Question

Q: Briefly explain why it was important that the beakers in which you prepared the dye solutions for Part 3 of the experiment were clean and dry before you began.

PROCEDURE:

Operating a Spectrometer

Part 1. Turning on the power to the instrument

Rotate the left-hand knob, the amplifier control, clockwise. On some models a red LED will light at this point. Allow about 15 minutes for the instrument to warm up before recording any measurements.

Cleaning cuvettes

While waiting fir the instrument to warm up, rinse two cuvettes with distilled or deionized water, Then rinse one of the cuvettes three times with the solution to be measured. Fill this cuvette three-quarters full with the solution to be measured. Fill the other cuvette three-quarters full with distilled water or the solution you are using as the reference solution. Do not handle the lower portion of the cuvettes, because smudges or droplets of solution will affect the passage of the light beam through the cuvette. Wipe off the outside with an absorbent tissue before inserting the cuvette clamp holder.

Setting the wavelength

Turn the wavelength control knob, on the top of the instrument, until the appropriate wavelength setting appears on the scale visible at the left of the knob. Do not change the setting during the experiment unless specifically instructed to do so.

Setting 0% transmittance

Make the following adjustment with no cuvette in the sample holder; under this condition, no light strikes the phototube. About 15 min after turning the instrument on, adjust the left-hand knob, the amplifier control, so that the needle on the meter points to zero on the percent transmittance scale.

Setting 100% transmittance

Turn the right-hand knob, the light control, counterclockwise almost to its limit before inserting a cuvette into the sample holder. Insert the cuvette containing the reference solution into the sample holder. Match exactly the index line on the cuvette with the index line on the holder. Close the top of the holder tightly. Turn the right-hand knob clockwise until the needlepoints to 100%. Immediately remove the cuvette to avoid fatiguing the phototube, and proceed to the sample measurement.

Check 0 and 100% transmittance

After the cuvette is removed from the sample holder, an occluder automatically drops into the light beam path, the needle should then point to zero. Each time the wavelength is changed, and during any extensive series of measurements at the same wavelength, the 0 and 100% transmittance settings should be checked. If necessary, reset these two settings using the procedures described above.

Part 2: Determining Analytical Wavelength of a Food Dye

In a clean, dry 100 mL beaker, obtain from your lab instructor about 50mL of your assigned food dye solution. Record the code number of the solution and its concentration and color on Data Sheet 1.

Rinse a cuvette three times with distilled water. Fill the cuvette 3-quarters full with distilled water.

Rinse another cuvette 3 times with small portions of your dye solution. Fill this cuvette three-quarters full with the dye solution. Be careful to avoid leaving your fingerprints on the lower part of the cuvette.

Carefully wipe the outside of the cuvette with an absorbent tissue. Check to make certain that you have removed all fingerprints.

Set the wavelength at 350 nm on the spectrophotometer. Set the instrument 100% T using the cuvette containing the reference solution, distilled water.

Place the cuvette containing the dye solution in the sample holder. Match exactly the index line on the cuvette with the line on the holder. Close the top of the holder tightly. Immediately read the percent transmittance to 3 significant figures and record the percent transmittance on Data Sheet 1.

Remove the cuvette from the sample holder and rest the instrument to 360 nm. Rezero the instrument with the reference solution in place. After removing the reference solution, determine the percent transmittance of the dye solution and record the %T on Data Sheet 1.

Repeat this procedure, changing the wavelength setting in 10-nm increments. Zero the instrument with the reference solution each time. Read the percent transmittance for each wavelength and record these data on Data Sheet 1. Continue this procedure until you have readings in 10-nm increments through 660nm.

Discard the dye solution following the directions of your lab instructor. Rinse the cuvette 3 times with small amounts of distilled water.

Before proceeding to Part 3 of this experiment, determine the wavelength region in which your dye solution has maximum absorbance or minimum transmittance. Convert your percent transmittances to equivalent absorbances and record them on Data Sheet 1. Select the wavelength with the highest absorbance as the analytical wavelength for your dye. Record this wavelength for your dye. Record this wavelength on Data Sheet 1.

Part 3: Preparing Food Dye Solutions

Rinse a clean, 10-mL pipet with about 1 mL of your assigned dye solution. Draw the solution into the pipet using a rubber bulb. Quickly disconnect the rubber bulb and place your index finger over the top opening to prevent the water from draining out of it. Hold the pipet in a nearly horizontal position. Rotate the pipet so that the rinse solution contacts as much of the inner surface of the pipet as possible. Remove your finger briefly during this process to allow the solution to enter the upper stem of the pipet. Drain the solution to enter the upper stem of the pipet into a beaker and discard the rinse solution, following the directions of your lab instructor.

Repeat the rinsing procedure with 2 additional portions of the solution. Discard the rinse solution following the directions of your lab instructor.

Use the pipet to transfer 10.0 mL of the stock dye solution to a clean, dry 50-mL beaker. Use a second pipet to add 10.0 mL of distilled water to the dye solution in the beaker to form Solution A.

Wash the first pipet and rinse it with 3 portions of distilled water. Transfer 10.0 mL of Solution A to a second clean, dry 50-mL beaker. With the second pipet used to transfer the distilled water, add 10.0 mL of distilled water to Solution A in the second beaker. Mix the solution by carefully swirling the beaker and its contents. Label this beaker Solution B.

Repeat the procedure used to prepare Solution B and 10.0mLof water

Solution D from 10.0 mL of Solution C and 10.0mL of water and

Solution E from 10.0 mL of Solution D and 10.0 mL of water.

Carefully label each beaker.

Part 4 Preparing a Beer’s Law Plot for a Food Dye

Set the wavelength control at the analytical wavelength that you determined for the dye solution in Part 2.

Calibrate the spectrometer by setting 0% transmittance with the amplifier control while the sample holder is closed and empty. Set the 100% transmittance with the reference solution.

Rinse a second cuvette 3 times with small amounts of the most dilute solution, Solution E. Discard the rinse solution following the directions of your lab instructor. Fill the cuvette 3-quarters full with this solution. Read the percent transmittance of Solution E and record this transmittance on Data Sheet 2.

Discard the dye solution in your cuvette, following the directions of your lab instructor. Rinse this cuvette 3 times with small portions of Solution D. Discard the rinse solutions as directed. Read the percent transmittance of Solution D and record this transmittance on Data Sheet 2.

Repeat this procedure until you have determined all 5 solutions.

Part 5 Analyzing a Dye Solution of Unknown Concentration

Obtain a sample of unknown concentration of your assigned dye solution from your lab instructor. Record the code number on Data Sheet 3.

Set 100% transmittance with the reference solution. Rinse a clean cuvette 3 times with small amounts of your unknown solution. Discard the rinse solutions as directed. Fill the cuvette 3-quarters full with your unknown. Read the percent transmittance of your unknown solution at the appropriate analytical wavelength. Record this transmittance on Data Sheet 3.

Discard all of your solutions following the directions of your lab instructor. Rinse your cuvettes three times with distilled water.

Answer 1

We wish to ascertain the concentration of a food dye in a sample of food by spectrophotometric determination. For this purpose, a series of standard solutions of known concentrations are prepared and the absorbance of the standard solutions are plotted against the concentration to prepare a calibration curve. The concentration of the unknown food dye is obtained from the calibration plot.

It is important that the beakers used in the experiment are clean and dry. Contaminations or impurities in the dye can significantly alter the absorbance. Infact, the impurities may absorb at the same wavelength as the dye and the readings will be erroneous. Therefore, it is definitely imperative that the beakers used are clean.

Moisture often decomposes food dye, since these dues contain N=N mostly and such bonds are susceptible to fission by traces of acid or moisture. Hence, the beakers must be dried before the experiment starts.