Dye Chromatography Lab

Figure 2: Dye percents versus absorbance in a control, 10%, 20%, and 30% azide solutions.

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

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%.

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 procedures for this “Chromatography Lab” include eight steps, which are talked about down

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.

Get a spinach leaf to obtain pigments from. Set the chromatography paper on top of

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.

The objective of this experiment was to investigate the concept of paper chromatography and attempt to achieve a clear separation of food dyes found in M&M’s and to compare and evaluate the dyes found. Chromatography is derived from the greek words “chroma” meaning color and “graphein” meaning to write.

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

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

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

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: