Disperse Dye Chromatography Optimization The MS, HPLC, and UV–Vis, optimization process was repeated for the disperse dye class. A toluene/water extraction was performed on each disperse dye standard in order to isolate the dye molecules from other interfering components of the dyestuff. The aqueous layer was then evaporated to concentrate the dye, and the residue was reconstituted with the 1:1 acidified acetonitrile and water system. The reconstituted dyestuffs, after extraction, contained the dye molecules as the primary components. Mass spectrometer parameters were optimized with the purified dye, and a disperse dye mix was made by combining the 13 purified dye standards. The HPLC was then optimized, in the same manner as the basic mixture, for separation of these dyes, followed by UV–Vis optimization. The parameters for the disperse dye class are summarized in Table 2. TABLE 2—Optimized gradients for basic dye separation, disperse dye separation, and the master method.Time (min) % ACN versus acidified water Flow rate (mL/min) Basic dye method 0 34 0.225 2 34 0.225 7 42 0.225 28 48 0.225 31 98 0.225 35 98 0.400 Disperse dye method 0 54 0.225 2 54 0.225 27 78 0.225 32 98 0.400 Time (min) % ACN versusacidified water* Temperature (°C) MS (V) Master method 0 34 35 CV=−3375 2 34 35 CEV=188 7 42 35 TD=60 28 48 35 30 54 35 34 54 45 CV=−4000 59 78 45 CEV=154 64 98 45 TD=56 *Flow Rate was 0.225 mL/min until 98% ACN when it switched to 0.400 mL/min.CV, capillary voltage; CEV, capillary exit voltage; TD, trap drive; MS, mass spectrometry; ACN, acetonitrile. Disperse Dye Extraction System Development and Evaluation As the classical extraction system for disperse dyes contains pyridine, a solvent not on Agilent's list for use in the ion trap (14), a novel extraction system was developed. The pH of the pyridine and water system was determined to be 8.9. Other low-molecular-weight polar solvents were selected from the Agilent list of compatible solvents (14), including acetonitrile and triethylamine. The acetonitrile and water system was found to have a pH of 8.9 at a volume ratio of 4:3. In the case of the triethylamine solution, the pH had to be adjusted to 8.9 using formic acid. Both systems were used to extract fiber swatches at 120°C for 60 min. The extracts were visually compared, and it was determined that the acetonitrile and water system yielded more color. After the new solvent system was selected, the adequacy of dye extraction relative to the pyridine system was evaluated to verify it as a reasonable substitute. This was performed using a UV–Vis spectrophotometer. Three dyestuffs representing various colors and chemical structures (see Table 1) were selected. Dyed fabric swatches, as well as undyed fabric blanks, were extracted with both the pyridine and acetonitrile solvent systems. For each dye, a wavelength range that covered the area under the peak maximum was selected for comparison. Relative extraction efficiency analysis was also performed using the DAD of the LC/MS. To establish a calibration curve, an estimate of dye concentration in 0.005 g of dyed fabric extracted with 0.5 mL of solvent was made assuming that a fiber contained less then 1% dye. Dyestuffs of unknown dye concentration were used as received, without the additional purification step. To enable full solvation of the dye standards, the solvent system for the calibration curves required the addition of methanol. The acetonitrile system was 45:45:10 acetonitrile, water, and methanol and the pyridine system was 45:45:10 pyridine, water, and methanol. The absorbance of each dye peak was calculated through automatic integration under the curve and manual integration for the lowest concentration dilutions. Absorbance versus concentration was plotted and linear regression was calculated using the method of least squares. The next step involved extraction of dye from the swatches and analysis. The swatches were extracted at 120°C for 90 min with both 4:3 acetonitrile:water and pyridine:water solvent systems. The extracts were then injected, and the absorbance of each dye peak was measured. The concentration of dye in the extracts was then calculated using Beer's Law.
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