A researcher runs a CE experiment at standard buffer pH and observes both cationic and anionic analytes arriving at the cathode-end detector in the same run. How is it possible for anions to reach the detector, given that they should migrate toward the anode?
AAt standard buffer pH, anions are protonated and temporarily neutral, so they do not migrate toward the anode
BThe high voltage reverses anion migration direction at CE field strengths
CElectroosmotic flow carries the bulk solution — including anions — toward the cathode at a rate that exceeds the anions' electrophoretic migration toward the anode
DAnions bind to the positively charged capillary wall and are swept toward the cathode by convection
This is the defining feature of electroosmotic flow (EOF) in CE. The silica capillary wall carries negative charges (deprotonated silanols) that attract a layer of cations from the buffer. When voltage is applied, this cation layer is driven toward the cathode, dragging the bulk solution with it. EOF is typically strong enough that it exceeds the electrophoretic velocity of anions moving in the opposite direction, so the net displacement of anions is still toward the cathode — just slower than for cations. This means cations, neutrals, and anions can all be detected in a single pass, with cations arriving first (moving with EOF + own electrophoresis) and anions last (moving with EOF − own electrophoresis).
Question 2 Multiple Choice
What is the primary reason CE achieves far higher theoretical plate counts — and therefore better resolution — than HPLC for comparable separation lengths?
ACE uses much higher voltages than HPLC, which drives analytes through the column faster and prevents diffusion
BCE uses smaller sample volumes, so there are fewer analyte molecules to separate
CEOF in CE produces a flat (plug-like) flow profile that minimizes band broadening, whereas HPLC's pressure-driven parabolic flow profile causes significant dispersion
DCE operates at higher temperatures that increase diffusion coefficients and speed up mass transfer
In pressure-driven flow (HPLC), the velocity of the mobile phase is fastest at the center of the column and zero at the walls — a parabolic profile. Analyte molecules near the wall spend more time in the column than those in the center, causing band broadening. EOF has a flat profile: the entire bulk solution moves at nearly uniform velocity from wall to center, because the driving force (the charged wall attracting buffer cations) acts at the surface and the resulting plug-like flow is nearly dispersion-free. This eliminates a major source of band broadening, enabling plate counts of 100,000 to 1,000,000 compared to 10,000–100,000 for typical HPLC.
Question 3 True / False
In CE, two molecules with identical charge but different sizes will migrate at the same velocity because separation is based solely on charge.
TTrue
FFalse
Answer: False
CE separation is based on electrophoretic mobility, which depends on the charge-to-size ratio (specifically, charge divided by hydrodynamic radius). A large molecule with the same charge as a small molecule will migrate more slowly because friction with the surrounding solution increases with size. Two molecules with the same charge but different sizes will have different charge-to-size ratios and therefore different mobilities — they will be resolved. This is analogous to falling through a viscous medium: a larger sphere falls more slowly than a smaller one despite the same gravitational force, because hydrodynamic drag increases with size.
Question 4 True / False
Electroosmotic flow in CE results from the migration of cations attracted to the negatively charged capillary wall, which drags the bulk solution toward the cathode.
TTrue
FFalse
Answer: True
The mechanism is straightforward: deprotonated silanol groups on the inner silica surface create a negative wall charge at typical buffer pH. Buffer cations are attracted to this surface, forming an electrical double layer. When a high voltage is applied, these surface-bound cations are driven toward the cathode, and because they are solvated and interact with adjacent water molecules, they drag the bulk solution along with them in a plug-like flow. The strength of EOF depends on the surface charge density (related to pH) and the zeta potential — which is why adjusting buffer pH is a primary tool for controlling EOF in CE.
Question 5 Short Answer
Explain why the flat flow profile of electroosmotic flow leads to higher resolution in CE compared to the parabolic flow profile in conventional liquid chromatography.
Think about your answer, then reveal below.
Model answer: In pressure-driven HPLC, flow velocity is highest at the column center and zero at the walls, creating a parabolic profile. Molecules in the fast-moving center traverse the column faster than molecules near the slow walls, so a narrow band of injected analyte spreads out into a broad zone over time — this is called axial dispersion or band broadening. In CE, EOF generates plug flow: the entire cross-section of the capillary moves at nearly the same velocity, because the driving force acts uniformly along the wall rather than in the center. There is no differential velocity across the capillary radius, so injected bands remain narrow. Fewer theoretical plates are 'lost' to flow dispersion, and the total plate count (a measure of separation efficiency) is dramatically higher.
The flat-profile insight also explains why CE can achieve its performance in narrow capillaries rather than packed columns. The geometry (very small diameter, thin annular region) combined with plug flow means that diffusion across the capillary diameter is rapid relative to the time analytes spend in the capillary, which further suppresses band broadening. This is the physical basis for CE's exceptional resolution with minimal sample volume.