PharmPK Discussion - Zwitterionic compound and HPLC

• On 14 Aug 2007 at 21:54:05, Tai Ly (twly911.-a-.yahoo.ca) sent the message
  

  
  I have a zwitterionic compound with a carboxylic acid on one end and
  a free aromatic amine on the other.  The compound initially elutes
  very close if not dead on the void peak area.  I have managed to get
  it to elute 2 minutes past the void time by slowing down the flow
  rate and starting the gradient with even less organic (5%) out the
  gradient but I am now seeing 2 peaks, one at or near the void time
  and the other 2 minutes later.  LC/MS indicates it to be the compound
  in question.  On other samples which I believe are more dilute, only
  the peak eluting 2 minutes later is observed which leads me to
  believe that the earlier peak is due to saturating the column's
  capacity to absorb the compound.  Any thoughts on how to tweak the
  method further?  I cannot really change the column.  The specifics of
  the method is as follows:
  
  Column:  C18, 150 x 4.6 mm, 5 um
  MPA:  0.05% formic acid in water
  MPB:  0.05% formic acid in methanol
  gradient:  5% B to 90% B over 15 min.
  flow rate:  0.5 mL/min
  
  thanks
  
  tw
  
  

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• On 15 Aug 2007 at 11:57:25, "RAJAN K S" (komburajan.aaa.gmail.com) sent the message
  

  
  Hi Tai,
  I work with amino acids which are zwitterionic.
  What I have observed is the reconstitution buffer contributes more in
  peak splitting or appearance of another peak. Please make sure that
  you have the starting mobile phase composition in your reconstitution
  solution.
  All the best
  Rajan Kombu
  
  

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• On 15 Aug 2007 at 11:13:18, "Michele Sanders" (mes.-a-.sparhawklabs.com) sent the message
  

  
  The following message was posted to: PharmPK
  
  Can you change to a longer column or smaller particle size (3-um)?
  
  Can you change to a TFA type reagent instead of formic acid?  TFA can
  ion
  pair and get more retention time than formic acid in some instances.
  
  What is the pka of your compound, and is your mobile phase too close
  to the
  pka?  Mobile phase pH should be more than 2 units away from pka.
  
  Have you changed pH of mobile phase?
  
  Can you shallow out your gradient or perform the gradient over a longer
  period?
  
  Can your C18 column tolerate 100% aqueous at the beginning of the
  gradient?
  
  Can you change to a C18 column that will tolerate 100% aqueous over a
  period
  of time?  Using TFA and 100% aqueous hold for a few minutes at the
  start of
  a gradient has bought me additional minute(s) for hydrophilic compounds.
  
  How much compound is on column, taking into account solution
  concentration
  and injection volume?
  
  Michele
  
  

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