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Acid Blue 9
Related Information: Chemical Sampling -
Acid Blue 9
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Method no.: |
PV2129 |
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Control no.: |
T-PV2129-01-0310-CH |
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Matrix: |
Air |
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Target Concentration: |
0.2 mg/m3. There is
no OSHA PEL for acid Blue 9. Neither NIOSH nor ACGIH has a recommended
standard for Acid Blue 9. For the purpose of this study, the target
concentration has been arbitrarily set at 0.2 mg/m3. It represents 100 x
the detection limit for the proposed method. |
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Procedure: |
Collection on a glass fiber filter,
extraction with methanol/water (1:1), and analysis by high performance
liquid chromatography (HPLC) with variable wavelength detector at 650 nm. |
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Recommended Air Volume and Sampling Rate: |
100 L at 1 Lpm |
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Detection Limit of the Overall Procedure Based on Recommended Air Volume: |
0.002 mg/m3 |
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Status of Method: |
This method has been only partially validated and is presented for information and trial use. |
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January 1982 |
Yihlin Chan |
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Methods Development Team
Industrial Hygiene Chemistry Division
OSHA Salt Lake Technical Center
Sandy UT 84070-6406 |
1. General Discussion
1.1. Background
1.1.1. History of Procedure.
Recently, the OSHA Analytical Laboratory received a set of field samples requesting analysis for Acid
Blue 9. The air samples had been collected on glass fiber filters, at 1 Lpm for a total of about 90
liters air. This report describes the analytical procedure developed and the preliminary validations
of the sampling method.
Acid Blue 9 is a widely used food dye. There has been many schemes proposed for the qualitative
analysis of food dyes, most of which depended on paper and thin-layer chromatography. Less attention
has been given to the quantitative analysis of dyes. Some of the methods attempted were:
(a) comparison of spot intensities on TLC plates with those of a range of standards, (b)
spectrophotometric quantitation, (c) titration with titanous chloride solution, and
(d) electrophoresis on polyacrylamide gel. More recently, HPLC has been applied for dye
analysis, using anion-exchange columns or, more satisfactorily, by ion-pairing. Paired-ion
HPLC affords a means of separating a mixture of food dyes in a single run (Ref. 5.2.). Preliminary search did not
reveal an air sampling method for Acid Blue 9. Judging from its physical properties, glass fiber
filters may be a suitable collection medium.
1.1.2. Toxic Effects.
(This section is for information only and should not be taken as the basis of OSHA policy.)
Acid Blue 9 is carcinogenic in rats after its subcutaneous injection: it produced fibrosarcomas
following repeated injections. It also produced an increased incidence of kidney tumors in mice
after its oral administration (Ref. 5.1.).
1.1.3. Potential Workplace Exposure.
Acid Blue 9 is an FDA certified food dye and is used in such products as gelatin desserts, ice cream and
sherbets, carbonated beverages, dry drink powders,
candy and confectionary products when they do not contain oils and fats, bakery products and cereal,
puddings, aqueous drug solutions, tablets, capsules, bath salts, and hair rinses (Ref.
5.3.). Acid Blue 9 has been produced in the U.S. for over sixty years. In 1975,
three U.S. companies produced 622,000 Kg of the general dye grade, and another
four companies produced 56,000 Kg of he food, drug, and cosmetic grade (Ref.
5.1.). Preliminary literature searches did not reveal any estimate on the extent
of worker exposure.
1.1.4. Physical Properties
Color Index Names: Acid Blue 9, Food
Blue 2
Color Index Number: 42090
Cas Reg. Number:2650-18-2 (3844-45-9 )
Chem. Abstr. Names:
N-Ethyl-N-(4[(4-(ethyl[(3-sulfophenyl)methyl]amino)
phenyl)-(2-sulfophenyl)methylene]-2,
5-cyclohexadien-1-ylidene)3-sulfobenzenemethanaminium hydroxide inner salt, disodium salt; C.I. Acid Blue
9, disodium salt; D and C Blue No.1; D and C Blue No.4; ethyl(4-(p[ethyl (m-sulphobenzyl)amino]-α -(o-sulphophenyl)benzylidene)-2,5-cyclohexadiene-l-ylidene)- (m-sulphobenzyl) ammonium hydroxide inner salt, disodium salt; FD and C Blue 1; FD and
C Blue. No.1; FDC Blue No.1; Acid Sky
Blue A; Acilan Turquoise Blue AE; A. F.
Blue No.1; Aizen Brilliant Blue FCF;
Aizen Food Blue No.1; Alphazurine; Alphazurine FG; Alphazurine FGND; Amacid Blue
FG; Amacid Blue FG Conc; 1206 Blue, 11388
Blue; Blue Dye Number 1 food additive;
Brilliant Blue; Brilliant Blue FCF; Brilliant Blue Lake; Bucacid Azure Blue; Calcocid Blue EG; Calcocid Blue 2G; Canacert
Billiant Blue FCF, Cogilor Blue 512.12;
Cosmetic Blue Lake; Dispersed Blue 12195;
Disulphine Lake Blue EG; Dolkwal Brilliant Blue; Edicol Blue Cl 2; Edicol Supra Blue
E6; Erioglaucine ; Erioglaucine A; Erioglaucine E; Erioglaucine G; Eriosky Blue; Fenazo Blue XI; Fenazo Blue XR; Food Blue 1;
Hexacol Brilliant Blue A; Hidacid Azure
Blue; Intracid Pure Blue L; Kjtoc Blue AR;
Kiton Pure Blue L; Maple Brilliant Blue
FCF; Merantine Blue EG; Neptune Blue BRA; Concentration; Patent
Blue AE; Patent Blue 2Y; Peacock Blue
X-1756; Usacert Blue No.1; Xylene Blue
VSG.
Appearance: Reddish-violet powder or granules with a
metallic luster.
Spectroscopy Data:
λ max 630 nm.
Chemical Formula and Molecular Weight:
![For problems with accessibility in using figures please contact the SLTC at (801) 233-4900. For problems with accessibility in using figures please contact the SLTC at (801) 233-4900.](structure.gif)
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Solubility: Soluble in water and ethanol; insoluble in vegetable oils.
1.2. Limit Defining Parameters
1.2.1. Detection Limit of the Analytical Procedure
The detection limit of the analytical procedure is 0.83 ng Acid Blue 9 per injection. This is the
amount of analyte which will give a peak whose height is approximately five
times the amplitude of the baseline noise. See Figure 1.
1.2.2. Detection Limit of the Overall Procedure
The detection limit of the overall procedure is estimated to be 0.2 µg per sample or 0.002 mg/cu m
based on the recommended air volume, assuming 100% recovery from the sampling device.
The recovery test at this level has not been performed.
1.2.3. Sensitivity
The sensitivity of the analytical procedure over a concentration range of 0.395 to 11.9
µg/mL is 19,280 area units per µg/mL of Acid Blue 9. The sensitivity is
determined by the slope of the calibration curve. See Figure 2.
1.3. Advantages
The analytical procedure is rapid, sensitive, and reproducible.
1.4. Disadvantages
The method has not been fully validated.
2. Sampling Procedure
2.1. Apparatus
2.1.1. An air sampling pump with a flow rate which can be calibrated to within
±5% of the recommended 1 Lpm flow rate while the sampler is in line.
2.1.2. Glass fiber filter, 37-mm diameter, Gelman Type A, or equivalent.
2.1.3. Filter holder for 37-mm filters, Millipore M000037AO, or equivalent.
2.2. Sampling Technique
2.2.1. Assemble the filter in the two-piece cassette holder and close firmly. The filter is supported by a
backup pad. Secure the cassette holder together with tape.
2.2.2. Attach the outlet of the filter cassette to the personal sampling pump inlet
with flexible tubing.
2.2.3. Air being sampled should not pass through any hose or tubing before entering the filter cassette.
2.2.4. A sample size of 100 liters is recommended. Sample at a flow rate of 1.0 liter/minute. The
flow rate should be known with an accuracy of ±5%.
2.2.5. With each batch of samples, submit a blank filter from the same lot of filters used for sample
collection. This filter must be subjected to exactly the same handling as the samples except that no air
is drawn through it. Label this filter as the blank.
2.2.6. The cassette should be shipped in a suitable container designed to prevent damage in transit. The
samples should be shipped to the laboratory as soon as possible.
2.2.7. A sample of the bulk material should be submitted to the laboratory
in a glass container with a Polyseal cap. Never transport, mail, or ship the balk sample
in the same container as the sample or blank filter.
2.3. Retention Efficiency
Two glass fiber filters were spiked with 1.1.5 µg of Acid Blue 9. Humid air (87% relative humidity) 140 liters
was drawn through the filters at 1 Lpm. The average recovery of
the two filters was 101%.
Sample |
Spiked Amount |
Treatment |
Peak Height |
Recovery |
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YC5
YC6
YC7
YC8 |
41.5 µg on GFF
41.5 µg on GFF
41.5 µg; control
41.5 µg; control |
140 L humid air
140 L humid air
none
none |
138.0 mm
131.5 mm
131.0 mm
136.0 mm |
103.4%
98.5%
----
---- |
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Average recovery 100.9% |
2.4. Extraction Efficiency
The average extraction efficiency from the glass fiber filters spiked with 41.5
µg of Acid Blue 9 was 95.9%.
Sample |
Spiked Amount |
Peak Height |
Recovery |
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YC3
YC4
YC7
YC8 |
41.5 µg on GFF
41.5 µg on GFF
41.5 µg; control
41.5 µg; control |
126.5 mm
129.5 mm
131.0 mm
136.0 mm |
94.8%
97.0%
----
---- |
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Average recovery 95.9% |
2.5. Storage
Two glass fiber filters were spiked with 41.5 µg of Acid Blue 9 and stored at room temperature in the dark for two days. The
average recovery was 100.2%.
Sample |
Spiked Amount |
Storage Days |
Peak Height |
Recovery |
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YC3
YC4
YC7
YC8
YC1
YC2
YC7
YC8 |
41.5 µg
41.5 µg
41.5 µg
41.5 µg
41.5 µg
41.5 µg
41.5 µg
41.5 µg |
0
0
control
control
2
2
control
control |
126.5 mm
129.5 mm
131.0 mm
136.0 mm
168.0 mm
157.0 mm
162.0 mm
158.0 mm |
94.8%
97.0%
----
----
105.0%
98.1%
----
---- |
2.6. Recommended Air Volume and Sampling Rate
2.6.1. The recommended air volume is 100 liters.
2.6.2. The recommended sampling rate is 1 Lpm.
2.7. Interferences
There are no known interferences associated with the sampling procedure.
2.8. Safety Precautions
2.8.1. Attach the sampling equipment to the worker in such a manner that it will
not interfere with work performance or safety.
2.8.2. Follow all safety practices that apply to the work area being sampled
3. Analytical Method
3.1. Apparatus
3.1.1. High performance liquid chromatograph equipped with pump, sample injector, variable wavelength detector,
chart recorder, and other necessary hardware.
3.1.2. HPLC reverse phase C18 analytical column. Dupont Zorbax ODS column was used for this study.
3.1.3. An electronic integrator or other suitable method to measure detector response.
3.1.4. Microliter syringe or automatic sampling device for making sample injections.
3.1.5. Volumetric flasks of convenient sizes for preparing standards.
3.1.6. Shaking device for extraction of samples.
3.2. Reagents
3.2.1. Acid Blue 9 (Erioglaucine)
3.2.2. Tetrabutylammonium phosphate, reagent grade
3.2.3. Methanol, HPLC grade
3.2.4. Water, HPLC grade
3.2.5. Phosphoric Acid
3.3. Sample Preparation
3.3.1. Remove the filter form the cassette clean tweezers and place it in a
20-mL scintillation vial.
3.3.2. Add 5 mL of methanol/water (1:1) to the vial and cap it.
3.3.3. Shake the vials vigorously on a shaker for 30 minutes.
3.4. Standard Preparation
3.4.1. Standard of Acid Blue 9 is prepared by dissolving 8 to 12 mg (accurately weighed) of Acid Blue 9 in
water in a 10-mL volumetric flask and making it to volume.
3.4.2. Dilute to the working range of 0.1 to 12 µg/mL with water.
3.4.3. Store standards in dark bottles under refrigeration.
3.5. Analysis
3.5.1. HPLC Conditions
Column: |
Zorbax ODS (25 cm x 4.6 mm) |
Mobile phase: |
55% methanol, 45% water, 0.005 M
tetrabutylammonium phosphate |
Flow Rate: |
1.0 mL/minute |
Variable Wavelength Detector: |
650 nm |
Injection Volume: |
20 µL |
Retention Time: |
7.8 minutes |
3.5.2. Chromatogram
See Figure 1.
3.5.3. Peak magnitude is measured by electronic integrator or other means.
3.5.4. An external standard procedure is used to prepare a calibration curve from the analysis of at least
three different concentrations from two separate weighings.
3.5.5. Bracket the sample with analytical standards.
3.6. Interferences (Analytical)
3.6.1. Any collected compound that has the same LC retention time as analyte and absorbs at 650
nm is an interference.
3.6.2. HPLC parameters may be varied to circumvent most interferences.
3.6.3. Retention time alone is not proof of a chemical identity. Confirmation by other
means should be sought when possible.
3.7. Calculations
3.7.1. The integrator value in area units for each standard is plotted against its concentration in
µg/mL and a calibration curve using the best fit straight line through the points is obtained.
3.7.2. Sample concentration is calculated from the calibration curve.
3.7.3. The air concentration of Acid Blue 9 for a sample is calculated by
the following equation:
mg/m3 = |
(µg/mL in sample)(extraction volume, mL)
(Air volume, L) |
3.8. Safety Precautions
3.8.1. Confine the use of solvents to a fume hood.
3.8.2. Wear safety glasses in all laboratory areas.
4. Recommendations for Further Study
4.1. Preparation of pure standard
The commercially available Acid Blue 9 is not pure. The U.S. specification for the food grade is 85% minimum. Purification
of the standard should be attempted either by preparative TLC or preparative HPLC.
![For problems with accessibility in using figures please contact the SLTC at (801) 233-4900. For problems with accessibility in using figures please contact the SLTC at (801) 233-4900.](image01.gif)
Figure 1. Chromatogram of Acid Blue 9 at Target Concentration and at
Detection Limit. |
![For problems with accessibility in using figures please contact the SLTC at (801) 233-4900. For problems with accessibility in using figures please contact the SLTC at (801) 233-4900.](image02.jpg)
Figure 2. Calibration of Acid Blue 9. |
5. References
5.1. WHO, International Agency for Research on Cancer, IARC Monograph
on the Evaluation of the Carcinogenic Risk of Chemicals to Man. Some Aromatic
Amines and Related Nitro Compounds -- Hair Dyes, Colouring Agents and
Miscellaneous Industrial Chemicals. Vol. 16, pp. 171-86.
5.2. J. Chudy, N.T. Crosby, and I. Patel, J. Chromatogr., 154, (1978), p 306-312.
5.3. A Standen, ed., Kirk-Othmer Encyclopedia of Chemical Technology,
Second Ed., Vol. 5, pp. 865-66. Interscience Publishers, New York, N.Y., 1963.
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