Chromatography Advanced Version 1

[deleted-37821]

Hi. I am following the experiment for Chromatography Advanced Version 1 and have a few questions. First, I wanted to test the effect of different combinations of subtstrate, solvent, and markers have on the performance of ink separation. How do you determine which combination is best at separating ink? Is it the greatest Retention factor value that determines this? How do you determine polarity and what is its role in the performance of ink separation?

Also, based on this experiment's results, I wanted to apply Forensic ink studies. By this I mean using chromatogrphy to find out who's marker wrote a ransom note at a crime scene. How do substrates (like chalk versus paper) and solvents (like rubbing alcohol versus water) apply to this?

If I have too many variables here, please advice me in which ones to eliminate.

Thank you.

[deleted-71417]

Hi,

The optimal separation condition is the separation is the condition that separates each pair of components most completely. Here are sites describing the way resolution is computed:

http://www.chemistry.adelaide.edu.au/ex ... solut1.htm

http://teaching.shu.ac.uk/hwb/chemistry ... chrom1.htm

You need to experiment to find conditions in which the various components are completely separated into individual completely separted spots if possible. This can be often done either by changing the pH of the solvent and/or the proportions of water and the solvent(like ethanol or isopropanol) in the eluent. Sometimes it is not possible to find conditions that completely separate all the components. When this happens people sometimes do the best separation they can, dry out the paper, then turn the paper 90 degrees and rechromatograph with a different solvent system(this is called 2D chromatography or two dimensional chromatography). Alternatively they develop the second dimesion by passing an electric current in the second direction (electrophoresis). It is also possible to do the electrophoresis at the same time as the first chromatographic separation(sometimes called hanging curtain electrophoresis).

Chromatography depends on the relative ability of the sample components (spots) to stick to the stationary phase (paper) vesus disolve in the liquid phase. Hopefully this relative ratio is different for each component. Spots can move while they are in the liquid phase, but are stationary when stuck to the stationary phase. Roughly, the polarity of the solvent correlates to how soluble a component is in the liquid phase. Ideally you want the average of all components in the sample to spend half the time stuck to the stationary phase, and half the time dissolved in the eluent(mobile phase) so the Rf is 0.5, but what is most important is that the different sample components have as different Rfs as possible. Usually you adjust this by changing the liquid phase and leave the stationary phase constant, because that is experimentally the easiest thing to do. In your shoes I think I would experiment with eluents made of varying mixtures of water, vinegar, and rubbing alcohol or acetone. If all goes well you will probably find that all your sample components travel with the solvent front with pure acetone, and barely move with water as the eluent. What you might do is try ro find a water - acetone or water-alcohol mix that gives the average spot an Rf of about 0.5, and then if you need to add small amounts of vinegar to the mix to change the separation of your most poorly separated spots.

I hope this ansers most of your questions.

Good luck, and have fun!

Barrett L Tomlinson

[deleted-71417]

Hi,

I realized on thinking about it that I had not completely answered your questions, so here is an expansion of my prior answer.

People do chromatography to answer two kinds of questions - what/how many different compounds are in the sample, and how much of each compound is in the sample.

Amber Hess, who works with Science Buddies, did a great project to help answer how much of a compound is in a thin layer chromatogram, which is extremely similar to paper chromatography like your project:

https://www.sciencebuddies.org/science- ... rticle.pdf

If all you are interested in is the answer to the first question- what or how many compounds are in the sample, it is less important to completely optimize the separation. If you are trying to determine your forensic question - which marker out of a set of markers was used to make a mark, all you have to find is an eluent (separation condition) which allows you to unambiguously match a chromatogram of your unknown to a chromatogram done under the same conditions to one your standard samples and none of the other standard samples. This probably does not require completely resolving all the peaks in every chromatogram/reference sample, as long as all the standard sample chromatograms are clearly different from one another.

Complete separation of peaks is mainly important if you are trying to determine how much of each compound is present in the sample, because if peaks(spots) are partly merged it is very difficult if not impossible to separate accurately the amount that is present of each of the overlapped components.

Finally, if you wish to eliminate as many variables as possible, stick with using one good quality paper and just vary the eluent composition to get your best chromatographic separation.

This is a really good project. I hope you have lots of fun with it!!

Best regards,

Barrett L Tomlinson

[deleted-37821]

Thank you! This really helped. The link about retention factor and selectivity factor showed how to measure how well a solute was separated. I'll do the experiment to see which combination works best. I still need to figure where a control comes into the experiment. And to determine which marker matches the unknown, I plan to simply compare the measured retention and selectivity factors, as well as the distinct colors that separated.