Turkish Chemistry

Column chromatography is one of the most common methods of protein purification. Like many of the techniques on this site, it is as much an art form as a science. Proteins vary hugely in their properties, and the different types of column chromatography allow you to exploit those differences. Most of these methods do not require the denaturing of proteins.

To be very general, a protein is passed through a column that is designed to trap or slow up the passing of proteins based on a particular property (such as size, charge, or composition).

There are three main steps to protein purification:

1. Capture. You need to get your protein into a concentrated form. If, for example, you are trying to isolate a protein you have synthesized in an E. coli cell, you could be looking at a protein to junk ratio of 1:1,000,000. For capture purification you need a high capacity method that is also fast. You need a speedy method because your crude solution is very likely to contain proteases in addition to your protein of interest that can quickly chew up your protein.

2. Intermediate. Intermediate purification requires both speed and good resolution.

3. Polishing. For the final step of purification you need a system that has both good resolution and speed. Capacity is usually irrelevant at this stage.

Some of the more common columns include:

• IEX: Ion exchange chromatography. Good for capture, intermediate, and polish.
• HIC: Hydrophobic interaction column. Good for intermediate purification.
• AC: Affinity chromatography. Good for capture and intermediate purification.
• GF: Gel filtration (size exclusion) chromatography. Good polishing step.

Let’s look at these types of columns in more detail.

Ion exchange chromatography

Ion exchange chromatography is based on the charge of the protein you are trying to isolate. If your protein has a high positive charge, you’ll want to pass it through a column with a negative charge. The negative charge on the column will bind the positively charged protein, and other proteins will pass through the column. You then use a procedure called “salting out” to release your positively charged protein from the negatively charged column. The column that does this is called a cation exchange column and often uses sulfonated residues. Likewise, you can bind a negatively charged protein to a positively charge column. The column that does this is called an anion exchange column and often uses quaternary ammonium residues.

Salting out will release, or elute, your protein from the column. This technique uses a high salt concentration solution. The salt solution will out compete the protein in binding to the column. In other words, the column has a higher attraction for the charge of salts than for the charged protein, and it will release the protein in favor of binding the salts instead. Proteins with weaker ionic interactions will elute at a lower salt, so you will often want to elute with a salt gradient. Different proteins elute at different salt concentrations, so you will want to be sure you know the properties your protein well for best results.

Also be aware that changes in pH alter the charges in proteins. Be sure you know the isoelectric point of your protein (the isoelectric point is the pH at which the charge of a protein is zero) and make sure the pH of your system is adjusted and buffered accordingly.

The basic steps in using an ion exchange column are:

1. Prep the column. Pour your buffer over the column to make sure it has equilibrated to the required pH.

2. Load your protein solution. Some proteins in the solution don’t bind and will elute during this loading phase.

3. Salt out. Increase the salt concentration to elute the bound proteins. It is best to use a salt gradient to gradually elute proteins with different ionic strengths. At the end bump the system with a very high salt concentration (2-3M) to make sure all proteins are off the column.

4. Remove salts. Use dialysis to remove the salts from your protein solution.

Temperature doesn’t have a huge effect on column chemistry. However, it is better to work cold since proteins are more stable cold.

Hydrophobic interaction chromatography

Where ion exchange chromatography relies on the charges of proteins to isolate them, hydrophobic interaction chromatography uses the hydrophobic properties of some proteins. Hydrophobic groups on the protein bind to hydrophillic groups on the column. The more hydrophobic a protein is, the stronger it will bind to the column.

Load the proteins in the presence of a high concentration of ammonium sulfate (not ammonium persulfate). Ammonium sulfate is a chaotropic agent. It increases the chaos (entropy) in water, and thereby increases hydrophobic interactions (the more disordered the water, the stronger the hydrophobic interactions). Ammonium sulfate also stabilizes proteins. So as a result of using an HIC column you can expect your protein to be in its most stable form.

The hydrophobic column is packed with a phenyl agarose matrix. In the presence of high salt concentrations the phenyl groups on this matrix binds hydrophobic portions of proteins. You can control elution of different column-bound proteins by reducing the salt concentration or by adding solvents.

Affinity chromatography.

Affinity chromatography relies on the biological functions of a protein to bind it to a column. The most common type involves a ligand, a specific small biomolecule. This small molecule is immobilized and attached to a column matrix, such as cellulose or polyacrylamide. Your target protein is then passed through the column and bound to it by its ligand, while other proteins elute out. Elution of your target protein is usually done by passing through the column a solution that has in it a high concentration of free ligand.  This is a very efficient purification method since it relies on the biological specificity of your target protein, such as the affinity of an enzyme for a substrate.

Gel filtration, or size exclusion, chromatography separates proteins on the basis of their size. The column is packed with a matrix of fine porous beads.

It works somewhat like a sieve, but in reverse. The beads have in them very small holes. As the protein solution is poured on the column, small molecules enter the pores in the beads. Larger molecules are excluded from the holes, and pass quickly between the beads.

These larger molecules are eluted first. The smaller molecules have a longer path to travel, as they get stuck over and over again in the maze of pores running from bead to bead. These smaller molecules, therefore, take longer to make their way through the column and are eluted last.

A Hexagonal Molecule on which is Founded Aromatic Chemistry

Benzene is one of the most interesting organic chemicals of all because it took so long for chemists to understand the structure. It was a mystery for so long but once its structure was determined it proved to be the key to a host of large molecules.

Discovery
The famous chemist, Michael Faraday discovered benzene in 1825 while distilling crude oil. Its empirical formula was found but its structure was not determined until 1865 when German chemist, Friedrich August Kekulé had a day-dream or “reverie” while thinking about the structure. He saw a snake curl round and bite its own tail, forming a ring structure. Kekulé immediately knew that benzene was a ring of six carbons connected by alternating double and single bonds (see image 2)

Hybridisation
The problem with this structure was that double and single bonds have different lengths, but X-ray crystallography studies have shown that the bond-lengths in benzene are the same. This led some scientists to suggest that the structure resonated between double and single bonds. Eventually Linus Pauling used his molecular orbital theories to propose a structure in which all the bonds are equal with a hybridised circular orbital above and below the benzene ring (see image 1).

Arenes
Benzene is the simplest of the group of organic chemicals known as arenes. The hydrogen atoms attached to each carbon in the ring can be substituted for other functional groups to form other chemicals many of which are important in industry for example. Benzene rings can be fused together to make structures such as naphthalene. Graphite is made up of a whole hexagonal network of carbon atoms in which all the hydrogen atoms have been replaced by other carbon atoms.

Lead-Free Petrol
One of the most common uses of benzene is as an additive to petrol or gasoline in cars. It improves what is known as the octane rating and reduces knocking. In the drive to reduce lead-containing additives in petrol benzene has been used as a replacement, although there are some health issues related to benzene itself. It is used in many parts of the chemical industry as a raw material in the manufacture of plastics, lubricants, drugs and pesticides.

Carcinogen
In the early twentieth century benzene was used as an after-shave lotion because of its pleasant smell, but it has since been discovered to be seriously hazardous to the health. It can be carried through the blood and damage bone marrow and red blood cells. It is also carcinogenic and can cause leukaemia.
The copyright of the article The Chemistry of Benzene in Organic Chemistry is owned by Simon Davies. Permission to republish The Chemistry of Benzene in print or online must be granted by the author in writing.

Molecular Size

Alternative fuels tend to be made up of small, fairly simple molecules; for example, here are schematic chemical diagrams (C denotes a carbon atom, H is hydrogen, and O is oxygen) of
methane (CH₄), the primary constituent of liquefied or compressed natural gas, and
propane (C₃H₈), the primary constituent of liquified petroleum gas.

Petroleum fuels are blends of lots of different chemical species; in general, the molecules of a liquid petroleum fuel are pretty big and complex. Here is
isooctane (C₈H₁₈), typical of the molecules found in gasoline (I had to spread out the structure a bit to get all the hydrogen atoms to fit in the picture–all of these molecules are, of course, three-dimensional, but some squish into a plane better than others!), and

this monster is cetane, or n-hexadecane (C₁₆H₃₄), typical of diesel fuel.

Incomplete Combustion

When a hydrocarbon fuel (that is, one that is made up of hydrogen and carbon) burns completely, the oxygen in the air combines with the hydrogen to form water (H₂O) and with the carbon to form carbon dioxide (CO₂). If the burning is not complete, then some of the carbon atoms only combine with one oxygen atom rather than two, to form carbon monoxide (CO), a highly poisonous gas.

Some of the carbon atoms may remain stuck together with each other and with some of the hydrogen atoms as well, so that unburned hydrocarbon molecules (mostly smaller than the ones in the original fuel) can also come out the tailpipe. These unburned hydrocarbons (plus any fuel hydrocarbons that evaporate from the fuel system before getting into the engine to be burned at all) react with nitrogen oxides (another pollutant from combustion) in the presence of sunlight to form ozone, which is a lung irritant (the “ozone layer” in the stratosphere is a shield against the sun’s ultraviolet light, but at ground level ozone is the main component of “photochemical smog”). Carbon atoms can also remain stuck to one another with few or no hydrogen atoms attached, especially during incomplete combustion of diesel fuel, producing soot.

This is one of the reasons alternative fuels are less polluting than gasoline and diesel: their simpler molecules are easier to burn more completely in an engine, so that less carbon monoxide, soot, and unburned hydrocarbons come out the tailpipe. In addition, any unburned hydrocarbons that are produced are less reactive than those that come from incomplete burning of gasoline or diesel fuel, and so they produce less ground-level ozone; methane in particular is almost incapable of forming smog.

Oxygen Content

Some alternative fuels are not hydrocarbons; alcohols and biodiesel contain oxygen atoms as well as carbon and hydrogen. Here are the chemical structures of the common alcohol fuels,
methanol (CH₃OH) and
ethanol (C₂H₅OH).
(Biodiesel molecules are “monoalkyl esters”, but I haven’t been able to trace down anything more specific. The “ester” part of that name, however, indicates that the molecules include oxygen atoms.)

In many parts of the USA, gasoline is “oxygenated” during at least part of the year; this means that oxygen-bearing compounds are added to the fuel mixture. The reason for doing this is that having some oxygen as part of the fuel molecules to start with promotes more complete combustion, so that less carbon monoxide, soot, and unburned hydrocarbons come out the tailpipe, as described above. Alcohol fuels and biodiesel carry this one step further, in that the oxygen-bearing compound is not an additive at the 5 to 10 percent level, but a major constituent of the fuel, which increases the benefits of oxygenation.

Carbon Content

Even if, with the aid of electronic engine controls and efficient catalytic converters, a hydrocarbon fuel is burned completely to water and carbon dioxide, there is now growing concern about carbon dioxide as a greenhouse gas. Measures to cut back on production of carbon dioxide by automobiles without sacrificing performance can focus on efficiency, i.e., getting as much useful propulsive power out of a given amount of fuel as possible, which typically involves replacing the traditional drivetrain of a piston engine driving the wheels through a gearbox with a more efficient design.

However, some fuels inherently produce less carbon dioxide when burned completely than gasoline or diesel fuel. For example, counting the numbers of oxygen atoms it takes to burn up an isooctane molecule and a methane molecule (typical of gasoline and natural gas respectively), one can calculate that 100 oxygen atoms will combine with four isooctane molecules to produce 32 carbon dioxide molecules and 36 water molecules, while the same number of oxygen atoms will combine with 25 methane molecules to produce 25 carbon dioxide molecules and 50 water molecules. That is, a given amount of air (oxygen) will produce about 25% less carbon dioxide if used to burn natural gas than if used to burn gasoline. (Of course, this advantage will be reduced if you have to open the throttle wider and burn an additional amount of air with natural gas to get the same amount of power, but in the real world the 25% figure turns out to be about right.)

Avoiding Carbon Dioxide Emissions Entirely

The other thing to consider is the source of the carbon in the fuel; if it came from the carbon dioxide in today’s air to begin with, like an alcohol fuel produced by fermenting biomass (as opposed to a fossil fuel, whose carbon came out of the air when the dinosaurs were around!), then returning it to the air now adds nothing to the net flow of carbon dioxide into the atmosphere. Alcohol fuels or biodiesel produced from plants, when burned, just return to the air the carbon dioxide that those plants took out of the air while growing.

Finally, there’s one fuel that, in itself, produces no carbon dioxide at all when burned, namely
hydrogen; there’s no carbon there to produce carbon dioxide!
Of course, since free hydrogen molecules don’t occur in nature, it is typically produced by “reforming” a hydrocarbon or alcohol fuel or by using electricity to split water into hydrogen and oxygen. Then the size of the contribution of hydrogen fuel to carbon dioxide emissions depends on the source of the hydrocarbon fuel that was reformed or the source of the electricity used to split the water.

If a fossil fuel was the ultimate source of the energy that is, in effect, stored in the hydrogen, then you can still gain a large improvement in carbon-dioxide production if the hydrogen is used in an efficient drivetrain, as noted above; the same is true for the electrical energy stored in a battery-powered electric vehicle. In order to obtain the full benefits of reduction of carbon dioxide (or of ordinary air pollutants like carbon monoxide), of course, the energy used to split the hydrogen or charge the battery can be obtained from a renewable source like wind power or photovoltaics.

The nice thing about hydrogen- or battery-powered vehicles is that they can run on whatever is available–efficient natural-gas-burning powerplants today, with an increasing contribution from renewable energy as time goes on and the price of photovoltaic cells (solar cells) and other renewable energy sources continues to decline. As renewable energy becomes an ever larger part of the power generation mix over the next few decades, hydrogen- and battery-powered vehicles can switch over to the new power sources without a hiccup–it’s all electricity to them!