Lab #8 -Spectrophotometric Analysis of a Mixture: Name_________________________Caffeine and Benzoic Acid in a Soft Drink(after Harris “Exploring Chemical Analysis”, Partner(s)_____________________2nd ed, p. 516-517)

PART I: Introduction

In this experiment, we use ultraviolet absorbance to measure two major species in soft drinks. Caffeine is added as a stimulant and sodium benzoate is a preservative. All solutions will contain 0.10 M HCl, so the sodium benzoate is protonated to make benzoic acid. Caffeine has no appreciable basicity, so it is neutral at pH 2.

We restrict ourselves to non-diet soft drinks because the sugar substitute aspartame in diet soda has some ultraviolet absorbance that slightly interferes in the present experiment. We also avoid darkly colored drinks because the colorants have ultraviolet absorbance. Mountain Dew, Mello Yello, and, probably, other lightly colored drinks are suitable for this experiment. There is undoubtedly some ultraviolet absorbance from colorants in these beverages that contributes systematic error to this experiment.

PART II: Preparing Standard Solutions of Benzoic Acid and Caffeine (use distilled water)

1. In the data sheet go to the section labeled Preparing Standard Solutions for the Calibration Curve. Enter the concentrations of the benzoic acid (BA) and caffeine (C) stock solutions in mg/L. Calculate how many millileters of the stock solutions are needed to produce 100 mL each of the indicated concentrations of BA and C using the dilution equation M1V1 = M2V2.

2. Prepare the standard BA samples by adding the calculated volumes of stock solution, 10 mL of 0.10 M HCl, and diluting to 100 mL in the provided volumetric flasks.

CaffeineFM 194.19

3. Prepare the caffeine standards WITHOUT 0.10 M HCl.

4. Calculate the molarities of the standard solutions.

5. Warm approx. 20 mL of soft drink in a beaker on a hot plate to expel CO2. After cooling to room temperature in ice, pipet 4.00 mL into a 100-mL volumetric flask. Add 10.0 mL of 0.10 M HCl and dilute to the mark.

PART III: Obtaining the Absorbance Spectra of Benzoic Acid and Caffeine

1. Familiarize yourself with the basic components of the spectrophotometer by referring to Figure 1. The U2000 is a dual-beam instrument, so the background absorbances present are automatically ratioed out.

Figure 1

2. You will be using the Hitachi U2000 dual-beam UV-Vis spectrophotometer. The U2000 has a cathode ray tube (CRT) for display and a keyboard. Most of the operation of the spectrophotometer is microprocessor controlled. We can access the instrument controls by using the keyboard (Figure 2). Read through the keyboard description before proceeding.

Figure 2

3. The instrument should have the following screen displayed after we turn it on (Figure 3). This is the MAIN MENU screen. NOTE: the figure numbers in the illustrations do not match the figure numbers in the instructions.

Figure 3

4. Press the down arrow cursor movement key to highlight selection number 2 or "wavelength scan". After selection press the ENTER key. The "WL SCAN" menu will appear (Figure 4).

Figure 4

5. Menu option #1 will be DATA MODE. It should already be set for DATA MODE = ABS. If not, change it to ABS by selecting the option displayed in the lower left-hand portion of the screen. Now move the highlighted area to choice #2 or TEST SETUP. A new screen will appear (Figure 5).

Figure 5

6. Enter a value of 350 for the START WL (nm). Press enter.

7. Enter a value of 210 for the STOP WL (nm). Press enter.

8. Enter 1.20 for the UP SCALE. A maximum absorbance of 1.20 will be displayed when we scan the spectrum.

9. Enter 0.00 for the LO SCALE.

10. Set the SCAN SPEED to 200 nm/min, the INIT DELAY to 0 sec, the NUM CYCLES to 1, the CYCLE TIME to 0 sec, and the DISPLAY FORMAT to OVERLAY.

11. Press the RETURN key to return to the WL SCAN menu. Now move the cursor down to the INSTR SETUP menu and enter. The screen in Figure 6 will appear.

Figure 6

12. Enter the following parameters:

Baseline: UserResponse: MediumLamp change: 340VIS Lamp: OnUV Lamp: OnGraph print: OffText print: OffList interval: 1.0

13. Return to the WL SCAN menu again and this time select USER BASELINE. You are going to record the background spectrum, store it in memory, and this background will be subtracted from all subsequent spectra. Fill two quartz cuvettes with distilled water and place them in the sample and reference slots.

14. Press the AUTO ZERO key to start with an absorbance of 0.00 at 350 nm. Now press the START key to measure the background.

15. We are now ready to record a spectrum. Press the FORWARD key on the keyboard. A screen similar to Figure 7 will appear.

Figure 7

16. Remove the sample cuvette and fill it with the lowest concentration BA sample (always take data from lowest to highest concentrations). Press the START key on the keyboard to obtain the spectrum.

17. After the spectrum is obtained, select the RESCALE option at the bottom of the screen. A screen similar to Figure 8 will appear. Select option 1) CURSOR ENTRY and then use the arrow keys to move the cross-hair over the spectrum until you can read the absorbance at 229 nm. Enter the absorbance in the data sheet. Repeat at a wavelength of 272 nm.

Figure 818. Press the return key twice to return to the screen shown in Figure 7. It’s very important that

you do not lose the data in the window by pressing the return key too many times! This is because we want all of the spectra to be displayed together in the data window.

NOTE: your spectrum will look different from this - it's meant to be diagrammatic.

19. Repeat steps 16-18 for each BA standard. After the last spectrum is obtained, print out the display by selecting option 4 in Figure 7.

20. Repeat steps 16-19 for the caffeine standards.

21. Measure the spectrum of the prepared soda and enter the absorbances at 229 and 272 nm in the data sheet.

22. OPTIONAL: If your instructor chooses, measure the spectrum of a synthetic, unknown mixture of benzoic acid and caffeine prepared by the instructor.

PART IV: Analyzing the Data Using Microsoft Excel

A. Theory

In lecture an equation will be derived that allows you to calculate the concentration of each component in a binary mixture using Beer’s Law (A = bc). The two equations are:

[X] = (A Y - A Y) / D use this equation for benzoic acid

[Y] = (A X - A X) / D use this equation for caffeine

where D = b(X Y - Y X)

A = total absorbance at wavelength 1 (229 nm)A = total absorbance at wavelength 2 (272 nm)X = molar absorptivity of molecule X (benzoic acid) at wavelength 1 (229 nm)X = molar absorptivity of molecule X (benzoic acid) at wavelength 2 (272 nm)Y = molar absorptivity of molecule Y (caffeine) at wavelength 1 (229 nm)Y = molar absorptivity of molecule Y (caffeine) at wavelength 2 (272 nm)

B. Procedure

1. Download the Excel analysis file from the class webpage by right-clicking on the link and saving to a floppy disk (the file name is Lab#6_UV-Vis.xls).

2. Start Microsoft Excel and enter the file. The spreadsheet will look similar to this:

3. From the UV-Vis Data section of the data sheet, enter the benzoic acid molarities and absorbances fom the data sheet into the top half of the spreadsheet. Do the same for the caffeine data in the bottom half of the spreadsheet. Add a trendline to each set of data by right clicking on a data point and selecting the Add Trendline option. The resulting linear trends in absorbance will be plotted in the graphs along with the linear least squares best fits.

4. The slopes of the calibration curves are equal to the molar absorptivities (). Notice that the equations are keyed to the wavelengths (y' and y" refer to 229 and 272 nm respectively). Enter the molar absorptivities in the spaces below:

X for benzoic acid at 229 nm = ___________________ cm-1M-1

X for benzoic acid at 272 nm = ___________________ cm-1M-1

Y for caffeine at 229 nm = ___________________ cm-1M-1

Y for caffeine at 272 nm = ___________________ cm-1M-1

5. Click on the tab on the bottom of the spreadsheet labeled "calculations" and a new sheet appears. In the upper left-hand corner of the sheet enter the molar absorptivities from step 4 (in the sheet they are labeled with an "e" rather than a ).

6. In the space below the molar absorptivities enter the absorbance of the soda sample at 229 and 272 nm (A' and A" respectively).

7. The spreadheet will calculate the concentrations of BA and C in the diluted sample. Enter the values for the molarities of BA and C in the soda sample:

[BA] = ______________ M

[C] = ______________ M

8. Calculate the molarity of BA and C in the soda before dilution:

calculations:

[BA] = ______________ M

[C] = ______________ M

9. Now calculate the grams/liter of BA and C in the soda:

calculation: benzoic acid MW = 122.12, caffeine MW = 194.19

[BA] = ______________ g/L

[C] = ______________ g/L

10. A 20 fl. oz. bottle is 592 mL. Calculate the grams/bottle for each component:

calculation: benzoic acid MW = 122.12, caffeine MW = 194.19

[BA] = ______________ g/bottle

[C] = ______________ g/bottle

Ch 312: Lab #8 - Spectrophotometric Analysis of a Mixture: Caffeine and Benzoic Acid in a Soft Drink

DATA SHEET

Preparing Standard Solutions for the Calibration Curve

Concentration of BA stock solution = ______________ mg/L

Concentration of C stock solution = ______________ mg/L

[BA], ppm Volume BA Stock

Solution

[BA], mol/L [C], ppm Volume CA Stock

Solution

[C], mol/L

2 44 86 128 1610 20

UV-Vis Data

[BA], M Abs @ 229 nm Abs @ 272 nm

[C], M Abs @ 229 nm Abs @ 272 nm

Soda Abs @ 229 nm Abs @ 272 nm4:100 dilution

synthetic sample