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... ... middle of paper ... ...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 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.
The GCMS uses a combination of gas chromatography and mass spectrometry to measure and identify the ingredients present in the gas over the liquid, otherwise known as the “headspace.” In gas chromatography, the sample of gas is injected into a port where it is carried through a mobile phase. The mobile phase passes the sample through a long column that also contains a stationary phase. Gas chromatography, also called “elution chromatography” because the various components of the sample are eluted from the column sequentially, is composed of a stationary and mobile phase. The stationary phase is usually a viscous liquid with a high boiling point that coats the inside of the column and the mobile phase is generally a gas. In this experiment a 30 meter long capillary GC column was used and the mobile phase gas was Helium. The different parts of the sample are separated based on their affinities for the mobile and stationary phase. A sample that has a high affinity for the stationary phase and also has a high boiling point will spend more time in the stationary phase. The longer a component spends in the stationary phase, the longer it takes the component to be eluted. The temperature of the column can be used to optimize the separation of the sample.
...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.
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
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 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.
Dondelinger, R. M. (2012). THE FUNDAMENTALS OF ... liquid chromatography systems. Biomedical Instrumentation & Technology, 46(4), 299-304. Retrieved from
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
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.
Stationary phase is of extreme importance in an HPLC analysis, as the chemical nature of the same and its compatibility with the analyte of interest is extremely significant for efficient separation. The most commonly used stationary phase is silica packed column which acts as a adsorbent. Each component in the sample interacts with these silica particles and gets eluted out in different time intervals. These silica columns may be of C14 or C18 type depending on the component of interest and also the columns themselves come in various dimensions each with a specific purpose of analysis.
Chromatography is a method of separating and analyzing complicated substances. This is done in two phases, a mobile phase and a stationary phase .During the stationary phase, said substance is stationary, while during the mobile phase, the substance moves in a specific direction. During the mobile phase, the substance is filtered through the stationary phase. The stationary phase in necessary in order for the substances to be separated even though it doesn?t involve movement of the substance because it filters the substance through the stationary phase.. Since the substance is made of different, specific substances, each can go though the process of chromatography at different rates. This causes the components of the substance to be moved over materials made for absorption at different times. This makes the different components of the substance absorb at different rates. This is done numerous times and is a very precise method of separation. This process can be used to separate a wide variety of things, and can be used to separate most volatile or soluble substances. This process is used many like because it is gentle enough to separate delicate solutions, like those of proteins.
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.
Last but not least, CE applications have been used for the analysis of particles, organelles, microorganisms, and eukaryotic cells, therefore also for their interactions, based mainly on the inhomogeneous nature of the analytes (Kremser, Blaas and Kenndler 2004).