In my spectrophotometer specifications say that the linear spectrophotometric range is 0-3 AU. Then why it can not give an accurate measurement above 1 AU? it gets saturated. I guess spectrophotometers cannot overcome 1 AU. Then why HPLC can reach up to 2 AU linearly? Did I work all my life with bad spectrophotometers?
Dear Alfonso,
The photometric range first of all depends on the stray light level in monochromator. Many routine spectrophotometers have the stray light of 0.05 - 0.1% and even higher. Thus, if You will measure the absorbance of absolutely non-transparent object (for example, steel) with 0.1% stray light you'll obtain the 3 AU value instead of infinity, you called it "saturation". The stray light level depends on wavelength and in short wave region can increase dramatically, often to 1% ("saturation" at 2 AU). As for your sample with the maximum measureable absorbance of 1 AU, I think this value is obtained using the reference cuvette with the same solvent, which absorbance is subtracted with the instrument. The background absorbance of solvent at the working wavelengths can be high, so the real value for your sample can exceed 2-3 AU. There are several solutions for problem: to use the spectrophotometer with double monochromator (ultra low stray light level), to use more transparent solvent, to use short lightpath, to work at another wavelength.
I suggest some of tips for problem solving...
1) you have to calibrate ur spectrophotometer according to pharmacopeia....
2) your solvent selection should be right
3) your have to dilute ur sample
4) contact ur service provider
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