Amount of sample to be loaded in Ion exchange column

[sboopathi]

I have to use Ion Exchange column to remove the excess 2-Aminopyridine labeling reagent from PA-Sugar. The ion-exchange capacity of the strong acid cation exchange is 1.7 meq/mL. ie, 1.7mM / 1 charge site, now I know the amount of sample to be loaded for eg. if 1mg is the sample amount, then how to calculate the respective amount of resin to be used. Please kindly help me to calculate this.

[tom jupille]

Convert milligrams to millimoles (divide by the molecular weight).
Divide the millimoles by the capacity (millimoles/mL) to get the volume (mL of resin).
Double that to allow a safety margin.

[sboopathi]

Thanks Tom for your reply. The resin capacity is 1.7 meq/ml, so for 1 exchange site is 1.7mM. How to calculate the no. of ion exchange sites in a given volume of resin (eg. 100ml).

[tom jupille]

1.7 mEq/mL means 1.7 millimoles of charge per milliliter of resin bed.

[sboopathi]

I use 1x10 cm column in which 7.85ml volume of resin can be packed. so I assume that it will have 13.35mM exchange sites for 7.85ml (based on 1.7mM/ml of resin bed) of Ion-exchange resin. I loaded respective amount (13.8mM)of Maltobiose (2.5mg) labeled with 100-fold excess 2-AP. But the recovery of Maltobiose from the ion-exchange column is only less than 10%. Is it because of the resin volume is too high or the sample amount is too low. Microscopic view of resin showed they are in good shape with no damage. So I want to know why the sample recovery is too low.

[tom jupille]

Two possibilities:

1. you didn't get it all on the column.
- did you start with the resin in an appropriate (easily displaced) ionic form (counterion more weakly bound than your analyte)?
- does your loading solution contain anything that is more strongly bound than your analyte?

2. you didn't get it off.
- is your eluant based on a more stronly bound counterion?
- is the ionic strength of the eluant high enough to ensure displacement?

Chromatography is an experimental science. Try loading a much smaller sample (say, 2 - 3% of your bed capacity) and calculate the recovery. If that looks good, then scale up from there to see how much you can get away with.

[sboopathi]

The initial resin was in H+ form but it was converted to NH4+ form by using 1M Ammonium Acetate pH 6.8 solution. Then the resin was equilibrated with 20mM Ammonium Acetate buffe,r pH 6.8 and loaded into the column. The loading solution doesnt have anything that can strongly bound than the analyte.

The elution buffer is 20mM Ammonium Acetate buffer (pH 8.5) which has been proved to elute the PA-Maltose and retain the excess PA. The excess PA retained in the column is then removed by eluting with 0.5M Ammonium Hydroxide solution. The ionic strength was was found to be enough to displace PA in previous studies.