I’m trying to quantify genistein in my test samples by alcl3 method using UV spectrophotometer. In order to quantify this, I first need to select wavelength where maximum absorption of isoflavone genistein and alcl3 complex occurs and hence I purchased standard genistein from sigma and tried to screen the optimum wavelength using these two protocols.

Method 1 & 2 followed is as below

1)      0.4ml of genistein(1mg in 5ml methanol) + 0.3ml of  5% NaNO2+0.3ml of 10% alcl3+2ml of 1M NaOH. Made up to 5ml with 90% methanol.

2)      0.4ml genistein+ 0.3ml of alcl3. Made up to 5ml with 90% methanol.

When I carried out my experiment I got maximum absorption at 325nm(1.525A) by following method 1 and with method 2, two peaks were observed. The first peak showed maximum absorption at 283nm(0.871A) and second peak at 377nm(0.293). later I added 1.5ml of alcl3 in excess thinking genistein concentration may be high but the results did not change.(genistein alone(0.2ml, Made up to 3ml with 90% methanol) showed maximum absorption at 284nm(0.847A))

The principle behind quantification of flavonoid by alcl3 method is there will be a bathomic shift after addition of alcl3. Literature says genistein and alcl3 complex maximum absorption occurs at 382nm.

So I’m facing problem with wavelength selection, could anyone help me in this?