Quinn Little
CHEM 4614
Dr. Jennifer Jamison
4/30/2014
Countercurrent Chromatography
Separations are important techniques in chemistry that are used to separate various components of a mixture. They are carried out by mixing two immiscible liquids containing certain solutes together in a separatory funnel, allowing them to separate, then extracting the distinct layers that form. The ratio of the concentration of solute present in the upper layer to the concentration in the lower layer is called the partition coefficient. The efficiency of a separation is described by this partition coefficient. If the coefficients for the two layers are largely different, then the separation can be carried out in a single step. If they aren’t, a more complex process is necessary.1,2 Countercurrent chromatography is a technique used carry out separations in these kinds of cases. It uses a continuous liquid-liquid partitioning process to streamline the usual extraction procedure.
The technique of countercurrent chromatography is based off of an older technique called countercurrent distribution.3 This method is a multi-step extraction technique that uses something called a Craig apparatus. It works by first adding a specific quantity of one solvent containing a solute of interest to each of the specially designed as in Figure 1, interconnected tubes in the series, then adding a second, lighter solvent that provides a partition coefficient greater than 1 to the first tube only. The two solvents are then mixed together, then allowed to separate into layers. The lighter top solvent is then transferred to the second tube in the series by tilting the apparatus, while the heavier bottom layer is retained. A fresh quantity of the lighter sol...
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...d to mix the contents of the column. Aside from these commonalities, the instrumentation used varies greatly depending on the type of countercurrent chromatography used.
The first type of countercurrent chromatography ever designed was helix countercurrent chromatography. In this form, a helical column is first filled with the stationary phase, then the second phase is continuously injected into one end of the tube. The mobile phase passes through the stationary phase, which is trapped at the bottom of each turn of the coil by gravity. The two phases equilibrate in the stationary segments to varying degrees. The degree of equilibration is controlled by a few factors, including the thoroughness of mixing and physical properties of the two, such as surface tension. At the end the mobile phase is eluted, usually along with a small amount of the stationary phase.
The purpose of this experiment was to learn and preform an acid-base extraction technique to separate organic compounds successfully and obtaining amounts of each component in the mixture. In this experiment, the separation will be done by separatory funnel preforming on two liquids that are immiscible from two layers when added together. The individual components of Phensuprin (Acetylsalicylic acid, Acetanilide, and Sucrose as a filler) was separated based upon their solubility and reactivity, and the amount of each component in the mixture was obtained. Also, the purity of each component will be determined by the melting point of the component.
Once the mixture had been completely dissolved, the solution was transferred to a separatory funnel. The solution was then extracted twice using 5.0 mL of 1 M
Put 1mL of 0.1M cobalt (II) chloride hexahydrate dissolved in 95% ethanol into a test tube. Then add 1mL of deionized water. Tap the end of the test tube to mix the solution and record the pertinent data in section 2 of the Data Table. Discard the solution in the appropriate container as directed to you by your lab instructor.
The objective of this experiment was to perform extraction. This is a separation and purification technique, based on different solubility of compounds in immiscible solvent mixtures. Extraction is conducted by shaking the solution with the solvent, until two layers are formed. One layer can then be separated from the other. If the separation does not happen in one try, multiple attempts may be needed.
As the components of the sample were eluted from the column they were passed over a detector which determines the quantity present and plots a peak on a chromatogram at a specific retention time.
...lications in the future. This is due to the fact that this method has become rough, not complicated and it can be performed in a conventional way without being mandatory the investigation into depth for every application (Tetala and van Beek, 2010). New forms are going to be operated in order to recognize bacteria and also aptamers are going to be used more often. Moreover, the investigation of new types of monoliths will also include the study of present or alterative types of polymers, in order to come out with a wider range of pore sizes, surface areas and new morphologies that can be used in this type of affinity chromatography (Pfaunmiller et al., 2013). Finally, monolithic stationary phases are expected to have a great impact on future applications, for instance if organic monolithic supports will be combined with hybrids of silica (Pfaunmiller et al., 2013).
The first component in the Mobile phase is phosphate buffer (pH 2.5) and the second component is methanol. The elution was isocratic eluting at 30% of the buffer and 70% of the methanol at a flow rate of 1mL min−1, The wavelength used for UV-detector was 280 nm, HPLC column C18 (150 mm×4.6 mm, 5µm), and the injection volume was 20 µL.
Dondelinger, R. M. (2012). THE FUNDAMENTALS OF ... liquid chromatography systems. Biomedical Instrumentation & Technology, 46(4), 299-304. Retrieved from
The purpose of this lab was to extract chlorophyll and carotenoid pigments from fresh spinach leaves and separate and analyze these pigments using column chromatography and thin layer chromatography. Acetone was used as a polar solvent to dissolve the more polar pigments first (Xanthophylls, chlorophylls), while hexane was used as a nonpolar solvent to dissolve the more nonpolar pigments such as the carotenes. In addition to being used as the polar solvent, acetone was used to remove the spinach components that were not pigments such as cellulose which is insoluble. The column chromatography worked by eluting the nonpolar carotene pigments first because the alumina is polar and doesn’t absorb the nonpolar carotene. The polar components such
One significant advantage of capillary electrophoresis (CE) is the separation of a broad range of analytes at the same moment. Affinity Capillary Electrophoresis (ACE) is a technique used in order to separate substances which participate either in specific or in non-specific affinity interactions during the electrophoresis process, by using a capillary electrophoresis format. The molecules can be free in solution or they can be immobilized to a solid support (Heegaard, Nilsson and Guzman, 1998).
The extraction procedure isolates the pigments in spinach that will be used in the TLC analysis by leaving the insoluble properties behind. Each step serves a purpose in doing so, for example, the spinach is blended into a puree in order facilitate the centrifuge process. 75/25 hexane/acetone solution is used in the blending process because hexane alone is not enough and acetone alone is miscible in water. Anhydrous sodium sulfate is used to dry the organic solution after it has been through the centrifuge tube and separated into a test tube. The purpose of the alumina column is to filter unwanted inorganic chemicals while letting the desired organic chemicals to pass through.
HPLC technology works on the principle of conventional chromatography where in there is a stationary phase and a mobile phase. The sample containing the mixture of components is introduced in a column packed
There are many types of chromatography, the types of which are as follows: Gas Chromatography, Liquid Chromatography, Ion Exchange Chromatography, and Affinity Chromatography . Gas Chromatography uses a pressurized gas camber to filter gasses by either thermal conductivity or flame ionization. There are three types of gas chromatography: capillary gas chromatography, gas adsorption chromatography and gas-liquid chromatography. Capillary gas chromatography used more often than any other type of gas chromatography. In this form of chromatography...
It is used for separation of polar/charged/hydrophilic molecules. We can separate macromolecules like proteins, amino acid or nucleotides through ion chromatography. Mobile phase and liquid phase can be of different type i.e., it can be liquid, gas or solid but here Mobile phase is liquid and stationary phase is solid. Mainly the column chromatography is used for this purpose. Column chromatography means we construct columns of polymers like cellulose or
... tested in the same manner for a specified purpose in order to maintain consistency and validity within results.