HPLC Method Development around the World: Why it is so hard

Taking a method that was developed and published from another country and trying to use it in another country – why should that be so hard? Well, once, I was trying to reproduce an HPLC method from a Japanese group published in an Analytical Chemistry Journal (Honda et al 1991 Anal Biochem 199: 256). It turns out the columns used, in the paper, were not available NOR for sale in the US. I tried many columns, with similar characteristics (packing material, coating, etc.) and none worked (even with help from companies selling columns based on their knowledge). I basically had to start from scratch and develop my own system, which I did and it was totally different and more complex.

Another time, I was using an older method, which used high pressure solvent mixing, versus what I had, which was low pressure mixing and could not reproduce the separation. Again with modifications, I was able to get the right results. As it turns out, high pressure mixing, involves solvent compressibility calculations in its mixing algorithm and if not calculated properly can actually throw off the solvent mix the column sees.

So the take home points:

  1. Make sure all the instruments are the same (avoids void volume issues, etc.)
  2. Make sure Solvent and water quality are the same (this is easier now).
  3. Make sure the columns are the same and available.
  4. Use set standards and if possible the same standards

David Slomczynski, Ph.D; Geometrick Enterprises


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