If you were using a methanol-water mixture as the solvent, you would therefore have to use a wavelength greater than nm to avoid false readings from the solvent. Note: If you are interested, there is a whole section about UV-visible spectroscopy on the site.
This explores the question of the absorption of UV and visible light by organic compounds in some detail. The output will be recorded as a series of peaks - each one representing a compound in the mixture passing through the detector and absorbing UV light.
As long as you were careful to control the conditions on the column, you could use the retention times to help to identify the compounds present - provided, of course, that you or somebody else had already measured them for pure samples of the various compounds under those identical conditions. But you can also use the peaks as a way of measuring the quantities of the compounds present. Let's suppose that you are interested in a particular compound, X.
If you injected a solution containing a known amount of pure X into the machine, not only could you record its retention time, but you could also relate the amount of X to the peak that was formed. The area under the peak is proportional to the amount of X which has passed the detector, and this area can be calculated automatically by the computer linked to the display.
The area it would measure is shown in green in the very simplified diagram. If the solution of X was less concentrated, the area under the peak would be less - although the retention time will still be the same. For example:.
This means that it is possible to calibrate the machine so that it can be used to find how much of a substance is present - even in very small quantities. Be careful, though! If you had two different substances in the mixture X and Y could you say anything about their relative amounts?
Not if you were using UV absorption as your detection method. In the diagram, the area under the peak for Y is less than that for X. That may be because there is less Y than X, but it could equally well be because Y absorbs UV light at the wavelength you are using less than X does. There might be large quantities of Y present, but if it only absorbed weakly, it would only give a small peak. Note: You will find a useful industry training video which talks through the whole process by following this link.
Linking to other sites is always a little bit hazardous because sites change. If you find that this link doesn't work, please contact me via the address on the About this site page.
This is where it gets really clever! When the detector is showing a peak, some of what is passing through the detector at that time can be diverted to a mass spectrometer.
There it will give a fragmentation pattern which can be compared against a computer database of known patterns. That means that the identity of a huge range of compounds can be found without having to know their retention times. Note: If you have forgotten about mass spectrometry, explore the mass spectrometry menu - particularly how a mass spectrometer works, and the formation of fragmentation patterns.
If this is the first set of questions you have done, please read the introductory page before you start. Use the BACK button on your browser to return quickly to this page. Carrying out HPLC Introduction High performance liquid chromatography is basically a highly improved form of column chromatography. The column and the solvent Confusingly, there are two variants in use in HPLC depending on the relative polarity of the solvent and the stationary phase.
The most powerful approach is the use multiple detectors in series. This provides, from a single injection, more comprehensive information about an analyte. HPLC Operation A simple way to understand how we achieve the separation of the compounds contained in a sample is to view the diagram in Figure G. Mobile phase enters the column from the left, passes through the particle bed, and exits at the right. Flow direction is represented by green arrows.
First, consider the top image; it represents the column at time zero [the moment of injection], when the sample enters the column and begins to form a band. The sample shown here, a mixture of yellow, red, and blue dyes, appears at the inlet of the column as a single black band.
After a few minutes [lower image], during which mobile phase flows continuously and steadily past the packing material particles, we can see that the individual dyes have moved in separate bands at different speeds. This is because there is a competition between the mobile phase and the stationary phase for attracting each of the dyes or analytes.
Notice that the yellow dye band moves the fastest and is about to exit the column. The yellow dye likes [is attracted to] the mobile phase more than the other dyes.
Therefore, it moves at a faster speed, closer to that of the mobile phase. The blue dye band likes the packing material more than the mobile phase. Its stronger attraction to the particles causes it to move significantly slower. 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. Nitrogen is used as a nebulisation gas in Evaporative Light Scattering Detector ELSD where the solvent is evaporated from the sample leaving a mist as is measured. 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.
0コメント