Dye Chromatography Lab

And finally into test tube 3, I pipetted 1.0 ml turnip extract and 4.0 ml of water. The contents of test tube 1 was poured into a spectrometer tube and labeled it “B” for blank. “B” tube was now inserted it into the spectrometer. An adjustment to the control knob was made to zero the absorbance reading on the spectrometer since one cannot continue the experiment if the spectrometer is not zeroed. A combination of two people and a stop watch was now needed to not only record the time of the reaction, but to mix the reagents in a precise and accurate manner. As my partner recorded the time, I quickly poured tube 3 into tube 2. I then poured tube 2 into the experiment spectrometer tube labeled “E” and inserted it into the spectrometer. A partner then recorded the absorbance reading for every 20 seconds for a total of 120 seconds. After the experiment, a brown color in the tube should be observed to indicate the reaction was carried out. Using sterile techniques, any excess liquid left was disposed

The purpose of this experiment is to determine the specific pigments that are found in each of the chosen plant leaves, as well as, discover the intermolecular forces present in each leaf. This is accomplished by using a technique called chromatography, which splits a mixture or solution into its different parts based on the mixtures ability to dissolve in a chosen solvent. Chromatography works by placing a strip of chromatography paper, that has a small amount of mixture on it, into a cup of the specific solvent chosen. In this experiment, one hundred percent acetone was chosen to be the solvent. Once the small amount of the mixture is aligned with the solvent, the mixture will begin to separate and rise by capillary action based on the solubility,

Finally, the test tubes were placed in the the rack and we recorded the color of the solution for day one in table

We then placed the chromatography paper into a container filled with 9 parts of petroleum ether and 1 part acetone. We made sure that the tip of the paper was fully submerged in the solvent while keeping the pigment line above the solvent. We then closed off the top of the tube and watched the pigments rise. During this process we couldn’t make any movement because it could alter the results and cause us to redo the experiment. We removed the paper from the container before the solvent reached the top of the paper and then observed and drew the results in our lab book.

In this investigation, we are trying to find how far each of the dye traveled on the chromatography paper. In the solvent with the blue dye, the solvent traveled 97mm and the blue dye traveled 81mm. In the solvent with the yellow dye, the solvent traveled 97mm and the yellow dye traveled 70mm. The percent error of the solvent front is 5.8%. The percent error of the distance traveled by the blue dye is 16%. The percent error of the distance traveled by the yellow dye is 26%. The percent error of the retardation factor of the blue dye is 11%.

The “E” solution ended up having the most spots because it was the pigment fragments. The Rf values could be calculated for all of the spots by taking the distance traveled by the spot and dividing it by the total distance traveled by the solvent front. The calculations are as shown:

Agarose gel (2%) on gel tray, TBE running buffer 1X, 350 mL, 5 micropipettes, metric ruler, electrophoresis chamber, power supply. The dyes: Bromophenol blue,

Goal: The first goal of today’s laboratory is to separate components of spinach dyes using different eluants. The four eluants will be using are ethanol, chloroform, 9:1 petroleum ether : ethanol, and petroleum ether. The second goal of the lab is to separate fluorine and fluorenone by column chromatography. Thin layer chromatography (TLC) was used to measure the polarity and separate the components in the mixtures. TLC was chosen because of its simplicity, high sensitivity, and speedy separation. For each part of the lab, we measured the retention factor on the TLC plate. To measure the retention factor, we used the formula:

The dye was removed from M&M’s by adding water on to them. The dye was then put on a 1 ½ / 6 inch piece of paper using capillary tubes. After a sample of all six dye combinations were added, three to each end of the paper, the ends of the paper were placed in a few drops of salt solution. The solution was then left to spread over the paper and separate the dye as it went. After the

Chromatography is the process by which a chemical mixture is separated, so it can be studied further. The objective to this lab was to separate

Concentrations of the dyes were recorded. Two burets were used, each with their respective dyes, the analyte and the internal standards. A calibration of the two solutions was created in dry test tubes. This calibration used 5 samples and in each sample taken there was an increase in volume of analyte dye stock added while there was a decrease in internal standard dye stock solution added (the table for exact measurements was given in the referenced lab manual and is exemplified in the results- Table 3. 40 mL of stock internal standards dye to the beaker containing a dissolved Peep solution.

For the Plant Pigments I and II- Pigment Extraction lab, the objective was to find a solution that separated pigments and allowed them to travel up the chromatograph. The results of the experiment found that a 1:5 Acetone to Hexane ratio was the best solvent to use. In order to execute the lab we had to roll a penny over the spinach, which would in turn extract the spinach pigment. Even though this is how the experiment was carried, it was also a limitation (and will affect the outcome of the experiment) to the experiment since the same amount of pigment was not always rolled on to the chromatograph. Not all of the 1:5 solutions produced similar results even though they had the same solvent ratio (as seen in Table 1 and Figure 2). There were

In the part one the experiment the objective was to use measurements in biology and properly measure small volumes using a micropipette. To also learn and understand the application of the spectrophotometry technique. Discover a standard curve by using a known concentration of nitrate. Then finally utilize the standard curve to determine the concentration of the unknown solution. The first step in conducting the experiment is using an indicator brilliant blue dye to add to the concentrated protein solution. The solution will then turn reddish brown because of the high volume in the reagent is unstable. Therefore the reagent binds to the protein in the solution and becomes stable. Using a spectrophotometer, the equipment will measure the protein and absorbance that’s in the solution. To find the measurement of each one must first create a blank cuvette and set the spectrophotometry wavelength to 595nm. Next the student should mix the dye with the reagent and add 1ml of the dye reagent to 3 cuvettes. Then using a 20 µl pipette the student will add 0.250 mg/ml to the 2nd cuvette and followed by the other 3 cuvettes using a new tip each time. After the cuvettes have been filled, para film should be used to cover the solution for at least 5minutes no more than 60mintues. Once they have set for 5 minutes maximum the spectrophotometry will come into place. Use the blank cuvette

Step 13- Measure the RF value by dividing “distance travelled by component” with “distance travelled by solvent

Following, placing the Gatorade sample into a cuvet and place it in the spectrometer and look for the wavelength that was recorded in the stock dye samples