1. General Discussion
1.1 Background
1.1.1 History of procedure
The OSHA Analytical Laboratory received a set of air samples
requesting the analysis of rhodamine B. The samples had been collected
on polyvinyl chloride filters (FWS-B) with air volumes around 970
liters. FWS-B filters and glass fiber filters were both evaluated since
glass fiber filters are more commonly used. This report describes the
analytical method developed for glass fiber filters.
1.1.2. Toxic effects (This section is for information only and should
not be taken as the basis of OSHA policy.)
Rhodamine B has been tested in mice and rats by subcutaneous
injection and, in inadequate studies, by oral administration. It was
carcinogenic in rats when injected subcutaneously, producing local
sarcomas. The intravenous LD50 in rats is 89.5 mg/kg. (Ref.
5.1)
1.1.3 Potential workplace exposure
The following information is taken from the IRAC Monographs
on the Evaluation of the Carcinogenic
Risk of Chemicals to Man. (Ref. 5.1)
Rhodamine B can be used to dye silk, cotton, wool, bast fibers,
nylon, acetate fibers, paper, spirit inks and lacquers, soap, wood
stains, feathers, leather and distempers on china clay. In the U. S., it
has been used as a drug and cosmetic colour in aqueous drug solutions,
tablets, capsules, toothpaste, soap, hair-waving fluids, bath salts,
lipsticks and rouges. This color has also been used as a tracing agent
in water pollution studies, as a dye for waxes and antifreeze and as an
analytical reagent for antimony, bismuth, cobalt, niobium, gold,
manganese, mercury, molybdenum, tantalum, thallium and tungsten.
No estimate of worker exposure to rhodamine B could be found.
1.1.4 Physical properties (Ref. 5.1 to 5.3)
Molecular weight: |
479 |
Molecular formula: |
C28H31ClN2O3
|
CAS number: |
81-88-9 |
IMIS number: |
0848 |
Melting point: |
165°C |
Solubility: |
very soluble in water and alcohol;
slightly soluble in hydrochloric acid and sodium
hydroxide |
Chemical name: |
N-[9-(2-carboxyphenyl)-6-(diethylamino)
-3H-xanthen-3-ylidene]-N-ethylethanaminium
chloride |
Synonyms: |
tetraethylrhodamine; D & C Red No.
19; rhodamine B chloride; C.I. Basic Violet 10; C.I.
45170 |
Description: |
green crystals or reddish-violet powder
|
Structure: |
|
|
UV data: |
546 nm maximum
(Ref. 5.1)
556 nm maximum |
|
Figure 1. UV Scan of Rhodamine B in Mobile Phase
|
1.2 Limit defining parameters
The detection limit of the analytical procedure is 0.013 ng per injection
with a fluorescence detector or 0.42 ng per injection with a UV detector. This
is the amount of analyte which will give a peak whose height is approximately
five times the baseline noise. (Figure 2) |
Figure 2.
Detection Limit Chromatogram of Rhodamine B on a Fluorescence
Detector |
2. Sampling procedure
2.1 Apparatus
2.1.1 A personal sampling pump that can be calibrated to within ± 5%
of the recommended flow rate with the sampling device in line.
2.1.2 Glass fiber filters, 37-mm diameter, Gelman Type A or
equivalent.
2.1.3 Cassette filter holders for 37-mm filters, Millipore M000037A0
or equivalent.
2.2 Reagents
No sampling reagents are required.
2.3 Sampling technique
2.3.1 Immediately before sampling, remove the plastic plugs from the
cassette.
2.3.2 Attach the cassette to the sampling pump with flexible tubing.
2.3.3 Attach the cassette vertically in the employee's breathing zone in such a manner that it does not impede work performance.
2.3.4 After sampling for the appropriate time, remove the cassette
and replace the plastic plugs.
2.3.5 Wrap each cassette end-to-end with an OSHA seal (Form 21).
2.3.6 Record the air volume for each sample, and list any possible
interferences.
2.3.7 Submit at least one blank for each set of samples. Handle the
blank in the same manner as the samples, except no air is drawn through
it.
2.3.8 Submit bulk samples for analysis in a separate container. Do not ship
them with air samples.
2.4 Extraction efficiency
Six glass fiber filters were each liquid spiked with 6 µL
of a 2.21 mg/mL rhodamine B standard. After drying, these filters
were each extracted with 5.0 mL of methanol, shaken for 30 min and
then analyzed as in Section 3. The results are listed in Table 2.4.
|
Table
2.4
Desorption Efficiency
|
amount spiked,
µg |
amount found,
µg |
% recovered |
|
13.26
13.26
13.26
13.26
13.26
13.26 |
14.24
13.47
13.26
13.19
13.28
13.42 |
107.4
101.6
100.0
99.5
100.2
101.2 |
|
|
101.6 |
|
|
2.5 Retention efficiency
Eighteen glass fiber filters were each liquid spiked with 6
µL
of a 2.21 mg/mL rhodamine B standard. These filters were dried and
then 240 L of humid air (~80%
relative humidity) were drawn though each filter at approximately
1.3 L/min. Six of the filters were then each extracted with 5.0 mL
of methanol, shaken for 30 min and analyzed as in Section 3. The
results are listed in Table 2.5. The rest of the filters were kept,
6 in a drawer at ambient temperature and 6 in a refrigerator, for
storage studies.
|
Table 2.5
Retention Efficiency
|
amount spiked,
µg |
amount found,
µg |
% recovered |
|
13.26
13.26
13.26
13.26
13.26
13.26 |
12.23
12.59
11.88
12.33
11.58
11.37 |
92.2
94.9
89.6
93.0
87.3
85.7 |
|
|
90.4 |
|
|
2.6 Sample storage
After 2 days of storage 6 samples, 3 from ambient storage and 3 from
refrigerator storage, were each extracted with 5.0 mL of methanol, shaken
for 30 min and then analyzed as in Section 3. After 7 days of storage, the
remaining samples were extracted and analyzed. The results are given in
Tables 2.6.1 and 2.6.2.
Table 2.6.1
Ambient Storage
|
days
stored |
amount
spiked,
µg |
amount
found,
µg |
%
recovered |
|
2
2
2
7
7
7 |
13.26
13.26
13.26
13.26
13.26
13.26 |
10.71
11.14
10.68
12.38
12.06
11.64 |
80.8
84.0
80.5
93.3
91.0
87.7 |
|
|
of 2
of 7 |
81.8
90.7 |
|
|
Table 2.6.2
Refrigerator Storage
|
days
stored |
amount
spiked,
µg |
amount
found,
µg |
%
recovered |
|
2
2
2
7
7
7 |
13.26
13.26
13.26
13.26
13.26
13.26 |
11.89
11.91
12.13
11.44
11.89
12.07 |
89.6
89.8
91.4
86.3
89.7
91.0 |
|
|
of 2
of 7 |
90.3
89.0 |
|
|
2.7 Recommended air volume and sampling rate
2.7.1 The recommended air volume is 240 L.
2.7.2 The recommended flow rate is 1.0 L/min.
2.8 Interferences (sampling)
It is not known if any compounds will interfere with the collection of
rhodamine B. Any suspected interferences should be reported to the
laboratory.
2.9 Safety precautions (sampling)
2.9.1 Attach the cassette in such a manner that it will not impede
work performance or employee safety.
2.9.2 Follow all safety practices that apply to the work area being
sampled.
3. Analytical procedure
3.1 Apparatus
3.1.1 A balance capable of weighing to the nearest tenth of a
milligram. A Mettler HL52 balance was used in this evaluation.
3.1.2 A mechanical shaker.
3.1.3 An HPLC with UV and fluorescence detectors. A Hewlett
Packard 1090 liquid chromatograph with a diode array detector and an
ABI Analytical (980G) fluorescence detector were used in this
evaluation.
3.1.4 An HPLC column capable of separating rhodamine-B from any
interferences. A 100 mm × 2.1 mm i.d. Hypersil ODS, 5 µm,
column was used in this evaluation.
3.1.5 An electronic integrator or some other suitable means for
measuring detector response. The Hewlett-Packard (HP) 1090 Chem
Station and HP 3357 data system were used in this evaluation.
3.1.6 Volumetric flasks and pipets of various sizes.
3.1.7 Scintillation vials, 20-mL.
3.1.8 Vials, 2-mL.
3.2 Reagents
3.2.1 Methanol, HPLC grade.
3.2.2 Water, HPLC grade.
3.2.3 Acetonitrile, HPLC grade.
3.2.4 Phosphoric acid (H3PO4), reagent grade.
3.2.5 Rhodamine B. An Aldrich standard of 99% purity was used in this
evaluation.
3.3 Standard preparation
Prepare rhodamine B stock standards by weighing 10 to 15 mg of
rhodamine B. Transfer the rhodamine B to separate 10-mL volumetric
flasks and add methanol to the mark. Make working range standards of
0.01 to 5.0 µg/mL by pipet dilutions of the stock standards with methanol. Store stock and
dilute standards in a freezer.
3.4 Sample preparation
3.4.1 Transfer the glass fiber filter to a scintillation vial.
3.4.2 Add 5.0 mL of methanol to each vial and seal with a
Teflon-lined cap.
3.4.3 Shake the vials for 30 minutes on a mechanical shaker.
3.4.4 Transfer the sample to a 2-mL vial for use in an HP autosampler.
3.5 Analysis
3.5.1 Instrument conditions
Column: |
100 mm × 2.1 mm i.d. Hypersil ODS, 5 µm |
Oven temperature: |
40°C |
Mobile phase:
|
85% acetonitrile 15%
water with 0.005M 1-heptanesulfonic acid
and the pH adjusted to 3.5 with H3PO4
|
Flow: |
0.2 mL/min |
Wavelength: |
556 nm |
Fluorescence detector:
|
Excitation: 210 nm
Emission: 550 nm cut off filter
|
Injection volume: |
1.0 µL
|
Retention time:
|
4.5 min
|
3.6 Interferences (analytical)
3.6.1 Any collected compound having a retention
time similar to that of rhodamine B is an
interference.
3.6.2 Generally, HPLC conditions may be varied to
circumvent interferences.
3.6.3 Retention time on a single column is not
proof of chemical identity. Analysis by an alternate
HPLC column, comparison of detector responses or
confirmation by mass spectrometry are additional means
of identification.
|
Figure 3.
Chromatogram of Rhodamine B on a UV Detector |
3.7 Calculations
3.7.1 Construct a calibration curve by plotting
detector response versus concentration (µg/mL)
of rhodamine B.
3.7.2 Determine the µg/mL
of rhodamine B in each sample and blank from the
calibration curve.
3.7.3 Blank correct, if necessary, the sample by
subtracting the µg/mL
found in the blank from the µg/mL
found in the sample.
3.7.4 Determine the air concentration by using the
following formula.
|
Figure 4. Calibration Curve |
3.8 Safety precautions (analytical)
3.8.1 Avoid skin contact and air exposure to rhodamine
B.
3.8.2 Avoid skin contact with all solvents.
3.8.3 Wear safety glasses at all times.
4. Recommendation for further study
This method should be fully validated.
5. References
5.1 IARC Monographs on the
Evaluation of the Carcinogenic
Risk of Chemicals to Man;
International Agency for Research on Cancer: Lyon, 1978;
Vol. 16, pp 221-231.
5.2 Registry of Toxic Effects
of Chemical Substances 1985-86
Edition; U.S. Department of Health and Human
Services: Cincinnati, OH, 1987; DHHS(NIOSH) Publication
No. 87-114, p 313.
5.3 Merck Index, 10th ed.; Windholz, Martha
Ed.; Merck: Rathway, N.J., 1983; p 1180.
|