Cromatography

this is what a chromatography experiment will come out like

Hello, everyone, and welcome to the wonderful world of chromatography! What is chromatography, you ask?? Well, quite simply, it is a broad range of physical methods used to separate and or to analyze complex mixtures. The components to be separated are distributed between two phases: a stationary phase bed and a mobile phase which percolates through the stationary bed.

A mixture of various components enters a chromatography process, and the different components are flushed through the system at different rates. These differential rates of migration as the mixture moves over adsorptive materials provide separation. Repeated sorption/desorption acts that take place during the movement of the sample over the stationary bed determine the rates. The smaller the affinity a molecule has for the stationary phase, the shorter the time spent in a column.

In any chemical or bioprocessing industry, the need to separate and purify a product from a complex mixture is a necessary and important step in the production line. Today, there exists a wide market of methods in which industries can accomplish these goals. Chromatography is a very special separation process for a multitude of reasons! First of all, it can separate complex mixtures with great precision. Even very similar components, such as proteins that may only vary by a single amino acid, can be separated with chromatography. In fact, chromatography can purify basically any soluble or volatile substance if the right adsorbent material, carrier fluid, and operating conditions are employed. Second, chromatography can be used to separate delicate products since the conditions under which it is performed are not typically severe. For these reasons, chromatography is quite well suited to a variety of uses in the field of biotechnology, such as separating mixtures of proteins.

Gas Chromatography

Gas chromatography makes use of a pressurized gas cylinder and a carrier gas, such as helium, to carry the solute through the column. The most common detectors used in this type of chromatography are thermal conductivity and flame ionization detectors. There are three types of gas chromatography that will be discussed here: gas adsorption, gas-liquid and capillary gas chromatography.
Gas adsorption chromatography involves a packed bed comprised of an adsorbent used as the stationary phase. Common adsorbents are zeolite, silica gel and activated alumina. This method is commonly used to separate mixtures of gases.
Gas-liquid chromatography is a more common type of analytical gas chromatography. In this type of column, an inert porous solid is coated with a viscous liquid which acts as the stationary phase. Diatomaceous earth is the most common solid used. Solutes in the feed stream dissolve into the liquid phase and eventually vaporize. The separation is thus based on relative volatilities.
Capillary gas chromatography is the most common analytical method. Glass or fused silica comprise the capillary walls which are coated with an absorbent or other solvent. Because of the small amount of stationary phase, the column can contain only a limited capacity. However, this method also yields rapid separation of mixtures.

Liquid Chromatography

There are a variety of types of liquid chromatography. There is liquid adsorption chromatography in which an adsorbent is used. This method is used in large-scale applications since adsorbents are relatively inexpensive. There is also liquid- liquid chromatography which is analogous to gas-liquid chromatography. The three types that will be considered here fall under the category of modern liquid chromatography. They are reverse phase, high performance and size exclusion liquid chromatography, along with supercritical fluid chromatography.
Reverse phase chromatography is a powerful analytical tool and involves a hydrophobic, low polarity stationary phase which is chemically bonded to an inert solid such as silica. The separation is essentially an extraction operation and is useful for separating non-volatile components.
High performance liquid chromatography (HPLC) is similar to reverse phase, only in this method, the process is conducted at a high velocity and pressure drop. The column is shorter and has a small diameter, but it is equivalent to possessing a large number of equilibrium stages.
Size exclusion chromatography, also known as gel permeation or filtration chromatography does not involve any adsorption and is extremely fast. The packing is a porous gel, and is capable of separating large molecules from smaller ones. The larger molecules elute first since they cannot penetrate the pores. This method is common in protein separation and purification.
Supercritical fluid chromatography is a relatively new analytical tool. In this method, the carrier is a supercritical fluid, such as carbon dioxide mixed with a modifier. Compared to liquids, supercritical fluids have solubilities and densities have as large, and they have diffusivities and viscosities quite a bit larger. This type of chromatography has not yet been implemented on a large scale.

Ion Exchange Chromatography

Ion exchange chromatography is commonly used in the purification of biological materials. There are two types of exchange: cation exchange in which the stationary phase carries a negative charge, and anion exchange in which the stationary phase carries a positive charge. Charged molecules in the liquid phase pass through the column until a binding site in the stationary phase appears. The molecule will not elute from the column until a solution of varying pH or ionic strength is passed through it. Separation by this method is highly selective. Since the resins are fairly inexpensive and high capacities can be used, this method of separation is applied early in the overall process.

Affinity Chromatography

Affinity chromatography involves the use of packing which has been chemically modified by attaching a compound with a specific affinity for the desired molecules, primarily biological compounds. The packing material used, called the affinity matrix, must be inert and easily modified. Agarose is the most common substance used, in spite of its cost. The ligands, or "affinity tails", that are inserted into the matrix can be genetically engineered to possess a specific affinity. In a process similar to ion exchange chromatography, the desired molecules adsorb to the ligands on the matrix until a solution of high salt concentration is passed through the column. This causes desorption of the molecules from the ligands, and they elute from the column. Fouling of the matrix can occur when a large number of impurities are present, therefore, this type of chromatography is usually implemented late in the process.

there is a much more complicated way of Affinity Chromatography and this is it, get your thinking hat on again!!!

An intriguing chromatographic technique based on the natural specificity of some biopolymers is affinity chromatography.
There are a number of proteins and other biological macromolecules that complex with some other biological entity with a high degree of specificity. This fact is made use of in product recovery operations via the use of affinity chromatography.
Suppose a certain biomolecule (a) is attached to a solid used to pack a chromatographic column. Now consider a molecule (b) in solution, which has a specific affinity for (a). It is but natural that (b) will want to get out of solution and bind to (a), right? It's this attraction of (b) for (a) which is defined as the partition coefficient 'K'. Now since 'K' for (b) is going to be much higher than that of any other proteins in solution, it will bind to the column while the rest of the complex solution will merely pass through the column with insignificant amounts of non-specific binding occuring.
What are some examples of molecules which may be used for this technique?
ENZYME + INHIBITOR <=> ENZYME-INHIBITOR COMPLEX
ANTIBODY + ANTIGEN ---> ANTIBODY-ANTIGEN PRECIPITATE
LECTIN + CELL WALL -----> LECTIN-CELL-WALL COMPLEX

hang on theres more

With the advent of Monoclonal Antibody production, which allows the synthesis of a single type of antibody with a very high specific binding constant to its corresponding antigen (a particular protein or other molecule), the preparation of affinity columns has become not only routine, but commercial. With such 'immunosorbent' column seperation, some important practical and theoretical differences arise compared to more conventional forms of chromatographic resolution. They are as follows:
1. The dominant cost in the process is the antibody needed to make the immunosorbent column. Generally speaking, this is much more costly than the antigen-containing broth itself. As a result,
2. A small column of repeated, high capacity use is required.
3. Elution of the adsorbed product requires breaking the antigen-antibody complex. Now this means that denaturing conditions must be employed. Since the antibodies themselves are proteins too, loss of some antibody binding affinity typically occurs, resulting in gradual loss of column capacity.
4. A first cycle on a new column gives poorer recovery than successive operations, apparently due to some irreversible binding.
5. A major economic goal in designing any affinity chromatography setup is determination of optimal elution buffer wash volumes and concentrations.

and more...

Now let us look at a specific example where affinity chromatography is used, namely, in the purification of human leukocyte interferon made in E. coli.
Proteins synthesized in genetically engineered organisms and intended for injection into animals must be stringently purified. Pyrogens from E. coli, including the outer envelope lipopolysaccharide (LPS) must be removed or inactivated. Hence product recovery operations such as affinity chromatography are an important step in the manufacturing process.
Given below is a schematic representation of the typical steps involved in processing human leukocyte interferon produced by recombinant DNA techniques. This will give you an idea of where exactly affinity chromatography is usually involved in the realm of bioprocessing.
HUMAN LEUKOCYTE INTERFERON

E. coli EXTRACTION BY MECHANICAL BREAKAGE

POLYETHELYNEIMINE PRECIPITATION

AMMONIUM SULFATE PRECIPITATION OF SUPERNATENT

DIALYSIS OF PELLET

* IMMUNOADSORBENT COLUMN (MONOCLONAL ANTIBODIES)

CATION EXCHANGE CELLULOSE CHROMATOGRAPHY

SO, Thank you everybody, for reading so patiently to what I had to say about affinity chromatography....

I hope you enjoyed the show...

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