A typical column has an internal diameter of 4. Non-polar compounds in the mixture will pass more quickly through the column, as polar compounds will stick longer to the polar silica than non-polar compounds will. The column is filled with silica particles which are modified to make them non-polar. This is done by attaching long hydrocarbon chains 8—18 C atoms to its surface. A polar solvent is used, for example, a mixture of water and an alcohol such as methanol.
Polar compounds in the mixture will pass more quickly through the column because a strong attraction occurs between the polar solvent and the polar molecules in the mixture. Non-polar molecules are slowed down on their way through the column. This includes:. Aside from these three, stearic selectivity, or shape selectivity, can sometimes play a role. Using the example of tapentadol as a typical small-molecule pharmaceutical compound, let us explore the 3 major mechanisms of interaction.
This molecule has polar, hydrophobic, and ionic components. Hydrophobic Interactions In RP-HPLC, the primary mechanism dictating retention behavior is hydrophobic interaction between the non-polar stationary phase ligand e. C18 and the hydrophobic nature of the sample molecule e. A fair estimate of retention can be predicted based on Log P value, which is the octanol; a water partition coefficient distribution ratio between octanol and water in a liquid- liquid extraction.
Polar Interactions These are interactions that occur between polar function groups of analytes, residual silanols, embedded polar groups, surface polar groups, or polar end-capping groups in the stationary phase.
They interact with the analyte through hydrogen bonding and dipole-dipole interactions. These interactions are relatively weak and transient compared to ion-exchange interaction. Ion-Exchange interactions Most RP media is based upon silica bonded with a non-polar stationary phase such as C Resulting in residual surface silanol groups Si-OH that are hidden. These silanols can become deprotonated and acquire a negative charge, then can interact ionically with positively charged basic analyte molecules.
These ion-exchange interactions are very strong and slow in contrast to hydrophobic and polar interactions. Sample retention time will vary depending on the interaction between the stationary phase, the molecules being analyzed, and the solvent, or solvents used. As the sample passes through the column it interacts between the two phases at different rate, primarily due to different polarities in the analytes.
HPLC can separate and detect each compound by the difference of each compound's speed through the column. There are two phases for HPLC: the mobile phase and the stationary phase.
The mobile phase is the liquid that dissolves the target compound. The stationary phase is the part of a column that interacts with the target compound. In the column, the stronger the affinity e. On the other hand, the stronger the affinity with the stationary phase, the slower it moves through the column.
The elution speed in the column depends on the affinity between the compound and the stationary phase. The word "chromatogram" means a plot obtained via chromatography.
The chromatogram is a two-dimensional plot with the vertical axis showing concentration in terms of the detector signal intensity and the horizontal axis representing the analysis time.
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