The question asks to identify separation processes that are based on molecular size. Let's analyze each option:
Analyzing Separation Processes by Molecular Size
- A. Size-exclusion chromatography: This technique explicitly separates molecules based on their hydrodynamic volume or size. Larger molecules elute first because they cannot enter the pores of the stationary phase, while smaller molecules enter the pores and elute later. This process is directly based on molecular size.
- B. Ion exchange chromatography: This method separates molecules based on their net surface charge. Molecules bind to oppositely charged resins in the stationary phase. It does not primarily rely on molecular size.
- C. Membrane ultrafiltration: This process uses membranes with specific pore sizes (rated in Daltons or kDa) to separate molecules. Molecules larger than the pore size are retained (retentate), while smaller molecules pass through (permeate). This separation is fundamentally based on molecular size.
- D. Ultracentrifugation: While sedimentation in ultracentrifugation is influenced by mass, shape, and density, separation often occurs based on differences in these properties, including size. Larger and/or denser particles sediment faster. Techniques like density gradient centrifugation can separate based on size and density differences, making it a valid method where molecular size plays a role in separation.
Conclusion on Molecular Size Separation
Based on the analysis, the separation processes that rely on molecular size are:
- Size-exclusion chromatography (A)
- Membrane ultrafiltration (C)
- Ultracentrifugation (D)
Therefore, options A, C, and D are correct.
Important Questions from Ion Exchange Gel Filtration Hydrophobic Interaction and Affinity Chromatography
- 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 ____________________.
- 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
