Question

Another labmate is performing ion exchange column chromatography to purify a mixture of proteins. She monitored the A20 of the solution eluting from the column over time, and plotted the data as shown in the following graph A280 דודודודו 2 4 6 8 10 12 14 16 18 20 Elution Time (min) Your labmate would really like to resolve her proteins better. How might she modify the efficiency oLine column, and how would the protein peaks of the graph change? How could she modify the selectivity of the column, and how would the protein peaks of the graph change? (4 points) We can acheive it by reducing the wilth of the peak This can be done by increasing tint. She can also change the pore size). By doing so the width between the peak will get reduce of each and can increase the effeciency. 4. Carlos and Camryn were told to purify a particular enzyme from yeast. Carlos purified the enzyme from the periplasm while Camryn purified it from protoplasts. Both fractions contained 24 U of total activity. After purification, the students verified that their enzyme was free from other contaminants by polyacrylamide gel electrophoresis. However, Carlos reported 200-fold purification of the enzyme while Camryn reported 400- fold purification. Both followed the same chromatography methods and obtained the same final amount of purified enzyme activity. Why did they find different fold purifications? (3 points)

both the questions those are related to each

Answer 1

In an Ion chromatography chromatography seperation of protein is done on the basis of affinity of the molecule to bind to the exchanger. Every charged protein can be seperated by this method whether it is large protein, small protein or amino acid. Protein seperation generally depends on the isoelectric point of the protein.

For improving the efficiency of the column and making better protein resolution following steps may be taken into consideration:-

1. By decreasing the sample load will help in increase the resolution of proteins to be seperated: It means that the amount of sample load generally depends on the binding capacity of column resin, high the sample load less binding capacity to the resin and vice versa. So we can load sample to minimum value to obtain high resolution in our graphs.

2. By decreasing the flow rate will help in increasing the resolution rate: Flow rate can be defined as how fast the buffer run from the column. By decreasing the flow rate we can achieve better seperation between the proteins.

3. By selecting smaller resin particles will also improve efficiency as it helps in giving higher resolution but mean average runtime of column is increased in choosing smaller particle size.

4 By selecting an appropraite mobile phase will help in improving the efficiency of the proteins.for eluting cation and anions seperate mobile phase will be used which thus provides a hgh resolution.  

  

• Decreasing the sample load will increase resolution
• Decreasing the flow rate will increase resolution
• A shallower elution gradient will increase resolution