10. All chromatography techniques typically consist of a mobile phase and a soli
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10. All chromatography techniques typically consist of a mobile phase and a solid phase. The individual components of a mixture bind to the solid phase differently depending on their structure are then separated as they compete with their interaction with the mobile phase. If you were given a mixture of the three peptides, A, B, and C, outline a chromatography step that would separate them. Identify which peptide will be retained most on the column and which peptide will move the fastest t the column. In most cases, the mobile phase is modified through the addition of salt (NaCI) or a water-soluble organic compound to "release" hrough the compounds that are retained most strongly. For e menod you chose, propose how the mobile phase could be modified to release the bound material. Finally, briefly explain why you chose chose functions to release the retained peptide. Assume physiological pH, 7.4. Peptide A: SWEETY Peptide B: KINKY this step and why the mobile phase modifier you Peptide C: LILLY Mix of A, B, and C Column Type: Retained Best: Retained Least: Mobile Phase Modifier: Explanation:Explanation / Answer
For purification of peptides, reverse phase HPLC is routinely used. In this method, the molecules are separated based on their polarity. The hydrophilic polar molecules elute out sooner and the hydrophobic ones are retained.
Column type:
For this C18 columns are used, which have as stationary phase, silica particles functionalized with alkyl chains (C18-carbon length).
In this mixture of peptides, we have 3 peptides:
A) SWEETY - With two glutamic acid, peptide A is negatively charged. It also has OH contatining aminoacids serine and threonine. Even though they have a tryptophan and a tyrosine, the peptide would still be polar. At neutral pH, they carry a 2 -ve charges.
B) KINKY - With two lysines (K), peptide B is positively charged at neutral pH. It also has an asparagine with a polar amide side chain. So, even though it has an isoleucine and a tyrosine, the peptide is quite polar.
C) LILLY - Peptide C has three leucines, one isoleucine and one tyrosine. It is totally hydrophobic.
In RP-HPLC, Peptide C would be retained the most.
Peptides A and B, on the other hand, would be less retained.
In order to separate the two we could use organic modifiers uch as alkylammonium formates. Formates would form complex with the positively charged amine functional groups (in lysine) of the peptide B and render them neutral. This could increase their retention time. So, if we have an alkyl ammonium formate as organic modifier in the mobile phase (typically Water/Acetonitrile, or Water/Methanol system), we would have the negatively charged, peptide A elute first, then the polar peptide B elute out next and therefore the two could be separated. The non-polar peptide C would be the last to elute from the column.
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