Inter-molecular binding titration using spectroscopic techniques like UV absorbance spectroscopy, fluorescence emission spectroscopy, etc require small amounts of samples in micromolar to nanomolar range. For this type of work, it is essential to accurately calculate the concentrations of both the ligand and the receptor.
Most common method of concentration calculation is the volumetric method in which one correctly weighs the compounds and make a solution by dissolving them in a suitable solvent. This method, however, can accumulate errors at various stages.
To accurately measure the concentration of biological molecules and drugs, it is often advisable to calculate the same using spectroscopic method. Spectroscopic method utilizes the Lambert-beers (LB) law with volume correction as appropriate.
LB law can be written as concentration C = A/E
where A is absorbance and E is the molar extinction coefficient of the compound.
Let’s see how the spectrometric method works. For this, we need the following:
A. Molar extinction coefficient of the compound of which the concentration is required.
B. Intensity of the Absorbance maxima (in a.u.) of the compound at a particular volume.
C. UV spectrometer.
1) First take some compound (without measuring) and make a homogenous dilute solution using a suitable solvent.
2) Initialize the double beam UV spectrometer.
3) Clean 2 absobance-free quartz cuvettes* and fill them with known volume of solvent.
4) Place the cuvettes appropriately in the UV spectrometer and scan the wavelength range.
5) Now take 2-4 microliters of the solution prepared as above (1) and add it to the sample cuvette already having solvent (as in step 2).
6) Now scan the cuvettes the full UV-Visible range to find out the peak maxima.
7) Record the peak maxima wavelength and its intensity.
8) Now calculate the concentration as shown below.
C is the concentration.
Volume of cuvette comprises the initial solvent volume + 2 or 4 microliters of compound solution added.
E is the molar extinction coefficient of the compound at peak maxima (this value is often reported in the literature).
Absorbance denotes the intensity of the peak maxima obtained after scan.
After calculating by the above method, the resulting concentration will be in micromolar unit.
*In a double beam UV spectrometer, one cuvette is used as a reference and the other is used as a sample cuvette.