Gel Filtration Chromatography Elution Order
Gel filtration chromatography, also known as size-exclusion chromatography, separates molecules based on their size and shape.
Principle:
- Larger molecules, unable to enter the pores of the stationary phase beads, travel faster through the column in the mobile phase and elute first.
- Smaller molecules can enter the pores, taking a longer, more tortuous path, and thus elute later.
Protein Molecular Weights
The proteins and their approximate molecular weights are:
- Albumin: 66 kDa
- Thrombin: 37 kDa
- Myoglobin: 17 kDa
- Insulin: 6 kDa
Determining Elution Order
Based on the principle of gel filtration, proteins elute in order of decreasing molecular weight (largest first).
- First Elution: Albumin (66 kDa) - Largest molecule, elutes first.
- Second Elution: Thrombin (37 kDa) - Second largest, elutes second.
- Third Elution: Myoglobin (17 kDa) - Third largest, elutes third.
- Fourth Elution: Insulin (6 kDa) - Smallest molecule, elutes last.
Therefore, Myoglobin (17 kDa) would elute third from the mixture.
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 ____________________.
