Sieveportive Chromotography

Sieveportive Chromotography - Sieveportive Chromatography:...

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Sieveportive Chromatography: Size-Exclusion and Ion Exchange Separation of Compounds Anna Krajec September 27, 2011 Lab Section 01
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Narration/Results 1.0 M Potassium Acetate Solution We prepared the columns of packed resin and immediately added some 1.0 M Potassium Acetate buffer to the top in order to keep the resin surface from going dry. Then, 5.0mL of the sample was added to the resin bed. This began to move through the column. For the rest of the experiment, 1.0 M Potassium Acetate buffer was added to the resin bed to keep it from going dry. The Blue dextran dripped out first almost immediately after adding the sample to the resin. We collected that in a test tube. The yellow and red DNP-glycine and Cytochrome C began to mix together and it was impossible to tell where one ended and the other began. Rather than distinct yellow and red samples following the Blue dextran, we collected a moderate amount of orange solution that contained both DNP-glycine and Cytochrome C. The Blue dextran dripped out first because it is nonionic and was not bound by the resin. After collecting the Blue dextran, the DNP-glycine and Cytochrome C mixed together. These two flowed out without separating because the 1.0M buffer made the column far too basic for the Cytochrome C to bind. At a pH higher than 10.7, the Cytochrome becomes anionic, just as the DNP-glycine is at any pH greater than 3.0. Since both were negatively charged in the highly basic environment created by the 1.0 M Potassium Acetate buffer, they did not separate from one another and eluted together. 0.1 M Potassium Acetate Solution Similarly, we prepared the columns of packed resin, but instead added a 0.1 M Potassium Acetate buffer to keep the resin from drying out. Once that liquid level nearly met the resin bed, we added 5.0mL of the sample to the resin bed, followed again by the 0.1 M Potassium Acetate buffer. Once again, the Blue dextran came out first, with a similar volume to that of the 1.0 M experiment. At this point in the experiment, there was a very stark distinction between the yellow DNP-glycine and the red Cytochrome C and the DNP-glycine was moving through the column while the Cytochrome C stayed near the surface. Once all the Blue dextran was collected, we began to run 1.0 M Potassium Acetate buffer through the column.
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Sieveportive Chromotography - Sieveportive Chromatography:...

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