HPLC

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sarika sawant
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HPLC

Quantitaive Analysis of Folic acid in Premix .
I have modified IP method  :-Modified one

1)  System Suitability :-
      Column :- C18 , 250 x 4.6 mm, 5 µ     (Applicable for 200 mm x 4.6 mm , 10 µ    )
      Flow rate :-  2.5 ml /min    ( It was 2.0ml/min ) Applied Formula 1    (Given below)

      Wavelength :-  283 nm
      Injection Qty = 20 µl
      Mobile phase :- Buffer 93 part and water 7 part

  2)   Mobile phase :- 0.05 M of KH2PO4  and  ACN ( 93: 7) ,Adjust pH 6.0 by using 1 M NaOH sol.

  3)   Standard solution :- 20 mg dissolve in 100 ml of 0.1 M NaOH ,5 ml of it dissolve in 100 ml of     
         Mobile  phase i.e 10ppm solution .

  4)   Sample solution :- Equivalent to 20 mg of folic acid premix  dissolve in 100 ml of 0.1 M NaOH ,    
         centrifuge than 5 ml of supernatant dissolve in 100 ml of mobile phase . i.e 10 ppm solution .  
     
  Observed   Result : -  Assay 94.56 %,   RSD % (n= 5 replicate ) :- Std  = 3.40 % and Spl = 3.32 %
                            
  Formula :- 1 :- Reference column (desired one ) ,2 :- Second column (undesire one )
                      r :- radius , L :- length  F:- Flow rate

                     (Column dimension 200 mm x 4.6 mm  to desired one 250 mm x 4.6 mm achieve only by   Adusting Flow rate )
                     F2 = F1 x L2 / L1 x ( r2/r1) 2       ;  As    ( r2/r1) 2  = (4.6/4.6)2 =1

        So,       2 = F1  x 200/250 x 1     ; F1    = 2.5 ml / min..

I have performed this method ,  Can i assign it a develop method by giving a RSD limit ?.

"Dr Analytical sir"  please  give your comment on this article .
  I am Eagerly waiting for your reply. 
  Thank you !!!!!

sarika sawant
sarika sawant's picture
 If anybody like to give

 If anybody like to give there suggestion ,regarding to this method are welcome .
 
Dr. Analytical sir ,pls give your comments on this Article.

With Regards,
Sarika.

Dr. Analytical
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Dear Sarika Sawant:

Dear Sarika Sawant:
Your equation for the new flow rate is not correct.  Flow rate is adjusted only when you change column diameter (no change here) or particle size (change dp from 10 um to 5 um).  Usually, we assume that your existing column is column 1 and the new column is column 2.

The equation should be

F2 = F1 X (r2/r1)2 X (dp1/dp2) = 2 X (4.6/4.6)2 X (10/5) = 4

However, I would not use the new column at such a high flow.  Usually, when we change to smaller particles (form 10 to 5 to 3), we also reduce the column length.  You should get similar results if you use a 100 mm column with 5 um particles.  

Also, a flow of 2 mL/min is very large for a 10 um column.  A typical flow is less than 1 mL/min.  A typical flow for a 5 um column is about 1 mL/min.  I think the flow was very high becase the retention time was very long.  

If you have good results for the new coluns and new settings, then you can use the new method, but you must document all changes and validate the change. 

However, this is not a good method for modern HPLC instruments.  I could develop a method which is much faster and may give better results and would be less expensive to operate.

sarika sawant
sarika sawant's picture
 "Dr.Analytical Sir " Thanks

 "Dr.Analytical Sir " Thanks a lot for explaining in such a beautiful manner . I will always follow this

  suggestion while experimenting a new method .

  As well as i 'll go on maintaining the records and also validate the new method.

  And once again thanks a lot "Sir"

  With Reagard's
  Sarika

Dr. Analytical
Dr. Analytical's picture
Sarika:

Sarika:
You are very welcome.  I wish you success with your development work.

Thank you for using ScientistSolutions.