Principles of Chromatography Ulrich Bergmann 2017

History Chromatography (the word means something like writing with colours) reflects it’s historical roots, since it was developed during studies on ”plant-greens” by Mikhail Semyonovich Tsvet in the early 19ths century. Plants contain the green, yellow and red substances in their green parts. All are soluble in non polar solvents, but to extract the green stuff from the plant material, polar solvents are required.

Tsvet’s hypothesis: Something in the plant matrix absorbes (from the PE solution) the different dyes with different affinity. This property can be exploited to separate the compounds He tested all kind of materials for their absorption His final version and first chromatoprapgy system was CaCO3, run with petrol ether and eluted with ether or alcohol.

The basis of chromatography Two phases Analytes must have a different affinity for the different phases Sample is allowed to distribute (equilibrate) between the phases Phases are separated and analytes are enriched in one of the phases compared to the starting composition

And a slightly older technique http://www.jochen-beier.de/Gallery/destillery.jpg

Destillation The two phases are liquid and vapour For mixtures, the amount of each component is different in vapour and liquid. Depending on the vapour pressure (volatility) Concetration in the liquid. And for ideal 2 compound mixtures we get the well known diagrams

From : http://www.chemguide.co.uk/physical/phaseeqia/bpcompi7.gif

How to increase separation? The distribution of a compound between two phases is a feature of the system. If we want to separate compounds, the distribution must be very different between the different components. But usually, goal is to separate compounds that are very similar. What to do? Repeat the process as much as necessary/possible. Ideally in a somewhat automatic way.

Multiple phase-separations In destillation, it’s achieved with fractionation towers. From : Wikipedia Plate: ideally one round of evaporation/condensating-equilibration

In liquid liquid extraction, there is the craig apparatus http://www.jbc.org/content/vol280/issue7/cover.dtl http://www.theliquidphase.org/index.php?title=Image:CCDMachine1.jpg

How does it work? http://www.chem.uoa.gr/Applets/AppletCraig/Appl_Craig2.html

And in theory: http://www. chem. uoa Everything depends on the distribution coefficient: The concentration of a given compound in two phases is constant (at equilibrium) C is concetration, p the fraction of compound A in the upper, q in the lower phase – In our models volumes are the same After equilibration, we have fraction p above and q below And we go to the second round, transfer the upper phase to Tube 2, and replace it with virgin liquid in tube 0. Try to figure out the distribution, remember the sum of Everything has to be 1, and the ratio in each tube equals D= p/q.

So we end up with a binomial distribution Fraction f in tube r after n rounds of extraction http://www.chem.uoa.gr/applets/AppletCraig/Appl_Craig2.html

From multiple extaction towards chromatography Thinking model: the craig machine is converted into a continuously working apparatus. The mobile phase is pumped continuously over the stationary phase Compounds dissolved in the mobile phase are in constant equilibrium with the stationary phase

2-Phase systems in chromatography Stationary phase Mobile phase Chromatography type liquid Countercurrent (CCC) Centrifugal partition (CPC) Droplet Countercurrent (DCCC) Solid (liquid derivatization) Thin layer (TLC) ”normal” liquid (LC) Liquid (on solid carrier) Inert gas Gas (GC)

Isocratic and gradient mode If the chromatographic conditions remain constant, it’s called isocratic chromatography Advantage: fast, more simple hardware, little baseline drift Disadvantage: separation power in many cases too low, elution system must be developed to some extent Solvent easy to recycle Gradient mode: Chromatographic condidtions (usually mobile phase composition in LC and temperature in GC) are gradually changed during the run. a wider spectrum of compounds can be analyzed in one run, especially for compounds with poor diffusuion (macromolecules) more hardware required Eluent recycling not practical (in laboratory scale) Baseline drift in certain modes of detection

What determines how good separation we get? Answer: the number of vessels used (number of plates) And the affinity of the compounds for mobile/ stattionary phase Meaning the difference in affinity for the different compounds

What determines how sharp the peaks are? Answer 1: volume in the tubes (the higher the broader the peaks) But volume needs to meet the solubility of the sample Answer 2: Affinity for the stationary phase (the higher the broader)

In chromatography We also have to consider the sample volume! Obvioulsy the sample volume (and amount) has to meet the capacity of the column or separation is poor and peaks are broad. But beyond that we have other phenomena that can spoil the chromatography.

1. Diffusion To have ideal peaks, all sample molecules should take the same flow path through the column and the system This is of course not the case and the learned name for the abberation is:

Eddy- Diffusion From: http://images.google.fi/imgres?imgurl=http://www.chemgapedia.de/vsengine/media/vsc/de/ch/3/anc/croma/basics/saulen_chr/deemter/anschaul/eddydiff000di0300_neu_altref.jpg&imgrefurl=http://www.chemgapedia.de/vsengine/vlu/vsc/de/ch/3/anc/croma/chromatographie_

2. Diffusion (longitudinal diffusion) At flow zero, sample moleucels should stay where they are. Of course they don’t and instead start to diffuse. Diffusion up or down the column causes peak broadening. From: http://www.chromatography-online.org/Principles/Principles_files/image040.jpg

3. Flow distribution All mobile phase moleucels should flow at the same speed. But they don’t, since they are pressed through a densily packed column bed, and close to the matrix the flow is zero, while reaching a maximum in the middle of the channels

Modified from: http://static. msi. umn

4. Too little diffusion Now we look at the mass transport between mobile and stationary phase, which should occur at infinite rate to achieve equilibrium at any flow rate. Of course this is not the case and if equilibria are lagging behind, we will get band broadening and especially tailing.

From: http://www.chem.uoa.gr/applets/Applet_Index2.htm

5. Dead volume in the chromatographic system The plumbing in the chromatopraphic system behind the column will act as mixing chamber, distributing the sample molecules with the mobile phase and cause band broadening.

6. Heterogeneity in the column material Idea is to have pure and controlled interaction between mobile and stationary phase If column particles are irregular in shape, size or surface chemistry, we shall get a ”diffuse” interaction and peaks will broaden

Van Deemter equation describes the performance as function of the flow rate Peak braodening From: http://www.restek.com/graphics/figure_pharm_016-1.gif mass transfer Eddy diffusion Longitudinal diffusion

In plain words: How to optimize chromatography 1. Find the optimal flow rate! Too slow, and the longitudinal diffusion will spoil the cake (in real life hardly ever an issue). Too high, and mass transfer is poor and equilibira are uncomplete. This problem will gain importance when working with macromolecules and viscosious mobile phases. A more trivial limit comes from what the pump can deliver and the column stand in terms of pressure (higher flow rates will cause higher back pressure)

In plain words: How to optimzie chromatography 2. Find the optimal temperature. The crucial mass transport is usually better at higher temperature, and performace will increase (at least higher flow rates are possible) with increasing temperature. Limits are naturally the stability of the sample and solvents as well as column material. Distribution coefficients may change with temperature, and difficutl separation problems may require a specific temperature window.

In plain words: How to optimize chromatography? 3. Pack column with sperical particles and unique chemistry, and pack it well Spherical particles have lowest backpressure and best properties concerning eddy diffusion and flow distribution. Material should be derivatized homogeniously (secret of the manufacturers) Packing has to be homogenious and without dead volume (art of manufacturing)

In plain words: How to optimize chromatography? 4. Pack with small spherical particles Size of the column packing is most important for getting quick equilibration. Limits are mostly that small particles will cause higher backpressure. Hence the desire for HP-systems

In plain words: How to optimize chromatography? 5. Avoid dead volumes. Choose tubing and components (especially detector flow cells) that suit the scale of the chromatography. Take care when making connections.