Dear Olushola

You don't give any description of your procedure so it is impossible to have a definite answer for your problem. Nevertheless, in any case:

1) you have to set as zero absorbance your reference blank (for example, in the below protocol the reference blank is butanol) before you execute any sample measurements

2) you have to subtract from your sample measurements the appropriate blanks.

A Lipid Peroxidation (LP) protocol for plant samples

The samples were ground at 4oC under dim light (to prevent artificial LP) in a porcelain mortar with homogenization buffer containing 50 mM Na2HPO4 pH 7.2, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM butylated hydroxyanisole (BHA) and 0.15% ethanol. The homogenization was carried out using 5 mL buffer per g fresh sample weight. The homogenate was assayed for LP products by a modified thiobarbituric acid (TBA)-based method (Buege and Aust, 1978). Specifically, 0.5 mL homogenate was mixed with 0.5 mL TBA reagent containing 0.5% (w/v) TBA, 20% (w/v) trichloroacetic acid (TCA) and 0.33 N HCl. To the resulting mixture was added 5 μL 2% (w/v) of the lipid antioxidant BHA (in absolute ethanol) to prevent artificial LP during the assay. The mixture was incubated at 100 °C for 15 min and brought to room temperature (RT). To that was added 1 mL 1-butanol, mixed by vigorous vortexing, centrifuged at 15,000 g for 3 min, and the absorbance determined with a Shimadzu UV-1601 spectrophotometer (Shimadzu Corp., Japan) of the upper butanol layer was measured at 535 and 600 nm (subtracting the non-specific absorbance) against a sample blank (0.5 mL sample mixed with 0.5 mL 20% TCA in 0.33 N HCl and with 5 μL 2% w/v BHA) and a reagent blank (0.5 mL homogenization buffer mixed with 0.5 mL TBA reagent and 5 μL 2% w/v BHA). Absorbance difference A(535–600) was converted to malondialdehyde (MDA) equivalents with the molar extinction coefficient 1.56 . 105. M-1. cm-1. LP was defined as TBA-reactive substances (TBARS) and was expressed in nmol MDA equivalents per g dry weight.

Thank you for your help. The procedure i used in brief; 15g of trichloroacetic acid, 0.375g of thiobarbituric acid and 100ml of 0.25N hydrochloric acid were mixed as the stick reagent. 1 ml of samples were put in test tubes. 2ml of the stock reagent was added to the test tubes and the blank test tube and the put in a 100°C water bath for 15minutes. It was then cooled and centrifuged at 1000rpm for 10 minutes. The supernatant was then put in a cuvette and absorbance read at 535nm after zeroing with the blank.

I think you have to reconsider all the procedure to find the possible error.

1) what is your sample and how is it prepared?

2) is your sample properly diluted before the TBA reaction?

3) what is the blank to which you add 2 ml of the reaction reagent?

4) do you zero the spectophotometer with the above blank?

5) do you use sample blanks? if you use how are they prepared?

6) I suppose that the final centrifugation precipitates any undesirable material. However, are the samples (supernatants) transparent in the cuvette or is there any turbidity? Do you follow the same procedure for the sample blank for each different sample?