Biochem Question

[fly1346]

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Just wondering why the answer is what it is, when textbooks say that the bigger AA will elude first

ALA and PHE from a column of unmodified polystyrene beads, eluted with pH 7 aqueous buffer. Which will elude FIRST?

A. ALA
B. PHE
C. both emerge simultaneously
D. neither can be eluted
[FONT=Times New Roman,Times New Roman][FONT=Times New Roman,Times New Roman]The best answer is A

WHY????
..

 

[fly1346]

Anyone??

 

[Bernoull]

I'll throw my hat in for this one, and I'll preface my comments by saying I'm not a chemistry major so I'm possibly off with some of the chemistry here. I'm a B.Sc. Biology, M.Sc (candidate) Biomedical Eng and a Pharmaceutical Scientist so I'm familiar with Chromatography especially HPLC.

Anyway here we go, I'm thorough in my explanation (my apologies) because I believe people learn best when you understand the fundamental concepts and this is key on the MCAT.

First I'm assuming you mean "elute" rather than "elude" and I'm assuming this is not size-exclusion chromatography or electrophoresis. For size exclusion, smaller means faster elution provided things are smaller than the "holes" they need to pass through.

Secondly, the statement "when textbooks say that the bigger AA will elude first" must be qualified by those textbooks. This cannot be true across the board. Polarity can have a stronger impact on elution rates in certain conditions.

Typically with chromatography and even electrophoresis, elution rates are determined by differential polarity, mass and their interaction with the stationary phase (polysterene in this case). To comprehend this explanation, find diagrams of the structure of PHE, ALA and styrene.

Between Ala and Phe the only difference is their side group (of course) methyl and phenyl groups respectively. In my view, two factors explain why ALA elutes before PHE.

1. PHE is obviously more massive than ALA and if the other factors mentioned above were negligible, then ALA will elute faster. Think of diffusion rate and its dependence on mass or simply put, diffusion rate is directly proportional to Velocity (rms) and inversely proportional to mass.

2. This is a more complicated answer, but sometimes this can be a stronger factor than mass, therefore it's equally important. Think about polarity its cause - separation of centers of positive and negative charges. Also remember that polarity is not an absolute measure but rather a relative one (By this I mean Michael Jordan is rich compared to me, but probably poor compared to Bill Gates). Even though both PHE and ALA are classed as nonpolar AAs, ALA is more polar than PHE. Here's why:

Think of polarity in terms of electronegativity. C has an EN of 2.55 and H has EN = 2.20 which make C-H bonds somewhat polar. Also remember that polarities can cancel (dipole moments are vectors), thus methane is nonpolar despite its polar bonds. With this in mind, look again at the methyl group of ALA, it has a dipole because its C-C bond -b/t methyl alpha C- has no dipole and thus cannot cancel the dipole of the C-H bond opposite it.

Also look at PHE and firstly, it has an aromatic ring (and resonance) and it has no dipole moment.

This relevance of this in answering your question is that polar compounds interact strongly (hydrogen bonds etc) whereas nonpolars interact weakly (van der waals forces) therefore given that the column has polysterene beads (which is essentially a benzene and ethyl groups attached). The polysterene is as nonpolar as PHE and thus they interact. This interaction slows down PHE's elution (Think of this as you running a marathon and the spectators at the side of the road grab you periodically hence slowing you down).

For ALA because of its strong interaction with other ALA molecules, it doesn't interact with the polysterene and proceeds unhindered. You can think of this in terms of surface tension and adhesive/cohesive forces. PHE has greater adhesive forces with the column and moves more slowly whereas ALA have greater cohesive forces with other ALA and moves unimpeded.

The net effect of factrs 1 & 2 is greater elution rate for ALA compared to PHE. I hope this helps 👍!!

 

[fly1346]

Thanks alot dude, really appreciate your time and effort, I reasoned the same after I read my prof's. explanation, however, the thing that troubled me was that all our professor said was large ones come out first, as that is what the textbook says

Biochem at my school is really a pain in the *****, prof, doesnt want us memorizing but understanding concepts, same professor teaches the med school biochem, and he basically teaches us the same he teaches them, same exams same everything

 

[TieuBachHo]

So what is your prof.'s explanation?

By looking at the 2 AAs (ALA and PHE), I couldn't find any strong evidence as to why they should be different except for size and aromaticity. By size alone, Phe should elute first as a common sense; however, the pi-pi interaction is obvious because the polystyrene is also an aromatic compound. This is called stacking interaction due to overlapping of pi-pi orbitals. If the nature of the beads are not restricted, then the pi-pi interaction override this. Just my 2 cents.

 

[BerkReviewTeach]

Thanks alot dude, really appreciate your time and effort, I reasoned the same after I read my prof's. explanation, however, the thing that troubled me was that all our professor said was large ones come out first, as that is what the textbook says

Biochem at my school is really a pain in the *****, prof, doesnt want us memorizing but understanding concepts, same professor teaches the med school biochem, and he basically teaches us the same he teaches them, same exams same everything

Double check the textbook and make sure the column is polystyrene beads. That might be the crux of the problem.

When using porous agarose beads, small proteins get stuck in the pores more than the larger proteins, thus they are hindered as they fall. It is size exclusion via gel-filtration chromatography where smaller ones get slowed down by the resistance as they pass through the pores. Thus, bigger proteins elute from the column before smaller proteins.

The question the professor gave involves a single amino acid rather than a protein, and polystyrene beads are used rather than porous dextran or agarose beads.

Polystyrene beads are not known for being porous, and certainly not pores small enough to distinguish single amino acids. Polystyrene is an aprotic molecule, so there are no ionic interactions to consider between the amino acids and the beads. It comes down to this being strictly a migration rate comparison, exactly as the previous poster pointed out. Smaller molecules travel faster than larger molecules, when they travel the same path.

Personally I think the professor was being tricky in trying to catch people who blindly memorized the gel-filtration method without knowing the medium it typically uses (agarose or detran) and that is applied to proteins and not amino acids. Also, he or she chose to use polystyrene because the side chain is often functionalized (made to be charged), so he or she could have tricked students that way too. The professor probably talked about sulfonated polystyrene columns that separate particles based on their charges. But the question involves plain ol' polystyrene beads and the separation of a small amino acid (Ala) from another amino acid that is about twice as large (Phe).

Gotta love trick questions that make you think, unless you miss them on the test, at which time that energy should be spent hating the professor.