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Reverse Phase Chromatography


What is reverse phase liquid chromatography?

Reverse phase liquid chromatography is the separation of molecules based upon their inter-action with a hydrophobic matrix which is largely based on their polarity. Molecules are bound to the hydrophobic matrix in an aqueous buffer (polar) and eluted from the matrix using a gradient of organic solvent (non-polar). The matrix usually consists of spherical silica beads (3-5 micron) which have linear octadecane groups (C18) attached to the surface via co-valent bonds. These beads are usually porous in order to increase the surface area of the beads available for binding. The C18 groups are very hydrophobic (non-polar) and can bind quite polar molecules such as charged peptides in a highly polar solvent such as water. The name "reversed phase" is derived from the opposite technique of "normal phase" chromato-graphy which involves the separation of molecules based upon their interaction with a polar matrix (silica beads without octadecane groups attached) in the presence of a non-polar sol-vent.


Reverse Phase Chromatography

To perform a chromatographic separation the C18 coated beads are packed into a linear column such that liquid from a pumping system may be pushed over and through the beads. The column walls and fittings are usually made from glass, plastic or stainless steel. The column is packed such that there is a minimum of unoccupied space between the beads and that the beads are evenly distributed along the column. Molcules (peptides) to be separated by their polarity are injected onto the column in a polar solvent (usually referred to as Buffer A) and bind to the C18 groups on the beads. The flow of buffer A is continued until all mol-ecules which cannot bind are washed away.


Reverse Phase Chromatography

A non-polar solvent (usually referred to as Buffer B) is then usually mixed with buffer A in slowly increasing proportion. The proportion of this mixture is usually represented as a per-centage of the buffer mixture derived from buffer B (%B). When the mixture of buffer A and buffer B matches or exceeds the non-polarity of a molecule bound to the C18 groups, the molecule will elute into the buffer flowing over the column at that time. As the buffer flows over the entire column the same molecule will elute at the same point (in the same polarity of solvent). This will elute each molecule individually at its specific polarity and generate a peak of each molecule eluting from the column. The image below shows the blue molecules elut-ing as the solvent within the column matches or exceeds the blue molecules non-polarity.


Reverse Phase Chromatography

If a detector of some kind (mass spectrometer, UV absorbance, fluorescence, etc...) is examining the flow of liquid from the column these peaks can be observed and shown graphically in a chromatogram. In a chromatogram the y-axis represents the signal gener-ated by a molecule(s) within the detector and may be propotional to the molecule(s) concen-tration. The x-axis represents time after the injection of the molecules onto the column or the time after the gradient of buffers A and B was started. The image below shows the blue mol-ecules eluting as a peak early in the gradient (at a lower %B). The other molecules (green and red) elute later in the gradient (at higher %B)


Reverse Phase Chromatography

Using a long gradient with a slow increase in % B a better separation of molecules with simi-lar polarity may be achieved. However this will occur with a loss of signal intensity as the peak itself will be wider. In contrast a short steep gradient will improved signal intensity with a resulting loss of resolution.


Reverse Phase Chromatography

Columns which are long and this will have a better ability to separate analytes of similar po-larity as there will be more time for molecules to interact with the matrix during the gradient elution process. In contrast short fat columns will have lower resolution.


Reverse Phase Chromatography

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