A Word About
Liquid Chromatography
Chromatogram representing a typical ion exchange chromatography process (by GE). The blue line
represents the continuous measurement of UV light absorbance of the liquid flowing out
of the chromatography column.
Liquid chromatography is one of several general types of chromatography and is the type used most in biopharmaceutical production. In liquid
chromatography, liquid is pumped through a container called a chromatography column. The column is packed tightly with a medium, usually
tiny synthetic beads known as "gel", an arrangement that bears some resemblance in concept to a sand filter on a swimming pool. Materials dissolved
in the liquid are separated based on relative differences in how fast they move through the medium.
Mechanically, liquid chromatography can be performed using several media formats, including:
- High Pressure Liquid Chromatography (HPLC, aka. High Performance Liquid Chromatography) - With extremely tiny beads, the pressure required to push
the liquid through the column can be hundreds of pounds.
- Low Pressure Liquid Chromatography - With larger beads, the pressure required to pump the liquid can be low enough to allow the use of peristaltic
pumps.
- Expanded Bed Chromatography - By not packing the beads tightly, leaving space at the top of the column, weighting the individual beads, and flowing
the liquid from bottom to top, this format causes the beads to spread out, avoiding problems with debris in the liquid clogging the column.
- Membrane Chromatography - Putting the same sort of functionality (aka. "mode", described below) on a membrane instead of on a bead improves
the flow characteristics of the system and avoids the need for any column. This is sometimes a single-use item (although a very expensive one at
that).
Large-scale liquid chromatography can be performed in several modes, including:
- Gel Filtration (size exclusion chromatography)- in this mode, the dissolved substances are separated by their relative sizes (molecular weights).
- Ion Exchange Chromatography (IEC) - in this mode, the concentration and acidity of the liquid is adjusted to control the relative speeds of the
molecules, based on their relative charges.
- Hydrophobic Interaction Chromatography (HIC) - in this mode, the salt concentration of the liquid is adjusted to control the relative speeds
of the molecules based on how hydrophobic they are. Oil, for example, is very hydrophobic, while sugar is very hydrophilic.
- Reversed Phase Chromatography - in this mode, the molecules are dissolved in organic solvents, such as alcohol, and separated based on how much they
are attracted to organic molecules on the beads.
- Affinity Chromatography - in this mode, relatively specific chemistries are added to the beads to select specific modecules under certain
conditions.
Page updated 7/6/17 |
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