Chromatography

[shinny]

For retention time and retention factor, what are the most influential factors affecting each one? Different exams prioritise different things, and some are ambiguous given that they don't state the mobile phase and stationary phase used or how the components interact with them (e.g. I've seen NEAP give statements like 'given that the components' solubility in the stationary phase increases with increasing molar mass'), so what general assumptions are we meant to make?

[Mao]

Generally:

mobile phase is usually non-polar
stationary phase is usually polar

hi Rt = low Rf = strong attraction to stationary phase

GC -> mobile = gas, stationary = solid/liquid, sample = gas
HPLC -> mobile = liquid, stationary = solid/liquid, sample = liquid
TLC -> mobile = liquid, stationary = solid, sample = liquid
PC -> mobile = liquid, stationary = paper, sample = liquid

higher temperature = more volatility [mobile phase] = lower Rt higher Rf
lower temperature = less activity = higher Rt lower Rf

i think that's really it?

[shinny]

Generally:
mobile phase is usually non-polar
stationary phase is usually polar

i question that. if the mobile phase and stationary phase werent both polar or both non-polar, then the solute could only adsorb/desorb into one of them.

and my main question was about the factors, particularly polarity vs molar mass, as this one always screws me up on things like GC. usually i can get away with logic by molar mass (lower M=lower Rt), but sometimes they assume i should be taking into account polarity of the molecules. i had another question today which gave no details of the interactions in a HPLC and asked which compound would have a lower Rt. can't remember which factor they took into account though nor can i find that question again o_O

[Mao]

Generally:
mobile phase is usually non-polar
stationary phase is usually polar

i question that. if the mobile phase and stationary phase werent both polar or both non-polar, then the solute could only adsorb/desorb into one of them.

that is the point of chromatography

if both were polar and there were non-polar components, it'll be stuffed.
if both were non-polar and there were polar elements, it'll be stuffed as well.

[shinny]

what about paper chromatography? thats certainly between two polar phases =\

paper is a polar substance because its made primarily from cellulose - a glucose polymer. all the hydroxyls hanging off makes it quite polar, and even more polar than water it seems since the more polar something is, the less it advances in PC. if u dont trust me, then explain why paper soaks and breaks =T

edit: whoops; major typo regarding how PC works

[Mao]

what about paper chromatography? thats certainly between two polar phases =\

paper is a polar substance because its made primarily from cellulose - a glucose polymer. all the hydroxyls hanging off makes it quite polar, and even more polar than water it seems since the more polar something is, the less it advances in PC. if u dont trust me, then explain why paper soaks and breaks =T

edit: whoops; major typo regarding how PC works

by that definition, paper would break apart when you soak [basically dissolve], but to break water you actually have to tear it.

cellulose cannot be digested by human, it is often referred to as fibre, found in celery for example. we do not have the enzymes to break it apart, and it is not readily dissolvable in water [overall NON-polar]
this is because of its structure [linear, and very compact], and water molecules cannot attack it to separate it [hence dissolve]

the mobile phase, usually acetone, is polar (C=O)

but then paper in PC is also polar. so the degree of polarity is used to separate the moleculess.

i think your understanding of polysaccharides need this:

starch is also a polysaccharide, but it has side chains [irregular structure] and can dissolve in water. hence can be used for energy in our body. glycogen has many side chains [highly irregular structure], and is readily dissolved i water, hence is stored in muscles and liver for energy.

[shinny]

hmm i see ur point; but then why does water not just run off it like a piece of plastic? and furthermore, then why would substances which are LESS polar travel further - wouldn't they stick to the stationary phase more if so? (correct me on this statement if im wrong - i am quoting NELSON CHEMISTRY - so im not held liable ;])

[Mao]

okay i'm wrong. apparently cellulose is polar?
.... then why does it not dissolve in water, I ask?


i take back everything that has been said - no comments on PC

[shinny]

oh, guess my assumption that paper was cellulose was wrong - missed that from ur first stating of it; sorry =P

damn my incompetent teachers

edit: oh u edited ur post...well..if i recall correctly from bio last yr, cellulose is glucose chains which are almost lined up ontop of one another like a wall of bricks, and i assume that maybe these bonds cannot be hydrolysed? hence why we need raw force to tear them apart?

[Collin Li]

Polysaccharides are not very soluble due to their size. Remember that the mechanism of dissolving requires water molecules to encapsulate the molecules and lift them out of their normal structure. The massive size of the molecules makes this difficult to achieve, despite there being many -OH groups.

That said, however, it is still a polar molecule and it will still attract other polar molecules.

[jeremykleeman]

HPLC factors:
          - length of the column (the longer the column is the longer all retention times will be and more spreading)
          - amount of stationary phase packed into column (more solid, longer retention times, more spreading) - however this may also slow down the mobile phase due to increased resistance, compounding to the longer retention times)
          - temperature (higher temperature, more viscous, shorter retention times)
          - flow rate and pressure of the mobile phase

But can someone explain, using good chemical language, why substances with higher molar mass in the same homologous series take longer to pass through? Is it because they cannot pass through the solid stationary phase as easily? Is it because (when containing an OH group) the shorter ones (hence smaller mass) are more polar and hence are more soluble?

[polky]

Isn't it just because they are physically bigger than the smaller compounds, that's why it takes them longer to get out?  Something like gel electrophoresis.

Might be very wrong, kinda sounds like a too-simplistic answer but that's what I reckon.

[jeremykleeman]

Yeh, It's probably just the fact that they can't travel through the finely packed particles in the column as easily.

In gel electrophoresis the larger particles can't travel as fast through the gel as they weigh more. Whereas in HPLC seperation is dependent on size. Am I right in that statement?

[shinny]

Gel electrophoresis is still size i'ld say as 'weight' has very little effect at a molecular level. It's more about the large molecules having to weave through the agarose gel matrix, which is obviously harder if your DNA strand is longer

[jeremykleeman]

Okay, yeh I suppose weight would only have a minor effect. Too fat, too slow.