The purpose of this lab was to better understand the process of chromatography by separating different mixtures. In one experiment, students had to separate b-carotene and chlorophyll in spinach. In the seconds experiment, students were expected to separate the different colors or compounds within several dyes. In this second experiment, students not only took a paper chromatography of the different dyes, but also of the spinach juice. Furthermore, students also calculate the Rf values for the paper chromatography experiment; students collected the wavelengths of the two bands that were collected from the first experiment (separating separate b-carotene and chlorophyll in spinach). I hypothesized that we would be able to better separate the …show more content…
We took pictures of each other’s data once finished with the lab. For the paper chromatography, students began by grinding 5g of spinach along with 2g of anhydrous magnesium sulfate. Students added hexanes and acetone as specified by the lab protocols. Once, the solvent was a dark green color, we placed it in a centrifuge and transfer the liquid portion of the solution into a test tube. Throughout this portion of the experiment, students used weighting paper as a funnel poring the indicated solution as stated by the protocol, for instance pouring silica gel and sand into the column. After, we poured about 3ml of Hexanes into the column, making sure not to let the column dry. We then added, spinach extract to the column—after, we added about 1ml of hexanes. Adding hexanes caused the solution to gain a yellow colored band. We added hexanes until the yellow band reached the bottom of the column, thus began to collect all the yellow pigment into a test tube. Once the elutant become colorless, we once again placed a waste basket under it. Finally, we collected the green pigment into another test tube by a 70%/ 30% mixture and a bit of acetone. Once the two colored bands were collected, we obtained the wavelengths of each colored band using the …show more content…
We then placed several dots as specified by the lab protocols, for instance placing a pencil dot in from the edge. Then, using a capillary, we spotted the 3cm spot about five times with spinach extract, that was obtained in column chromatography. Students, the placed a spinach leaf on the paper, covering 7 and 10cm dots. Using a quarter, students rolled over the leaf from about the 7 to 10cm mark several times. We then rolled the paper into a cylinder and stapled it. Once the paper was placed in a beaker containing 30ml of 90/10 hexanes to acetone mixture, foil was placed on top. Once the solvent reached about 1cm from the top of the paper, we took it out and marked the solvent front and again outlined every spot. We again, measured the distance to the solvent front and to the center of each spot—and calculated the Rf values for each spot. The colors for each spot, for both this and the paper chromatography were
A spectrum is a group of light wavelengths that are ordered in relation to their wavelength length. The electromagnetic spectrum consists radio waves, microwaves, infrared, visible, ultraviolet, X-rays and gamma rays. (1)Specifically, this lab looks at the visible light part of the spectrum because one of the colors in the visible light spectrum is shine through the sample. The visible light spectrum consists of colors of red, orange, yellow, green, blue, indigo, and violet. The color chosen to be shine through the sample is affected by the color of sample when mixed with the indicator Ammonium Vanadomolybdate (AMV). The color on the color wheel that is opposite of the solution’s color is the color that is shined through the
To test for this, DCIP (a chloroplast isolation buffer) was used to The hypothesis for this experiment was that the cell fraction in the cuvette marked P2 will have more chloroplast activity because it will exhibit greater color change and differences in the absorbance readings compared to the other cuvettes when exposed under the condition of light; moreover, this notion was believed to be so because the more a cell fraction is centrifuged, the more intact chloroplasts we’ll find (Leicht and McAllister, This meant that this cuvette (tested under light) should display a higher decrease in DCIP due to the reduction in absorbance (dependent variable) opposed to the other cell fractions tested depending on a sixteen minute period (independent variable). The overall goal was to provide proof, through data, that the cell fractions put under the light during the sixteen minute period would indicate a higher set of chloroplast activity versus the ones put in the
This experiment requires four tubes with an enzyme solution, chelating agent and deionized water. Also a fifth tube that is the calibration tube for the spectrophotometer, which only has 5ml of dH2O. The calibration tube is used to level out the spectrophotometer to zero before each trial. The spectrophotometer was set at 540 nm, “since green is not a color seen with the conversion of catechol to benzoquinone.” The enzyme solution was made by using potato that was peeled so that the golden color of the skin wouldn’t react or interfere with the red color needed in the spectrophotometer. After it was peeled, it was cut into chunks to minimize excess heat created while it was blended. It was put in a chilled blender and 500ml of deionized water was added. Chilled, deionized water was used because it created a hypotonic environment that caused the cells from the potato to burst and release the catecholase. It was chilled
PURPOSE: The purpose of the experiment is to determine the specific types of pigments found in water-soluble marker pens by using paper chromatography and water as a solvent.
The independent variable for this experiment is the enzyme concentration, and the range chosen is from 1% to 5% with the measurements of 1, 2, 4, and 5%. The dependant variable to be measured is the absorbance of the absorbance of the solution within a colorimeter, Equipments: Iodine solution: used to test for present of starch - Amylase solution - 1% starch solution - 1 pipette - 3 syringes - 8 test tubes – Stop clock - Water bath at 37oc - Distilled water- colorimeter Method: = == ==
using the control (“Blank”) as you did in the previous experiments. The contents of test tube 2 and 3 were mixed in a clean (clear of fingerprints) and the absorbance changes at 15 second intervals for 60 seconds were measured. The results were recorded in Table 9.
Materials used in the experiment included 5-7 g of the potato tissue, 50ml of 2.0M phosphate buffer coffee filter and guaiacol dye.
We were not given any instructions either to shake or not to shake the test tubes with the coloured solutions before inserting them in the spectrophotometer to read the absorbance. By shaking each test tube a certain number of times before putting it in the spectrophotometer could have improved the accuracy of the of absorbance of the solutions.
The data from the chromatography portion of the experiment showed that the least polar of the pigments would travel the most and the most polar would travel the least; chlorophyll b was the most polar and carotene was the least polar. The spectrophotometric portion of our experiment support this as well by showing us what wavelengths the pigments reflected and absorbed. With any experiment, however, are there sources of error. One source of error with this experiment would include not cleaning the cuvettes before placing them in the spectrophotometer. The smallest fingerprints or particles can lead to an inaccurate transmittance reading. Also, not using the reference cuvette when changing wavelengths is a source of error because it will lead to an inaccurate reading. Sources of error when using the chromatography paper include, too much or too little time for the solvent to ascend up the paper and the possibility that the solvent level may be too high. When studying photosynthetic wavelengths and pigments, it is known that, depending on the plant, some pigments are absorbed during photosynthesis while others are not. Pigments absorb only the light energy that is necessary in carrying out photosynthesis. This knowledge can assist in determining what areas
The system involved in this lab was L-dopa as a substrate, enzyme was Tyrosinase, and the product was Dopachrome. Tyrosinase is commonly known as polyphenol oxidase, an enzyme that present in plant and animal cell (#1 Boyer). In plant cell, the biological function if Tyrosinase is unknown, but its presence is readily apparent. Tyrosinase is also involved in the browning of fruits, tubers, and fungi that have been damaged. In mammalian cell, Tyrosinase is involved in melanin synthesis, which gives skin its color. It will act on the substrate L-dihydroxyphenylalanine (L-Dopa) and convert to Dopachrome, which is the product that has color, and it can measure at 475nm using the Spectrophotometer. This work based on the Beer-Lambert’s Law (A=εlc), A stands for Absorbance, ε is extinction coefficient or the molar absorptivity (M-1 cm-1), and l is the path length (distance) that light passes through the sample (cm), c is a concentration of solution (M) (#3 Ninfa, Ballou, Benore). Beer- Lambert Law predicts a linear relationship between absorbance and the concentration of a chemical species being analyzed. It states that the absorbance (A) of a sample solution is directly proportional to the concentration (c) of the absorbing colored
the amount of sugar increases. Colorimeter Instruments for the analysis of coloured solutions include the colorimeter. The colorimeter is a colorimeter. In both these instruments, light is emitted from a source.... ... middle of paper ... ...
1. What is the difference between a. and a. Background MCB 253 Evan London 2/5/16 A Comparison of Size Exclusion and Affinity Chromatography In molecular cell biology experiments, accuracy is one of the most important factors in obtaining reproducible results. A luxury not afforded to molecular cell biologists is the ability to easily manipulate samples since the material they deal with is at a microscopic level.
This experiment demonstrated the ability of agarose gel electrophoresis to separate the mixture of dyes into their individual components by the application of a combination of dyes to the same sample well. The experiment effectively demonstrated that the dyes where different in structure, energy, and composition. Most of the dyes where negatively charged at neutral pHs and only one with positive charge. The positive charge one moved an opposite direction compared to the other dyes.
As seen on many crime shows and at real-life crime scenes, it is necessary to be able to identify DNA. Most of the time, this is done using a technique known as gel electrophoresis. Gel electrophoresis is a method used to separate the macromolecules that make up nucleic acids, such as DNA and RNA, along with proteins. Gel electrophoresis is significant because it has given scientists insight on what cells cause certain diseases and has led to advancements in DNA and fingerprint identification. My experiment will use gel electrophoresis to compare samples of natural and synthetic food dyes. The background for this experiment broaches the following subjects: inventors, real-world uses, necessary components, separation, and information on food dyes.
In this experiment, we worked with paper chromatography. Before discussing the experiment in full detail, we must first review what exactly paper chromatography is. Paper chromatography is a certain technique used for separating different mixtures. It uses a specific type of paper made up of cellulose, and has a solution that separates the mixture into the multiple substances that make up that mixture1. Paper chromatography can also test the purity of the mixtures as well2.