f v a r i a n c e a t t h e 0.05 l e v e l o f s i g n i f i c a n c e . S i g n i f i c a n t d i f f e r e n c e s were observed w i t h the r e c o v e r i e s from C.I. D i r e c t Red 28. Duncan's m u l t i p l e range t e s t i n d i c a t e d that the recovery on day 1 was s i g n i f i c a n t l y d i f f e r e n t from the other three analyses a t days 7, 14 and 28. Analyses a t days 7, 14, and 28 were not s i g n i f i c a n t l y d i f f e r e n t . With C.I. D i r e c t Blue 6, C.I. D i r e c t Black 38 and C.I. D i r e c t Brown 95 there were no d i f f e r e n c e s between r e c o v e r i e s a t days 1, 7, and 28 f o r each dye. However, the a n a l y t i c a l c o e f f i c i e n t s o f v a r i a t i o n became l a r g e r on day 28 f o r these three dyes.


2.


Benzidine-Based

Dyes

31

Table IV E v a l u a t i o n of the S t a b i l i t y of F i l t e r Samples Containing Benzidine-Based Dyes Stored Up to Twenty-Eight Days Loading (yg)


Dye C.I. D i r e c t Red 28 C.I. D i r e c t Blue 6 C.I. D i r e c t Brown 95 C.I. D i r e c t Black 38

13.7 15.0 12.1 6.2

a

Day 1 101.0° (+ 8.7) 103.0° (+ 12.7) 71.4 ( + 7.0) 78.1° (+ 16.1)

Average Recovery ($) Day 14 Day 28 Day 7 83.8° (+ 16.0) 105.0 (+ 7.0) 73.9 (+ 8.6) 68.8 (+ 8.6)

86.4

Development of an Analytical Method for Benzidine-Based Dyes

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