20-Bio-B10 Biomechanical Device Design & Human Factors · May 2015
Nivaar worked solution (AI-drafted; not reviewed by a licensed engineer)
Paper format: National Exams, May 2015 — 04-Bio-B10 Analytical Biochemistry. Three hours, closed book, any non-communicating calculator. Six questions of equal value (20 marks each); five constitute a complete paper and only the first five appearing in the answer book are marked. All six are solved here, because this set is a study resource rather than an examination script. Every question is essay/descriptive (technique principle, interpretation of an instrument trace or image) rather than numerical, except Question 2(d), which asks for a short exponential-growth calculation from PCR cycle theory.
Reference texts (the books a candidate should have reviewed for this subject):
Question text not reproduced: the examination questions are © Engineers and Geoscientists BC. Open the official past paper (linked at the top of this page) to read the question, then follow the worked solution below.
A weak ion exchanger carries functional groups that are only partially ionized, over a limited working pH range — for example, a weak cation exchanger built on carboxymethyl (–COOH/–COO−) groups (pKa ≈ 4–5) or a weak anion exchanger built on diethylaminoethyl (DEAE, –NR2H+/–NR2) groups (pKa ≈ 9–10). Because its charge density varies strongly with pH near the group's pKa, a weak exchanger's binding capacity and selectivity are pH-tunable but also narrower and less robust than a strong exchanger (e.g. sulfonate –SO3− or quaternary ammonium –NR3+ groups), which stays fully ionized across essentially the whole practical pH range and so gives a more constant, predictable binding capacity regardless of small pH shifts.
The most direct fix is to shallow the salt (ionic-strength) gradient — increase the ionic strength more gradually, over a longer elution volume/time — which spreads out proteins with similar binding affinities so they desorb from the column at more distinct points and improves resolution between closely eluting species. Complementary strategies include adjusting the mobile-phase pH (shifting the net surface charge of the co-eluting proteins differently, since they rarely share an identical pI, which changes their relative retention), using a higher-resolution resin (smaller, more uniform particle size) or a longer column, and reducing the sample load (column overloading broadens and can merge adjacent peaks). Given that the problem here is specifically overlapping peaks on an otherwise-working linear gradient, flattening the gradient slope is the first and cheapest change to try.
A step gradient replaces the smooth, continuous rise in ionic strength with a series of sudden jumps to fixed salt concentrations. The main practical benefits are speed and concentration: each bound species desorbs abruptly and in a single, sharp, concentrated peak as soon as the salt concentration exceeds its elution threshold, and the overall run is much faster than a slow linear ramp. The cost is resolution: proteins with similar (but not identical) binding affinities that would have separated gradually along a linear gradient can instead be swept off together in the same step (co-eluting), and a species whose true elution point falls just below a step may "streak" or bleed into the following step rather than eluting cleanly. A step gradient therefore trades separating power for speed and peak sharpness, and is generally the wrong choice when the goal is exactly what part (a)(ii) was trying to fix — resolving closely related, overlapping species.
Reverse-phase chromatography uses a nonpolar (hydrophobic) stationary phase — typically silica particles derivatized with C18 (or shorter C4/C8) alkyl chains — together with a polar mobile phase (an aqueous buffer to which an organic modifier, commonly acetonitrile or methanol, is added in an increasing gradient). Separation is governed by hydrophobicity: more hydrophobic analytes partition more strongly into the nonpolar stationary phase and are retained longer, eluting only once the mobile phase has become sufficiently organic (non-polar) to out-compete that hydrophobic interaction; less hydrophobic species elute earlier, at lower organic content. The name "reverse" phase refers directly to this being the inverse polarity arrangement of classical (normal-phase) liquid chromatography, which uses a polar stationary phase and a nonpolar mobile phase. Reverse-phase HPLC is widely used for peptide and protein separation/analysis precisely because it resolves species by hydrophobicity, a property that (unlike net charge, which ion exchange exploits) is largely independent of the buffer pH used elsewhere in a purification scheme.
