Team:UFlorida/Experiments
Sample Preparation
The standard stock solution (15 mg mL-1) of TOL was prepared in methanol. The standard Trp stock solution (15 mg mL-1) was prepared in water. Series (n = 5) of working solutions for each standard were prepared (0.0625 - 125 μg/ml)by appropriate dilution of the stock solution with methanol. To determine the recovery of the studied indolic compounds from bacterial broth during sample preparation, standards were spiked into sterile LB broth at five different concentrations. Bacterial broth was processed with the sample preparation procedure described below.1. The bacteria were cultivated for 72 h in LB broth with 0.5, 1.0, 2.0, 3.5 and 5.0 mM Trp supplementation
2. Bacterial cultures were then centrifuged, and the bacterial culture supernatants were processed
3. Sample preparation consisted of a single centrifugal filtration step using 3-kDa cut-off membrane centrifugal filters.
4. 0.5 mL of bacterial culture supernatants or spiked sterile bacterial broths were transferred to the sample chamber of a 0.5 mL centrifugal filter tube and centrifuged at 14,0009*g at 4 C for 30 min.
5. The filtrates were directly analyzed by HPLC.
HPLC Conditions
A C18 column was used after a previous run with a C8 column showed that tryptophan eluted too rapidly and early in the gradient method used.Eluent A
2.5 : 97.5 % (v/v) acetic acid: H2O, pH 3.8
*pH was adjusted by addition of 1 mol L-1 KOH
Eluent B
80 : 20 % (v/v) acetonitrile: H2O
Gradient Elution (A>B)
80:20
50:50
0:100
80:20
Time Scale(min) @ FR (1ml/min)
0-25
25-31
31-33
33-36
Analysis
Initial Standard Run of Tryptophan
MW of compounds:Tryptophan MM: 204.225 g/mol
Indole-3-pyruvic acid MM: 203.197 g/mol
Indole-3-acetaldehyde MM: 159.188 g/mol
Tryptophol MM: 161.20 g/mol
