Multicomponent UV Analysis Determination of Caffeine and Benzoic Acid in Soft Drinks 

READING (FROM HARRIS TEXT):

• • 444-450 (Properties of Light; Absorption of Light; Measuring Absorbance)

  • 472-476 (Analysis of a Mixture)

OBJECTIVES:

• • To determine the amounts of caffeine and benzoic acid in a soft drink.

  • To learn a method for determining the concentrations of multiple compounds in a mixture using UV-Vis spectrophotometry, even when the spectra of the compounds overlap.

Spectrophotometry is one of the most commonly used instrumental methods in all of science. While the term is used to refer to any technique that uses light to measure the concentration of a chemical, most spectrophotometric measurements rely on the absorption of light. The basic components of an absorption spectrophotometer are shown in Fig. 1. Polychromatic (many colors) light passes into a monochromator where one small band of wavelengths is selected. This monochromatic (one color) light then passes through the sample compartment and on to a detector where it is converted into an electrical current, i, proportional to the intensity of the light. (Intensity is more correctly referred to as irradiance, P.)

Figure 1. Components of a simple absorption spectrophotometer.

Some of the light passing through the sample may be absorbed by the molecules in the sample. When a molecule absorbs a photon of light, the energy of the molecule increases. If the light is from the ultraviolet (UV) or visible portions of the electromagnetic spectrum, this absorption results in electrons being promoted to higher energy levels. We say the electron has gone from the lowest energy state (the ground state) to a higher energy state (an excited state).  This process is illustrated in Fig. 2 below. A photon of light can be absorbed when it has an energy (hν) exactly equal to the energy difference between the ground state (E1) and the excited state (E2).

Figure 2. The absorption process.

To begin an absorbance measurement we must first measure the initial irradiance,P0, by placing solvent in the sample holder.  This is called the blank measurement.  The solvent is then replaced with the real sample, and the irradiance, P, is measured.  As stated in your Harris text, transmittance (T) is the fraction of the original light that passes through the sample.

It should make intuitive sense that transmittance can vary from 0 to 1 (or 0 to 100% for %T).  Transmittance is related to absorbance, A, by the following:

Finally, we see the relationship between absorbance and concentration, c, by the familiar Beer’s law equation:

Where c is the concentration of the analyte in the sample, ε is the molar absorptivity of the analyte (sometimes called the extinction coefficient), and b is the sample cell pathlength shown in Fig. 1.  You should recognize that Beer’s law predicts a linear relationship between absorbance and concentration.  Experimentally, this is shown by generating a Beer’s law plot of A (y-axis) vs. c (x-axis).

Figure 3. Beer's Law plot

    Analytical measurements should typically be made an the absorbance range from 0.3 to 2. At high absorbance values, too little light is transmitted and it is hard to measure with high precision. At low absorbance values, there is little difference between the sample and the blank.

MULTICOMPONENT ANALYSIS:

In the past you've probably used Beer's Law to determine the concentrations of species which absorb either UV or visible light. This procedure was pretty straightforward. You made a series of standard solutions which were used to generate a (hopefully) linear Beer's Law plot (A vs. c). Then you measured the absorbance of your unknown and plugged it into the equation of your line to yield the concentration of the unknown. Simple.

But now let's add a little twist and say that our sample consists of two compounds (X and Y) that we wish to determine, and that these compounds have individual spectra which overlap. If we obtain a spectrum from a mixture of these compounds, then this spectrum is just the sum of the spectra from the individual components X and Y. This means that at any wavelength on the spectrum of our mixture the total absorbance is just equal to the sum of the absorbances of the individual components:

Atot = AX + AY

Let's choose two specific wavelengths, λ1 and λ2, at which both compounds show significant absorbance. We can then write expressions for the total absorbance at each of these wavelengths:

Atot,λ1 = AX,λ1 + AY,λ1 and Atot,λ,2 = AX,λ2 + AY,λ2

Using Beer's Law, these equations can be expanded to:

Atot,λ1 = εX,λ1bcX  +  εY,λ1bcY and Atot,λ2 = εX,λ2bcX  +  εY,λ2bcY

If all the molar absorptivities are known, then we have a simple system of two equations and two unknowns, cX and cY. (We can use standard solutions of X and Y to determine the four ε values from the slopes of four Beer's Law plots:  A vs. c at λ1 and λ2 for standard solutions of both X and Y.)

As your textbook points out, a system of two equations and two unknowns is adequate if the individual spectra of compounds X and Y are fairly well-resolved. However, if there is significant overlap in the spectra then we add more Atot equations at different wavelengths.

In this experiment you’ll be determining the amount of caffeine and benzoic acid in a soft drink sample. Caffeine, as you know, is a stimulant, and benzoic acid is commonly used as a food preservative since it inhibits the growth of mold, yeast, and bacteria. The UV spectra of caffeine and benzoic acid overlap, although you’ll see there are wavelength regions where one component dominates. It’s important that we don’t use a diet drink since aspartame absorbs in the same region of the UV. Also, colas generally don’t work well since a colorant absorbance band extends too far into the UV.

CARY 60 SPECTROMETER

The instrument we'll be using is the Agilent Cary 60 UV-Vis spectrophotometer, the components of which are shown in Fig. 4 below. You'll notice the similarity to Fig. 1 above. The light source is a xenon flash lamp. Unlike most other light sources, this lamp only turns on when it needs to take a measurement. So this should extend its lifetime to ten years or more. The polychromatic light from the lamp then enters the monochromator (containing mirrors and a diffraction grating). The now monochromatic light now passes through the sample and on to the detector.

Figure 4. Components of the Cary 60

Figure 5 shows a photograph of the instrument in our laboratory. You'll notice two extra components: a Peltier temperature controller (which we won't use in this experiment), and a fiber optic dip probe. The dip probe replaces the traditional sample holder we see in Fig. 4. So instead of filling and refilling a cuvette, we'll simply dip the probe in our solutions to record the absorbance.

Figure 5.  The Cary 60 in our laboratory.

Figure 6 shows how the dip probe works. Light comes in through one fiber optic cable, passes through a lens, then moves through the sample solution until it is reflected off the mirror. From the mirror it passes back through the lens and through another fiber optic cable which carries it to the detector. Notice that the pathlength (b) is equal to twice the distance between the lens and the mirror.

Figure 6. Fiber optic dip probe configuration.

REFERENCE:

McDevitt, V.L, Rodriquez, A., and Williams, K.R.  J. Chem Educ.  1998, 75, 625-629.