General Biology Lab 1 (Bsc1010l)

Exercise 5: Identifying Solutes and Determining their Concentration ______

OBJECTIVES: 1. Understand how a spectrophotometer works and be able to operate one 2. Construct an absorption spectrum for multiple compounds 3. Construct and use a standard curve to determine the concentration of an unknown chemical ______

INTRODUCTION: Different materials absorb and reflect different wavelengths of light, and therefore have different colors. The color an object appears is determined by the color(s) that it reflects; thus a red object reflects red wavelengths to your eyes and absorbs the other wavelengths. Visible light is only a small part of the total electromagnetic spectrum (Fig. 1), and is what we’ll be working with in today’s lab.

Figure 1: Electromagnetic spectrum

Question

Is the absorbance of light critical for the function of molecules or just a consequence of their molecular structure (Think about an organelle like a chloroplast)?

Spectrophotometry is a technique for determining the concentration of solutes (dissolved material) within a solution and identifying the potential identity of a solution. This technique is based on the principle that all atoms, molecules, and chemical bonds absorb and reflect unique wavelengths of light. As a result, each has a unique pattern, or “fingerprint” of various wavelengths that it absorbs and/or reflects. In this exercise you will (a) determine the pattern of absorption for two common molecules, and (b) measure the concentrations of these molecules. A spectrophotometer measures the amount of light absorbed and transmitted by a dissolved chemical. By measuring absorbance or transmittance we can identify a chemical and

1 Courtesy McGraw-Hill, Inc. its concentration. The spectrophotometer separates white light into a spectrum of colors (wavelengths), and then directs a specific wavelength of light at a tube containing a solution to be measured. The light is either absorbed by the dissolved substance or transmitted through the solution and exits the sample tube. The spectrophotometer compares the amount of light exiting the tube with the amount entering the tube and calculates transmittance - the more solute (dissolved material), the lower the transmittance. The spectrophotometer also calculates the amount of light absorbed - the more solute, the higher the absorbance.

sample holder

wavelength selection

calibration

Figure 2: Colorimeter

In this lab, we will use a Vernier Colorimeter. This device operates using the same principles as a spectrophotometer, but only takes absorbance measurements at a small number of wavelengths. Light from a LED light source passes through a cuvette containing a solution sample (Fig. 2). Some of the incoming light is absorbed by the solution. As a result, light of a lower intensity strikes a photodiode. Cuvettes containing a blank (only solvent) are used to calibrate the spectrophotometer for the solution being tested. For today, the blank will be distilled water. It is important that when handling the cuvettes, you only touch the areas where the light does not pass through (the sides with lines in the plastic) - if you touch the areas light will touch (sides without lines), it may leave a mark that increases the amount of light absorbed. The photodetector receives light transmitted through the cuvette and converts the light energy into electrical energy - the more light transmitted the more electricity produced. You will read the absorbance values on the display unit attached to the colorimeter. Absorbance is the amount of radiation retained by the sample, and transmittance is the amount of radiation that passes through the sample. Thus, transmittance is the intensity of light exiting the sample divided by the amount of light entering the sample:

Percent Transmittance = (It/Io) * 100 where It = transmitted (exiting) light intensity and Io = original (entering) light intensity

Absorbance is the logarithm of the reciprocal of transmittance, and is expressed as a unit-less number:

Absorbance = log10 (Io/It)

2 Courtesy McGraw-Hill, Inc. Based on mathematics, a twofold increase in absorbance indicates a twofold increase in the concentration of the sample. To measure unknown concentrations of dissolved chemicals, two procedures are carried out: 1) determine the chemical’s absorption spectrum, and 2) build a standard curve. You will do both today. ______

Task 1 - ABSORPTION SPECTRUM OF COBALT CHLORIDE

Your first task is to learn to operate a spectrophotometer while deriving the absorption spectrum of a common chemical, cobalt chloride (CoCl2). The pattern of wavelengths absorbed by CoCl2 is its “fingerprint” because it is unique to that chemical.

Procedure

1. Prepare seven solutions in the designated test tubes with the mixtures of distilled water and CoCl2 stock solution listed in table 1.

Volume of CoCl2 Volume of distilled Concentration of Tube # (mL) water (mL) CoCl2 (mg/mL) 0 0 10.0 0 1 0.1 9.9 1 2 1.0 9.0 10 3 2.0 8.0 20 4 3.0 7.0 30 5 4.0 6.0 40 6 5.0 5.0 50

Table 1: Volumes and concentrations used to prepare CoCl2 solutions

2. Cap the tubes and label 0-6.

3. Connect the colorimeter to the Lab Quest display unit and turn on the unit.

4. The software should immediately identify the colorimeter. If it does not, select the Sensor tab (upper left), the select Sensor Setup. On the new screen, select the arrow for the channel the colorimeter is plugged into. On the dropdown menu, select “colorimeter.”

5. Allow the colorimeter to warm-up for at least 5 minutes before calibrating.

6. Using a clean transfer pipette, fill a clean cuvette with the blank solution (tube 0) and place in the sample holder.

7. Adjust colorimeter to the lowest wavelength (430 nm) using the < or > buttons and read the absorbance value on the meter. The distilled water blank has no color and should not absorb any light.

3 Courtesy McGraw-Hill, Inc. 8. If absorbance is not zero, press the “Cal” button and wait for absorbance to read 0.000.

9. Remove the blank and replace it with a cuvette filled with solution from tube 6 (50 mg/mL) - this is the sample you will use to determine the absorption spectrum of CoCl2.

10. After the meter has stabilized (5-10 sec), read the absorbance value and record the absorbance value in Table 2.

11. Remove cuvette with tube 6 solution and adjust the meter to 470 nm.

12. Put the blank back into the colorimeter, and readjust for zero absorbance at the new wavelength. The colorimeter should be recalibrated every time you change the sample tube and/or adjust the wavelength emitted.

13. Insert solution from tube 6 and measure its absorbance at the new wavelength. Record the absorbance in table 2.

14. Repeat, such that the absorbance of CoCl2 is measured for each wavelength in table 2

15. Plot the data from table 2 below and connect the points with a smooth line.

Wavelength (nm) Absorbance 430 470 565 635

Table 2: Absorbance of CoCl2 at 50mg/mL

4 Courtesy McGraw-Hill, Inc. Questions

a. What wavelength is the peak absorbance of CoCl2?

b. Why is it important to recalibrate with the blank often?

c. Would you expect a similarly shaped curve for another chemical like chlorophyll?

5 Courtesy McGraw-Hill, Inc. Task 2 - THE STANDARD CURVE

A graph showing a chemical’s concentration versus it absorbance of a wavelength of light is called a standard curve - the relationship is a straight line. You will construct a standard curve and then use it to determine some unknown concentrations of CoCl2 solutions. Use the six dilutions that you prepared in Task 1 to construct a standard curve for CoCl2. These are standard solutions, because their concentrations are known. The absorbance of each standard is measured at the peak wavelength of the absorption spectrum for CoCl2 determined from Task 1.

Procedure 1

1. Determine the wavelength of peak absorbance for CoCl2 from Table 2, and set the filter of the spectrophotometer to this wavelength.

2. Insert the blank (tube 0) and adjust the spectrophotometer for zero absorbance.

3. Replace the blank with tube 1 (1.0 mg CoCl2/mL), measure its absorbance and record it in Table 3.

4. Repeat for the rest of the tubes (2-6).

5. Plot the data below with concentration on the x-axis and absorbance on the y-axis.

6. Draw a straight line (best fit), rather than connecting the points as in Task 1

Concentration of Cobalt Chloride (mg/mL) Absorbance 1 10 20 30 40 50

Table 3: Absorbance values of CoCl2 standards at peak absorbance wavelength

6 Courtesy McGraw-Hill, Inc. Procedure 2:

1. Obtain a tube with an unknown concentration and record the tube name in Table 4.

2. Use the blank (tube 0) to zero the spectrophotometer to the wavelength of peak absorbance of CoCl2.

3. Measure the absorbance of the unknown solution and record this in Table 4.

4. Find the absorbance value on the y-axis of the standard curve created for CoCl2, and draw a line parallel to the x-axis until it intersects the standard curve.

5. Draw a line from the intersection straight down until it intersects the x-axis. This is the concentration of the unknown solution - record this value in Table 4.

6. Repeat this for three more tubes of unknown concentrations.

Tube number Absorbance Concentration (mg/mL)

Table 4: Absorbance and concentrations of unknown CoCl2 solutions

7 Courtesy McGraw-Hill, Inc. Task 3 - ABSORPTION SPECTRUM OF CHLOROPHYLL

You will now plot the absorbance spectrum of chlorophyll and determine its peak absorbance as you did with CoCl2.

Procedure:

1. Obtain a tube of the chlorophyll extract and prepare a blank.

2. Using the procedure for CoCl2, measure the absorbance of chlorophyll for the wavelengths in Table 5 using the spectrophotometer.

3. Record your results in table 5.

4. Plot your data and create the absorbance spectrum for chlorophyll below

Wavelength (nm) Absorbance 430 470 565 635 Table 5: Absorbance values for chlorophyll

8 Courtesy McGraw-Hill, Inc. Question

The color our eyes see is the light that is reflected by an object. Explain how you would be able to predict the colors/wavelengths with the highest absorbance for chlorophyll.

9 Courtesy McGraw-Hill, Inc.