UV detection in HPLC

[imranali]

I can`t find the solution about the saturation of Peak in UV detection, I know that the peak will saturated when we inject the higher concentration but what is the resion behind that? And one more thing is that when we are appling the gradiant why the base line goes in nagative. the mobile phase is 10 mM ammonium acetate with acetonitrile in a linear gradiant starts with 10% acetonitrile and goes to 90% and again comes at 10%.

[tom jupille]

I can`t find the solution about the saturation of Peak in UV detection, I know that the peak will saturated when we inject the higher concentration but what is the resion behind that?

UV detectors measure transmittance (the fraction of light that gets through the flow cell). They use that to calculate absorbance (the negative log of transmittance) which is what we want since it is proportional to concentration. The catch is that an absorbance of 2.0 is the same as a transmittance of 0.01 (1%). It's hard to make a perfectly light-tight system, so by the time you get down to 1%, you are measuring mostly stray light. Any signal more than about 2 absorbance units essentially "flat-tops".

And one more thing is that when we are appling the gradiant why the base line goes in nagative. the mobile phase is 10 mM ammonium acetate with acetonitrile in a linear gradiant starts with 10% acetonitrile and goes to 90% and again comes at 10%.

Depending on the wavelength, the ammonium acetate solution has a higher absorbance than does acetonitrile (acetate has a UV cutoff around 210 if memory serves, but it does have measurable absorbance at longer wavelength).