HPLC quantification of salicylic acid
Once you have extracted your SA its most likely you want to know how much you have in your samples.
Thaw your samples and place 200 mL in appropriate tubes for your HPLC.
Use 10 cm long X 4.6mm radius column containing 3.0 mM ODS2 Spherisorb beads (Waters part number PSS832112), adjust flow rate to 1 ml/min and protocol length to 10 minutes.
Prior to analyzing sample make sure the HPLC has been purged and the line properly washed with running buffer.
Inject sample and run.
Once all samples are run calculate the area under the curve of the peak that correspond to SA (determined by the recovery control which should have a single peak).
In an Excel sheet enter the values as suggested in the table below.
|Sample name||Sample weight||Area under the curve||g of SA||g of SA/g of tissue|
Column B. Enter leaf weight only (not tube !)
Column C. As determine using HPLC
Column D. Here the 0.000000138 correspond to the amount of SA added in the recovery control. Modify this value if you use different amount. All this is divided by C4 that’s the area under the curve of the recovery control, basically that’s what was detected of the 0.000000138g of SA you extracted.
Your extraction should be done in triplicates, you can then plot average and error bars on the graph.