HPLC CALIBRATION

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benazir
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HPLC CALIBRATION

Is it ok to use uracil standard solution for hplc calibration? also i am using methanol 100% as mobile phasse

Rajiv Dua
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Calibration Standards (varying amounts of aspirin and caffeine plus internal standard in methanol) If your unknown has acetaminophen in it, make sure you don't overload your column. For example, although Exedrin may contain 250 mg of acetaminophen, we will get a split peak caused by column overloading at this level. Use 25 mg of acetaminophen instead. Suggested values for aspirin and caffeine will probably work as specified.

Use four 125 mL Erlenmeyer flasks, rinse them first with methanol. Dissolve the solids in the 30 mL of methanol before adding the internal standard. The amounts given for Aspirin and Caffeine are for example only. You should choose amounts that fit the tablet you are analyzing. For example, a bottle of "Extra Strength" Exedrin states that each tablet has 250 mg of Acetaminophen, 250 mg of Aspirin, and 65 mg of Caffeine. Assume we chose to dissolve half a tablet. Reasonable choices for our standards would then be:
 

Flask #

Aspirin

Caffeine

Methanol

Internal Standard

1

100 mg

40 mg

30 mL

10.0 mL

2

125 mg

30 mg

30 mL

10.0 mL

3

150 mg

20 mg

30 mL

10.0 mL

4

175 mg

10 mg

30 mL

10.0 mL

Unknown

1/2 Tablet

1/2 Tablet

30 mL

10.0 mL

 

To avoid weighing out individual portions of solids as small as 10 mg, prepare standard solutions of Aspirin and Caffeine in methanol, add the appropriate volumes, then enough methanol to reach about 30 mL. (You can use a larger volume than 30 mL if you need to, just keep the final volume about the same for all solutions so that we will get similar detector responses.) If you have problems getting the Caffeine to dissolve, try using some of the acidfied water/methanol mobile phase as a solvent. The water and lower pH should turn Caffeine into a cation that will readily dissolve. Then add the 10.0 mL of Internal Standard. The composition of the aspirin and caffeine solutions is left as an exercise for the student.
Remember too that the detector measures the absorbance at 250 nm. These substances will not all have the same molar absorptivity at this wavelength, resulting in different peak sizes for similar amounts. You may have to adjust the concentrations if your detector cannot accurately measure the full range of peak sizes for the concentrations you have chosen. (See the warning about acetaminophen given above.) A general rule is that structures showing extensive networks of conjugated double bonds should have higher molar absorptivities than those that do not.

Analysis of the Data

After running the Unknown and the Calibration Standards and getting retention times and peak areas for all the peaks, prepare working curves by plotting Peak Area/Internal Standard Peak Area vs. mg of Standard from your calibration runs, then use Peak Area/Internal Standard Peak Area from the peaks of your unknown run to read the amounts from the calibration curves. Use the usual statistical methods on your working curves. You should be able to deduce which peak is which from the way the peak areas change when you run the standards. Make the appropriate calculations to compute the mg of each ingredient in the original tablets.

benazir
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i am talking specifically

i am talking specifically about hplc system calibration ....why do we need aspirin and paracetamol?

ytrao
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it is not suggestble to use uracil standard for calibration because It  is unretained.  

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Uracil has been added to the standard mixture to provide a dead time marker .

Thanks,
Tirumal

Dr. Analytical
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The compound that you use for

The compound that you use for calibration will depend on what type of calibration you are doing.

Detectors are often calibrated using caffeine.  The flow rate can be checked using any compound that produces good chromatography, or you can just measure the volumetric output.

If this does not answer your question, please write back with more information about what you want to do.