Unmixing: Chromatography


There are a variety of common ways of separating one or more solutes from a solution. A few are:

  • Precipitation
  • Crystallization
  • Distillation
  • Solvent extraction
  • Chromatography

Chromatography is a powerful technique used to separate mixed components of liquid or gas mixtures. In fact, a large fraction of all of the money spent on chemical research are spent on chromatographic equipment and supplies. It's a hugely-important method.

The basic idea in any chromatographic experiment is that the mixed sample is or is part of some mobile phase — liquid or gas that can move, and is forced (by pumping, gravity or capillary action) through a stationary phase, something with a known chemical composition that does not move.

There are three ways that the components of a mixture can be separated by moving a mobile phase over/through a stationary phase.

Adsorption

Differences, sometimes very small, in attractive interactions between the molecules of the mobile phase and those of the stationary phase will cause the various components of the mobile phase to lag by different amounts as they transit over the stationary phase.

Those attractive interactions arise from weak intermolecular forces like dipole-dipole interactions, induction and dispersion forces, or from stronger interactions like ionic attraction and repulsion.

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.

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aqueous

Of or containing water, typically as a solvent.

An aqueous solution is one in which the solvent is water (root = "aqua"). Typically, but not always, aqueous solutions are ionic salts dissolved in water.

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chimera

In biology or genetics, a chimera is an organism composed of two distinct genotypes — basically the genes of two separate organisms (of the same species).

In molecular biology a chimera can be fragments of two proteins fused together as one via a peptide bond.


The basic chromatography setup


There are many ways to do chromatography experiments and chromatographic separations, but we'll start with the figure below, showing a cross section of a tube (column, in gray) filled with a stationary phase (blue spheres).

The precise chemical nature of the stationary phase is important, but it won't be for the purpose of this illustration. Let's just say that whatever mixture we run through the column, its components (solutes) interact differently with it the stationary phase.

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.



Separation


As components of a mixture are passed across the stationary phase, we're hoping that they will separate, either because they adsorb, weakly or strongly, onto the stationary phase, or because they partition between two phases along the way. We'll be more specific about the difference between adsorption and partitioning later.

Now whether we're talking about adsorption or partitioning, we can think of a chromatography experiment in terms of the time it takes a sample to flow across the stationary phase. Some solutes (top of the figure below) move across rapidly in some time t1.

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.



Zone migration & zone spreading


Like all things in life, we can't get something for nothing. A main complication in all chromatographic separation is zone spreading.

As any solute begins to transit the stationary phase, it is a little spread out in time because it (1) occupies a finite volume,and (2) because the process of diffusion happens in all three dimensions, regardless of any flow. We can limit the volume by injecting as concentrated a sample as possible, but it does have some finite volume.

Diffusion

All atoms and molecules are always in constant thermal motion, and that motion is random, meaning that there will be just as much forward (with respect to the column & flow) thermal motion as backward motion.

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).

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Diffusion

Diffusion is the intermingling of substances by natural movement of their particles. All substances are in random thermal motion that increases with temperature.

An example is the diffusion of an odor. If a bottle of strong perfume is opened in a corner of a room, its molecules will soon diffuse throughout the room so that it can be detected (smelled) uniformly everywhere.



Ideal separation


In performing a chromatography experiment, our goal is separation of the components of a mixture, so we'd like to make that a complete separation. Imagine that we are running a chromatography experiment in which a mixture making up a mobile phase is injected into one end of a column containing a stationary phase, and that we can monitor what comes out of the other end of the tube in some manner, perhaps by the absorption or emission of light. The particular method of detection doesn't really matter right now. We'll just call the detection signal "signal."

Here is an example of a good separation of a two-component mixture containing solutes A and B.

Good separation

Notice that both components A and B are well separated in time. Component A exits the column first and can be collected before any B emerges. That's a good separation. There's little reason to be worried, in this case, about cross contamination of our two emerging samples.

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.



Chromatography experiments — some examples

In the next four sections we'll look at some common kinds of chromatography experiments and how they work in practice. Afterward, we'll develop some mathematical theory so that some of the details of how a separation might work can be predicted, and so we can choose the best method.



I. Paper chromatography


Paper chromatography can be a valuable tool for separating a wide variety of liquid mixtures. It's also often a student's first experience with this technique.

Here is a sample set up. It consists of a beaker containing a bit of some solvent (the mobile phase), perhaps an alcohol, over which is hung a strip of cellulosic paper (the stationary phase), just submerged in the liquid. The paper is hung in some manner; here it's a binder clip around a glass rod — very simple.

Before immersing the bottom of the paper in the solvent the samples are applied by blotting onto the paper near the bottom, and the blots are dried.

Once the paper is immersed (up to a line below the samples), capillary action pulls the solvent up the paper, and a partitioning of the components of the samples (two samples in this case — black and red) between the hydrophilic paper and the solvent occurs. If the partitioning is different for the various components of each sample, a separation will occur.

Once the solvent front (the leading edge of wetness) has advanced sufficiently to provide adequate separation of the components of each sample, the paper can be removed and dried. the capillary action will cease and the component spots will be fixed on the paper. Here we've labeled them A - E.

Notice that in this made-up example, there is a green spot from each sample at the same height on the paper. It's likely that the same chemical component is a part of each sample, particularly if the samples are related in some logical way, such as different colors of pen ink.

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.

Spot distance % from
solvent front
front 5.79
A 3.55 0.61
B 2.34 0.40
C 1.55 0.27
D 4.29 0.74
E 3.67 0.63

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.


Cellulose is composed of glucose molecules linked together in such a way that the in-ring oxygen atoms alternate in their orientation with respect to the chain (up-down-up-down here). Cellulose chains can be thousands of units long. By the way, cellulose is not digestible by most mammals, but if the rings are all oriented with the in-ring oxygens in the same direction, the result is digestible starch. Chemistry!


Thin layer chromatography (TLC)

TLC is related to paper chromatography in that it is performed on a thin, flat stationary phase, and the solvent is pulled upward by capillary action. It differs in that the stationary phase can consist of a variety of substances (but usually silica gel and CaSO4) applied to a thin glass plate. TLC is widely used in organic chemistry, particular in synthesis, to check for reaction progress and the types of products formed.

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hydrophilic

Having a tendency to mix with, dissolve in or be wetted by water. Literally (Latin), it means "water loving."


II. Ion-exchange chromatography


This kind of chromatography is almost always performed on some sort of column, with the liquid mobile phase pumped or pulled by gravity through a solid stationary phase packed into a cylinder.

Here is an example of such a setup. An ion-exchange resin* is packed into a glass column and the mobile phase can either be pumped though or fed by gravity.

* "resin" here refers to any number of inert, chemically resistant, and usually organic compounds that can support various attachments to present to the stationary phase. These may be cation- or anion-rich groups or other ligands.

Many substances one might wish to separate from a mixture are electrically-charged (ionic) in certain circumstances, particularly in some pH range. Proteins are a good example. Any protein can have many charged side chains (in addition to its two ends) that can be charged or neutralized depending on pH.

Acidic conditions will protonate (and therefore positively-charge) certain chemical groups, and basic conditions will remove protons (and therefore negatively-charge) other groups. In this way the overall charge of a molecule can often be manipulated by careful selection of pH conditions, usually by using an appropriate pH buffer system.

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.

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ligand

A ligand is anything that bonds to an atom or molecule of interest. Which atom or molecule is in the context of the statement. The four hydrogen atoms of CH4 are ligands of the carbon atom. An oxygen molecule, O2, is a ligand for the heme group in hemoglobin, and so on.

Ion exchange chromatography

Here are the general conditions for performing ion-exchange chromatography on charged compounds, where the pI of the compound of interest is known. The pI is the negative log10 of the pH at which a multiply-charged molecule has no net charge. It's the equivalent of pH 7 (neutral) for a solution titration experiment.

Resin type Cation
exchange
Anion
exchange
Net charge
of component

+

-

Charge
of resin

-

+

pH
conditions
0.5-1.5 pH units
below pI of
solute molecule
of interest
0.5-1.5 pH units
above pI of
solute molecule
of interest

III. Gas chromatography

Gas-liquid & gas-solid chromatography


Gas chromatography is a versatile technique for separating and identifying mixtures of gases or volatile liquids. There are two main types of gas chromatography. Both are column methods in a closed system.

Gas-solid chromatography uses a solid stationary phase, and the various components of the gaseous mobile phase are separated by differential adsorption on that stationary phase column packing.

In gas-liquid chromatography, the particles of the stationary phase are wetted with some solvent, thus the liquid part. In this case, the separation is performed by partitioning of gases between the mobile phase and the liquid coating of the stationary phase.

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.


IV. High-performance liquid chromatography (HPLC)


High-performance liquid chromatography (HPLC) is another form of column chromatography that relies on partitioning of solutes between two phases (often an aqueous phase and an organic phase) to achieve separation in time as a mobile phase transits a stationary phase.

What makes HPLC interesting and highly useful is that instead of using low pump pressures, gravity or capillary action to advance the mobile phase, it employs high pressure pumps (sometimes hundreds of atmospheres of pressure) to push liquids through the stationary phase. There are two important results from using high pressure:

  1. The mobile phase can be moved more rapidly, leading to more rapid separations with less diffusion, and

  2. The stationary phase can be divided into finer particles, which leads to more precision in the separation.

A typical HPLC setup is diagrammed here. Two high-pressure pumps are used to pump combinations of two solvents through a column. The pumps can be precisely controlled to deliver various mixtures of the solvents to optimize the desired partitioning effect. Generally, conditions are found in which a desired solute binds to the column,

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.


V. Size-exclusion chromatography


Size-exclusion chromatography, also known as gel filtration, is a way of separating larger molecules (often proteins containing thousands of atoms) by 3-dimensional size (not mass).

Here is a schematic diagram of what happens in a size-exclusion chromatographic separation.

The gray blob represents the stationary phase packed into a column. It consists of some inert material that is very porous; each particle contains many small pores of various sizes.

The green, pink and blue balls represent solute molecules (usually macromolecules such as proteins) of different sizes. As the mobile phase transits the stationary phase, small molecules like the green balls can fit inside the smaller pores of the mobile phase, while larger proteins like the pink balls are limited to passing into and out of larger pores—they are excluded from smaller pores, and still-larger molecules won't fit into any of the pores.

We say that these sizes are completely excluded from the stationary phase.

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.


A typical size-exclusion (gel filtration) chromatogram




VI. Electrophoresis

Moving the stationary phase using an electric field gradient


Our final example of chromatography is electrophoresis, the separation of molecules in a solution by differences in their charge, either a charge that is inherent to the molecules or one that is added to them artificially.

A typical electrophoresis setup is shown below.

The stationary phase in this case usually consists of some gel, which can be made of a variety of relatively inert materials, from agarose to polyacrylamide, a soft, porous plastic polymer. The gel is immersed in a pH-buffered solution.

Sample wells notched into the gel hold small bits of samples. Once the gel is loaded with sample, an electric field is generated across the gel. In this case, negatively-charged samples in wells on the right will be repelled by the negative electrode (immersed in the buffer solution), and drawn toward the positive electrode.

The strength of the electric field gradient formed in this way can be adjusted using an external power supply, as shown.

Electrophoresis separations are typically used on samples of biological macromolecules like DNA, RNA and proteins. The phosphate backbones of RNA and DNA are negatively charged, so the setup depicted here will work just fine.

Protein separation might require swapping of the electrodes, depending on the overall charges of the proteins to be separated. The pH of the buffer is a factor in determining the charge of a protein, too.

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.


Separation by partitioning — a closer look


Partitioning refers to the idea that a given solute will have different (we say differential) solubilities in different solvents. So if two solvents (which are usually, but not always, immiscible) are present, more of a given solute will dissolve in the solvent in which it is more soluble — that is, it will partition between the two solvents.

The figure below shows this schematically.

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.

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miscible

Two miscible liquids form a uniform solution when mixed together. Immiscible liquids do not. An example of immiscible liquids is oil and water. Even after vigorous mixing, oil tends to float as a layer on top of water.


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