Folic Acid Immunoaffinity Column

Additional Information

Enrichment Step IAC:
10ml extract (containing the quantity of folic acid from a 0.4g sample if above-mentioned sample preparation is followed) is diluted with a total volume of 30ml PBS and then applied in a reservoir on top of the B-TeZ IAC Folic Acid 3ml column.  The optimal flow rate through the gel is between 1 to 3 ml/min.

Wash:
After whole sample has passed through the gel the latter is washed with 5ml of PBS. Remaining liquids in the gel are removed by applying either pressure from top of the column or pressure from the bottom.

Elution:
The sample reservoir on top of the B-TeZ IAC Folic Acid 3ml column is removed, and an appropriate vial is placed below the affinity column. The bounded folic acid is eluted by using a total volume of 3ml of methanol-phosphoric acid solution (99.8/0.2 v/v). The elution process is performed in two steps. First, an amount of 1ml methanol is applied. Once this amount has passed through the column, there should be a waiting time of 30 seconds. After that, the second portion of 2ml of methanol is eluted through the column. The flow rate should lie below 3ml/min.  The remaining methanolic solutions should be eluted by application of slight under- or overpressure. All methanolic fractions are unified to give the column elute.

The column elute may be injected into the HPLC directly or, if concentrations are very low, concentrated by evaporation at 50°C for 1h (e.g. using VLM evaporator), re-dissolved in HPLC solvent and finally injected into the system. For the latter case, please see the sample calculation in which the sample concentrate is re-dissolved in 1.0ml HPLC solvent.