analytical method development

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humaira's picture
analytical method development

can I get the synopsis for analytical method development in UV-Vis Spectroscopy.I would like to carry out the work on drug formulations

Lynn E.
Lynn E.'s picture
I am not aware of any

I am not aware of any synopsis- it is something best learned by doing ?).

In method development, you start at the beginning. What is the compound? Does a lit search bring up any spectrophotometric methods for your compound of interest, or a related compound (one that might be expected to react similarly under the same chemical conditions)? What other components in your sample could interfere? Are there ways to reduce or eliminate this? (kinetic discrimination, SPE clean up, etc,)

Once you find a promising method candidate or two, the next thing is some quick and dirty test tube work. I usually make reagents, and start with a blank (whatever the sample will be in) and a high level standard. By starting high, I at least know that the colorimetry is functioning. ALWAYS include a blank. Or you may find down the road you get the same color formation regardless of concentration!

You can then set up a matrix experiment to vary reagents ratios, time, and temperature to determine where you get the best sensitivity and most stable end product. (Fast is no good, if you can not measure everything before it is too late). I always start with the literature, and go from there (no sense reinventing the wheel if you don't have to)

Next, work on diluting the high concentration standard down, and work your way down into the range in which you need to measure (which is often ppbs).

If the reaction is slow, heat or time will help. If you are trying to go very, very low you may find that reagent concentrations need to be increased. (This is simply to help kinetics- more reagent in solution increases the chances your analyte and reagents will interact). If (this is unusual) you have too much signal, you can often measure at a wavelength that is not directly at the absorbance max, but off to the side. (Calibrate under these conditions to be sure response remains proportional to concentration!)

Sometimes, you need to separate one compound from a group of related ones. In this case, IC or HPLC, especially in conjunction with post column derivatization can help. If your compound has double bonds, UV detection with HPLC can work BUT this is a bulk property (not specific) and you have to very carefully work out your separation (HPLC) to be sure you are measuring what you think you are (again, start with a solution of ONLY the compound of interest, at a high enough level that you will get a peak.)

Most important- have fun!

Html Color
Html Color's picture

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Jonh Braves
Jonh Braves's picture
thank your post

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