retention time in amino column

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mohd
mohd's picture
retention time in amino column

when we add buffer in mobile phase the retention time in amino column decreases but we add acetonitrile retention time increases.in case of c18 or c8 the case is reverse ,why?

Sami Tuomivaara
Sami Tuomivaara's picture
mohd,

mohd,

Both normal and reversed phase chromatography methods rely on partition of of the analyte between the stationary phase (in your case either polar amino or non-polar alkyl coated) and mobile phase (polar aqueous and non-polar organic solvents in some ratio).

The partition of the analyte between the stationary and mobile phases depends on which one the analyte "resembles" more. The best single determinant of "resemblance" is the polarity. If the analyte has lot of amino, hydroxyl, carboxyl or carbonyl groups it is polar. If the analyte has lot of methyl or longer alkyl groups (for example C8 or C18 chains) or aromatic rings, it is non-polar in character. The partition dictates whether the analyte elutes fast or slow - analyte which partitions well to the stationary phase elutes late and analyte which partitions well to the mobile phase elutes early.

It all boils down to the relative polarity of your analyte with respect to the polarities of the mobile and stationary phases. You can follow this basic logic to predict the behaviour of analytes of different polarities upon changing the mobile and stationary phase polarities.

Cheers,

Dr. Analytical
Dr. Analytical's picture
Here are some more thoughts.

Here are some more thoughts.

On the amino column, this is "normal phase" mode and water is a "strong" solvent (more water means less retention).  Acetonitrile is a "weak" solvent; more acetonitrile means more retention.

In a "reversed phase" column like C18, the situation is reversed - water is a weak solvent and acetonitrile is a strong solvent.  As Suola says, the molecule will go to the phase that it is most similar to.
 

efoconnor
efoconnor's picture
with buffer the AAs are more

with buffer the AAs are more soluble in the mobile phase than they are in the column with organicn, they are more soluble in the column than in the mobile phase.

mohd
mohd's picture
But my chromatography is

But my chromatography is reverse phase chromatography.So why the same?

Bryan Evans
Bryan Evans's picture
Aminopropyl phase is a

Aminopropyl phase is a combination of normal phase and anion exchange:
http://www.imtaktusa.com/site_media/files/technical_information/TI316E.pdf

So it makes sense that you see decreased retention with increased buffer concentration

rupalimahajancpn
rupalimahajancpn's picture
Sir

Sir
i have one molecule which analyse on amino colume already
but for the study of molecule i dont have amino colume
can i analysis this molecule by c8 or c18 colume ?

Dr. Analytical
Dr. Analytical's picture
See the post by Bryan Evans

See the post by Bryan Evans above.  The amino column separates by "normal phase" (water is a strong solvent) and "anion exchange" (the buffer is a strong solvent).  On a C8 or C18 "reversed phase" column, water is a weak solvent. 

You may be able to get good retention on either column, but if you want to decrease retention on amino, you add more water or more buffer.  If you want to decrease retention on C18, you add less water.

The order of elution will not be the same, so if you have other components in your sample, they will not elute in the same order on the two different columns.