Column chromatography
The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process.
[1] For the wet method, a slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column.
The composition of the eluent flow can be monitored and each fraction is analyzed for dissolved compounds, e.g. by analytical chromatography, UV absorption spectra, or fluorescence.
The stationary phases are usually finely ground powders or gels and/or are microporous for an increased surface, though in EBA a fluidized bed is used.
For silica column chromatography, this ratio lies within 20:1 to 100:1, depending on how close to each other the analyte components are being eluted.
It is chosen so that the retention factor value of the compound of interest is roughly around 0.2 - 0.3 in order to minimize the time and the amount of eluent to run the chromatography.
Common mobile phase solvents, in order of increasing polarity, include hexane, dichloromethane, ethyl acetate, acetone, and methanol.
[3] A common solvent system is a mixture of hexane and ethyl acetate, with proportions adjusted until the target compound has a retention factor of 0.2 - 0.3.
Contrary to common misconception, methanol alone can be used as an eluent for highly polar compounds, and does not dissolve silica gel.
This information allows users to select optimal parameters for preparative-scale separations before the flash column itself is attempted.
Many manufacturers like Biotage, Buchi, Interchim and Teledyne Isco have developed automated flash chromatography systems (typically referred to as LPLC, low pressure liquid chromatography, around 350–525 kPa or 50.8–76.1 psi) that minimize human involvement in the purification process.
Automated systems will include components normally found on more expensive high performance liquid chromatography (HPLC) systems such as a gradient pump, sample injection ports, a UV detector and a fraction collector to collect the eluent.
The resolution (or the ability to separate a mixture) on an LPLC system will always be lower compared to HPLC, as the packing material in an HPLC column can be much smaller, typically only 5 micrometre thus increasing stationary phase surface area, increasing surface interactions and giving better separation.
The separate curves in the diagram represent different sample elution concentration profiles over time based on their affinity to the column resin.
Even smaller particles such as proteins, carbohydrates, metal ions, or other chemical compounds are conjugated onto the microbeads.
[8] Using this as a basis, three different isotherms can be used to describe the binding dynamics of a column chromatography: linear, Langmuir, and Freundlich.
The linear isotherm occurs when the solute concentration needed to be purified is very small relative to the binding molecule.
Thus, the equilibrium can be defined as: For industrial scale uses, the total binding molecules on the column resin beads must be factored in because unoccupied sites must be taken into account.
