主题：HPLC/UV–Vis/ESI MS

Experimental Procedure   
HPLC/UV–Vis/ESI MS
The instrument was an 1100 series Agilent HPLC/MS (Fremont, CA) with an atmospheric pressure electrospray interface (AP-ESI). The HPLC system consisted of a binary pump solvent-delivery system with a 100 μL sample loop. The column was a narrow bore Agilent Zorbax SB-C18 column (2.1 × 150 mm) with an Agilent Eclipse XDBC8 narrow bore guard column (2.1 × 2.5 mm). Chromatography was developed using a gradient of acidified acetonitrile (pH 2.5) (HPLC Grade Acetonitrile; Burdick and Jackson, Muskegon, MI) and acidified nanopure water (pH 2.5) (Barnstead Thermolyne, Dubuque, IA). The pH was adjusted using formic acid (Formic Acid, EM Science, Darmstadt, Germany). The flow rate was 0.225 mL/min for a 1-μL injection sample with a column oven temperature of 35°C for basic dyes and 45°C for disperse dyes, with an 8 min postrun. The MS trap (Bruker Esquire 3000 Plus, Fremont, CA) acquired data in the range 50–1000 mass-to-charge (m/z) at a maximum accumulation time of 200 ms with a target of 30,000 ions in the positive-ion ESI mode. Nebulizing gas was set at 35.0 psi, with the drying gas being pumped at 12.0 L/min, and the temperature in the ion source was held at 350°C. The diode array detector (DAD) was set to collect the highest signal in the visible region.
Spectrophotometer
Analysis was performed using an A U-3000 Hitachi spectrophotometer (Hitachi Instruments Inc., Danbury, CT). Dyed and undyed extracts were placed in cuvettes, with the latter used as the blank. Scans were collected from 350 to 800 nm at a rate of 300 nm/min with a slit width of 2.0 nm.
Dye Standards
Dyes were obtained from Clarient Corporation (Charlotte, NC), Dyestar (Charlotte NC), Classic Dyestuff (High Point, NC), and Sigma-Aldrich (St. Louis, MO). Standard solutions were made by dissolving 0.005 g of dyestuff in 50 mL of the solvent system (1.0 × 10 4 g/mL). Basic dye standards were dissolved in a 50:50 solution of acidified acetonitrile (pH 2.5) and water (pH 2.5), while the disperse dye standards were dissolved in a 45:45:10 solution of acidified acetonitrile (pH 2.5), water (pH 2.5), and methanol (HPLC Grade Methanol, Burdick and Jackson). Additional information on dye standards is summarized in Table 1.