The fingerprint character and high sensitivity of 3D UV-vis fluorescence spectra offer special advantages for identification of dyes in a museum or forensic setting. However, the extraction process is likely to affect the pH of the medium and, in some cases, may alter the dye itself. We report a study of 65 dyes extracted from wool fibers that are part of the Schweppe Collection of Important Synthetic Dyes. The 3D fluorescence spectra of the dye extracts at pH 1 and pH 14 are compared with the same dyes from the Schweppe solution library, run under the same conditions, as well as with the 3D fluorescence spectra of the dyes taken directly from the solution library without pH control. This analysis leads to guidelines for the use of such spectra in identifying unknown dye samples.
We recently reported “fingerprint” patterns obtained from 3-dimensional fluorescence spectrometry [
Because of the trifluoroacetic acid extraction procedure, we employed results in a highly acidic solution; we examined the effect of pH on the 3D fluorescence spectra of these dyes and found that many of the spectra are pH dependent. Therefore, we developed a standard protocol in which the fluorescence spectrum of each dye extract is recorded at pH 1 and then again at pH 14. Dye samples from the Schweppe solution library were subjected to the same protocol, and their fluorescence spectra were compared with those from the wool extracts. This comparison led to recommendations on the interpretation of such spectra for problems involving dye identification.
We used a Hitachi F 4500 fluorescence spectrophotometer with a Hitachi 650 - 0116 microcell for running fluorescence spectra; this cell requires 200 μL of solution. The excitation wavelength EX was scanned from 250 - 380 nm and the emission wavelength EM was scanned from 395 - 700 nm. These two ranges intentionally do not overlap in order to avoid exposing the photomultiplier to 1st order scattering from the Xe light source. The scanning rate was 1200 nm/min, and the response time was set at 0.1 sec. With these conditions, a spectrum could be obtained in less than 8 minutes with no apparent loss of detail, compared with slower scan speeds.
Approximate pH measurements with narrow-range pH paper were spot-checked and confirmed using a Fisher Accumet Model AB-15 pH meter with an Accumet glass combination electrode (Fisher 13-620-285).
The original solution Schweppe Library of Synthetic Organic Dyes [
For extracting the dyes, we diluted Aldrich spectrophotometric grade trifluoroacetic acid (TFA) (Aldrich 302031) with Sigma-Aldrich Chromasolv-Plus HPLC water (Sigma-Aldrich 34877) to 2 M and also used Sigma-Aldrich Chromasolv HPLC MeOH (Sigma-Aldrich 34860). For adjusting the pH, we made a 6 M solution of NaOH using Fisher NF/FCC sodium hydroxide (Fisher S 320) and HPLC water. 100% pure medical grade USP modified lanolin (CVS Lanolin Cream, SKU 148868) was used for the lanolin spectrum, as described below.
Since the original Schweppe collection is over 50 years old, some of the library solutions show obvious decomposition, indicated by an absence of color: Acid Black 1 (CI 20470), Basic Green 4 (CI 42000), Acid Blue 93 (CI 42780), Acid Black 2 (CI 50420), Murexide (CI 56085), Mordant Red 3 (CI 58005), and Acid Blue 74 (CI 73015). For these dyes, the fluorescence spectra reported were obtained using fresh dye samples.
The issue of dye concentration has been discussed in [
The extraction of both synthetic and natural dyes from textile samples for analysis has been a subject of prior investigation [see for example 3 and 4]. Souto [
To extract a sample, we clipped a fragment of wool (about 2 mg) from a Schweppe library wool sample and placed it at the bottom of a 0.5 mL polypropylene microcentrifuge tube. We added 20 μL 2 M TFA, agitated with a glass rod, and let stand at room temperature for 2 min. We then added 30 μL MeOH and let stand for another 4 minutes at room temperature. The sensitivity of fluorescence spectrometry is such that sufficient dye was extracted in most cases even under these gentle conditions. Finally, the extract was diluted with 250 μL H2O. The dyes insufficiently extracted under these conditions were Acid Violet 7 (CI 18055), Acid Black 1 (CI 20470), Basic Yellow 2 (CI 41000), Acid Green 6 (CI 42075), and Mauveine (CI 50245).
The dye solution as extracted from the wool fibers with TFA is highly acidic, even after dilution, with a pH of 1 indicated by narrow range pH paper and confirmed using a glass electrode pH meter. After the 3D fluorescence spectrum was run at pH 1, 10 μL of 6 M NaOH solution was added to the microcuvette and thoroughly mixed. If necessary, additional NaOH solution was added to bring the pH to 14. The pH 14 spectrum was then run.
The Supplementary Information—
Certain distinct artifacts characteristic of fluorescence spectra need to be taken into account when examining the spectra reported here. These artifacts include Rayleigh scattering and Raman scattering from the solvent. These artifacts have been discussed in detail in [
Care needs to be taken to avoid confusion due to a peak centered near λEX = 300 nm λEM = 400 nm, often seen in the pH 14 wool extract spectra (
While a detailed correlation of fluorescence spectra with molecular structure is a complex matter [
Of the 65 dyes in the Schweppe collection, the 3D
fluorescence spectra for only 7 of the dyes were unaffected by the change from pH 1 to pH 14. Twenty-four of the Schweppe dyes are actually acid/base indicators [
S 51 (Solvent Red 49,
In contrast, S 41 (Basic Green 1,
It is known that S 60 (Murexide) irreversibly decomposes at pH values lower than 4.5 and higher than 9 [
To employ the methods described here for identifying an unknown dye, one should extract the dye, adjust the pH, and run the spectra as described in Section 2. Then:
1) Compare the pH 14 and pH 1 spectra with the 46 library (“vial”) spectra in the Supplementary Information—
2) If no match is found in step (1), repeat the comparison against the 19 spectra in the Supplementary Information—
[http://simmons.academia.edu/LenSoltzberg]. Compare the spectra of the unknown sample against only the library spectra, since in
because of insufficient extraction.
Over 75% of the 65 synthetic organic dyes in the Schweppe collection can be successfully extracted from wool yarn under mild conditions and identified from their 3D fluorescence spectra at pH 1 and/or pH 14. Only 2 mg of wool is a sufficient sample size. There are three pairs of structurally similar dyes for which the spectra are too similar to allow differentiation (S 19/S 20, S 23/S 24 and S 25/S 26); in these cases, MALDI-TOF mass spectra would resolve the identity of the dyes [
We thank the Camille and Henry Dreyfus Foundation for a Senior Faculty Mentor grant supporting the participation of Kirch and Flynn. We also thank the National Science Foundation for grant CHE-0216268.