Analytical Chemistry Question:
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How do we fix the sample concentaryion in hplc method development. what is the basis?

Answers:

Answer #1
IF ALL IMPURTITIES AT SPEC LEVEL ARE MEETING S/N RATIO MORE
THAN 60 AT THAT PARTICULAR CONCENTRATION AS WEL AS NO
COLUMN OVERLOAD.

Answer #2
first run the our sample with approx sample concentration and run in HPLC, if we get any saturated peak in them than dilute sample concentration and again run in HPLC, when finally we can\\'t get any saturation and also we get our interested analyte peak than finallize sample concentration and also check our interested analyte recovery also.

Answer #3
How can we tell that the column is overload

Answer #4
Its fully based on scope of the method (AY, RS or Other)

If its AY/Content Method - The fixed test concentration must meet recovery (Which must be taken into consideration are system load, column load etc,.)
If its RS method - the method must give S/N Above 10 for the impurity which has less response against active at the level of 50% w.r.t impurity spec levels.

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