GATE 2025 BT Q.23: solution and answer

GATE 2025 · BTQ.23 · 1 mark · MSQ

Which of the following separation processes is/are based on molecular size?

(A)
Size-exclusion chromatography
(B)
Ion exchange chromatography
(C)
Membrane ultrafiltration
(D)
Ultracentrifugation

See the question in the paper

Answer and solution

ANSWER

A, C and D. Size-exclusion chromatography, membrane ultrafiltration and ultracentrifugation separate by molecular size. Ion exchange chromatography separates by charge.

Official answer: A, C, DSource: Official answer key, GATE 2025 BT (IIT Roorkee)

Take the options one at a time and ask which physical property each process uses to tell molecules apart.
(A) Size-exclusion (gel-filtration) chromatography: small molecules can enter the pores of the beads, so they travel a longer path and elute later. Large molecules are shut out of the pores and elute first. The elution volume $V_{e}$ depends on hydrodynamic size, so A is size-based.
(B) Ion exchange chromatography: proteins bind to the charged resin by electrostatic attraction and come off when the salt concentration or pH is changed. What decides the separation is net surface charge, which depends on the protein's pI and the buffer pH, not on size. B is not size-based.
(C) Membrane ultrafiltration: a pressure-driven membrane with a molecular-weight cut-off (MWCO, typically $1$ to $1000 kDa$ ) holds back molecules larger than its pores and lets smaller ones pass. This is sieving by molecular size, so C is size-based.
(D) Ultracentrifugation: in a centrifugal field, the settling velocity of a sphere follows Stokes' law, $v = \frac{d _{p}^{2} ( ρ _{p} - ρ _{f} ) ω ^{2} r}{18 μ}$ where $d_{p}$ is the particle (molecule) diameter, $ρ_{p}$ and $ρ_{f}$ are the particle and fluid densities, $ω$ is the angular speed, $r$ is the radial distance from the axis and $μ$ is the fluid viscosity.
Because $v \propto d_{p}^{2}$ , larger molecules sediment faster. The sedimentation coefficient $S = v / (ω^{2} r)$ (in svedbergs) therefore rises with molecular size and mass. This is why differential and rate-zonal (sedimentation-velocity) ultracentrifugation separate macromolecules by size, so D is size-based. (Isopycnic density-gradient ultracentrifugation separates by buoyant density instead, but the standard sedimentation-velocity mode is size-based, and that is the sense the question uses.)
Only ion exchange works on a property other than size (charge). The correct options are A, C and D.

KEY IDEA

Name the physical property each process uses to tell molecules apart. Gel filtration uses pore exclusion, ultrafiltration uses a membrane MWCO, and ultracentrifugation uses sedimentation rate ( $v \propto d_{p}^{2}$ ). All three are size-based. Ion exchange uses net charge.

WATCH OUT FOR

Leaving out ultracentrifugation on the grounds that centrifugation is 'density-based'. Only isopycnic (equilibrium density-gradient) runs separate by buoyant density. In differential and rate-zonal runs the sedimentation velocity scales with $d_{p}^{2}$ , so the separation is by size and mass. The other common slip is to think ion exchange depends on size because larger proteins carry more charges, but the separation is set by net charge (pI relative to pH), not size.

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GATE 2025 BT Q.23: solution and answer

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