R, Q, P
Understanding Peptide Elution in Anionic Ion-Exchange Chromatography
Ion-exchange chromatography separates molecules based on their net charge. An anionic resin is negatively charged and serves as the stationary phase. This type of resin attracts positively charged molecules (cations) and repels negatively charged molecules (anions). The degree of repulsion or attraction determines how quickly a molecule moves through the column and thus its elution order.
Peptide Charge Analysis
- Peptide P: Carries a positive charge.
- Peptide Q: Carries a weak negative charge.
- Peptide R: Carries a strong negative charge.
Determining Elution Sequence
When a mixture containing these peptides is passed through a column packed with an anionic (negatively charged) resin:
- Positively charged molecules (like P) will be attracted to and bind strongly with the negatively charged resin.
- Negatively charged molecules (like Q and R) will be repelled by the negatively charged resin.
- The strength of repulsion dictates the elution order for negatively charged molecules. Molecules with a stronger negative charge will be repelled more strongly and elute earlier.
- Molecules that bind strongly will require specific conditions (like increased salt concentration) to be eluted and will therefore elute last.
Based on these interactions, the elution order is determined as follows:
- R (Strongly Negative): Experiences the strongest repulsion from the anionic resin, thus eluting first.
- Q (Weakly Negative): Experiences weaker repulsion compared to R, eluting second.
- P (Positive): Is attracted to and binds strongly to the anionic resin, eluting last.
The order of elution is therefore R, Q, P.
Important Questions from Ion Exchange Gel Filtration Hydrophobic Interaction and Affinity Chromatography
- Which of the following separation processes is/are based on molecular size?
- Match the stationary phase (Column I) with its corresponding chromatography technique (Column II).
Column I Column II P. Protein A 1. Size exclusion chromatography Q. Sephadex 2. Ion-exchange chromatography R. Phenylsepharose 3. Affinity chromatography S. Diethylaminoethyl cellulose 4. Hydrophobic interaction chromatography - Two monomeric His-tagged proteins of identical molecular weight are present in a solution. pIs of these two proteins are 5.6 and 6.8. Which one of the following techniques can be used to separate them?
A protein is to be purified using ion-exchange column chromatography. The relationship between HETP (Height Equivalent to Theoretical Plate) and the linear liquid velocity of mobile phase is given by:
$H = \frac{A}{u} + Bu + C$
where H is HETP (m) and u is linear liquid velocity of mobile phase ($m.s^{-1}$). The values of A, B and C are $3\times10^{-8} \ m^2.s^{-1}$, $3 \ s$ and $6\times10^{-5} \ m$, respectively. The number of theoretical plates based on minimum HETP for a column of 66 cm length will be ____________________.
- Match the entries in the Group I with the elution conditions in Group II.
Group I Group II P. Ion-exchange chromatography 1. Isocratic solvent Q. Hydrophobic column chromatography 2. Ampholytes R. Gel filtration chromatography 3. Increasing gradient of salt S. Chromatofocusing 4. Decreasing gradient of polarity
