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
Matching Chromatography Techniques with Elution Conditions
This question requires matching four chromatography techniques listed in Group I with their corresponding elution conditions from Group II.
Analysis of Group I Entries:
- P. Ion-exchange chromatography: Separates molecules based on their net surface charge. Elution is typically achieved by increasing the ionic strength (salt concentration) of the mobile phase, which competes with the bound molecules for the charged stationary phase.
- Q. Hydrophobic column chromatography (HIC): Separates molecules based on their hydrophobicity. Molecules bind to a hydrophobic stationary phase in high salt concentrations (low polarity mobile phase). Elution is performed by decreasing the polarity of the mobile phase, often by reducing the salt concentration or increasing organic solvent content.
- R. Gel filtration chromatography (Size Exclusion Chromatography): Separates molecules based on their hydrodynamic volume (size and shape). The mobile phase composition remains constant throughout the separation, meaning an isocratic solvent system is used.
- S. Chromatofocusing: Separates molecules based on their isoelectric points (pI). This technique relies on creating a pH gradient within the column, often using buffering components called ampholytes, to elute proteins at their respective pIs.
Matching Group I with Group II:
- P. Ion-exchange chromatography matches with 3. Increasing gradient of salt.
- Q. Hydrophobic column chromatography matches with 4. Decreasing gradient of polarity.
- R. Gel filtration chromatography matches with 1. Isocratic solvent.
- S. Chromatofocusing matches with 2. Ampholytes (used to establish the pH gradient for elution).
Conclusion:
The correct matching based on the principles of each chromatography technique is:
- P - 3
- Q - 4
- R - 1
- S - 2
This corresponds to the combination P-3, Q-4, R-1, S-2.
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 - A mixture contains three similarly sized peptides P, Q and R. The peptide P is positively charged, Q is weakly negative and R is strongly negative. If this mixture is passed through an ion-exchange chromatography column containing an anionic resin, their order of elution will be
- 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 ____________________.
