HPLC is essentially an adaptation of column chromatography - so it might be a good idea to have a very quick look at that as well. High performance liquid chromatography is basically a highly improved form of column chromatography.
Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to atmospheres. That makes it much faster. It also allows you to use a very much smaller particle size for the column packing material which gives a much greater surface area for interactions between the stationary phase and the molecules flowing past it. This allows a much better separation of the components of the mixture.
The other major improvement over column chromatography concerns the detection methods which can be used. These methods are highly automated and extremely sensitive. Confusingly, there are two variants in use in HPLC depending on the relative polarity of the solvent and the stationary phase. This is essentially just the same as you will already have read about in thin layer chromatography or column chromatography. Although it is described as "normal", it isn't the most commonly used form of HPLC.
The column is filled with tiny silica particles, and the solvent is non-polar - hexane, for example. A typical column has an internal diameter of 4. Polar compounds in the mixture being passed through the column will stick longer to the polar silica than non-polar compounds will. The non-polar ones will therefore pass more quickly through the column. In this case, the column size is the same, but the silica is modified to make it non-polar by attaching long hydrocarbon chains to its surface - typically with either 8 or 18 carbon atoms in them.
A polar solvent is used - for example, a mixture of water and an alcohol such as methanol. In this case, there will be a strong attraction between the polar solvent and polar molecules in the mixture being passed through the column. There won't be as much attraction between the hydrocarbon chains attached to the silica the stationary phase and the polar molecules in the solution. Polar molecules in the mixture will therefore spend most of their time moving with the solvent.
Non-polar compounds in the mixture will tend to form attractions with the hydrocarbon groups because of van der Waals dispersion forces. They will also be less soluble in the solvent because of the need to break hydrogen bonds as they squeeze in between the water or methanol molecules, for example. They therefore spend less time in solution in the solvent and this will slow them down on their way through the column. A chromatogram is a representation of the separation that has chemically [chromatographically] occurred in the HPLC system.
A series of peaks rising from a baseline is drawn on a time axis. Each peak represents the detector response for a different compound. The chromatogram is plotted by the computer data station [see Figure H].
In Figure H, the yellow band has completely passed through the detector flow cell; the electrical signal generated has been sent to the computer data station. The resulting chromatogram has begun to appear on screen.
Note that the chromatogram begins when the sample was first injected and starts as a straight line set near the bottom of the screen. This is called the baseline; it represents pure mobile phase passing through the flow cell over time. As the yellow analyte band passes through the flow cell, a stronger signal is sent to the computer.
The line curves, first upward, and then downward, in proportion to the concentration of the yellow dye in the sample band. This creates a peak in the chromatogram.
After the yellow band passes completely out of the detector cell, the signal level returns to the baseline; the flow cell now has, once again, only pure mobile phase in it.
Since the yellow band moves fastest, eluting first from the column, it is the first peak drawn. A little while later, the red band reaches the flow cell. The signal rises up from the baseline as the red band first enters the cell, and the peak representing the red band begins to be drawn. In this diagram, the red band has not fully passed through the flow cell.
The diagram shows what the red band and red peak would look like if we stopped the process at this moment. Since most of the red band has passed through the cell, most of the peak has been drawn, as shown by the solid line. The main advantage of an uHPLC is speed.
These systems are faster, more sensitive, and rely on smaller volumes of organic solvents than standard HPLC, resulting in the ability to run more samples in less time.
Figure 1 shows what happens to a sample containing a mixture of compounds after injection into the column. The compounds bind to the column and are eluted out at different times, depending on their polarity. Figure 1. The principle behind HPLC. Compounds of differing polarities indicated as darkening shades of blue are injected into the HPLC column entire cylinder.
The mobile phase is pumped through the column, and the addition of solvent along a concentration gradient shown as a black dotted line continuously decreases the overall polarity of the mobile phase Y-axis. Compounds are able to stick to either the column or the mobile phase, depending on their polarity.
The compounds will then dissociate from the column and will be eluted at a particular time X-axis during the run. This time is known as the Rf for that compound. The horizontal series of peaks seen in the chromatogram represent compounds eluted from the column with different R f values.
Modern HPLC equipment is often coupled to a diode array detector DAD , allowing the user to look at the resulting chromatogram of separated compounds in wavelengths from nm to nm. If the compounds under investigation are known, the user can choose to look only at one or a few selected wavelengths. For instance, cocaine can be observed at nm. Figure 2. A typical HPLC chromatogram. This chromatogram shows the separation of compounds from a chemical reaction, and the chromatogram is viewed at nm.
Two main peaks occur at 8.
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