Chemical Forums
Chemistry Forums for Students => Analytical Chemistry Forum => Topic started by: Kemistry Kaiser on August 15, 2015, 08:44:56 PM
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I'm starting a new analytical research project this semester in which we are utilizing a gel permeation chromatography technique. Following my research professors recommendation, I've been doing some research on the technique. What I can't seem to understand is the precise difference between the two. I know gel-filtration chromatography specifically utilizes water as the solvent, whereas gel-permeation chromatography utilizes an inert organic solvent as the mobile phase. Why is water considered only a solvent in GFC but in several definitions I've read GPC an organic solvent is also the mobile phase as well. I guess it comes down to what the specific difference is between solvent and mobile phase is. I know what each one is, but I thought they're always both solvent and mobile phase when analyzed in a chromatograph. Thank you.
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Both techniques are basically forms of the same thing, size exclusion chromatography (SEC), where molecules are differentiated by size on the basis of their ability to penetrate porous materials. I think the difference is mainly historical; GFC was developed by biochemists to separate biochemical molecules like proteins; GPC was developed by polymer chemists to analyse the MW distribution of polymers (mainly synthetic and non-water-soluble). You can also have aqueous GPC, for water-soluble polymers, e.g. PEG; and when it is done by polymer chemists, it is called aqueous GPC, not GFC. The difference, I think, is not really in the solvent/mobile phase, but in the context, the analytical focus, and the specific expertise. The sort of polymers that chemists typically study have varying molecular weights and MW distributions, and GPC is ideal for studying these. A specific protein, say, has a definite MW, so there is no question of determining its distribution; the biochemist may be interested in analysing a complex mixture, identifying specific molecules, and perhaps separating them on a preparative scale. Along with this goes expertise specific to each context, e.g. choice of solvent, column material, and sample preparation (e.g. for proteins, denaturing or non-denaturing conditions).
Thus, although the physical basis of the techniques is the same, the distinction of name is useful because expertise in one doesn't mean you're an expert in the other. I have done a fair bit of GPC in my time, but that doesn't mean I could walk into a biochem lab and start doing GFC on proteins without learning the trade.
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Both techniques are basically forms of the same thing, size exclusion chromatography (SEC), where molecules are differentiated by size on the basis of their ability to penetrate porous materials. I think the difference is mainly historical; GFC was developed by biochemists to separate biochemical molecules like proteins; GPC was developed by polymer chemists to analyse the MW distribution of polymers (mainly synthetic and non-water-soluble). You can also have aqueous GPC, for water-soluble polymers, e.g. PEG; and when it is done by polymer chemists, it is called aqueous GPC, not GFC. The difference, I think, is not really in the solvent/mobile phase, but in the context, the analytical focus, and the specific expertise. The sort of polymers that chemists typically study have varying molecular weights and MW distributions, and GPC is ideal for studying these. A specific protein, say, has a definite MW, so there is no question of determining its distribution; the biochemist may be interested in analysing a complex mixture, identifying specific molecules, and perhaps separating them on a preparative scale. Along with this goes expertise specific to each context, e.g. choice of solvent, column material, and sample preparation (e.g. for proteins, denaturing or non-denaturing conditions).
Thus, although the physical basis of the techniques is the same, the distinction of name is useful because expertise in one doesn't mean you're an expert in the other. I have done a fair bit of GPC in my time, but that doesn't mean I could walk into a biochem lab and start doing GFC on proteins without learning the trade.
Thank you for the clarification mjc! You obviously have a lot of experience with these chromotagraphy techniques, so could you please explain the difference between the solvent and the mobile phase? Obviously the solvent is the solvent, but is the mobile phase the whole solution moving through the chromatograph, or is it just the solute moving in the solvent. The specific distinction between the two is very unclear to me. Thanks!
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The mobile phase is a term used to distinguish itself from the stationary phase, and your sample dissolved in the solvent/mobile phase is carried through the column where in other forms of chromatography such as reversed or normal phase (SEC/GPC/GFC ideally should have no interactions), the compounds from your sample partition between the mobile phase and stationary phase. The more the compound partitions into the stationary phase (the more alike it is with the column material ie C18 since like-dissolves-like), the more theoretical plates it will have and hence better resolution.
A analogy I was taught with is if a big family go into a mall (the column) at one end and walk through going through different stores (injection of a mix), the men elute first at other end only going into a few of the stores (fewer plates), the teenagers elute next, and then finally with the longest retention time, the women elute after going through every single store!
I guess to possibly extend the mall analogy to SEC, then the stores are the beads and the pores are the doors of the store, and I guess very big people cannot get into the door so elute out other side of mall very fast, while the smaller people can make it in some stores and get slowed down, while the smallest people can make it into all stores and therefore go into every single store.
The difference is in the first example with adsorption or partition chromatography there is selectivity based on the chemical properties while with SEC the selectivity should only be based on the size of the molecules being excluded from the bead pores (ie the men have no choice but to go in all stores if they are small enough to fit!).