assay by hplc

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thirupathi
thirupathi's picture
assay by hplc

hello guys
can i do the assay by hplc for reaction mass samples .plz provide the  assay calculation procedure

Dr. Analytical
Dr. Analytical's picture
Please give us more

Please give us more information about what you are trying to calculate?
If you are trying to evaluate the results from a synthetic reaction, then HPLC is a very good way to do this.  The HPLC experiment can tell you how many reaction products are in the sample.  However, there are some cautions.
You can see how many peaks appear in the chromatogram, and this will be the minimum number of different compounds present.  However, each peak may not be only one component.  Two or more compounds can elute at the same time.  You must adjust your separation conditions so that the molecules stay on the column long enough to separate from each other.  A "gradient" separation is usually a good way to analyze an unknown sample.
Calculating how much of each compound is present can be difficult.  You must have a standard to calibrate the system.  Only then can you calculalte % yield.  The percent peak area only tells you the relative from the detector, and this can change with detector settings and type of detector.
Does this answer your questions?
 

Santosh@India
Santosh@India's picture
Assay estimation by HPLC is

Assay estimation by HPLC is possible , the things that you should know is
1)You should know the impurity profile ,as any rection can creats new side products/impurities eluting at same rT as that of analyte.
2)The final volume of the rection mixture.
3)Should know the responce factor of individual components in the reaction mixture to know their exact concentraction.