Partitioning

Another form of chromatographic separation involves the idea that a solute partitions or distributes between the mobile phase and the stationary phase according to some solubility property.

For example, if one component (solute) of a solution is more soluble in water (a polar solvent) and another is more soluble in a non polar (usually organic) solvent, then as an aqueous mixture containing that solute is passed over a stationary phase coated with non polar solvent, there is a concentration gradient between them— a gradual transition between organic and aqueous phases. Where the solute resides within that gradient — in the stationary phase or in the mobile phase, determines the speed with which it will pass through the system.

In practice, every solute that will remain in solution in the solvent on the stationary phase will remain there until it is eluted using the same or similar non polar solvent.

We'll talk a little more about partitioning below.

Affinity

Finally, sometimes we take advantage of specific affinities between solutes and certain stationary phases. For example, in protein purification, proteins are often engineered to include a protein, attached to one end, called glutathione S-transferase (GST). This "tagging" with GST allows the chimera to bond to a special resin the mimics the binding site of GST in cells. With the chimeric protein adsorbed strongly onto the resin, it can be washed thoroughly to remove impurities, then released by elution with a solution containing a high concentration of the same binding site.

In this section we'll take a closer look at chromatography and discuss several kinds.

The mobile phase (green), containing the sample to be separated is passed over/across the stationary phase at some rate that is slow enough so that it essentially allows the system to be at equilibrium at every step. That will be our basic assumption later in developing some theory of chromatography.

This movement can be achieved by pressure if the mobile phase is a gas, by pumping if it's a liquid, by gravity (or sometimes centrifugation — artificial gravity) or even by capillary action, such as when water "climbs" up a sponge or a paper towel.

Other solutes in the same mixture (bottom of the figure) will have stronger (but not permanent) interactions with the stationary phase, and take a longer time, t₂, to transit the column.

This kind of chromatography is basically a separation of solute flow in time. The first solute can be collected when it exits the column, while the second lags behind and can be collected afterward.

Strictly speaking, this is the picture only for adsorption chromatography, but the result is similar for partition chromatography, as we will see later.

This motion, called diffusion (like how a sponge soaks up water over time), happens in addition to the flow of the mobile phase, and will thus cause the solute zone to spread out over time.

By analyzing simple diffusion, it can be shown that zone spreading scales as the square root of the distance moved by the solute zone. So if the zone moves forward across the stationary phase by 4 units, it should spread no more than 2 units in width. In principle, then, it should be possible to separate most solutes with a long enough chromatography column, a concentrated-enough sample, and by adjusting flow rates and temperature (to slow down the rate of diffusion).

Now let's consider a poor separation of the same components. Here the retention times for the components might be closer (they are not in this example) and the zone spreading might be large enough to cause overlap of the samples as they emerge from the column.

Poor separation

In this case, it would be difficult to recover pure samples of solute A and solute B. Each would be likely to be contaminated with the other. The blue line in this figure approximates the detector signal that we'd actually see.

Below is another picture of good vs. poor separation using our schematic column chromatography experiment.

Measuring the positions of spots

We can actually use paper chromatography to identify components if we have some sort of standard with known components. In order to do that we need some reliable and reproducible measure of how far a component migrated up the paper. The simplest way to do that is to calculate the ratio of the travel distance of each spot to the solvent-front distance.

Here a ruler has been placed along the paper:

The table below gives the results. The distance from the samples (it helps to make a light pencil mark before the experiment begins) to the solvent front (make another mark before it evaporates!) was 5.79 cm. The distances to all of the other spots, and their ratios with 5.79 are recorded in the table below, and the migration distance of each spot is represented as a fraction (percent) of the distance that the pure solvent rose.

From this experiment it is clear that (1) the black sample is composed of three distinct compounds, (2) the red sample is also composed of three compounds, and (3) both samples seem to have one compound (the green one) in common.

If we had good guesses for what these compounds might be, we could run parallel samples of each compound alone — as controls — up the chromatography paper and check for identical migration distances. Otherwise, we have still isolated these compounds on the paper for further analysis.

What is paper?

Just for completeness, the structure of cellulose, of which most paper is made, is shown below. Cellulose is composed of glucose molecules linked together as shown, and alternating in whether the oxygen atom within the 6-membered ring is pointing up or down. Notice that cellulose is quite hydrophilic due to all of the hydroxyl (OH) groups, which mimic the structure of water.

Once a set of conditions that will lead to the preferred charging of solutes is found — usually the maximum amount of charge, either positive or negative, an appropriate chromatography resin can be chosen. Cation-exchange resins are negatively-charged and can therefore bind to cations, while anion-exchange resins are positively-charged and will bind to anions.

Once a set of target solutes has been bound to an ion exchange resin, the column can be washed with a quantity of pure buffer solution to get rid of impurities that don't bind to the resin. On average, that's about half of all impurities — a pretty good deal. This step is often called washing the resin.

Next we need to release our target compound(s) from the resin. This is called elution, and is usually accomplished by flowing a pH-buffered solution in which the concentration of some simple salt, such as NaCl is steadily increased — a salt gradient.

The figure above shows schematically how this works. We assume that we can somehow monitor what emerges from our column. That might be looking for light absorption, a color change in some stain or another method. It produces the blue trace in the graph. As the salt concentration of the eluent solution is increased, salt ions crowd out binding sites on the resin and release the bound solute molecules. Some bind more tightly than others, and that's how we get our separation. The magenta curve shows our salt gradient. The salt concentration is low during loading and washing, then it is increased steadily in order to elute the solutes.

The table in the box below gives guidelines for the kinds of resins (cation or anion exchange) and the pH values to use in order to bind a given solute to an ion-exchange column.

A typical gas chromatography setup is shown here schematically.

It consists of a supply of some carrier gas, usually an inert gas like helium, a flow regulator to ensure a constant flow rate across the column, a sample injection port that allows the sample to be mixed into the flowing carrier gas in a very small volume, the column, which may be several meters long (coiled object in the figure), and some means of detecting and recording the presence of solute gases (gas mixtures can be considered to be solutions).

The column is generally heated to keep components with higher boiling points in the gas phase.

Combination with MS

Detection in an MS experiment is often done using a mass spectrometer, which has the benefit of being able to determine the masses of solutes as they emerge from the column. The gas-chromatograph mass-spectrometer (GCMS) is a powerful tool of any analytic chemistry lab because of the extreme sensitivity of the mass spectrometer and the fact that it can receive relatively pure samples of solutes.

is washed with a quantity of pH-buffered solvent mixture, then eluted, often by running a gradient, in which the concentration of one of the solvents is increased, over the column.

HPLC columns are usually encased in steel because of the high pressures involved. A wide variety of columns can be purchased. Unlike other kinds of column chromatography, these are made with a great deal of precision and generally not "home built."

A detector, often a UV-visible spectrometer is used to monitor the output of the column, and that signal is recorded. A fraction collector can be used to collect individual solutes as they emerge from the column.

This last group will emerge from the column first, because there are relatively few pathways for them to meander, while the smallest particles are hung up in the medium for longer times because the number of random paths open to them is larger.

In general, SEC chromatography separates solutes by size, with larger components passing through the stationary phase faster than smaller components.

Stationary phases

Typically, stationary phases for SEC consist of "beads" made of long polymers of an inert sugar compound called agarose. The material is quite stable, it's easy to manipulate the range of pore sizes, and they are relatively chemically inert.

Sepharose is a common trade name for SEC columns, which are usually purchased pre-prepared. Sepharose columns can be made with various pore-size ranges, depending on the sizes of molecule that need to be separated.

Size-exclusion chromatography is often a final step in purification of proteins prior to other experiments in molecular and structural biology.

The figure below illustrates the trace of a typical SEC separation. For proteins, UV absorption at a wavelength of 280 nm, caused by the absorption of tryptophan and tyrosine amino acids, is commonly used to detect the eluted proteins. All of the components in the separation below are well-separated in time, thus they can be collected independently as relatively pure solutions.

One important technique relies on the detergent sodium doedecyl sulfate (SDS). Samples of proteins are denatured (unfolded) in the presence of SDS, making them, for the most part, blobs of size proportional to the number of amino acids in the chain. The SDS also imparts the proteins with a negative charge, so they can be separated (usually in a polyacrylamide gel) using the setup shown here. The technique is call SDS-PAGE, for SDS polyacrylamide gel electrophoresis.

Many aspects of experimental molecular and structural biology rely heavily on electrophoresis experiments, which are just chromatography experiments in which the driving force on the mobile phase is just an electric field gradient.

An example

Here's a nice example of one kind of experiment that can be done with a polyacrylamide gel electrophoresis experiment. This gel has 8 wells, loaded from the top, as the gel appears below. The experiment is trying to show whether a protein complex binds specifically to a short segment of DNA.

Lane 1 was loaded with only the DNA. Lanes 2-8 contained the same amount of DNA, but also increasing concentrations (listed on the top in namomolar — nM) of the protein that is thought to bind to it. It would have been nice, in this experiment, to see a "control" lane containing only the protein.

Notice that in lane 2, the DNA band isn't quite as dark as lane 1, and that as we go to the right it gets fainter. Moreover, the protein band at the top of each lane gets darker and moves upward. This technique mostly separates the protein and DNA based upon their size, so the fact that in lanes 6, 7, 8 is shifted upward a bit, coupled with the "missing" DNA at the bottom, shows that a complex is being formed between the protein and the DNA — cool.

In the figure, there are 20 red spheres and 20 blue spheres, dissolved in a yellow solvent and a blue one. The red solute is equally soluble in both solvents; it partitions equally between both. The blue solvent is 9 times more soluble in the blue solvent than in the yellow. The partition coefficients of the blue solute are 0.1 for yellow solvent and 0.9 for blue solvent.

We won't go into calculation of partition coefficients in this section, but suffice to say that our goal in chromatography is to find systems of solvents, stationary and mobile phases that maximize the differences in partitioning of the various solutes in a solution.

Sample solutes which partition into the liquid associated with the stationary phase of the chromatography experiment will be retained longer than those that flow through more easily, being dissolved more readily in the mobile- phase solvent.