Laboratory Tools: The spectrophotometer Biology 185, Winter 2020

Objective of laboratory: Become familiar with spectrophotometers Determine the concentration of an unknown dye Determine the absorption spectrum for two dyes, separately and in a mix

The spectrophotometer

Supplies Obtain the following supplies from the back benches for each group:

o 1 rack spec tubeso 10 mL of concentrated yellow food dye

(4 drop yellow dye)o diH2O

o Dye: red, blue, red/blue (spec tubes)o Liquid waste beakero Serological pipetteso Tip waste beaker

Experiments throughout the semester require the use of a spectrophotometer, which passes a specific wavelength of light through a liquid sample. If the sample contains a substance that absorbs light at that wavelength, a detector opposite the sample detects less light coming through the fluid than was originally present on it. The spectrophotometer provides a reading in terms of: 1.) Percent transmittance (%T), a measure of the ratio of light passed through the sample verses the amount of light shown on it, or 2.) absorbance (Abs), also called optical density, (O.D.), a reading that is essentially the reverse of percent transmission. We will use the absorbance scale and refer to the units as “A” units.

Throughout the semester, either the analog Spectronic 21 (Spec 21) or the digital Spectronic 200 (Spec200) will be used. While these are different makes, with more or less sophistication, the same operating principles apply. Instructions are provided below for operating both types of spectrophotometer.

Operating the Spec21 (analog): 1. Turn the instrument on and let it warm up for at least 5 minutes or until the readings remain steady for at

least 15 sec.2. With the sample chamber empty, close the lid, and turn the power-on knob until the needle on the gauge

reads infinite absorbance. A shutter prevents light from reaching the photosensor, letting the instrument know what an opaque sample looks like without sample in the chamber.

3. Fill spectrophotometer tube (or cuvette) to fill-line with the liquid used to dissolve the sample (or solvent; e.g., for samples in water, use water; for samples in a 50:50 mix of alcohol and water, use that same solution). This is called a blank and will be a negative control (a sample lacking only the tested substance).

a. Handling note : Glass, dirt, scratches and fingerprints also absorb light, thus care should be taken to handle the spectrophotometer tubes from the top. Carefully wipe the tubes, especially the bottom, to get rid of particles that might alter the readings. Do this for ALL tubes.

4. Close the chamber door and set the wavelength (read in nanometers; nm) of the light beam with the dial on the upper right hand of the instrument. For today, read the initial sample at a wavelength of 420nm.

5. Turn the Zero Absorbance control knob until the instrument gauge reads A = 0. This is called zeroing (or blanking) the spectrophotometer and lets the instrument know what to expect if nothing in the sample absorbs light at the specific wavelength setting.

Note: This procedure calibrates the instrument, i.e., readies it to receive a specimen. Next, you can replace the blank with a spec tube filled to the fill-line with the solution to be tested and record the value from the meter for each sample. Make sure you close the door of the sample chamber before reading the absorbance value.

Operating the Spec200 (Figure 1): 1. Turn the instrument on and allow it to perform a series of initialization self tests.

a. After initialization the instrument will display the Home Menu. If not, press the “home” button.2. In the Home Menu:

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a. Select the SPEC 200 Modern Interface option by pressing the “enter” button (situated in the center of the four directional arrow buttons).

b. For “Application” select Live Display. This allows measurements to present without prompting. c. Use the up/down arrow keys to move to “Measurement mode.” The text will turn green to indicate

which option is selected.i. Click the right or left arrow to change the measurement mode from %T to Absorbance (Abs).

3. Use arrow keys to select GO, press Enter. The Live Display mode should be visible on the screen.

4. To determine 100% transmission or 0% absorption use a blank.

a. Fill spectrophotometer tube (or cuvette) to fill-line with the liquid used to dissolve the sample (or solvent; e.g., for samples in water, use water; for samples in a 50:50 mix of alcohol and water, use that same solution). This is called a blank and will be a negative control (a sample lacking only the tested substance).

b. Handling note : Glass, dirt, scratches and fingerprints also absorb light, thus care should be taken to handle the spectrophotometer tubes from the top. Carefully wipe the tubes, especially the bottom, to get rid of particles that might alter the readings. Do this for ALL tubes.

c. Place the blank in the sample chamber.d. Select the autozero function (the middle button) to set 100% T or 0% Abs. This is called zeroing (or

blanking) the spectrophotometer and lets the instrument know what to expect if nothing in the sample absorbs light at the specific wavelength setting.

e. The Spec200 is now calibrated and ready for samples.

Note: This procedure calibrates the instrument, i.e., readies it to receive a specimen. Next, you can replace the blank with a spec tube filled to the fill-line with the solution to be tested and record the value from the meter for each sample. Make sure you close the door of the sample chamber before reading the absorbance value.

Concentration of an unknown dye

The spectrophotometer will be used to determine the concentration of yellow dye in a solution. In research and medical diagnostic laboratories, this procedure is often used to determine concentrations of compounds such as DNA, proteins and sugars in samples.

The traditional method for determining the concentration of an unknown sample is to generate a standard curve of absorbance verses concentration from standards of known concentration. The standards are samples containing concentrations that span the range of the expected unknown sample. In addition to the four standards, an unknown sample (“U”) with an unknown quantity of dye will be measured. At the end of the experiment, the standard curve will be used to deduce the concentration of dye used in the unknown.

Preparation of standards1. A concentrated yellow food dye was previously prepared. This concentrated solution will be used to

prepare the remaining dilutions for the standard curve.a. The concentrated yellow food dye was prepared using a 1:100 dilution of stock yellow dye in diH2O,

namely 1mL of stock dye was added to 99mL of diH2O.b. The concentrated yellow food dye will be referred to as the 4 drop sample.

Figure 1. Spectronic 200 display screen.

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2. Each group is responsible for preparing the remaining three dilutions necessary for generating the standard curve. All three dilutions will be prepared from the 4 drop sample

a. Calculate the amount of concentrated yellow dye and water necessary for each of the dilutions: 1 drop solution

The one drop solution is a 1:4 dilution of the concentrated yellow dye Determine how many mL of 4 drop solution and diH2O are needed to prepare 4mL of

total solution (Table 1) 2 drop solution

The two drop solution is a 1:2 dilution of the concentrated yellow dye Determine how many mL of 4 drop solution and diH2O are needed to prepare 4mL of

total solution (Table 1) 3 drop solution

The three drop solution is a 3:4 dilution of the concentrated yellow dye Determine how many mL of 4 drop solution and diH2O are needed to prepare 4mL of

total solution (Table 1)

Table 1. Preparation and absorption of standard and unknown solutions“Drop”

numberDilution of 4

dropVolume of 4 drop

to use (mL)Volume of diH2O (mL)

Total volume

A420 of sample Drops

1 drop 1:4 4mL 1 drop

2 drop 1:2 4mL 2 drops

3 drop 3:4 4mL 3 drops

4 drops Previously prepared

Previously prepared

Previously prepared 4mL 4 drops

Unknown Previously prepared

Previously prepared

Previously prepared 4mL

b. Prior to preparing the dilutions have the study group leader or instructor confirm dilution volumesc. Prepare the three dilutions

a. In three separate spec tubes use a serological pipette to combine the calculated amounts of 4 drop solution and diH2O to prepare the 3, 2, and 1 drop solutions

Procedure:

**Note: Adjust the protocol depending on whether the Spec21 or Spec200 is available. The Spec21 is simpler to use but requires a “blank” every time the wavelength is changed.

1. Adjust the wavelength to 420 nm using the two-stage wavelength knob (λ, for the Spec200)a. Turning the λ knob changes the wavelength in steps of 10 nm for the Spec200.b. Press the knob down and turn to change the wavelength in steps of 1 nm.

2. Measure the four standards and the sample of unknown concentration using the approach described above.

a. Note: As the Spec200 autozeros at all wavelengths, it is not necessary to zero the instrument at each wavelength. The Spec21 must be “zeroed” or “blanked” with each wavelength change.

3. Record the four readings, as well as the reading for the unknown (U) (Table 1). a. Note: The “Drops in ‘U’ from Excel Graph” cannot be completed until all class data is entered into

the Data collection: Spectrophotometer spreadsheet posted on Moodle. 4. Enter all absorbance values into the Data collection: Spectrophotometer spreadsheet posted on Moodle .

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5. The data from the class will be plotted in a graph labeled “Yellow dye (drops)” (x-axis) and “A420” (y-axis), with a line fit to the data (which is the average from the class). This produces a standard curve.

6. As part of the write-up, determine the number of drops in the unknown using the following approach:a. Locate the position on the y-axis for the absorbance value of the “U” sample; draw a horizontal line

from that point to the standard curve.b. Draw a vertical line from the point identified on the standard curve to the x-axis and read the value

from this axis. Note that this will probably produce a fractional number of drops.

Absorbance spectra of dyes

Colored substances absorb visible light at specific wavelengths, leaving the remaining wavelengths of light to be transmitted or reflected into your eyes and perceived as a color. For instance, plants are green because blue and red light wavelengths are efficiently absorbed by chemicals in plant tissues, leaving those corresponding to green to be observed. In this exercise, determine the absorption spectrum, profile of light absorbance by a sample over a wide range of specific wavelengths, for two dyes, separately and in a mix.

Procedure:

**Note: Adjust the protocol depending on whether the Spec21 or Spec200 is available. The Spec21 is simpler to use but requires a “blank” every time the wavelength is changed.

1. Adjust the wavelength to 400nm using the two-stage wavelength knob (λ). Record the absorbance values for the red, blue, and red/blue mix samples (Table 3).

1. Adjust the wavelength to 420nm, and record the new readings for each of the samples. Again, record the values (Table 3).

2. Repeat this procedure of increasing the setting by 20nm, and reading the values for each sample through 700nm, producing a total of 16 readings for each color.

a. Note : If the analog spectrophotometer is used (Spec21), the spec must be re-zeroed/re-blanked between every change in wavelength.

2. Enter all absorbance values into the Data collection: Spectrophotometer spreadsheet posted on Moodle . The data for the entire class will be used to write the lab write-up.

Table 2. Absorbance values for red, blue, and mixed samplesAbs Red Blue Red/

BlueAbs Red Blue Red/

BlueAbs Red Blue Red/

Blue400 nm

520 nm

640 nm

420 nm

540 nm

660 nm

440 nm

560 nm

680 nm

460 nm

580 nm

700 nm

480 nm

600 nm

500 nm

620 nm

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Glossary

Absorbance (Abs, also called optical density, O.D.): Measure of transmitted light absorbed by the sample; essentially the negative log of transmission

Absorption spectrum: Profile of light absorbance by a sample over a range of specific wavelengths Blanking (zeroing): Acts as a negative control; setting a spectrophotometer to 0% absorbance (100%

Transmission) “tells” the instrument what to expect if nothing in the sample absorbs light at the given wavelength setting

Calibration: check the graduation Negative control: Group in which no outcome or change is expected

o Example: For samples in water, use water; for samples in 50:50 mix of alcohol and water, use the same mixture

Percent transmittance (%T): Measure of the ratio of light passed through the sample versus the amount of light that initially fell on the sample

Spectrophotometer: Device that measures the amount of light a sample absorbs. Functions by passing a light beam through a sample and measuring the light intensity reaching a detector

Standard curve: Line graph plotting the absorption versus concentration for a series of standard solutions with known concentrations

Scientific writing

The full scientific write-up will include:

Goal: What was the reason the experiment(s) was/were performed (what was discovered/studied/identified)?

Checklist: Is it brief? (Typically one sentence per unique experiment) Is it descriptive? (Does the goal mention the tested variables?)

Results: What were the major findings of the experiments performed (include means, SD, and t-test values when appropriate)? What information do the figures present (figures must be cited in the text)? If t-tests were performed, what is indicated, does a significant difference exist between the treatments?

Checklist Findings in paragraph form, including

o Descriptive statistics (Mean, SD) for each treatment are included?o If a t-test was performed, do results include whether the means are significantly different or

not?o Do the results avoid interpretation or analysis?o Does the results cite the figure (s) (at the end of sentences)?

Pertinent figures from the Excel spreadsheet, with captions

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Discussion: How do the results help meet the goal(s)? How should the results be interpreted? Are trends present? If t-test was performed, what do the significance and non-significance mean?

The questions below should be addressed in the Discussion section: Concentration of an unknown dye1. Discuss the relationship between concentration of dye and optical density at 420nm; in other words,

is there a correlation between the concentration of a dye and its absorbance?2. How might the relationship above be used in future labs? Absorption spectra of dyes1. Describe the absorption spectra of each colored dye based on the figure in the spreadsheet.

Where is each color dye’s peak absorbance? 2. Are there differences between red, blue, and purple? Why do the dyes not produce the same

spectrum, and discuss the unique absorption spectrum of the purple dye. 3. Use the internet to find a figure of the electromagnetic spectrum. Discuss the electromagnetic

spectrum in relation to the figure drawn from the class data. If the electromagnetic spectrum is included in the scientific writing, a citation for the source must be provided and a caption must be writing.

Citation formats: Journal article: General format: Author(s). Year published. Article title. Journal title. Volume

(issue): page numbers. Books: General format: Author/editor last name and first initial. Year published. Title of work.

Edition (if provided). Publication city: publisher name. Total number of pages in book (followed by p.)

Online: General format: Title of article [Internet]. Date of last update. Publisher; Date accessed. Available from: Internet URL.

Final Tips and Suggestions:1. Read over the report at least twice before submitting to ensure major points are addressed following

correct grammatical rules. 2. Use proper units throughout (mL, μL, g, nm, etc.).3. Do not do the report at the last minute; give yourself a few days to come up with an outline. 4. Come see me if you need assistance.

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