acarbose for dissolution

[tradam]

Dear All ,
I want to make analysis acarbose dissolution for HPLC. My conditions
UV 210 nm Mobil phase 75/25 MeCN/Waters NH2 column, 20 ul injection.
However I have limit problems
İf any sugestions please send to me
thanks

[tom jupille]

Can you be more specific as to the "limit problems":

What mass are you injecting?
What detection limit do you need?
What column dimensions?
What flow rate?
What is the noise specification for the detector you are using?
How does your actual noise compare with the specification?

[tradam]

Dear Tom
First Thank you for your reply.
Lets explain prepare sample;
100 mg Acarbose disolve 500 ml water and 20 ul injection Column dimesion 250/4.6 100-5 NH2 flow 2ml/min and Shimadzu PDA detector.I give signal 4-5 mAbs
If I incrase to injection than more 20 ul , Peaks will bording

Thanks

[tom jupille]

Detection limit is ultimately controlled by signal/noise ratio. There are only two ways to improve this:
- more signal
- less noise

You have already established that you can't increase the signal by increasing the mass-on-column.

What happens if you go to a lower wavelength? Both the signal and the noise should increase (the question is which one increases more!)?

The other approach is to decrease the noise. I'll repeat the last parts of my earlier question:
- what is the manufacturer's specification for noise under these conditions?
- how much noise are you actually seeing?

If you are meeting Shimadzu's specifications, then there's not much you can do except to look at reducing noise computationally (averaging or bunching measurements to damp out short-term noise). If you are not meeting the specifications, then you need to establish why. Possibilities include:
- dissolved air in mobile phase
- aging lamp
- ambient temperature variations

[Uwe Neue]

Water is a strong solvent in this application. This means that the peak broadening probably comes from the water injected instead of the analyte.

On the other hand, the solubility of the analyte in the acetonitrile-water mixture may be limited. However, I would try this first: dissolve the sample in mobile phase instead of in water. If the sample is really much less soluble in mobile phase, you can always use this lower concentration and inject more. Now I expect that the peak broadening problem will be less than with the sample dissolved in water.