A technician checks the column bed volume on a column that is supposed to be 2ml. If the bed volume is not 2ml, is this a problem? Why or why not? The column drips very slowly. Suggest a method to increase the rate of flow through the column. How can you check to ensure that this method does not compromise the separation? A scientist would like to know the charge of a certain protein at pH 7.2. How could this column be used to help determine the overall charge of the protein at pH 7.2? What problems might occur using the proposed approach, and how might they be addressed?

Column chromatography is a separation and purification technique. Column chromatography requires a mobile phase and a stationary phase. The separation of the components of a mixture occurs here as the mobile phase carries the mixture through the stationary phase.1. Column bed volume is the volume of adsorbent (like resins) in the column. Changing the column bed volume will affect efficiency of column chromatography. Too low column bed volume means that there isn't enough packing in the column for the components in the mixture to get separated out. Too high column bed volume would mean the void volume is too low because of excessively tight packing of resins. This means the rate of elution will be very very low as the mobile phase will find it harder to pass through the excessively tight packing. 2. To increase the rate of flow you can exert external pressure on the column from the top . The size of adsorbent particles like resins used to pack the column is another important factor that influences the rate of flow. A very low rate of flow can be because of very fine resins. So the rate of flow can be increased by using resins of a larger size. This would increase the void volume and hence the rate of flow. Increasing the rate of flow beyond a limit, can lead to non-laminar flow within the column. This could lead to different substances taking paths of different lengths within the column and this decreases the resolution of separation. This can compromise the separation process. A low resolution would lead to band broadening in the chromatogram. So to ensure that the increase in flow rate did not affect the separation process, make sure that band broadening is not observed in the chromatogram. If band broadening is observed, then it means that increasing the flow velocity compromised the separation process. 3. To determine the overall charge of a certain protein at a pH (like pH 7.2) , we need to change the column to an ion-exchange column. For this we need to modify the resin used in the column to a charged resin. For this charged species should be attached to the resin. If we attach positively charged DEAE (diethylaminoethyl) on to resin, then the resin becomes an anion exchange resin. If the protein is negatively charged at pH 7.2 , then it will get attracted to the resin and hence it wont elute out easily. We will have to change the pH to elute out the protein. If the protein is positively charged at pH 7.2, then it will elute at a very fast rate. If the protein is neutral at pH 7.2 , it will elute at rate lower than that if the protein was positively charged. If the protein has the opposite charge as that of the resin, then the protein will get attached to the resin. In-order to elute the protein we need to change the pH of the column to the pI of the protein. When pH= pI , the protein becomes neutral. Thereby it looses its attractive force with the column and gets eluted out. But at pI, most proteins looses their native structure to some extend. This can be overcome by using salts to elute the protein, rather than changing pH.

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