Hplc chromatography how does it work

In other words, it is the most retained compound in this sample mixture. The red dye band has an intermediate attraction for the mobile phase and therefore moves at an intermediate speed through the column. Since each dye band moves at different speed, we are able to separate it chromatographically.

What Is a Detector? As the separated dye bands leave the column, they pass immediately into the detector. The detector contains a flow cell that sees [detects] each separated compound band against a background of mobile phase [see Figure H]. A choice is made among many different types of detectors, depending upon the characteristics and concentrations of the compounds that need to be separated and analyzed, as discussed earlier.

What Is a Chromatogram? 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. 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.

Note: I have been a bit careful about how I have described the attractions of the non-polar molecules to the surface of the stationary phase. In particular, I have avoided the use of the word "adsorpion".

Adsorption is when a molecule sticks to the surface of a solid. Especially if you had small molecules in your mixture, some could get in between the long C 18 chains to give what is essentially a solution.

You could therefore say that non-polar molecules were more soluble in the hydrocarbon on the surface of the silica than they are in the polar solvent - and so spend more time in this alternative "solvent".

Where a solute divides itself between two different solvents because it is more soluble in one than the other, we call it partition. So is this adsorption or partition? You could argue it both ways! Be prepared to find it described as either.

Injection of the sample is entirely automated, and you wouldn't be expected to know how this is done at this introductory level. Because of the pressures involved, it is not the same as in gas chromatography if you have already studied that. The time taken for a particular compound to travel through the column to the detector is known as its retention time. High-performance liquid chromatography HPLC is an analytical technique to separate, identify, and quantify components in a mixture.

It is the single biggest chromatography technique essential to most laboratories worldwide. In column chromatography a solvent drips through a column filled with an adsorbent under gravity. HPLC is a highly improved form of column chromatography.

A pump forces a solvent through a column under high pressures of up to atmospheres. The column packing material or adsorbent or stationary phase is typically a granular material made of solid particles such as silica or polymers.

The pressure makes the technique much faster compared to column chromatography. Instrumentation: Main components in an HPLC system include the solvent reservoir, or multiple reservoirs, a high-pressure pump, a column, injector system and the detector. Helium sparging is an effective method of degassing the mobile phase to avoid unstable baselines caused by dissolved air.