Bioprocess engg. & technology

msbo309

Experiment 5

Aim of the Experiment

Bio-separations: Various chromatographic techniques and extractions.

Principle

Distribution coefficients: The basis of all forms of chromatography is the distribution or partition coefficient (Kd), which describes the way in which a compound (the analyte) distributes between two immiscible phases. The term effective distribution coefficient is defined as the total amount, as distinct from the concentration, of analyte present in one phase divided by the total amount present in the other phase. It is in fact the distribution coefficient multiplied by the ratio of the volumes of the two phases present. If the distribution coefficient of an analyte between two phases A and B is 1, and if this analyte is distributed between 10 cm3 of A and 1 cm3 of B, the concentration in the two phases will be the same, but the total amount of the analyte in phase A will be 10 times the amount in phase B. All chromatographic systems consist of the stationary phase, which may be a solid, gel, liquid or a solid/liquid mixture that is immobilised, and the mobile phase, which may be liquid or gaseous, and which is passed over or through the stationary phase after the mixture of analytes to be separated has been applied to the stationary phase. During the chromatographic separation the analytes continuously pass back and forth between the two phases so that differences in their distribution coefficients result in their separation.

Column chromatography

In column chromatography the stationary phase is packed into a glass or metal column. The mixture of analytes is then applied and the mobile phase, commonly referred to as the eluent, is passed through the column either by use of a pumping system or applied gas pressure. The stationary phase is either coated onto discrete small particles (the matrix) and packed into the column or applied as a thin film to the inside wall of the column. As the eluent flows through the column the analytes separate on the basis of their distribution coefficients and emerge individually in the eluate as it leaves the column.

ION-EXCHANGE CHROMATOGRAPHY

This form of chromatography relies on the attraction between oppositely charged stationary phase, known as an ion exchanger, and analyte. It is frequently chosen for the separation and purification of proteins, peptides, nucleic acids, polynucleotides and other charged molecules, mainly because of its high resolving power and high capacity. There are two types of ion exchanger, namely cation and anion exchangers. Cation exchangers possess negatively charged groups and these will attract positively charged cations. These exchangers are also called acidic ion exchangers because their negative charges result from the ionisation of acidic groups. Anion exchangers have positively charged groups that will attract negatively charged anions. The term basic ion exchangers is also used to describe these exchangers, as positive charges generally result from the association of protons with basic groups.

MOLECULAR (SIZE) EXCLUSION CHROMATOGRAPHY

This chromatographic technique for the separation of molecules on the basis of their molecular size and shape exploits the molecular sieve properties of a variety of porous materials. The terms exclusion or permeation chromatography or gel filtration describe all molecular separation processes using molecular sieves. The general principle of exclusion chromatography is quite simple. A column of microparticulate cross-linked copolymers generally of either styrene or divinylbenzene and with a narrow range of pore sizes is in equilibrium with a suitable mobile phase for the analytes to be separated. Large analytes that are completely excluded from the pores will pass through the interstitial spaces between the particles and will appear first in the eluate. Smaller analytes will be distributed between the mobile phase inside and outside the particles and will therefore pass through the column at a slower rate, hence appearing last in the eluate. The mobile phase trapped by a particle is available to an analyte to an extent that is dependent upon the porosity of the particle and the size of the analyte molecule. Thus, the distribution of an analyte in a column of cross-linked particles is determined solely by the total volume of mobile phase, both inside and outside the particles, that is available to it. For a given type of particle, the distribution coefficient, Kd, of a particular analyte between the inner and outer mobile phase is a function of its molecular size. If the analyte is large and completely excluded from the mobile phase within the particle, Kd ¼ 0, whereas, if the analyte is sufficiently small to gain complete access to the inner mobile phase, Kd ¼ 1. Due to variation in pore size between individual particles, there is some inner mobile phase that will be available and some that will not be available to analytes of intermediate size; hence Kd values vary between 0 and 1. It is this complete variation of Kd between these two limits that makes it possible to separate analytes within a narrow molecular size range on a given particle type.

AFFINITY CHROMATOGRAPHY

Separation and purification of analytes by affinity chromatography is unlike most other forms of chromatography and such techniques as electrophoresis and centrifugation in that it does not rely on differences in the physical properties of the analytes. Instead, it exploits the unique property of extremely specific biological interactions to achieve separation and purification. As a consequence, affinity chromatography is theoretically capable of giving absolute purification, even from complex mixtures, in a single process. The technique was originally developed for the purification of enzymes, but it has since been extended to nucleotides, nucleic acids, immunoglobulins, membrane receptors and even to whole cells and cell fragments.

GAS CHROMATOGRAPHY

The principles of gas chromatography (GC) are similar to those of HPLC but the apparatus is significantly different. It exploits differences in the partition coefficients between a stationary liquid phase and a mobile gas phase of the volatilised analytes as they are carried through the column by the mobile gas phase. Its use is therefore confined to analytes that are volatile but thermally stable. The partition coefficients are inversely proportional to the volatility of the analytes so that the most volatile elute first. The temperature of the column is raised to 50-300 oC to facilitate analyte volatilisation. The stationary phase consists of a high-boiling-point liquid material such as silicone grease or wax that is either coated onto the internal wall of the column or supported on an inert granular solid and packed into the column. There is an optimum flow rate of the mobile gas phase for maximum column efficiency (minimum plate height, H).

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

As the number of theoretical plates in the column is related to the surface area of the stationary phase, it follows that the smaller the particle size of the stationary phase, the greater the value of N, i.e. N is inversely proportional to particle size. Unfortunately, the smaller the particle size, the greater is the resistance to the flow of the mobile phase for a given flow rate. This resistance creates a backpressure in the column that is directly proportional to both the flow rate and the column length and inversely proportional to the square of the particle size. The back-pressure may be sufficient to cause the structure of the matrix to collapse, thereby actually further reducing eluent flow and impairing resolution. This problem has been solved by the development of small particle size stationary phases, generally in the region of 510 mm diameter with a narrow range of particle sizes, which can withstand pressures up to 40 MPa. This development, which is the basis of HPLC that was originally and incorrectly referred to as high-pressure liquid chromatography, explains why it has emerged as the most popular, powerful and versatile form of chromatography. Larger particle size phases are available commercially and form the basis of low-pressure liquid chromatography in which flow of the eluaent through the column is either gravity-fed or pumped by a low pressure pump, often a peristaltic pump. It is cheaper to run than HPLC but lacks the high resolution that is the characteristic of HPLC. Many commercially available HPLC systems are available and most are microprocessor controlled to allow dedicated, continuous chromatographic separations.

Procedure

Column chromatography

Basic column chromatographic components: A typical column chromatographic system using a gas or liquid mobile phase consists of the following components:

• A stationary phase: Chosen to be appropriate for the analytes to be separated.

• A column: In liquid chromatography these are generally 2550 cm long and 4mm internal diameter and made of stainless steel whereas in gas chromatography they are 13m long and 24mm internal diameter and made of either glass or stainless steel. They may be either of the conventional type filled with the stationary phase, or of the microbore type in which the stationary phase is coated directly on the inside wall of the column.

• A mobile phase and delivery system: Chosen to complement the stationary phase and hence to discriminate between the sample analytes and to deliver a constant rate of flow into the column.

• An injector system: To deliver test samples to the top of the column in a reproducible manner.

• A detector and chart recorder: To give a continuous record of the presence of the analytes in the eluate as it emerges from the column. Detection is usually based on the measurement of a physical parameter such as visible or ultraviolet absorption or fluorescence. A peak on the chart recorder represents each separated analyte.

• A fraction collector: For collecting the separated analytes for further biochemical studies.

ION-EXCHANGE CHROMATOGRAPHY

Matrices used include polystyrene, cellulose and agarose. Functional ionic groups include sulphonate (–SO and quaternary ammonium (–NþR3), both of which are strong exchangers because they are totally ionised at all normal working pH values, and carboxylate (–COO) and diethylammonium (–HNþ(CH2CH3)2), both of which are termed weak exchangers because they are ionised over only a narrow range of pH values. Examples are given in Table 11.3. Bonded phase ion exchangers suitable for HPLC, containing a wide range of ionic groups, are commercially available. Porous varieties are based on polystyrene, porous silica or hydrophilic polyethers, and are particularly valuable for the separation of proteins. They have a particle diameter of 525 mm. Most HPLC ion exchangers are stable up to 60 C and separations are often carried out at this temperature, owing to the fact that the raised temperature decreases the viscosity of the mobile phase and thereby increases the efficiency of the separation.

MOLECULAR (SIZE) EXCLUSION CHROMATOGRAPHY

Relative molecular mass determination: The elution volumes of globular proteins are determined largely by their relative molecular mass (Mr). It has been shown that, over a considerable range of relative molecular masses, the elution volume or Kd is an approximately linear function of the logarithm of Mr. Hence the construction of a calibration curve, with proteins of a similar shape and known Mr, enables the Mr values of other proteins, even in crude preparations, to be estimated.

AFFINITY CHROMATOGRAPHY

The procedure for affinity chromatography is similar to that used in other forms of liquid chromatography. The buffer used must contain any cofactors, such as metal ions, necessary for ligand–macromolecule interaction. Once the sample has been applied and the macromolecule bound, the column is eluted with more buffer to remove nonspecifically bound contaminants. The purified compound is recovered from the ligand by either specific or non-specific elution. Non-specific elution may be achieved by a change in either pH or ionic strength. pH shift elution using dilute acetic acid or ammonium hydroxide results from a change in the state of ionisation of groups in the ligand and/or the macromolecule that are critical to ligand–macromolecule binding. A change in ionic strength, not necessarily with a concomitant change in pH, also causes elution due to a disruption of the ligand–macromolecule interaction; 1M NaCl is frequently used for this purpose.

GAS CHROMATOGRAPHY

The majority of non- and low-polar compounds are directly amenable to GC, but other compounds possessing such polar groups as OH, NH2 and COOH are generally retained on the column for excessive periods of time if they are applied directly. Poor resolution and peak tailing usually accompany this excessive retention. This problem can be overcome by derivatisation of the polar groups. This increases the volatility and effective distribution coefficients of the compounds. Methylation, silanisation and perfluoracylation are common derivatisation methods for fatty acids, carbohydrates and amino acids.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

It is evident from equations 11.1 to 11.13 that the resolving power of a chromatographic column is determined by a number of factors that are embedded in equation 11.13. This shows that the resolution increases with:

• the number of theoretical plates (N) in the column and hence plate height (H). The value of N increases with column length but there are practical limits to the length of a column owing to the problem of peak broadening (Section 11.2.4);

• the selectivity of the column, a; and

• the retentivity of the column as determined by the retention factor, k.