UV-Vis spectrophotometer

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

See Appendix 1 for more details


pp. 394-400, and 419-424 in Harris text



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.  Light passes into a monochromator where one small band of wavelengths is selected.  This monochromatic 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.

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 () exactly equal to the energy difference between the ground state (E1) and the excited state (E2).

Figure 2.  The absorption process.

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, 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.

Figure 3. Beer's Law plot

Ocean Optics Spectrometers:

For our absorbance measurements we will be using an Ocean Optics Red Tide UV-Vis spectrophotometer.  One of the main advantages of these instruments is their portability due to their compact design.  The entire instrument fits easily in the palm of your hand.  A schematic diagram of the instrument is shown in Fig. 4.  Notice that there is no exit slit from the monochromator to the detector as there was in Fig. 1.  Instead, a charge-coupled device (CCD) detector, similar to what you have in your digital camera, is used to measure the entire spectrum at once.

Figure 4. Ocean Optics Red Tide UV-Vis spectrophotometer.

Figure 4. Ocean Optics Red Tide UV-Vis spectrophotometer.

Making absorbance measurements with the Red Tide spectrometers is very simple.  In addition to making a blank measurement, you must also measure the detector signal when no light from the source is present.  This is called the dark measurement.  The instrument software subtracts this value from both the reference and sample signals when calculating absorbance:

Equation 4

where S is the sample signal, R is the reference signal, and D is the dark signal.

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:

Atot1 = AX1 + AY1 and Atot,λ,2 = AX2 + AY2

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 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.