Hplc how does it work

The smaller particles have a much greater surface area for interactions between the stationary phase and the molecules flowing past it. This results in a much better separation of the components of the mixture. The components of a mixture are separated from each other due to their different degrees of interaction with the absorbent particles. This causes different elution rates for the different components and leads to the separation of the components as they flow out the column.

Compared to column chromatography, HPLC is highly automated and extremely sensitive. 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.

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. The number of absorbance units AU is shown on the Y-axis while the time of the run is shown on the X-axis. Within biology and medicine, HPLC is often used as an analytical tool to assay biological and environmental samples for the presence or absence of known compounds for example, metabolites, drugs, toxins, pesticides and can assist in the identification of unknown compounds.

Within chemistry, however, HPLC is routinely used to monitor chemical reactions and to determine the purity of the products. HPLC can come across as being very complicated, but rest assured—just like most other lab techniques, it makes a lot more sense when you actually do it. Has this helped you? Then please share with your network.

You must be logged in to post a comment. This site uses Akismet to reduce spam. Learn how your comment data is processed. Facebook Twitter LinkedIn More. Written by Dr. Karen O'Hanlon Cohrt. Image Credit: Doug Letterman. This was very simple and informative. Thank you! Log in to Reply.

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.

However, if the systems are run at typical pressures greater than bar, the columns age, or degrade quicker. Fast protein liquid chromatography FPLC : FPLC is a system similar to high-performance liquid chromatography that is used to separate or purify proteins and other biomolecules from complex mixtures.