Column Chromatography

Adsorption chromatography in biochemical applications usually consists of a solid stationary phase and a liquid mobile phase. The most useful technique is column chromatography, in which the stationary phase is confined to a glass or plastic tube and the mobile phase (a solvent or buffer) is allowed to flow through the solid adsorbent. A small amount of the sample to be analyzed is layered on top of the column. The sample mixture enters the column of adsorbing material and the molecules present are distributed between the mobile phase and the stationary phase. The various components in the sample have different affinities for the two phases and move through the column at different rates. Collection of the liquid phase emerging from the column yields separate fractions containing the individual components in the sample.

Specific terminology is used to describe various aspects of column chromatography. Poured or packed: When the actual adsorbing material is made into a column, it is said to be poured or packed.  Loading is Application of the sample to the top of the column. Developing or eluting: Movement of mobile phase through the loaded column is called developing or eluting the column. The bed volume is the total volume of solvent and adsorbing material taken up by the column. The void volume: is the volume taken up by the liquid phase in the column is the void volume. The elution volume is the amount of solvent required to remove a particular analyte from the column. This is analogous to Rf values in planar chromatography. In adsorption chromatography, solute molecules take part in specific interactions with the stationary phase. Herein lies the great versatility of adsorption chromatography.

Adsorbing material: A specific sorbent can be chosen that will effectively separate a mixture. from a large variety of available sorbing materials,  There is still an element of trial and error in the selection of an effective stationary phase. However, experiences of many investigators are recorded in the literature and are of great help in choosing the proper system.  Adsorbing materials come in various forms and sizes. The most suitable forms are dry powders or a slurry form of the material in an aqueous buffer or organic solvent. Alumina, silica gel, and fluorisil do not normally need special pretreatment. The size of particles in an adsorbing material is defined by mesh size. This refers to a standard sieve through which the particles can pass. A 100-mesh sieve has 100 small openings per square inch. Adsorbing material with high mesh size (400 and greater) is extremely fine and is most useful for very high resolution chromatography. For most biochemical applications, 100 to 200 mesh size is suitable.

Operation of A Chromatographic Column

A typical column setup is shown in Figure 5.4. The heart of the system is, of course, the column of adsorbent. In general, the longer the column, the better the resolution of components. However, a compromise must be made because flow rate decreases with increasing column length. The actual size of a column depends on the nature of the adsorbing material and the amount of chemical sample to be separated. For preparative purposes, column heights of 20 to 50 cm are usually sufficient to achieve acceptable resolution. Column inside diameters may vary from 0.5 to 5 cm.

Packing of Column

Once the adsorbing material and column size have been selected, the column is poured. If the tube does not have a fritted disc in the bottom, a small piece of glass wool or cotton should be used to support the column. Most columns are packed by pouring a slurry of the sorbent into the tube and allowing it to settle by gravity into a tight bed. The slurry is prepared with the solvent or buffer that will be used as the initial developing solvent. Pouring of the slurry must be continuous to avoid formation of sorbent layers. Excess solvent is eluted from the bottom of the column while the sorbent is settling. The column must never run dry. Additional slurry is added until the column bed reaches the desired height. The top of the settled adsorbent is then covered with a small circle of filter paper or glass wool to protect the surface while the column is loaded with sample or the eluting solvent is changed. Sometimes it is necessary to pack a column under pressure (5 to 10 psi). This leads to a tightly packed bed that yields more reproducible results, especially with gradient elutions sections).

Loading Of Column

The sample to be analyzed by chromatography should be applied to the top of the column in a concentrated form. If the sample is solid, it is dissolved in a minimum amount of solvent; p. 74. After the sample is loaded onto the column with a graduated or disposable pipet, it is allowed to percolate into the adsorbent. A few milliliters of solvent are then carefully added to wash the sample into the column material. The column is then filled with eluting solvent.

Eluting The Column

The chromatography column is developed by continuous flow of a solvent. Maintaining the appropriate flow rate is important for effective separation. If the flow rate is set too high, there is not sufficient time for complete equilibration of the analytes with the two phases. Too low a flow rate allows diffusion of analytes, which leads to poor resolution and broad elution peaks. It is difficult to give guidelines for the proper flow rate of a column, but, in general, a column should be adjusted to a rate slightly less than “free flow.” Sometimes it is necessary to find the proper flow rate by trial and error. One problem encountered during column development is a changing flow rate. As the solvent height above the column bed is reduced, there is less of a “pressure head” on the column, so the flow rate decreases. This can be avoided by storing the developing solvent in a large reservoir and allowing it to enter the column at the same rate as it is emerging from the column.

Adsorption columns are eluted in one of three ways. All components may be eluted by a single solvent or buffer. This is referred to as continual elution. In contrast, stepwise elution refers to an incremental change of solvent to aid development. The column is first eluted with a volume of one solvent and then with a second solvent. This may continue with as many solvents or solvent mixtures as desired. In general, the first solvent should be the least polar of any used in the analysis, and each additional solvent should be of greater polarity or ionic strength. Finally, adsorption columns may be developed by gradient elution brought about by a gradual change in solvent composition. The composition of the eluting solvent can be changed by mixing two different solvents to gradually change the ratio of two solvents.

Alternatively, the concen tration of a component in the solvent can be gradually increased. This is most often done by addition of a salt (KCl, NaCl, etc.). Devices are commercially avail able to prepare predetermined, reproducible gradients.

Collecting the Eluent

The separated components emerging from the column in the eluent are usually collected as discrete fractions. This may be done manually by collecting specified volumes of eluent in Erlenmeyer flasks or test tubes. Alternatively, if many fractions are to be collected, a mechanical fraction collector is convenient and even essential. An automatic fraction collector  directs the eluent into a single tube until a predetermined volume has been collected or until a preselected time period has elapsed; then the collector advances another tube for collection. Specified volumes are collected by a drop counter activated by a photocell, or a timer can be set to collect a fraction over a specific period.

Detection of Components

The completion of a chromatographic experiment calls for a means to detect the presence of analytes in the collected fractions. The detection method used will depend on the nature of the analytes. Smaller molecules such as lipids, amino acids, and carbohydrates can be detected by spotting fractions on a thin-layer plate or a piece of filter paper and treating them with a chemical reagent that pro duces a color. The same reagents that are used to visualize spots on a thin-layer or paper chromatogram are useful for this. Proteins and nucleic acids are conveniently detected by spectroscopic absorption measurements at 280 and 260 nm, respectively. Enzymes can be detected by measurements of catalytic activity asso ciated with each fraction. Research-grade chromatographic systems are equipped with detectors that continuously monitor some physical property of the eluent and display the separation results on a computer screen . The newest advance in detectors is the diode array . Most often the eluent is directed through a flow cell where absorbance or fluorescence characteristics can be measured. The detector is connected to a recorder or com puter for a permanent record of spectroscopic changes. When the location of the various analytes is determined, adjacent fractions containing identical components are pooled and stored for later use.