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Study Notes: RP-HPLC

Reversed-phase HPLC (or RP-HPLC) is a technique that is often used to analyse proteins. The term ‘reversed-phase’ relates to the polarity of the stationary and mobile phases. In RP-HPLC:

  • The stationary phase is nonpolar, often a hydrocarbon (for instance the C18 column material is coated with an 18 chain hydrocarbon).
  • The mobile phase is relatively polar (such as water, methanol or acetonitrile).

The most polar component elutes first and increasing the polarity of the mobile phase increases the elution time.

The stationary phase is classified as reversed-phase when the bonded coating is nonpolar in character.

Most modern HPLC is carried out using columns with such reversed-phase packing. In the case of C18 packings, the long chain hydrocarbon groups are aligned parallel to one another and perpendicular to the packing surface. A good way to visualise this is as a brush where the wooden handle is the stationary phase packing and the bristles are the C18 molecules standing out. A C18 column can be considered to have the structure of a tiny brush, which this looks like, the C18 strands running across the brush.

Generally the longer the chain length the better the separation, that is, longer chains produce packings that are more retentive, with better separation of components in a given sample.

Diagram shows that as the chain length increases (from C3 to C8 to C18) the separation (resolution) of the 6 bands is increased.

Additionally, longer chain length allows the use of larger sample volumes.

It is usual in RP-HPLC to use a highly polar mobile phase such as an aqueous solution containing various concentrations of solvents such as methanol, acetonitrile or tetrahydrofuran.

In such cases, the pH must be controlled to less than 7.5 because hydrolysis of the siloxane (which binds the C18 hydrocarbon to the solid support of the packing material) occurs above this pH value and would lead to degradation of the packing with consequent loss of separation ability.

The HPLC carried out at SimuLab is RP-HPLC conducted with the following standard conditions.

  • Column material - C18
  • Injection volume of 100 µL
  • Pressure of 4500 psi
  • UV absorbance detector
  • Temperature of 25°C.

The only factors that may be changed are the following.

  • Flow rate
  • Run time
  • Mobile phase - isocratic (constant composition) or gradient (changing composition).
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