Ultraviolet-Visible Absorption of a Two-Component Mixture
Introduction. An HPLC instrument was used to determine the UV-Visible Absorption measurements of a cola containing benzoic acid and caffeine. The absorbance wavelength was also measured for an unknown solution. Standards were prepared in order to graph calibration curves (absorbance vs. concentration). Four dilutions were made from the 50 mg/L benzoic acid standard. Four dilutions were made from the 100 mg/L caffeine standard.
Part 1. The UV-VIS spectra for the unknown sample are presented in Figure 1. The spectra showed two peak absorbance at wavelengths 228 nm and 273 nm.
Table 1. Unknown Sample, Peak Absorbance.
Figure 1. UV Absorbance (Peak Area) vs. Unknown Concentration (mMol/L)
Part 2. The spectra for the standard curves are presented in the appendix. The Benzoic Acid absorbances for the four standards are presented in table 1. Figure 2 and 3 present the standard curves of the benzoic acid. Some of the readings were negative indicating that they were below the detection limit. Based on the data presented in table 2, only the absorbance at wavelength 228 nm can be used. The slope of the standard curve which represents the molar absorptivity at 228 was found to be 8.0985 and R2 = 0.9947. The value of 0 will be used for the molar absorptivity at 273 since the measured values were negative.
Table 2. Concentration and absorbance for two wavelength for Benzoic Acid spectrum.
Figure 2. Benzoic acid Concentration (mM/L) vs UV Absorbance (Peak height) at wavelength 228.
Figure 3. Benzoic acid Concentration (mM/L) vs UV Absorbance (Peak height) at wavelength 273.
Similarly, Caffeine absorbances for the four standards are presented in table 1. Figure 4 and 5 present the standard curves of caffeine. Some of the readings were negative indicating that they were below the detection limit. Based on the data presented in table 3, only the absorbance at wavelength 273 nm can be used since 2 of the 4 absorbance values at wavelength 228 were negative. The slope of the standard curve which represents the molar absorptivity at 273 was found to be 5.0995 and R2 = 0.8191. The value of 1.4818 will be used for the molar absorptivity at 228 which is obtained after the elimination of negative values. The four molar absorptivities were:
Benzoic Acid
228 nm
ε11=8.0985 mM-1 cm-1
273
ε12=0 mM-1 cm-1
Caffeine
228 nm
ε21=1.4818 mM-1 cm-1
273
ε22=5.0995 mM-1 cm-1
Table 3. Concentration and absorbance for two wavelength for Caffeine spectrum.
Figure 3. Caffeine Concentration (mM/L) vs UV Absorbance (Peak height) at wavelength 228.
Figure 4. Caffeine Concentration (mM/L) vs UV Absorbance (Peak height) at wavelength 273.
Part 3. Using the two standard curves for Benzoic Acid and Caffeine the concentration of the unknown was determined as follows:
The unknown absorbances were A1=0627 and A2=0.118 mAU at wavelength 228 and 273 respectively.
[Benzoic Acid] =(A1*ε22-Α2*ε21)/(ε11*ε22-ε21*ε12)=0.076 mM
[Caffeine] =(A2*ε11-Α1*ε12)/(ε11*ε22-ε21*ε12)= 0.00067 mM
Accounting for a 5/1 dilution (5 mL yellow mello in 25 ml total volume)
The concentration of Benzoic acid and caffeine in the unknown were 0.38 mM and 0.0034 mM respectively.
Part 4. Improper selection of dilutions made some of the standards to measure close to the instrument detection limits. There was not good linearity in the caffeine which means the standards were not prepared accurately. Based on the final result the unknown was over diluted with respect to caffeine.
