If you don't see the expected response at the predetermined wavelength for UV absorbance measurement, think about where you may be losing your molecule of interest during the experimental process. Is it soluble? Did you attempt to isolate the molecule? Was the recovery poor, was it lost during the extraction process? Consider the HPLC system, is it performing properly? Are you using the correct column, or could it be your mobile phase or gradient? Is your molecule bonding irreversibly to the column or vials?
Although I don't have any specific experience with acrylamide, these are some of the things I'd consider.....
thank you for your response
actually I measured the UV absorbance of a certain Concentration of acrylamide (which i dissolved in water), and in parallel I measured the UV absorbance of a certain Concentration of a protein which i dissolved in water at the same wavelength.
and subsequently when I mixed the solutions, the absorbance of the mixture was significantly less than sum of two absorbance values.
all I know about the mixture is that there are only hydrogen bonds between acrylamide and the protein.
This may seem like a silly question, but are you taking into account any dilution which is taking place? From what you described above, the concentrations of both the protein and the acrylamide will decrease if you are mixing the two solutions together.