Detection Limits of Chemical Spot Tests toward Certain Carcinogens on Metal, Painted, and Concrete Surfaces R. W. Weeks, Jr.,* B. J. Dean, and S. K. Yasuda Industrial Hygiene Group, Health Division, Los Alamos Scientific Laboratory, University of California, Los Alamos, N.M. 87545
The formation of chromogenic and fluorogenic derivatives of certain aromatic amines designated by OSHA as cancersuspect agents has allowed these compounds to be detected at very low levels through chemical spot tests. Well-defined painted, metal, and concrete were used as standard surfaces to evaluate the limit of detection values of these compounds as a function of both the visualization reagent and the method of detection. The chromogenic reagent of choice was Ehrlich’s reagent and the fluorogenic reagents were either fluorescamine or o-phthalaldehyde. The limit of detection values for these compounds in terms of grams of analyte per cm2 of surface being analyzed ranged from the low nanogram to 5-pg level depending upon the compound, sampling technique, and surface involved. The combination of sampling technique and visualization reagent employed extend the limit of detection to levels considerably lower than previously reported.
T h e Occupational Safety and Health Administration (OSHA) has recently imposed strict procedural guidelines in t h e use and handling of 14 cancer-suspect agents (CSA’s) (1). Because of a need t o sample a n d analyze these compounds within t h e working environment, a project was undertaken t o fulfill this objective. As part of t h e program, sensitive spot tests and sampling methods were required for trace analyses of nonvolatile carcinogens found on metal, painted, and concrete surfaces which simulated those conditions and complications ( 2 )often encountered at industrial sites. Of primary concern were those carcinogens and other related compounds containing amino a n d nitro functional groups. Our investigation proceeded along two pathways, namely: (1)chromogenic spot tests readily recognized by the unaided eye, and (2) formation of fluorescent derivatives made visible by irradiation with an ultraviolet light source. In both cases, simplicity and sensitivity were prerequisites for the sampling and detection methods. T o meet these requirements, various chemical reactions were tested and they include: 1. Ehrlich’s reagent ( 3 )(p-dimethylaminobenzaldehyde) or ethyl Ehrlich’s reagent ( 4 ) (p-diethylaminobenzaldehyde) with primary amines. 2 . p-Dimethylaminocinnamaldehyde (3, 5 ) with primary amines. 3. Chloranil (6) (tetrachloro-1,4-benzoquinone) with primary aryl amines. 4. Fluorescamine (Fluram), (7-10) 4-phenylspiro(furan2 [3H]l’-phthalan)-3,3’-dione with primary amines. 5 . o-Phthalaldehyde (11, 12) with primary aromatic amines. Among these reactions, we have found several sensitive detection methods for the carcinogens of interest and report t h e conditions to achieve detection limits previously not accomplished with these compounds, i.e., nanogram quantities.
EXPERIMENTAL The limit of detection (LOD) for these chemical spot tests was evaluated on five different matrices, namely, paper, metal, concrete,
and painted surfaces and also silica gel thin-layer chromatography (TLC). Because of the nebulous nature of these generic names, it was necessary to define the terms for this investigation. The definitions will be given later in this section. Apparatus. Visualization of fluorescent spots was accomplished using a Model CC20 Portable UV Dark Room (Brinkmann Instruments) or a UVSL-25 Mineralight (Ultra-Violet Products, Inc.). Both instruments were capable of short (254 nm) and long (366 nm) wavelength radiations. Surface textures were measured in the laboratory with a surface roughness scale (Catalogue No. 8665947G1, General Electric Co., Schenectady, N.Y.). Borosilicate laboratory glassware was used throughout these studies for reagent preparation. Reagents were stored in either clear borosilicate glass or amber flint glass vessels. Rectangular Desaga tanks were used for TLC work. Spotting of plates and of filter papers was accomplished with a microliter pipet (Scientific Manufacturing Industries) equipped with disposable tips. Materials and Reagents. Model Compounds. Aniline, o-chloroaniline, p -chloroaniline, 4,4’-methylenedianiline, o -chlorotoluidine, 2-aminobiphenyl, o-tolidine, o-toluidine, m-toluidine, p -toluidine, chlorobenzene, and methanol were of reagent or analyzed grade and were obtained from J. T. Baker Chemical Co., Eastman Organic Chemicals, Aldrich Chemical Co., or Matheson, Coleman and Bell. m-Chloroaniline, m-chlorotoluidine, and N-methylaniline were Eastman Practical grade. N,N-Dimethylaniline and ethanol were purchased from Eastman and U.S.I. Industrial Chemicals Co., respectively. Visualization Reagents. The visualization reagents, p-dimethylaminobenzaldehyde, p -diethylaminobenzaldehyde, p-dimethylaminocinnamaldehyde, chloranil (tetrachloro-l,4-benzoquinone), and fluorescamine,were of reagent quality and were obtained from Aldrich Chemical Co., Pierce Chemical Co., Applied Science Laboratories, J. T. Baker Chemical Co., or Eastman Organic Chemicals Co. oPhthalaldehyde was Baker grade from J. T. Baker. m-Phthalaldehyde was from Aldrich and reported as 97% pure. p-Phthalaldehyde obtained from Matheson, Coleman and Bell had a melting point of 115-116 “C. Zinc powder was analytical reagent grade from Mallinckrodt Chemical Works. Cancer-Suspect Agents. 4,4’-Methylenebis(2-~hloroaniline), benzidine, a-naphthylamine, &naphthylamine, and 4-aminobiphenyl were reagent grade and purchased from either Litton Bionetics, Eastman Organic Chemicals, or ICN K&K Laboratories. 4-Dimethylaminoazobenzenewas indicator grade and was purchased from Aldrich Chemical Co. 3,3’-Dichlorobenzidine was technical grade material purchased from ICN K&K Laboratories. 4-Nitrobiphenyl was 98% pure and obtained from Sigma Chemical Co. Those plates used in TLC studies were 20 X 20 cm, 0.25-mm silica gel with a fluorescent indicator (Machery Nagel distributed by Brinkmann Instruments, Inc.). The filter paper of choice was 7-cm diameter Whatman 42, Whatman, Inc., Clifton, N.J. Visualization reagents were prepared as follows: Fluorescamine: 35 mg diluted to 100 ml with tetrahydrofuran. Isomeric phthalaldehydes: 1 mg/ml in glacial acetic acid. Chloramine-T: 5 g/lOO ml of water. p-Dimethylaminocinnamaldehyde (PDCA):1g of PDCA plus 8 g of trichloroacetic acid were diluted to a volume of 100 ml with methyl alcohol. Chloranil: 0.2 g/100 ml of chlorobenzene. p-Dimethylaminobenzaldehyde: 0.25 g/100 ml of 0.25 N ethanolic HCl. Metals. Metals used as standard surfaces in this study were American Iron and Steel Institute (AISI) austenitic stainless steel types 304,316, and 347 (13).Surfaces were milled to afford a number of different textures ranging from quite smooth to rather rough (14). These surface roughness values were determined quantitatively by measuring with a profilometer and are expressed in units of microinches (1 pinch = 25.4 nm) representing an arithmetic average between the mean and the actual surface of a material. Concrete. Portland cements meeting ASTM type I, IA, 11, or IIA specifications (15, 16) were used as standards for the concrete surfaces. The cement was blended with 20/40-mesh sea sand (Sargent
ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER 1976
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Table I. Color of Cancer-Suspect Agent Chromogenic Derivative Cancer-suspect agent
Ehrlich’s reagent derivative color
4,4’-Methylenebis(2-~hloroaniline) Benzidine 3,3’-Dichlorobenzidine @-Naphthylamine @-Naphthylamine 4-Aminobiphenyl 4-Nitrobiphenyl (after Zn/HC1 reduction)
Yellow Orange Orange Yellow Yellow Yellow Yellow
Chloranil derivative color
p -Dimethylaminocinnamaldehyde derivative color
Blue/purple Bludgray Turquoise Blue/purple Blue Blue/gray
Red/magenta Purple Magenta Red Magenta Red
...
...
Derivative upon addition of acid Dimethylaminoazobenzene
Magenta
Table 11. Color of Fluorogenic Derivative upon Irradiation with 366 n m Ultraviolet Light Fluorogenic reagent Cancer-suspect agent
Fluorescarnine Yellow Yellow Yellow Yellow Yellow Yellow Yellow
4,4’-Methylenebis(2-~hloroaniline) Benzidine 3,3’-Dichlorobenzidine a-Naphthylamine 0-Naphthylamine 4-Aminobiphenyl 4-Nitrobiphenyl (after Zn/HC1 reduction) Welsh) and water to form a concrete aggregate of uniform consistency. The concrete was molded in 10-cm plastic Petri dishes or cardboard 1-pint cylindrical ice cream containers. When removed from these vessels, the concrete surface was smooth, Le., U3 or better as defined in the US.Bureau of Reclamation’s Concrete Manual (17). These surfaces are not unlike typical smooth concrete floors in industrial settings. A surface roughness scale was used to determine the approximate texture of the concrete in microinches. A “rough” concrete surface was approximately 500 pinches and a “smooth” surface texture was determined to be approximately 125 pinches. Painted Surfaces. Because of the plethora of possible paint types, and the likelihood of reaction interferences in this type of work, it was necessary to define the paints rigidly. Accordingly, wood surfaces with at least two coats of the designated paint were used as test panels. Paints selected for use as standard surfaces were alkyd enamels and were specified as those meeting the following Federal Paint Specifications: Federal Specifications TT-E-489 (E or F) (18) or TT-E-291B (19).
Class A G or L Composition and meeting pigmentation requirements per Federal Standard Number 595 as follows: Color Number Color 15102 Medium blue 16187 Medium gray 17815 White An additional black low sheen paint meeting Federal Specifications TT-P-32a (20) Type I requirements was used as a defined black surface. This was a paint which may be used to coat blackboards. Procedures. Primary Aromatic Amines FILTERPAPER.Using a microliter syringe, a known amount of amine solution of a given concentration was applied directly to the center of a filter paper. To this were applied 10 drops of ethanol to disperse the model compound over an area of roughly 3 cm2. (The results were normalized such that they are reported in terms of 1cm2 of area.) After the ethanol evaporated, visualization reagent from a medicine dropper was streaked across the filter paper and spotted area. This provided a blank adjacent to the sample area. For those derivatives visible to the unaided eye, a reaction period of about 3 min was allowed to elapse before examination. For fluorescent derivatives, the reaction period was 6 min. 2228
o -Phthalaldehyde
... Blue
*..
... Blue Blue Blue
CONCRETE.For the CSA homologues and analogues, testing of concrete surfaces was effected by “leaching” the compound of interest from the surface with solvent impregnated filter paper. To do this, a filter paper was placed upon the surface and five drops of methanol were applied to the paper’s center. The paper was kept in intimate contact with the surface until it dried. It was then removed and visualization reagent applied (to the surface of the paper which had contacted the concrete) in the same fashion as for filter paper, Le., streaking from one edge of the filter paper across the spotted area and to the other edge. After a suitable reaction period, the paper was examined for color formation. This technique will be referred to as the “leaching” technique. METAL.Three techniques were tested in early studies to evaluate the techniques themselves on metal surfaces. The first, and so-called “direct” technique, involved the application of visualization reagents directly onto the metal surface. The second technique was the “leaching” procedure mentioned earlier in this report. The third, or “swipe”, method employed unidirectional wiping of approximately 10 cm2of the surface with the filter paper and then application of the visualization reagent. In each of the above methods, the filter paper was allowed to dry before examination. Later tests used only the “leaching” method because of its overall sensitivity relative to the other methods tested here. PAINTED SURFACES.Only the “leaching” procedure was used to obtain LOD values on painted surfaces. THIN-LAYER CHROMATOGRAPHY. Plates were spotted with 5 pl of solutions of given concentrations. Contrary to laboratory reference testing, in field survey and monitoring work, a combination of the swipe and leaching techniques is r e c o m m e n d e d k s of methanol or ethanol and then swiped over a relatively large area, Le., approximately 500 cm2.This effectively concentrated the analyte and gave a subsequent enhancement of sensitivity. 4-Nitrobiphenyl (4-NBP). FILTER PAPER.Evaluations were performed by dusting approximately 0.1 g of zinc powder onto one side of the 7-cm (diameter) filter paper and “brushing” it into the paper via a spatula. A 5-pl sample of the desired 4-NBP concentration was placed upon the zinc-coated surface of the filter and 2 drops of 0.1 N HCl were then added to the sample. Visualization was effected by appropriate application of the same reagents used for the primary aromatic amines. These reagents were applied to the zinc-coated surface and the filter paper was then examined on the uncoated surface.
ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER 1976
~~
~~
~~~~
~
~~
Table 111. Limit of Detection Values for Aromatic Amines Visualized on Filter Paper a via Chromogenic Derivatization Chromogenic visualization reagent, limit of detection values (ng/cm2) p-Dimethylaminobenzaldehyde (Ehrlich’s reagent)
Compound studied 1. Aniline
2. 3. 4. 5. 6. 7.
o-Chloroaniline m-Chloroaniline p-Chloroaniline o-Toluidine m-Toluidine p-Toluidine 8. o-Tolidine 9. 4,4’-Methylenedianiline 10. 4,4’-Methylenebis(2-~hloroaniline) (MOCA) 11. BenzidineC 12. 3,3’-Dichlor~benzidine~ 13. a-NaphthylamineC 14. P-NapthylamineC 15. 4-Aminobiphenylc Whatman 42 filter paper.
p-Diethylaminobenzaldehyde
Chloranil
30 150 30 30 150 30 30 30 30 150
150 800 80 80
30 30 800 30 30
... ... ...
800 800
...
800
p -Dimethylaminocinnamaldehyde
Chloroamine-T
30 30 30 30 30 30 30 30 150 30
...
30 30 30 30 30
30 30 800 800 800
800 800 800 800 800 800 800 800 150
...
80 150 150 80
...
800
800 800
...
...
...
..*
... ... ... ... ... b
Not detected a t the level of 800 ng/cm2 Compounds designated as Cancer-Suspect Agents by the
U.S. Occupational Safety and Health Administration (OSHA). Table IV. Limit of Detection Values for Aromatic Amines Visualized on Filter Paper via Fluorogenic Derivatization Fluorogenic visualization reagent, limit of detection values (ng/cm2) Isomeric phthalaldehydes Compound studied
Fluorescamine
0-
m-
3 30 3 3 3 3 3 5 3 3
30 b 30 30 800 30 30
b
3 3 30 3 3
1. Aniline 2. o-Chloroaniline
m-Chloroaniline p-Chloroaniline o -Toluidine m-Toluidine p-Toluidine o-Tolidine 4,4’-Methylenedianiline 4,4’-Methylenebis (2-chloroaniline) (MOCA) 11. BenzidineC 12. 3,3’-Dichlorobenzidine 13. a-Naphthylaminec 14. @-Naphthylamine 15. 4-AminobiphenylC 3. 4. 5. 6. 7. 8. 9. 10.
a
Whatman 42 filter paper.
Not detected at the level of 800 ng/cm’.
METALAND PAINTED SURFACES. The leaching technique was used for these determinations. The zinc-coated paper was placed zinc sidsup upon the surface being tested. Five drops of methanol were placed upon the paper to “leach” the 4-NBP from the surface. After drying, 2 drops of 0.1 N HC1 were added to the zinc-coated surface of the paper. This was allowed to dry and the appropriate visualization reagent was then applied to the zinc. The uncoated surface of the paper was then examined for color formation. CONCRETE. The LOD values for 4-NBP on concrete were determined by the swipe technique. The zinc-coated filter paper was swiped zinc side down over the surface being studied. Two drops of 0.1 N HCl were added to the zinc reductant and, after drying, the visualization reagent was applied directly to the zinc-coated side of the paper. The filter paper was then examined for color formation opposite the zinc-coated surfaces. 4-Dimethylaminoazobenzene ( N D A A B ) . FILTER PAPER.The limit of detection value of 4-dimethylaminoazobenzene on Whatman 42 filter paper was determined as follows: A 5-fil sample of the desired concentration of NDAAB was placed upon the filter. A 1-2 ml volume of 1 N HC1 was streaked across the filter paper and the paper then
Pb
... ...
... ...
b
b b
b
...
... ... ...
... ... ...
30 800
800 800
800 800
3 800 80 3 3
80 150 80 80 80
80 80 80 80 80
See Table 111, Footnote c.
allowed to dry. If NDAAB were present, a magenta color resulted. METALAND PAINTED SURFACES. T o determine the NDAAB limit of detection value on metal or painted surfaces via the leaching technique, 5 gl of the desired NDAAB concentration was placed upon the surface of interest. Filter paper was then placed atop the doped area and five drops of methanol were added to the center of the paper, while ensuring intimate paper-surface contact. A positive test was indicated through formation of a transient magenta color after the addition of 1N HC1. CONCRETE. Attempts to determine LOD values for this compound on concrete did not give values lower than those values at which the compound per se was visible to the unaided eye, Le., it was self-indicating. E’ualuatzon of Fzlter Papers. Using micro and submicro quantities of analyte, derivatives of 0-,m-, and p-chloroaniline were prepared with Ehrlich’s reagent on a variety of filter papers. Based upon color intensity of the developed spots, Whatman 42 paper was selected for our work. This is not meant as a product endorsement, but rather a statement of fact. Reagent Shelf-Life Eualuatzon. Chromogenic (chloranil,Ehrlich’s
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Table V. Aromatic Amine Cancer-Suspect Agent-Limit Technique
of Detection Values on Stainless Steel via Leaching Visualization reagent, limit of detection (ng/cm2) Fluorogenic
Cancer-suspect agent
Chromogenic, Ehrlich’s reagent
Fluorescamine
o-Phthalaldehyde
4,4’-Methylenebis(2-chloroaniline) Benzidine 3,3’-Dichlorobenzidine a-Naphthylamine /3-Napththylamine 4-Aminobiphenylc
150 30 30 150 30 30
6 6 6 30 6 6
... 6
... ... 15 6
a Identical values were obtained for a given analyte/visualization reagent pair on all stainless steels studied. These were shiny AIS1 Stainless Steel Types, No. 304,316, and 347 at surface textures of 0.5-3.5,12-22,30-72 and 90-260 winches. Also included was a “rough” aged (non-shiny oxidized) surface whose texture was 120-250 pinches. The “leaching” technique was performed per: 1. Place filter upon test surface. 2. Apply 5 drops of methanol to center of paper being certain paper maintains intimate contact with surface. 3. After methanol has evaporated from the paper, lift paper from surface and by using a suitable dropper, streak visualization reagent across the paper. 4. Examine after suitable time period. 4-Aminobiphenyl is of itself fluorescent with an excitation maximum at 290 nm (33). Under the conditions of the present work in which 254 or 366 nm irradiations were used, an enhancement of the LOD value was obtained by fluorogenic derivatizations with fluorescamine or Ehrlich’s reagent (ER) (31). Thus, the orange fluorescence obtained while irradiating the ER derivative at 254 or 366 nm allowed the ER to be either a fluorogenic or a chromogenic derivatization reagent.
Table VI, Limit of Detection Values for Aromatic Amines on Concrete via Leaching a Technique Visualization reagent, limit of detection values (ng/cm2) Compound studied
p -Dimethylaminobenzaldehyde
p-Dimethylaminocinnamaldehyde
15 30 15 15
15 150 30 150
C C
C
...
C C
...
Aniline o-Chloroaniline rn-Chloroaniline p-Chloroaniline o -Tolidine
4,4’-Methylenebis(2-~hloroaniline) (MOCA) Benzidined 3,3’-Dichlorobenzidine a-Naphthylamined P-Naphthylamined 4-Aminobiphenyld
I
.
.
... ...
C C
*..
C
Fluorescamine o-Phthalaldehyde 6 6 6 6 6 C C
C C
... ... ... ...
... ... C
...
...
C
C
C
C
No difference detected between rough and smooth concrete. Rough concrete in the present work is a See Table V, Footnote b. defined as having a surface texture of 500 winches. Likewise, smooth concrete is that which has a surface texture of 125 winches. ‘ Not detected at a concentration of 150 ng/cm2. See Table 111, Footnote c. reagent, and chloramine-T) and fluorogenic (fluorescamine and ophthalaldehyde) reagents were stored at room temperature in colorless and brown flint glass bottles, respectively. Their shelf-life limits were studied as a function of reagent age by determining their LOD values on Whatman 42 filter paper as mentioned earlier in this report.
R E S U L T S AND D I S C U S S I O N Aromatic Amines. Whatman 42 filter paper was used for this work as it gave the highest composite relative intensity of spots formed in the filter paper evaluation test. To evaluate limits of detection, various visualization reagents were analyzed for their sensitivity to aromatic amines on this filter paper. The color of the derivatives formed and results of these determinations are given in Tables I, 11, 111, and IV. Based upon their sensitivities as shown by these LOD values, certain of the visualization reagents were chosen for further evaluations on metal, concrete, and painted surfaces. Particularly, the chromogenic reagent of choice was Ehrlich’s reagent and the fluorogenic reagents were fluorescarnine and o-phthalaldehyde. These results will be reported later in this paper. 2230
I n each reaction based upon amino functionality, it was important to note the potential for “false positive” results. Interferences for either the fluorogenic or chromogenic derivatives may be caused by other primary amines (3,9,10, l l ) , amino acids (9, 12), isocyanates (21) (either directly or by , (23,24), hydrazines ( 2 5 ) , hydrolysis to amines ( 2 2 ) ) indoles hydrogen peroxide (26), tryptophan (27), pyrroles (23),or phenols (28, 29) which coexist with the primary analytes. Species which are, of themselves, normally capable of fluorescence without derivatization may, likewise, cause false positives. As might be expected from electronegativity and/or electronic steric effects, the o -substituted anilines have higher limits of detection than m-, p - , or nonsubstituted aniline. Conceivably, these effects hinder the extent of reaction and cause the limit of detection values t o be higher in the time frame of these experiments. An important observation relevant to these studies was that a given visualization reagent may form more than one color with a given analyte. As reported earlier (24,30),the color of the chromogenic derivatives formed has, in some cases, been
ANALYTICAL CHEMISTRY, VOL. 48, NO. 14, DECEMBER 1976
Table VII. Limit of Detection Values: Aromatic Amine Cancer-Suspect Agents on Concrete via Direct Technique a Visualization reagent limit of detection values (ng/cm2) p -Dimethylaminobenzaldehyde
Compound studied
Rough concreteb
4,4’-Methylenebis(2-chloroaniline) Benzidine 3,3’-Dichlorobenzidine a-Napthylamine @-Naphthylamine 4-Aminobiphenyl
Smooth concrete 3000 5000 2000 2000 5000 5000
C
5000 C C
5000 5000
Fluorescamine Rough concrete
Smooth concreteb 5000 500 5000 5000 500 500
500 200 500 500 500 200
a Direct application of visualization reagent to surface. Rough concrete: See Table VI, Footnote b. Smooth concrete: See Table VI, Footnote b. Not detected at the level of 5000 ng/cm2.
Table VIII. Limit of Detection Values: Aromatic Amines on Painted Surfaces via Leaching Technique a Limit of detection values (ng/cm2) Flat Black
Compound studied Aniline o-Chloroaniline m-Chloroaniline p-Chloroaniline o-Tolidine 4,4‘-Methylenedianiline 4,4’-Methylenebis(2chloroani1ine)f Benzidine/ 3,3’-Dichlorobenzidinef a-Naphthylamine! @-Naphthylaminef 4-Aminobiphenylf
p-Dimethylaminobenzaldehyde
Machine Gray‘ p-DimethylaminoFluobenzrescamine aldehyde
Blued p -DimethylaminoFluobenzrescamine aldehyde
Fluorescamine
30 30 15 15 30 150 150
30 e 150 150 150 15 15
15 30 15 15 15 30 150
30 e 150 150 150 5 15
30 . 150 30 30 30 150 150
30 e 30 150 150 5 15
150 150 150 150 150
15 15 30 15 15
150 150 150 150 150
15 15 30 15 15
150 150 150 150 150
15 15 30 15 15
See Table V, Footnote b. Flat black, Federal Specification TT-P-32A, Wellborn Paint Manufacturing Co., Albuquerque, N.M. Machine Gray, Federal Specification TT-E-491B, Class A, Composition G, The Glidden Company, Cleveland, Ohio. Blue, Color 15102, Federal Specification TT-E-489F, Class A, Composition G, Enmar, Inc. Wichita, Kansas. e Not detected at a concentration of 150 ng/cm2. f See Table 111, Footnote c . found to be a function of the ratio of the reactants in the system. I t is probable that the observed color depended upon the reaction product and indicated that the compound formed was a function of the reactant concentrations. In the present work, the analyte concentration was always a t low levels, Le., less than 5 pg/cm2 of surface being analyzed. This kept the analyte/visualization reagent (VR) mole ratio relatively constant and is likely the reason that for a given analytenTR pair only a single color was observed in this work. T o duplicate surfaces one would likely encounter in industrial sites, the work was extended beyond the “ideal” situation of laboratory filter paper. As previously mentioned, spot tests were performed on metal, concrete, and painted surfaces. Stainless steels of surface textures varying from quite smooth to quite rough were chosen in order t o provide standard metal surfaces. These were chosen to evaluate the effect of surface texture as a reaction parameter. Additionally, stainless steels of different transition element content were chosen to determine what effects these might have had on the LOD values. T o determine this, the four aromatic amines, o-chloroaniline, p-chloroaniline, o-tolidine, and 2-aminobiphenyl, were studied on various surface textures (0.5-260 pinches) by the three techniques mentioned earlier, Le., direct, leach, and swipe. As previously shown (321,the leaching method gave the
lowest LOD. In most cases, the LOD (31, 32) by a given method for a given compound was independent of surface texture and metal type. Exceptions were found, however, in swipe tests with o-tolidine and Ehrlich’s reagent on stainless steel 316 where LOD values were larger a t higher surface texture values (90-260 pinches). Other LOD inconsistencies were found on “rough” (a profilometer surface measurement of 120-250 pinches upon an aged, rather than a freshly machined surface) surfaces. Catalytic effects by transition metal oxides of the steels may have altered the LOD values relative to those of non-oxidized surfaces. Table V reports LOD values on stainless steel for those aromatic amines on the OSHA list (1) of cancer-suspect agents. As seen in this table, the values obtained with fluorescamine or o-phthalaldehyde (OPA) were all 6 ng/cm2, except for a- and @-naphthylamines.These species gave values of 30 ng/cm2 for a-napthylaminelfluorescarnine and 15 ng/cm2 for @-napthylaminelOPA.T h e OPA LOD values for MOCA, 3,3’-dichlorobenzidine and a-napthylamine were sufficiently high in earlier studies (Table IV) t h a t they were not investigated for use on metal surfaces. Ehrlich’s reagent LOD values for these compounds were routinely fivefold higher than those for fluorogenic derivatives. However, they were in all cases less t h a n 200 ng/cm2 and thus considered quite sensitive, Tables VI and VI1 report the limit of detection values for
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Table IX. Limit of Detection and RfValues for Representative Aromatic Amines a , b via Silica Gel Thin Layer Chromatography Visualization reagent Aniline o-Chloroaniline
m -Chloroaniline p -Chloroaniline o-Tolidine 4,4’-Methylenedianiline
Fluorescamine 0.01 1500 000 0.22 0.1 150 000 0.41 0.1 1 5 0 000 0.28 0.1 1 5 0 000 0.23 0.01 1:500 000 0.04 0.01 1500 000 0.03
Chloranil
p-Dimethylaminobenzaldehyde
p-Dimethylaminocinnamaldehyde
0.1 1:50 000 0.24 0.1 1:50 000 0.43 0.1 1 5 0 000 0.31 0.1 1:50 000 0.26 0.1 1 5 0 000 0.08 0.1 1:50 000 0.03
0.1 150 000 0.19 0.1 1:50 000 0.41 0.1 1 5 0 000 0.28
0.1 1 5 0 000
0.19 0.5 1 : l O 000 0.43 0.1 150 000 0.30 0.1 1:50 000 0.24 0.1 1 5 0 000 0.04 0.1 1 5 0 000 0.03
0.1
1 5 0 000 0.23 0.1 1 5 0 000 0.04 0.1 1:50 000 0.02
0.1; 1:50 000; 0.19 means a volume of solution containing 0.1 pg of the compound of interest at a dilution of 1:50 000 in methanol had an Rf value of 0.19. Solvent system: CHC13.
Table X. Limit of Detection Values for 4-Dimethylaminoazobenzene on Various Surfaces Using 1N HC1 as Visualization Reagent Limit of detection values (ng/cm2) Filter papera
Metal surface
...
Leaching technique Direct application of visualization reagent
Painted surface‘
15
...
30
...
30
Concrete
...
lOOOd
Whatman 42. See Table V, Footnote a . Paints used are defined in Table VIII, Footnotes b, c, and d. benzene was self-indicating at a concentration of 1000 ng/cm2 on the concrete surface.
4-Dimethylaminoazt
Table XI, Limit of Detection Values for 4-Nitrobiphenyl on Given Surfaces Using Zn/HCl Reduction Limit of detection values (ng/cm2)
Visualization reagent Ehrlich’s reagent Fluorescamine o-Phthalaldehyde
Filter paper” (Direct applicationd of visualization reagent)
Metals (Leaching techniqued)
Paints‘ (Leaching techniqued)
80 30 30
30 30 30
150 150 150
Concrete (Swipe techniquee) 5000 5000
5000
Table X, Footnote b. ‘ Table VIII, Footnotes b, c, and cl. See Table V, Footnote b. e A filter paper was wiped a Whatman 42. across the surface being monitored, removed from the surface, visualization reagent streaked across paper, and the paper then examined.
representative aromatic amines on concrete surfaces. Because of the complex chemical composition of concrete, the possibility of interferences was anticipated. However, for all compounds tested under the conditions reported here, it was possible to obtain results in the microgram or submicrogram per square centimeter range via a t least one combination of sampling technique and detection reagent. Interestingly, relative to the mononuclear aromatic amines, much higher LOD values were obtained on concrete surfaces for those amines containing two aromatic nuclei. Conceivably, relative to the mononuclear amines, the binuclear molecules had interacted (chemisorbed) with the concrete t o such a n extent t h a t the molecule was not as readily available for reaction with the visualization reagents. Certain K--A bondings 2232
of the concrete/analyte system could have led t o these interactions. For the present evaluation, the direct technique gave the lowest LOD value for the “cancer-suspect agents” on concrete surfaces. These results are given in Table VII. Table VI11 presents results obtained for aromatic amines on painted surfaces. As with other surfaces, the possibility for interference manifested itself. Particularly, the potential for quenching fluorescence decay modes with a subsequent elevating of the LOD for fluorescamine did exist. A shift in the fluorescence emission peak was noticed from the chartreuse to the purple spectral region for certain cases. T h e fact t h a t LOD values for a given compound and visualization reagent did vary from one paint to another showed t h a t secondary surface interactions were playing a role, albeit minor. With
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few exceptions, the fluorogenic detection mode afforded greater sensitivity t h a n the chromogenic mode on these painted surfaces. Thin-layer chromatographic results for certain homologues and analogues of the cancer-suspect agents are given in Table IX. For all compounds studied, LOD values well below 1 pg were readily detected by both chromogenic and fluorescent derivatization. This is in agreement within a n order of magnitude of the LOD value for similar species reported previously ( I O ) . 4-Dimethylaminoazobenzene. 4-Dimethylaminoazobenzene was readily protonated by 1 N HC1 to form a vivid magenta species on filter paper, metal, or painted surfaces. As shown in Table X, this procedure gave LOD values of 15 ng/cm2 on metal surfaces. Both the filter paper test and the painted surfaces gave LOD values of 30 ng/cm2. As observed for most compounds reported herein, the LOD value on concrete was considerably higher than on the other surfaces tested. T h e multitude of possible interfering reactions between HCl and the constituents of concrete may cause this higher LOD value. Fortuitously, the compound was self-indicating as a yellow-brown color a t the level of 1000 ng/cm2 on the concrete surface. 4-Nitrobiphenyl. 4-Nitrobiphenyl was visualized as a n aromatic amine after reduction with Zn/HCl. As shown in Table XI, both the chromogenic and fluorogenic derivatives were detected a t the sub-microgram level on all surfaces tested except concrete. Quite likely the values reported were slightly higher than expected because the chromogen's color was somewhat obscured by the unreacted zinc on the filter paper. Visualization Reagent Shelf-Life. Ehrlich's reagent, chloranil, p-dimethylaminocinnamaldehyde, fluorescamine, and o-phthalaldehyde were evaluated for sensitivity as a function of visualization reagent age. The evaluation consisted of measuring the LOD values for MOCA, benzidine, and 3,3'-dichlorobenzidine over a time period of six months. Except for a n increase after one month with fluorescamine and p -dimethylaminocinnamaldehyde, the reagents showed shelf lives of at least six months. The chromogenic reagent of choice, because of its sensitivity and long shelf-life was Ehrlich's reagent, p -dimethylaminobenzaldehyde in 0.25 N ethanolic HCl. Similar considerations implied fluorescamine and/or o -phthalaldehyde be employed as fluorogenic reagents in field situations.
SUMMARY Sensitive chemical spot tests employing a "swipe" technique have been developed for detecting a variety of primary aromatic amines and related compounds on painted, metal, and concrete surfaces. In addition to aromatic amines on the OSHA list ( I ) of "cancer-suspect agents", the compounds, 4-nitrobiphenyl (readily reduced to the amine) and 4-dimethylaminoazobenzene, were readily detected by the techniques reported here. For most of the compounds and surfaces studied, limit of detection values have been obtained a t the level of less than 200 ng of material per cm2 of surface being tested. The tests in which fluorescent derivatives were formed were generally more sensitive t h a n those relying upon highly colored derivatives. There did, however, exist situations where fluorescent quenching raised the fluorescence LOD value and, thus, showed the danger of relying upon a single test method. By employing both chromogenic and fluorogenic derivatization techniques as complementary methods, the analytical confidence of a test series was enhanced. Whereas the chromogenic tests are frequently not as sensitive as the fluorogenic, positive chromogenic tests are obtained a t concentration levels where quenching may obviate the fluorescence tests. Hence,
if a n initial test by one method has given a negative value, the importance of using both types of tests is seen. For field testing of laboratory and working envifonments, the swipe test with alcohol moistened filter paper will provide the lowest limits of detection. By sampling a large area (approximately 500 cm2), the analyte was effectively concentrated on the filter paper. A simple, yet sensitive thin-layer chromatographic scheme has been presented as have been shelf-life studies for the visualization reagents employed in these tests.
ACKNOWLEDGMENT T h e authors thank A. W. Teass, L. J. Doemeny, and R. H. Hill, Jr., of NIOSH (Cincinnati, Ohio), for helpful discussions during the course of this study.
LITERATURE CITED (1) Fed. Regist., 39 (20), 3756 (January 29, 1974). (2) F. Feigl (translated by R. E. Oesper), "Specific, Selective and Sensitive Reactions", Academic Press, New York, N.Y., 1949, p 433. (3) F. Feigl (translated by R. E. Oesper), "Spot Tests in Organic Analysis", 7th ed., Elsevier Publishing Company, New York, N.Y., 1966, p 243. (4) S. K. Yasuda, J. Chromatogr., 104, 283 (1975). (5) R. T. Morrison and R. N. Boyd, "Organic Chemistry", Allyn and Bacon, Boston, Mass., 1973, p 859. (6) F. Feigl, translated by R. E. Oesper, "Spot Tests in Organic Analysis", 7th ed., Elsevier Publishing Company, New York, N.Y., 1966, p 249. (7) S. Udenfriend, S. Stein, P. Bohlen, and W. Dairman, "New Fluorometric Procedure for Assay of Amino Acids, Peptides and Proteins in the Picomole Range" in "Chemistry and Biology of Peptides", Ann Arbor Science Publishers, Ann Arbor, Mich., 1972;~ 655. (8) P. Bohlen, S. Stein, W. Dairman, and S. Udenfriend, Arch. Biochem. Biophys., 155, 213-220 (1973). (9) K. Imai, P. Bohlen, S. Stein, and S. Udenfriend. Arch. Biocbem. Biophys., 161. 161-163 (1974). (10) J. Sherma and G. Marzoni, Am. Lab., 6 (lo), 21 (1974). (11) W. Troll and E. Rinde, "Metabolism of Benzidine and Benzidine Azo Dyes", National Cancer institute Carcinogenesis Program, Third Annual Coliaborative Conference, Orlando, Fla., February 2-5, 1975. (12) M. Roth, Anal. Chem., 43, 880 (1971). (13) "Stainless Steel Handbook", Alleqhenv . Ludlum Steel CorDoration. Pittsburgh, Pa., 1959, p 12. (14) J. P. Vidosic, "Metal Machining and Forming TeChnOlOQv", The Ronald Press -. Company, New York, N.Y., i964, p 244.(15) "Cement: Comparison of Standards and Significance of Particular Tests", American Society for Testing and Materials, STP 441, June 1967. (16) U.S. Department of Interior, Bureau of Reclamation, "Concrete Manual", 7th ed.. 1966, p 43. (17) Ref. 16, p 393. (18) General Services Administration, United States Government, Federal Specification TT-E491E or TT-E-489F, December 1970. (19) General Services Administration, United States Government, Federal Specification TT-E-491B, March 1972. (20) General Services Administration, United States Government, Federal Specification TT-P-32A, Augusi 1965. (21) D. B. Robinson, Am. lnd. Hygiene Assoc. J., 23,228 (1962). (22) J. D. Roberts and M. C. Caserio, "Basic Principles of Organic Chemistry", W. A. Benjamin, Inc., New York, N.Y., 1965, p 686. (23) Ref. 3, p 381. (24) B. L. Lowrance, P. Reich, and W. H. Traub, Appl. Microbiol., 17, 923-924 (1969). (25) R. W. Weeks, Jr., S. K. Yasuda, and B. J. Dean, Anal. Chem., 48, 159 (1 976). (26) B. R. DasGupta and D. A. Boroff, Anal. Chem., 40, 2060 (1968). (27) Ref. 3, p 372. (28) G. J. Kapadia, J. R. Mosby, G. G. Kapadia, and T.6.Zalucky, J. Chromatogr., 12,420 (1963). (29) G. J. Kapadia, J. R. Mosby, G. G. Kapadia, and T. B. Zalucky, J. Pharm. Sci,, 54, 41 (1965). (30) J. E. Barney 11, S. R. Harvey, and T. S. Hermann, J. Cbromatogr., 45, 82 (1969). (31) R. W. Weeks, Jr., and B. J. Dean, Los Alamos Scientific Laboratory, unpublished data, 1975 and 1976. (32) R. W. Weeks, Jr., R . Morales, S. M. Rappaport, H. J. Ettinger, and E. E. Campbell, "Development of Sampling and Analytical Methods for Carcinogens", Los Alamos Scientific Laboratory report LA-6042-PR, September 1975. (33) I. M. Jakovljevic, J. Zynger, and R. H. Bishara, Anal. Chem., 47, 2045 (1975).
RECEIVEDfor review May 24,1976. Accepted August 16,1976. Work performed for the National Institute for Occupational Safety and Health and performed a t the Los Alamos Scientific Laboratory operated under the auspices of the U.S. Energy Research and Development Administration. Contract W7405-ENG-36.
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