Introduction

Early in the semester, we did a Pigments and Dyes lab dealing with spectroscopy. After completing this lab, I became particularly interested in the correspondence of the results from the spectrophotometer (graphs based on readings from an instrument, the HACH DR/2500) and the results from the spectroscope (visual readings). Dr. Bordley suggested that I do something dealing with spectroscopy and dye dilutions for my project.

The purpose of my lab was to learn more about dyes and dye dilutions, and which dilutions would be best for readings. I wanted to observe the similarities and differences between the spectrophotometer and spectroscope readings, as well as the results of each involving colors transmitted and absorbed. I wanted to use the Leaf Green dye as a basis, as it is a dye-dye mixture containing some blue and some yellow from the PRO Chemical Dye company.

I hypothesized that "the more diluted the dye, the lower and less sharp the curves from the graphs of the spectrophotometer would be. If the dye is less diluted, the curves on the graphs will be sharper and taller. The wavelengths transmitted and absorbed will vary with the different dyes used." (from Project Proposal)

Background

During lab time, I tested the dilutions of 5 different dyes: Sun Yellow, Yellow 135, Brilliant Blue, National Blue, and Leaf Green. I created either three or four dilutions of each dye, depending on the graphs of absorption and percent transmittance given by the spectrophotometer. If the absorbance for the lowest dilution was above 1.00, I created a more dilute dye solution. I used standard ratios for the first three dilutions. Each time I diluted a dye, I created dilutions of 1 mL (36 drops) to 200 mL of RO water, 0.5 mL (18 drops) to 200 mL, and 0.25 mL (9 drops) to 200 mL. These dilutions worked well for each dye except for the blues: Brilliant Blue and National Blue, which required less concentrated dilutions. In this case, I created a new dilution based on the range of the level of absorbency (i.e. if the absorbance was significantly higher than 1.00, I would use less pure dye, and more RO water in the solution). I worked with these dilutions to do tests and obtain results using the spectrophotometer and spectroscope.

Procedure

MATERIALS:

-desired dyes (in this experiment: Sun Yellow, Yellow 135, Brilliant Blue, National Blue, and Leaf Green)

-at least three 200 mL volumetric flasks (need as many as the number of dilutions desired) with caps
-plastic cells for use in spectrophotometer
-Barnes bottles with cork tops
-droppers:  at least 2 (one for the dye and one for the RO water)
-RO water
-spectroscope
-spectrophotometer
-labels

BELOW: My work station

ABOVE: Barnes bottles with dye dilutions (which have already been tested)

PROCEDURE:

I began my experiment with Sun Yellow (119) dye. We only had Sun Yellow in the form of power, so I had to make the dye solution myself. I followed the simple instructions given by the PRO Chemical and Dye company. I measured 4.5 grams of dye powder and added that to 250 mL of RO water. I stirred until the solution contained no sediment, and was completely clear and homogenous.

After creating the dye solution, I began diluting. First I created the 1 mL / 200 mL dye. The first idea, as to what dilutions I would use, was that I would make a 1 mL / 200 mL dilution, and then do a dilution of 1 mL of the first dilution to 200 mL RO water. I tried this, however, and found that the second dilution was essentially colorless, and I would not get very good readings from dilutions of that nature. I then decided that I would do half as much dye for the second dilution, and so on and so forth.

As mentioned previously, I tested 3 dye dilutions for Sun Yellow: 1 mL / 200 mL, 0.5 mL / 200 mL, and 0.25 mL / 200 mL. When diluting the dyes, I simply used a dropper to place the given amount of drops into a 200 mL volumetric flask. I then added RO water to make 200 mL of solution and mixed the solution by lightly shaking the glassware. I did this for each dilution until I had all three prepared and ready to be tested.

The next step was to pour a small amount (about 10 mL) of each dye solution into a plastic cell to be inserted into the spectrophotometer. I prepared the instrument for readings by doing the following:

-press the power button
-go to the main menu
-press "wavelength scan"
-go to "options"
-wavelength range should be 380 – 750
-scale and units should be: Auto Abs (0.000 - 2.000)
-cursor mode: track
-Insert a blank cell filled with water into the cell holder
-press "zero"
-After the zeroing is done, place the sample cell with the dye dilution into the instrument and press "read". The graph of absorbance will appear when the reading is done. To see the graph of transmittance, press "options", select % T, press OK, and finally "return". The graph for percent transmittance will then appear.

I then sketched the graphs of both absorption and percent transmittance (including the numbers for wavelengths, and absorption/transmittance given). I created each graph (starting half way through the first dye) using 20 boxes downwards in my lab book, and 20 boxes across. This helped me to keep all graphs on the same scale.

After sketching the graphs, I added a sample of each dye dilution into a Barnes Bottle. As I transferred the dye dilutions, I labeled all bottles to keep each sample identified, as the different dilutions oftentimes looked similar.

I then made the room completely dark and turned on a lamp, which was my source of light for the spectroscope. I stood in front of the lamp looking directly towards the light through the slot in the spectroscope. I moved the Barnes Bottle of dye solution in and out of the light just in front of the spectroscope (as we did in our lab experiment). I recorded what I saw (the colors transmitted through the dye dilution) in similar charts in my lab book.

I followed this procedure for each dye I tested. I did, however, do an extra dilution for the National Blue and Brilliant Blue. The lowest dilution (0.25 mL / 200 mL) of the Brilliant Blue dye was still too concentrated. The peak on the graph given by the spectrophotometer was 1.154, and it should not be over 1.000. To solve this problem, I created a dilution of 6 drops / 200 mL RO water. I then followed the procedure above to do the spectrophotometer and spectroscope readings for that dilution. I had the same problem with the National Blue dye. The peak on the graph of absorption for the 0.25 mL / 200 mL dilution was 1.726. To correct this, I created a dilution of 3 drops / 200 mL RO water to ensure that the number dropped below 1.000.

BELOW: "My room" in the back of the lab where I conducted my experiment!

ABOVE: The lamp (my source of the light for the spectroscope readings) and 2 spectroscopes!

After I had created and tested the dilutions for each dye, I began studying the graphs in more detail. I copied the graphs onto transparency films and compared them. I held each blue with each yellow to see if any of these dyes might be part of the dye-dye mixture, Leaf Green. To do this, I simply cut each transparency film in half. I held the film over the graph of transmittance for the 0.25 mL / 200 mL dilution (which I used for each film), and followed the lines with a dry erase marker. I was then able to hold a certain blue over a yellow, and see if these graphs might come together to make the graph that I had gotten from the spectrophotometer for Leaf Green. The results will be discussed in Conclusions.

After I had made some guesses, the only way to test if I was correct was to create the mixtures. I created the mixtures in the plastic cells and placed them in the spectrophotometer for reading. First, I created 10 mL blue / 10 mL yellow mixtures, but the blue was much more concentrated than the yellow in each case and the green I created was very blue. I then made 7 mL blue / 13 mL yellow mixtures. These worked well and I got the desired readings. To create these mixtures, I simply measured the dye dilutions directly from the Barnes bottles (in which I kept a sample of each dye for the duration of the experiment) in 10 mL measuring glassware. I shook the mixtures and then placed them in the spectrophotometer.

Observations and Data

The similarities and differences in the graphs based on the dye dilutions were not as evident as I had expected. The peaks of the dilutions with a higher concentration were not necessarily taller and sharper. For example, the peak itself of Brilliant Blue is sharper for the 0.25 / 200 mL dilution than the 1 mL / 200 mL dilution. The more concentrated dye dilution peaks for longer (i.e. more wavelengths of that absorption number, 3.200, are absorbed). The peak then becomes more of a defined peak. The lines sloping towards the peak, however, are not as steep with the lower concentrations. For example, the line leading to the "peak" of the 1 mL / 200 mL dilution is very steep, versus the line leading to the peak of the 0.25 mL / 200 mL dilution.

The trend in the peaks is easier to read when observing the dye dilutions for the yellows. For example, it is easier to see that the peaks of Yellow 135 become less defined with the lesser concentrations. For the absorption, the peak goes from a defined peak with 1 mL / 200 mL to a more broad peak with 0.25 mL / 200 mL.

Below are graphs of percent transmittance for each dye. The dilution shown for each is 0.25 mL / 200 mL. The bottom right image shows the graphs of Yellow 135, Brilliant Blue, and Leaf Green, which I will discuss in Conclusions.

As I worked with the less concentrated dilutions, I found it much harder to read the transmitted colors through the spectroscope. It oftentimes looked as though nothing had changed as I moved the dye in and out of the light. This was particularly true for the yellow dyes. This may have caused some experimental error while transferring what I saw with the less concentrated dilutions of all dyes. The higher concentrations of the blue dyes usually showed a great deal of change on the visual spectrum, completely blocking out the red, orange, and yellow colors (as with the 1 mL / 200 mL dilution of National Blue). The highest concentrations of the yellow dyes sometimes did not even show notable changes.

Conclusions

I had originally planned to test other dyes if allowed the time (Caramel and Fuchsia, among other dyes). I did not find time to do this. I did, however, discover things that I had not originally intended to find, and added to my procedure because of this. I had planned on using the Leaf Green dye as a basis, but instead found possible components of the Leaf Green dye mixture! I cannot be sure, as I did not have sufficient time for thorough testing, but through comparisons of the graphs, spectroscope results, and the graphs of the dye mixtures that I created at the end of my experiment, it appears that Yellow 135 and Brilliant Blue are highly likely to be components. I discovered this as I was comparing the graphs which I copied onto the transparency films. When I held the Brilliant Blue over the Yellow 135, they looked as though they might come together to make a graph similar to that of Leaf Green (see the bottom right image in the table above)!

On the last day of experiment work, I had enough time and was able to create every possible mixture of the blues and yellows I had tested. I created 4 combinations. I took readings from the spectrophotometer of each of these dye-dye mixtures. Sure enough, the mixture with the graph closest to that of Leaf Green (in shape as well as in numbers for the wavelengths and percent transmittance of the peaks) was the mixture of 7 mL Brilliant Blue / 13 mL of Yellow 135! This mixture was also the one which LOOKED the most like Leaf Green in the plastic cells.

As discussed previously, I did not discover as much pertaining to the trends of shape and size for the peaks of different dye dilutions as I had anticipated. I did, however, still discover correlations between the graphs and concentrations. The higher concentrations of blues (both National Blue and Brilliant Blue) do not have distinct peaks. They instead have a flat line where the peak would be, i.e. the peak is not a small area, or a point; it is a long, flat area. This only means that more of the wavelengths between (in the example of absorption for the National Blue 1 mL / 200 mL dilution) 552 nm and 665 nm are absorbed.

Trends can also be seen with the yellow dyes. These were more like the trends I had expected. For example, the peaks for absorption of all Sun Yellow dilutions become less sharp with less concentrated dilutions. The peak also moves down in the absorption number given (2.406 to 1.372 to 0.664) while staying about the same in wavelength (420 nm to 419 nm to 419 nm).

In dealing with the spectroscope, I found that the higher the concentration, the more obvious the results (i.e. changes in the visible spectrum shown). Brilliant Blue is a good example of this. After working with the Sun Yellow and seeing essentially no change in the spectrum, I saw drastic changes with the higher concentrations of Brilliant Blue. Violets, blues, and greens were transmitted while yellows, oranges, and reds were absorbed. As I moved lower in concentration (0.5 mL / 200 mL), more violets and greens were transmitted. I then saw violets, blues, and greens, then oranges and reds for the 0.25 mL / 200 mL dilution (mostly yellow was absorbed). This corresponds with the graph from the spectrophotometer: the wavelengths around 380 nm to 457 nm (violets to greens) are transmitted. The wavelengths between around 550 nm and 650 nm (mostly yellow, with some green and orange) are absorbed. The wavelengths from 650 nm to 750 nm (oranges and reds) are transmitted. This is exactly what I saw through the spectroscope, and what is recorded in my lab book.

The higher concentrated dilutions are best for readings through the spectroscope. This is due to the limits of the human eye in observing change and color. The less concentrated dilutions are best for the spectrophotometer, as the instrument does not need such high concentrations to read the wavelengths absorbed and transmitted. A 1 mL / 200 mL dilution seems ideal for the spectroscope, while a 0.25 mL / 200 mL dilution (or even less concentrated) seems ideal for the spectrophotometer readings. As I was unaware of the limits of certain dilutions, the spectroscope was not as useful in this experiment as I had anticipated, although in some cases (as with the blues and somewhat with the Leaf Green) I was able to really see the correlations between the graph and the spectroscope reading of a dye dilution.

Links

PRO Chemical Home page:

Acknowledgements

I would like the thank Dr. Bordley for helping me with the idea for this project. I would also like to thank all other members of the Chemistry department who helped me to get the necessary supplies and answer any questions I may have had along the way (Ms. Fitz in particular)!