To determine a concentration gradient, the blank should be treated as zero and the absorbance of known concentrations (different dilution levels) at a specific wavelength should be to determine the standard curve. For this purpose, you need to make different dilutions. That is the standard procedure of making a calibration curve. So if there is any absorbance with the blank/standard, that should be subtracted. Once you make a standard curve with known concentration gradient, then you can calculate the unknown samples through the equation of the standard curve.

I hope it makes sense.

In addition to what Dr Muhammad Saad Khan said, I would suggest you consider abs values less than 0.5 for accuracy.

Also see attached papers for simple protocols to determine EE of liposomal / nanoliposomal formulations:

Colas, J. C., Shi, W., Rao, V. M., Omri, A., Mozafari, M. R., & Singh, H. (2007). Microscopical investigations of nisin-loaded nanoliposomes prepared by Mozafari method and their bacterial targeting. Micron, 38(8), 841-847.

------- and:

ElMeshad, A. N., Mortazavi, S. M., & Mozafari, M. R. (2014). Formulation and characterization of nanoliposomal 5-fluorouracil for cancer nanotherapy. Journal of liposome research, 24(1), 1-9.

Thanks for response. I did the experiment but there's an error somewhere because my drug recovery was 264%. I've attached a picture of my calculations and calibration curve but to summarise.

My blank was done using 0.5ml empty liposome + 3ml EthOH so 7-fold dilution (absorbance 0.088) and zeroed.

For drug recovery dissolved 0.5ml of loaded suspension + 3ml EthOH gave an absorbance of 1.408.

I did a second measurement using 0.25ml of loaded + 3.25ml EthOH (so 14-fold dilution)

Absorbance was 0.715, so both measurements were within my linear curve.

I used the 0.715 value to calculate drug recovery using equation on my calibration curve;

y=0.1513x-0.0025

making x the subject to find concentration where y is absorbance

0.0025+0.715/0.1513 = x

= 4.74ug/ml

I then multiplied 4.74 by the dilution factor of 14 to get a conc 66.36ug per 0.25ml.

My initial concentration was only 100ug per ml (100mg lipid and 5mg curc in 50ml).

So a drug recovery of 264% is clearly wrong.

Where have I gone wrong?

This puzzle has been bugging me all weekend!

I figured it out, my drug recovery is 14.2ug/ml after 14-fold dilution and thus recovery is about 66mg or 66% recovery.