HiTrap™ Columns are available for either cation exchange or anion exchange chromatography. SP Sepharose™ are cation exchange columns and Q Sepharose™ is an anion exchanger. These are prepacked columns and ready to use with the simple preparation of a few buffers. The packing material is based on patented Sepharose™ High Performance (HP) beads. As mentioned in the accompanying literature, the small particle size of 34 um allows fast adsorption and desorption even at high sample loadings and flow rates. These columns allow affordable extension of purification procedures in a fast and convenient manner. Since these columns can be operated with something as simple as a syringe or as complex as an FPLC system, multifarious applications of these columns can be assigned.
I have used these columns for the past year for adding a purification step to my protein purification protocol. I have used them both before and after affinity chromatography. Better results were observed in terms of purity and yield when these columns were used after the affinity chromatography. I used a syringe to operate columns with 1 ml packed slurry (5 ml columns for larger volumes are also available) and found that there was high back pressure. I have not tried other modes like a peristaltic pump or high-pressure systems to use these columns. To get accurate, reproducible results, a few important parameters and handling routines should be kept in mind:
1. Always use a buffering agent, which has a pKa within +\- 0.5 pH units of the operating pH. Otherwise, there is a risk of dramatic pH fluctuation due to the limited buffering capacity of the buffering agent. Refer to the booklet that comes along with columns for pKa values of buffers.
2. Check the buffer pH after addition of salt or other additives in binding and elution buffers, and adjust if necessary. Salt and other additives may alter the pH.
3. Remember that pH varies with temperature. Tris buffers are particularly temperature variable. Adjust pH value after buffers have been equilibrated to the desired running temperature.
These columns are very useful for proteins with a known isoelectric point (pI). They allow us to choose the purification procedure at a desired pH value. The ionic state of a biomolecule at a certain pH will determine the usage of either SP or Q columns. SP columns can be operated in buffers of pH range, 1.4 (Maleic acid buffer) to 8.8 (BICINE buffer). Q columns can be operated in buffers of pH range 4.5 (N-Methylpiperazine) to 11.6 (Piperidine). Extreme care should be taken in using high-purity salts for preparing buffers. All solutions and water should be filter sterilized. Attachment of the column to the pressure device should be prompt and continuous, so that no air bubble is trapped therein.
The elution buffer contains 1 M NaCl which generally necessitates the use of desalting columns after the use of ion-exchange columns. This brings in use of another column and may result in further dilution of protein.
My overall assessment of these columns is just satisfactory.
HiTrap™ Sepharose™ HP Ion Exchange Columns From GE Healthcare
Easy-to-use ion-exchange columns meant for quick clean up procedures for biomolecules.
High back-pressure in the columns while using with a syringe.
The Bottom Line
Good for ion-exchange chromatography of small amounts of charged biomolecules.