Scientist Solutions: International Life Science Community By Scientists For Scientists
    
Home » Forums » Analytical Chemistry » Chromatography » HPLC

Thanks to our sponsors who make this site possible

HPLC

RSS Feed

Would you like to save this topic, event, protocol or job so you can find it again easily?

Just click the "Save to My Lab Drawer" link and the item will be saved in the My Lab Drawer section of your bench space.

Available to members only. Please log in or register for your free account now.


Send PM
See Mini bio

Status: Frog Egg
Frog Egg
Topic Started by sarika sawant
on 7/21/2012 20:07 PM   
Reply to this post Go to the top of the page

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 !!!!!

Replies

Send PM
See Mini bio

Status: Frog Egg
Frog Egg
Posted By sarika sawant
on 7/27/2012 20:18 PM   
Reply to this post Go to the top of the page

 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
United States

Send PM
See Mini bio

Status: Analytical Chemistry Moderator
Frog Laureate
Posted By Dr. Analytical
on 8/1/2012 8:23 AM   
Reply to this post Go to the top of the page

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.



Send PM
See Mini bio

Status: Frog Egg
Frog Egg
Posted By sarika sawant
on 8/3/2012 21:08 PM   
Reply to this post Go to the top of the page

 "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
United States

Send PM
See Mini bio

Status: Analytical Chemistry Moderator
Frog Laureate
Posted By Dr. Analytical
on 8/9/2012 8:21 AM   
Reply to this post Go to the top of the page

Sarika:

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

Thank you for using ScientistSolutions.

 


As a Scientist Solutions member, you are able to register a positive vote for any topic which you believe is useful and relevant to our board or any reply which you believe is especially well worded and helpful.

By participating in the voting, you will be helping to identify the best topics & replies on the board.

You may vote once for any one post, and you may not vote for your own posts.

A post (topic or reply) will earn one "thumbs up" icon for every 10 votes received (up to 3 thumbs up), and the person who made the post will also earn two bonus points.

learn more about member points.



Click here to
Become a member & join our
community (It's easy & free)
Already a member? Please log in
User Name  
Password  
Forget Password?
Scientists
Not finding the answer you need?

Post a new topic

You must be logged in to post. Log in above.
Not a member yet? Click here to register
(it's free)
Thank You to Our Sponsor