HPLC method

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sarika sawant
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HPLC method

I have a problem in Calculation of Assay ,which is analyse by HPLC method .Retension Area of standard as well as sample is much more vary , it indicate unequal concentration.But due to back calculation ,i am not getting expected result .
I have a Query that i) The sample which i analyse have lower label claim or ii) Is there any factor is to be added while calculating % Assay .or iii) The instrument is not giving proper reading. Can anybody will help me ?  I am new to handle this system.
Looking forward of anyone's reply.....

sarika sawant
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   I would be glad if " Dr

   I would be glad if " Dr.Analytical sir" will give me reply.......

Dr. Analytical
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Dear Sarika Sawant:

Dear Sarika Sawant:
We need more information to completely solve this problem, including information on the mobile phase, HPLC system, column, and settings (flow rate, injection volume, detector settings).  An example of the differences in retention time and area would also be helpful.

If you do not have good reproducibility for both your standards and samples, then you will not get a good result.  You should make six injections of standard and six injections of a sample, and compare the average, standard deviation, and %RSD (100*Std Dev/Average).  The %RSD should usually be less than 1 %.  

If the %RSD is too large, then investigate problems in the instrument.  If the reproducibility is acceptable, then prepare another standard and another sample. Maybe you have a preparation error.

sarika sawant
sarika sawant's picture
Thanks Sir for your reply .

Thanks Sir for your reply .
I have notice about my developing method ;
As you said :- i) The Std as well as Spl should be reproducible but it get vary.
ii) As i have injected std & spl thrice which will not give precise result.
iii) My analysis is based of multivitamins Premixes ,the Std Area's are having % RSD of below 1% but Spl Area does not complies.

I 'll be modifying the method as per your suggestion . And I'll
be back with my result and regarding of system suitability
Thanking you,

sarika sawant
sarika sawant's picture
Quantitaive Analysis of Folic

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

1)  System Suitability :-




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      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 you reply. 
  Thank you !!!!!