Lab Life-Line Monthly
Practical Advice for the Busy Lab
from ACCTA, Inc.

Vol. 5, No. 1.                                                                        First Quarter, 2016

A C18 Column Is Not Always the Best Option

When I evaluate a method, I am usually not concerned about whether it is new or old, or fast or slow (I can fix "slow").  My main concern is whether or not the method does what it is supposed to do.  What should a good method do?  Here is a short list:
  • Provide an acceptable separation of all analytes of interest
  • Provide adequate separation of analytes from other unwanted peaks in the chromatogram
  • Produce acceptable peak shape (not always good, but good enough)
  • Generate peaks that can be reproducibly integrated (signal/noise> 10)
  • Produce adequate retention (k> 2)
  • Not operate near the operating limits for the column or instrument

Let's focus on the last two items in the list - retention and operating conditions.  One of the most disappointing things I see is methods that assume that a C18 column is the only option.  The result is usually a bad method that is either not efficient or not reproducible.

The most common scenario is a method that specifies a C18 column and a mobile phase with little or no organic solvent modifier.  Historically, we have always recommended that you use at at least 5% organic modifier (acetonitrile, methanol, THF, etc.).  To be fair, many manufacturers have developed "aqueous stable" stationary phases which do produce more retention for those analytes that a poorly retained on a traditional C18, and some other specific phases can be used with all aqueous mobile phases.  However, the problem methods do not usually specify such columns; they just assume that all C18 columns are alike. 

How can you predict if a molecule is likely to be difficult to separate on C18?  You can get some clues from examining the structure and properties of the molecule.  For example, these types of compounds may be poorly retained:
  • Highly water soluble, with much lower solubility in organic solvents
  • Ionic or a strong acid or base that is ionized in the pH 2 - 7 range
  • Carbon/heteroatom ratio less than 3.  (Count up the number of heteroatoms - oxygen, nitrogen - and compare to the number of carbons
  • log P (octanol-water partition constant) is negative

If your molecules have two or more of these properties, you will probably not see much retention on C18 unless you use little or no organic modifier.  Some examples include:
  • Water soluble vitamins
  • Nucleosides and nucleotides
  • Sugars and other saccharides
So, what are your options?  There are many, and in the next newsletter, we will look at some examples of problem separations, and show you better options.
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Thought For Today

"The basic texture of research consists of dreams, into which the threads of reasoning, measurement and calculation are woven."

– Albert Szent-Györgyi von Nagyrapolt,
Nobel Prize winner
In This Issue

Other Training Events
07 July 2016
29 September 2016
08 December 2016

·
OpenLab ChemStation Oparation (C versions) (4 days)
09 August 2016
18 October 2016
24 January 2017

Practical Guide to  Reversed-Phase HPLC (1 day)
EAS
Somerset, NJ
14-16 November 2016

Download Section
Minnesota Chromatography Forum, May, 2014:
Sometimes spending a little money can save you a lot!
Contact Details
Merlin K. L. Bicking, Ph. D.
ACCTA, Inc.
3534 Jessie Ct, Saint Paul, MN  55125 USA
Phone:  +1 (651) 731-3670+1 (651) 731-3670
Email: info@accta.com



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