Ion Exchange Chromatography Lab Report
Chromatography refers to a set of laboratory methods used in separating as well as purifying biomolecules. A variety of chromatography techniques exist, and all depend on the interaction between a stationary and a mobile state. Two types of chromatography methods were examined in this investigation. First, ion-exchange chromatography was used. This method separates ions and polar molecules based on their affinity to the ion exchanger [2]. Specifically, cation-exchange chromatography was performed, a technique that uses a negatively charged ion exchange resin with an affinity for molecules with a net positive charge. Anion-exchange chromatography relies on the same principal, but with a positively charged ion exchange resin with an affinity
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The pI values of aspartic acid, histidine and isoleucine are 2.77, 7.59 and 6.02 respectively. Therefore, at the initial pH of 3.25, the negatively charged aspartic acid eluted first as it did not bind to the negative resin. This occurred in fraction numbers 3 and 4. Next to elute was isoleucine. While uncharged at neutral pH, at a lower pH, it was slightly positively charged. As a result, it did not elute immediately. With increasing pH and ionic strength, it did not bind the negative resin anymore and eluted. This occurred at fraction numbers 7 and 8. Last to elute was histidine. Histidine was positive at the initial pH. As a result, it was bound to the negative resin and only eluted with increasing pH and ionic strength. This was evident at fraction number 12. When comparing these experimental observations to observations and patterns seen in literature, it is evident that the findings of this investigation were consistent with theory. As P. B. Hamilton determined during a study on chromatography of amino acids, negatively charged amino acids elute first in cation exchange, followed by uncharged amino acids and positively charged amino acids [3]. Elution rates were dictated by the alteration of pH and salt concentration. Figures 4 through 6 demonstrate the ionization states of the three amino acids examined in this
The pI values of aspartic acid, histidine and isoleucine are 2.77, 7.59 and 6.02 respectively. Therefore, at the initial pH of 3.25, the negatively charged aspartic acid eluted first as it did not bind to the negative resin. This occurred in fraction numbers 3 and 4. Next to elute was isoleucine. While uncharged at neutral pH, at a lower pH, it was slightly positively charged. As a result, it did not elute immediately. With increasing pH and ionic strength, it did not bind the negative resin anymore and eluted. This occurred at fraction numbers 7 and 8. Last to elute was histidine. Histidine was positive at the initial pH. As a result, it was bound to the negative resin and only eluted with increasing pH and ionic strength. This was evident at fraction number 12. When comparing these experimental observations to observations and patterns seen in literature, it is evident that the findings of this investigation were consistent with theory. As P. B. Hamilton determined during a study on chromatography of amino acids, negatively charged amino acids elute first in cation exchange, followed by uncharged amino acids and positively charged amino acids [3]. Elution rates were dictated by the alteration of pH and salt concentration. Figures 4 through 6 demonstrate the ionization states of the three amino acids examined in this