binding affinity for liquid chromatography

[enzymepurifier]

Is there any relationship between total resin binding capacity for a "target" molecule and when in a gradient elution that "target" molecule elutes off? In other words, if a molecule elutes off earlier in a gradient, is that an indication of lower binding capacity relative to a different target that would elute off higher in a gradient with the same conditions?

[CharonY]

I am not entirely sure what you are asking, as your title and OP actually refer to different things. Generally, the binding capacity (which most often refers to dynamic binding capacity) refers to how much target molecule a packed column can bind before breakthrough of unbound analyte happens. So in that regard, the capacity in itself does not influence elution behaviour if you load less than the total capacity. If you overload the column the result is that unbound analyte will elute fast, providing no real separation.

 

The strength of the affinity between analyte and stationary phase on the other hand, does determine the elution behavior (and is pretty much the basis of this separation technique).

[enzymepurifier]

Sorry, for the confusion - I am still a little confused I think... If we are hypothetically working with an ion exchange stationary phase (say SP resin), is the analyte:SP resin affinity in anyway correlated to to when it would elute off the column in a 0-1M NaCl gradient?

 

i.e. If the analyte elutes early in the gradient would that indicate weaker affinity than if it eluted late in the gradient?

Sorry, for the confusion - I am still a little confused I think... If we are hypothetically working with an ion exchange stationary phase (say SP resin), is the analyte:SP resin affinity in anyway correlated to to when it would elute off the column in a 0-1M NaCl gradient?

 

i.e. If the analyte elutes early in the gradient would that indicate weaker affinity than if it eluted late in the gradient?

I think you already answered this actually --- but, if this is true wouldn't binding capacity of a stationary phase be based on the strength of affinity to that specific analyte?

[CharonY]

Yes and no. The total capacity is somewhat dependent on binding affinity, but not solely. If you pack the column more or less dense for example, you will change the overall capacity but you will not change the affinity the stationary phase itself. Or if you change flow rate. Again, change in total capacity, but the material does not change.

 

Edit: that being said, keeping all else constant one could reasonably infer that capacity is a function of affinity. But while the elution time is somewhat dependent on affinity, it is again not the the sole factor. Additional effects such as interaction with column material (rather than the resin itself) can change the elution order, for example. This is why these analytical techniques typically require empirical validation as models usually are not perfect predictors.

 

But if your question is more whether there is a correlation at all, then the answer is yes, but depending on the effect you see, it may not tell you enough about what is happening in your system

Edited April 21, 2017 by CharonY