This causes each compound to move at a different speed, thus creating a separation of the compounds. Tswett coined the name chromatography [from the Greek words chroma , meaning color, and graph , meaning writing—literally, color writing ] to describe his colorful experiment. Figure A: Tswett's Experiment. Liquid Chromatography LC Techniques Liquid chromatography can be performed using planar [Techniques 1 and 2] or column techniques [Technique 3].
Column liquid chromatography is the most powerful and has the highest capacity for sample. In all cases, the sample first must be dissolved in a liquid that is then transported either onto, or into, the chromatographic device. Technique 1. The sample is spotted onto, and then flows through, a thin layer of chromatographic particles [stationary phase] fixed onto the surface of a glass plate [Figure B]. The bottom edge of the plate is placed in a solvent.
Flow is created by capillary action as the solvent [mobile phase] diffuses into the dry particle layer and moves up the glass plate.
This technique is called thin-layer chromatography or TLC. Technique 2. In Figure C, samples are spotted onto paper [stationary phase]. Solvent [mobile phase] is then added to the center of the spot to create an outward radial flow.
This is a form of paper chromatography. Notice the difference in separation power for this particular paper when compared to the TLC plate. The green ring indicates that the paper cannot separate the yellow and blue dyes from each other, but it could separate those dyes from the red dyes.
And the dead time t 0 is defined as the time for a non-retained molecular species to elute from the column. The retention volume related to the dead time is known as dead volume V 0.
The migration rate can be defined as the velocity at which the species moves through the column. And the migration rate U R is inversely proportional to the retention times. If only a fraction of molecules that are present in the mobile phase are moving. In the separation, the molecules running through the column can also be considered as being in a continuous equilibrium between the mobile phase and the stationary phase.
The most important aspect of HPLC is the high separation capacity which enables the batch analysis of multiple components. Even if the sample consists of a mixture, HPLC will allows the target components to be separated, detected, and quantified. Also, it has a high sensitivity while a low sample consumption. HPLC has one advantage over GC column that analysis is possible for any sample can be stably dissolved in the eluent and need not to be vaporized.
With this reason, HPLC is used much more frequently in the field of biochemistry and pharmaceutical than the GC column. Columns Different separation mechanisms were used based on different property of the stationary phase of the column. Reverse-phase Chromatography In reverse-phase RP chromatography the stationary phase has a hydrophobic character, while the mobile phase has a polar character. Ion Exchange Chromatography The ion exchange mechanism is based on electrostatic interactions between hydrated ions from a sample and oppositely charged functional groups on the stationary phase.
Size Exclusion Chromatography It is a chromatographic method that separate the molecules in the solutions based on the size hydrodynamic volume. Detectors Detectors that are commonly used for liquid chromatography include ultraviolet-visible absorbance detectors, refractive index detectors, fluorescence detectors, and mass spectrometry.
Parameters related to HPLC separation Flow Rate Flow rate shows how fast the mobile phase travels across the column, and is often used for calculation of the consumption of the mobile phase in a given time interval. Retention Time The retention time t R can be defined as the time from the injection of the sample to the time of compound elution, and it is taken at the apex of the peak that belongs to the specific molecular species.
Migration Rate The migration rate can be defined as the velocity at which the species moves through the column. Equilibrium Constant and Phase Ratio In the separation, the molecules running through the column can also be considered as being in a continuous equilibrium between the mobile phase and the stationary phase.
As the sample passes through the column it interacts between the two phases at different rate, primarily due to different polarities in the analytes. Analytes that have the least amount of interaction with the stationary phase or the most amount of interaction with the mobile phase will exit the column faster. Many organic compounds absorb UV light of various wavelengths.
If you have a beam of UV light shining through the stream of liquid coming out of the column, and a UV detector on the opposite side of the stream, you can get a direct reading of how much of the light is absorbed. The amount of light absorbed will depend on the amount of a particular compound that is passing through the beam at the time.
You might wonder why the solvents used don't absorb UV light. They do! But different compounds absorb most strongly in different parts of the UV spectrum. Methanol, for example, absorbs at wavelengths below nm, and water below nm. 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. 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.
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