Electrophoresis, chromatography and mass spectrometry

8 DOH Abu Dhabi (HAAD) lab licensing practice questions on Electrophoresis, chromatography and mass spectrometry, part of Clinical Chemistry. The first 8 are below; the first 5 with answers and explanations (tap Show answer after you have chosen), the rest in Learn with a free account.

1. Question 1 · Medium

   In capillary zone electrophoresis of serum in an uncoated silica capillary at alkaline pH, the negatively charged proteins still reach the detector near the cathode. Why?

   1. AThe negative silica wall repels them toward the cathode
   2. BPressure from the injector pushes them through
   3. CThe proteins become positive at alkaline pH
   4. DElectroosmotic flow toward the cathode is stronger

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   Answer: D · Electroosmotic flow toward the cathode is stronger

   At alkaline pH the silanol groups of the silica wall are ionized and negative. Positive buffer ions gather at the wall and are drawn to the cathode, dragging the whole buffer column with them; this is electroosmotic flow. It outweighs the proteins' pull toward the anode, so all proteins are carried to the cathode end, with the most negative ones, such as albumin, arriving last. Capillary electrophoresis gives fast, automated protein separations without staining.

   • A: The wall's charge creates electroosmotic flow; it does not push proteins along by repulsion.
   • B: Injection pressure loads the sample but does not drive the separation.
   • C: Serum proteins are negatively charged at alkaline pH, above their isoelectric points.

2. Question 2 · Medium

   In size-exclusion (gel filtration) chromatography, which molecules leave the column first?

   1. AThe smallest molecules
   2. BThe most positively charged molecules
   3. CThe largest molecules
   4. DThe most hydrophobic molecules

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   Answer: C · The largest molecules

   The column is packed with porous beads. Small molecules enter the pores and take a longer path, so they elute late, while large molecules are shut out of the pores and pass straight through between the beads. Separation therefore depends on size and shape, not on charge or polarity. Gel filtration is the reference method for separating macroprolactin from monomeric prolactin.

   • A: Small molecules enter the pores and elute last.
   • B: Charge is the basis of ion-exchange chromatography.
   • D: Hydrophobicity is the basis of reversed-phase chromatography.

3. Question 3 · Easy

   In isoelectric focusing, a protein stops moving when it reaches the point in the pH gradient where it:

   1. ABinds firmly to the gel matrix
   2. BIs too large to pass the gel pores
   3. CHas no net charge
   4. DHas its greatest negative charge

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   Answer: C · Has no net charge

   Ampholytes set up a stable pH gradient across the gel. A protein moves in the electric field until it reaches the pH equal to its isoelectric point, where its net charge is zero, and there it stops. If it drifts away it gains a charge and is pushed back, which keeps the bands very sharp. Isoelectric focusing of paired CSF and serum is the reference method for detecting CSF oligoclonal IgG bands.

   • A: Proteins stop because they become uncharged, not because they bind to the gel.
   • B: IEF gels have large pores so that size does not limit migration.
   • D: A protein with a net negative charge keeps moving toward the anode.

4. Question 4 · Easy

   In SDS-polyacrylamide gel electrophoresis (SDS-PAGE), proteins are separated mainly by their:

   1. AMolecular size
   2. BIsoelectric point
   3. CHydrophobicity
   4. DNative net charge

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   Answer: A · Molecular size

   SDS is a detergent that unfolds proteins and coats them with negative charge roughly in proportion to their length. Every protein then has a similar charge-to-mass ratio, so its own charge no longer matters and the gel's pores sieve the molecules by size; small proteins move farthest. Molecular mass is estimated by comparing bands with a ladder of standards. The same principle underlies immunoblot (Western blot) methods.

   • B: Separation by isoelectric point is isoelectric focusing, not SDS-PAGE.
   • C: Hydrophobicity is the basis of reversed-phase chromatography.
   • D: SDS masks the protein's own charge with a uniform negative charge.

5. Question 5 · Hard

   On today's serum protein electrophoresis run, every band has moved less far than usual, the bands are sharper, the gel is warm, and the current is higher than usual at the set voltage. The buffer most likely:

   1. AIs too dilute, with too low an ionic strength
   2. BHas a pH well above the usual 8.6
   3. CHas a pH close to the proteins' isoelectric points
   4. DIs too concentrated, with too high an ionic strength

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   Answer: D · Is too concentrated, with too high an ionic strength

   In a concentrated buffer more ions carry the current, so current and heat rise at a given voltage. Buffer ions also crowd around each protein and slow it, so bands travel less far, although they are sharper. A buffer can become too concentrated if it is made up wrongly or reused until water evaporates. A too-dilute buffer does the opposite: lower current, faster migration and broader bands.

   • A: A dilute buffer gives a lower current and faster, broader bands.
   • B: A higher pH makes proteins more negative, so they would move farther.
   • C: A pH near the isoelectric points would slow the proteins but would not raise the current.

6. Question 6 · Hard

   An LC-MS/MS urine drug confirmation accepts a result if the retention time is within 0.1 min of the calibrator and the qualifier/quantifier ion ratio is within ±20% of the calibrator's. Calibrator: 3.42 min, ratio 0.60. Sample: 3.44 min, ratio 0.31, quantified at twice the cutoff. How should the sample be reported?

   1. ANot confirmed, as the ion ratio fails the criterion
   2. BConfirmed, because the amount is above the cutoff
   3. CConfirmed, because the retention time matches
   4. DPositive, with a comment about the ion ratio

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7. Question 7 · Medium

   On a thin-layer chromatography plate, a drug spot has moved 3.6 cm from the origin and the solvent front 9.0 cm. What is the drug's Rf value?

   1. A2.50
   2. B0.60
   3. C0.40
   4. D5.40

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8. Question 8 · Easy

   In the quadrupole of a mass spectrometer, ions are sorted according to their:

   1. AIsoelectric point
   2. BPolarity on the column
   3. CMass-to-charge ratio
   4. DBoiling point

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