In size exclusion chromatography (SEC), a mixture contains proteins of 10 kDa, 100 kDa, and 500 kDa. In what order do they elute from the column?
A10 kDa first, then 100 kDa, then 500 kDa — smaller molecules interact less with the stationary phase
B500 kDa first, then 100 kDa, then 10 kDa — larger molecules cannot enter the pores and are excluded
CThey all elute simultaneously because SEC does not distinguish by size
D100 kDa first because mid-sized molecules partition optimally between pore and channel
In SEC, large molecules cannot enter the pores of the matrix and travel only through the solvent between particles, so they elute first. Small molecules diffuse in and out of pores, taking a longer path, and elute last. This is counterintuitive: unlike every other separation mechanism, larger molecules are NOT retained longer — they are geometrically excluded from the pores and experience no chemical interaction with the stationary phase at all.
Question 2 Multiple Choice
A chemist wants to separate a mixture of amino acids (zwitterionic at pH 7) by chromatography. Which mechanism offers the best selectivity for this task?
APartition chromatography, because amino acids dissolve in organic solvents
BSize exclusion, because amino acids differ in molecular weight
CIon-exchange chromatography, because amino acids bear different net charges depending on their side chains and the mobile phase pH
DAdsorption chromatography on a nonpolar stationary phase, because amino acids are hydrophobic
Amino acids differ in their pKa values and charge states. Ion exchange exploits this by selectively retaining charged species via electrostatic attraction to the resin. Partition relies on differential solubility in two phases — amino acids are largely water-soluble and partition poorly into organic phases. SEC separates by size, but amino acids are very similar in molecular weight. Nonpolar adsorption is a poor choice for polar, charged molecules. Selecting the mechanism based on the physical/chemical property that most distinguishes your analytes is the first decision in separation science.
Question 3 True / False
In size exclusion chromatography, larger molecules are retained longer than smaller ones because they adsorb more strongly to the stationary phase surface.
TTrue
FFalse
Answer: False
This is false on two counts. First, SEC involves NO chemical interaction between analytes and the stationary phase — separation is purely geometric. Second, larger molecules actually elute FIRST (earlier, not later) because they cannot enter the pores and take only the shortest path through the column. Smaller molecules are delayed by entering and exiting pores. If you applied the logic of other mechanisms (stronger interaction = longer retention) to SEC, you'd predict exactly the wrong elution order.
Question 4 True / False
Selectivity and efficiency are both important to the resolving power of a separation, but they can be improved independently: selectivity by choosing the right mechanism, and efficiency by minimizing band broadening.
TTrue
FFalse
Answer: True
Resolving power requires both analytes to interact differently with the system (selectivity) AND for the zones to remain narrow as they travel (efficiency). Selectivity is determined primarily by the mechanism and phase chemistry — which physical property of the analyte the system exploits. Efficiency is determined by kinetic factors like diffusion, flow rate, and particle size, which govern how much each band spreads. Optimizing one does not automatically optimize the other, and both must be addressed to achieve a good separation.
Question 5 Short Answer
Both adsorption and partition chromatography use a stationary phase and a mobile phase, yet they separate analytes by different mechanisms. What is the key physical distinction between them, and how does this affect how you would optimize each?
Think about your answer, then reveal below.
Model answer: Partition relies on differential solubility in two bulk phases — analytes dissolve into a liquid stationary phase and re-dissolve into the mobile phase, so retention reflects the analyte's partition coefficient between the two solvents. Adsorption relies on differential surface binding — analytes interact with the surface of a solid stationary phase (e.g., silica), so retention reflects surface affinity driven by polarity and functional group interactions. To optimize partition, you adjust mobile phase polarity to shift the partition equilibrium. To optimize adsorption, you adjust mobile phase composition to compete with analyte-surface interactions. The distinction matters because the same mobile phase change may improve one and worsen the other.
The key is where the analyte 'lives' when retained: dissolved in a liquid layer (partition) vs. bound to a surface (adsorption). This affects not just selectivity but also how temperature, flow rate, and solvent composition changes shift retention — the underlying thermodynamics differ between bulk dissolution and surface binding.