Column Chromatography Lab Report

calophrys was evaluated using qualitative and quantitative DPPH methods [28,29] with slight modifications. For qualitative analysis, sample was applied on a TLC plate within concentration range of 0.1–100 μg, developed using a suitable mobile phase, and dried at room temperature. The dried plate was then sprayed with DPPH solution (0.2% in MeOH) and incubated for 30 min in dark at room temperature. The observation of active samples was made based on the intensity of yellow spots against the purple background on the plate. Furthermore, for quantitative analysis, triplicate 1 mL aliquots of samples were mixed with 1 mL of DPPH solution (500 μM; 0.2 mg/m) and incubated for 30 min in dark at room temperature. The absorption was measured spectrophotometrically at 517 nm against methanol as a blank. The amount of sample at which the absorbance at 517 nm decreased a half from the initial value was determined as the IC50 value, expressed in μg/mL. Ascorbic acid was used as positive

During this experiment the His-tagged protein RNase H was purified using affinity chromatography. The source of over-expressed His-tagged RNase H used was 5.0 mL of E. coli lysate. The experiment was performed in partners using different materials in order to prepare the needed three buffers to an approximate pH value of 7.9. Once the three buffers were ready a minicolumn was packed with 0.5 mL of Ni-NTA agarose and then different solutions were used to wash the column, starting with DI water, followed by the loading buffer, then the E.coli lysate, the washing buffer, and finally the eluting buffer. Some of the flows through were collected for future analysis in the upcoming experiments. The first was after the washing with the E.coli lysate, then the first wash from the washing buffer, and finally the flow through obtained after the wash with the eluting buffer.

There are many methods in the literature about measuring the porosity of porous material29. In our experiment, straight pieces of Grade 1 chromatography paper of 20 ⅹ 1 cm2 are cut. These segments are laminated to reduce the evaporation effect of water and also minimize the paper swelling upon getting wet. They were first weighed dry on a sensitive weighing balance. The bottom section of the laminated film is open, and the paper strips are dipped into water and oleic acid in a vertical position. The top of the laminated section is pierced to allow atmospheric pressure to act on both the sides and therefore not increasing the pressure increase inside the film as shown in Figure 1A.

Liquid scintillation counters (LSCs) involve adding scintillation solution to a purified strontium sample in an LSC vial (Randolph, 1975). There are different methods that can be used to prepare samples for LSC counting and samples need to be concentrated, as well as decontaminated. Certain times during the chemical separation must be taken to calculate the final activities of each radioisotope and samples must be dark adapted to prevent counting errors from luminescence. Strontium and yttrium carriers, standard solutions with known concentrations of non-active strontium or yttrium, are added to the samples for the determination of the final recovery (Randolph, 1975).

acid (0.2%) were added and the reaction mixture was heated on a boiling water bath for time period of 30 min. The absorbance was measured at 570 nm using a UVvisible spectrophotometer (LAMBDA 1050, PerkinElmer). Dulcitol. was used as standard.

The prime goal in implementing preparative gas chromatography was to separate components of a mixture. Afterwards, the identity of the separated parts was observed by the use of infrared spectroscopy. In the experiment, the unknown was labeled with the number 138R, and the identity was predicted to be 4-methyl-2-pentanone due to the presence of the functional groups: alkanes, and carbonyls on the IR spectrum.

The solvents that were used in the column chromatography were hexanes, 75/25 hexanes/acetone solvent, and acetone. The adsorbent used in the column chromatography was alumina, which is polar. All of the pigments moved through the column based on their polarity. Hexanes were used because they are nonpolar due to their hydrocarbon chain and was used as the initial solvent before the extract was placed in the Pasteur pipette. Carotene, which is nonpolar hydrocarbon chain, was the first pigment extracted. The nonpolar solvent was used because it is soluble in the nonpolar carotene, thus moving it through the column quickly. To elute the next pigment of chlorophyll the solvent was the hexane acetone mix. This mixture is slightly more polar because

Afterward, PTEN purified using affinity chromatography. Since PTEN fused with His6 tag, Ni-NTA affinity chromatography would work to purify PTEN. Ni-NTA is a type of immobilized metal affinity chromatography that contain coupled Ni2+ nickel chelate forming ligand (imiondiacetic acid) that immobilized to agarose. The science behind using Ni-NTA affinity chromatography is that histidine bind specifically and reversibly to the nickel chalet thus washing step will remove other contaminate and then His-PTEN eluted using imidazole. Bradford assay and SDS-PAGE performed to quantify and analyze the progress toward PTEN purification. The calculated concentration of the samples presented in table 8. The Concentration of samples make since having the

The function of Columnar epithelial tissue is to surround glands and ducts, which then help in absorption, secretion of mucus, enzymes, and other substances. The columnar epithelial tissue is made of large epithelial cells that are specialized for absorption, and are commonly found in areas where there is a lot of wear and tear, such as in the digestive tract. The larger shape of the columnar cell allows this tissue to perform its function in two ways. First, it increases the number of organelles to synthesize needed material, and second, it increases surface area. Simple columnar tissue commonly surrounds glands and ducts, therefore the increased amount of organelles aid in the production of material that is secreted. Columnar epithelial

When observing the thin-layer chromatography, it was evident that two compounds from experiment 5 expressed no impurities. There was one yellow band from the ferrocene dot, one red band from the acetylferrocene dot, and two yellow and red bands from the standard consisting of both

Column Chromatography is common and useful separation technique in organic chemistry. This separation method involves the same principles as thin layer chromatography but can be applied to separate larger quantities than TLC. Column chromatography can be used on both a large and small scale. The applications of this technique are wide reaching and cross many disciplines including biology, biochemistry, microbiology, and medicine. The technique of TLC is useful in determining the type and number of ingredients in the mixture, but column chromatography also allows for collecting the separated components.

It can be concluded that column chromatography is an effective way of separating components of a mixture. The crude pigment was sent through the column however three distinctly separate layers were collected (yellow, green-blue and blue). Each coloured layer represented a different pigment. It was made clear by the column chromatography that the beta-carotene (yellow) is the least polar pigment, chlorophyll b (green) is the most polar and chlorophyll a (blue-green) is intermediately polar based on the order the pigments were eluted. In the column, the least polar pigment eluted first as it adhered best to the 8:2 pet ether: acetone mobile phase. The last pigment that passed through was chlorophyll b as it was the most polar and adhered greatly to the silica. The effectiveness of column chromatography was confirmed using thin layer chromatography. On the silica plate, a spot was added for each separated pigment and the crude pigment. A highly effective separation using column chromatography would show corresponding distances travelled for the three separate colours and the three spots the crude separated into on the TLC plate. This confirmed that the column separated the pigments into the correct three layers. On the TLC

As various constituents in the extract will show maximum absorbance at different wavelengths; the optimisation of the UV wavelength for detection of analytes is rerquired such that be able to detect maximum number of well resolved peaks.

PL spectrometer, gloves, 1000µL micropipette, deionized water, ethanol, 1 PL quarts cuvette, and cis-diisothiocyanato-bis(2,2’-bipyridyl-4,4’-dicarboxylic acid)ruthenium(II) dye (Solaronix)

The retardation factor or Rf value is the ratio of the distance the compound travels to the distance the solvent travels. The Rf value allows the experimenters to determine if the compound is polar or nonpolar. If the compound is nonpolar (the Rf value is close to 1), then it will have a little to no affinity in the stationary phase. If the compound is polar (the Rf value is close to 0), then the compound is very polar and has little to no affinity in the mobile phase. These determinants will help decide which eluent should be selected for the chromatography process.