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

Vol. 5, No. 2.                                                                        Second Quarter, 2016

When Your C18 Column Doesn't Work

In the last newsletter we talked about what I expect from a "good" LC method.  Two important criteria were:
  • Produce adequate retention (k> 2)
  • Not operate near the limits for the column or instrument
How can you predict if a molecule is likely to be difficult to separate on C18?  Problem analytes usually have one or more of these characteristics:
  • Highly water soluble
  • Ionic or a strong acid or base
  • Carbon/heteroatom ratio less than 3.
  • log P (octanol-water partition constant) is negative

The water soluble vitamins include several compounds that are difficult to separate on a traditional reversed phase column.  Ascorbic acid (Vitamin C), for example, is highly water soluble and difficult to retain on a C18 column.  I have seen many tricks (high salt or acid, no organic, ion pairing), but all are problematic in one way or another. 

No matter what you try, you are likely to experience difficulty because, quite simply, you are trying to make a very polar molecule stick to a very non-polar surface.  They don't like each other.

The alternative?  Use chemistry!  Attract your polar molecule with a polar phase.  Examples include diol/OH, cyano, and pentafluorophenyl (PFP).  There are other possibilities, but these are often now referred to as "HILIC" or aqueous normal phase.  Here is an example:

OK, it's a little messy, but let me point out some important results.  The lines connect the same molecules on each phase, and crossing lines indicate a change in relative retention order (selectivity change).  There are many, which is to be expected when you switch to a very different stationary phase. 

Now, focus on ascorbic acid and nicotinic acid, both barely retained on C18.  They are the last to elute on the polar column!  Notice that the other acids, pantothenic and folic, are also highly retained..  So, if you need to analyze these, or other organic acids, this column looks like a much better option.  Of course, there are still some problem analytes,thiamine, nicotinamide, and pyridoxine, but we are working on those now.

The take home message here, is simple: match the polarity of your analytes and stationary phase, and you will probably  have much better luck with retention!

In the next newsletter we will look at general retention trends on these HILIC columns.

As always, feel free to contact us if you have any questions.


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Thought For Today

"Truth is much too complicated to allow anything but approximations."

– John Von Neumann
In This Issue

Other Training Events

EAS Short Courses
Somerset, NJ
Troubleshooting Chromatography Systems
13-14 November, 2016
Sample Preparation
15 November, 2016
Practical Guide to Reversed Phase LC
16 November, 2016

Infinity Series HPLC Maintenance and Troubleshooting (2 days)

29 September 2016
08 December 2016

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


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Contact Details
Merlin K. L. Bicking, Ph. D.
ACCTA, Inc.
3534 Jessie Ct, Saint Paul, MN  55125 USA
Phone:  +1 (651) 731-3670
Email: info@accta.com