In the Laboratory
The Determination of Vanillin in a Vanilla Extract
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An Analytical Undergraduate Experiment Jozef L. Beckers Department of Chemical Engineering (SPO), Eindhoven University of Technology, STW 2.24, P.O. Box 513, 5600 MB Eindhoven, The Netherlands;
[email protected] Analytical chemistry in the second year at the Eindhoven University of Technology culminates with a final assignment over the course of four afternoons. The students do not get written instructions but they have to use their cognitive and manual abilities to solve an analytical problem. As part of a problem-posing approach to teaching we often give a group of four students a “real” sample with an unknown composition. The students deliberate how to solve the problem, discuss their approach with the instructor, and then set to work. A disadvantage of a “real” sample is that the composition of the sample is unknown and it is difficult to check whether the students work accurately. To get a better insight into the accuracy of the student results, the students must use at least two different analytical tools. Not all “real” samples are useful for final assignments. The most important conditions are that the sample component to be determined (i) can be analyzed with at least two different analytical methods, (ii) must be present in a matrix, not too complex, and (iii) the component should be present in the matrix at a concentration large enough so that pretreatments are not necessary. An example of such a final assignment was described recently (1) whereby UV–vis spectrophotometry, high-performance liquid chromatography (HPLC), and capillary electrophoresis (CE) were used for the determination of caffeine in coffee. The results obtained with HPLC and CE were similar whereas greater values of caffeine were obtained with UV–vis spectrophotometry because of the presence of other UV absorbing components in the sample matrix. In this article the student results are presented for another final assignment, the determination of vanillin in a vanilla extract. Vanillin (4-hydroxy-3-methoxybenzaldehyde) is an interesting compound for educational chemistry and is often used in bakery goods for its taste and aroma (Figure 1). The synthesis and production of vanillin have been discussed by several authors. Hocking described the production of vanillin from lignin-containing aqueous liquor obtained from acid sulfite pulping of wood (2) and Lampman et al. described the preparation of vanillin by converting eugenol, obtained from cloves (3, 4). Fowler presented five microscale experi-
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O
Figure 1. Structural formula of vanillin.
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Experimental Procedures
Reagents, Standards, and Materials All chemicals were analytical grade and deionized water was prepared with a Milli-Q system (Millipore, Bedford, MA). Standard stock solutions of 1.00 × 10᎑3 M vanillin and 1.00 × 10᎑3 M o-vanillin in water were prepared. From these solutions dilutions were made for the preparation of the calibration curves and separations. Dilutions were made using Eppendorf and Finn pipets. HPLC Equipment The HPLC equipment (Pharmacia-LKB, Bromma, Sweden) consisted of a Model 2150 pump and a VWM 2141 dual-wavelength UV detector. Chromatographic separation was obtained with a LiChrospher 100 RP-18 end-capped column (125 mm × 4 mm, 5 µm) from E. Merck (Darmstadt, Germany). Injections were made with a Model 7125 universal loop injector of 20 µL (Rheodyne, Berkeley, CA). Reversed-phase HPLC was performed at ambient temperature with an isocratic mobile phase consisting of water兾methanol (40:60 v兾v). The flow rate was 0.70 mL兾min. The mobile phase was degassed by vacuum filtration through a 0.45 µm filter and sparging with helium. Data analysis was performed using the in-house data analysis program DAX. CE Equipment For all CE experiments the P/ACE System 2200 HPCE (Beckman Coulter, Fullerton, CA) was used with a Beckman eCap capillary tubing (75 µm) with total length 36.7 cm and
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ments to study the properties of the multifunctional compound vanillin (5). Less attention has been paid, however, to the determination of the concentration of vanillin. Ainscough and Brodie discussed the determination of vanillin in vanilla extract using ultraviolet spectroscopy (6) including a solvent extraction. In this article the determination of the vanillin concentration in the Dutch product “Baukje vanilla extract” is described, but any commercially available vanilla extract should give good results. Because the composition of the extract is unknown, the students analyzed the sample with two different analytical methods: RP-HPLC (reversed-phase HPLC) and CE in the micellar electrokinetic capillary chromatography (MECC) mode. In this way, students can compare the results of different analytical tools and gain a better insight into the possibilities and accuracies of these methods. Representative results obtained by different groups of students are given. The separation of vanillin and its isomer o-vanillin (3-hydroxy-2-methoxy benzaldehyde) is taken into consideration.
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In the Laboratory
UV–Vis Spectrophotometer An LKB UV–vis spectrophotometer (Ultraspec II, Model 4040, LKB, Bromma, Sweden), with 1-cm quartz cuvettes, was used to measure the UV spectra of vanillin and o-vanillin. Hazards
1.5
Absorbance
a distance between injection and detection of 30.0 cm. The wavelength of the UV detector was set at 214 nm. All experiments were carried out at a constant voltage of 10 kV and an operating temperature of 25 ⬚C. Sample introduction was performed by applying pressure injections at 0.5 psi. Data analysis was performed using the laboratory-written data analysis program CAESAR. All experiments were carried out in the MECC mode with a background electrolyte (BGE) consisting of 0.010 M Tris and 0.040 M SDS adjusted to pH 8 by adding acetic acid and with the anode placed at the inlet and the cathode at the outlet.
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300
350
Figure 2. UV spectra for vanillin and o-vanillin. Measuring conditions were: 1.00 x 10᎑4 M of vanillin and o-vanillin in water and ambient temperature.
o-vanillin vanillin
Relative Absorbance at UV Detector
UV–Vis Spectrophotometry The students determined the UV spectra of vanillin and o-vanillin. The spectra of solutions of 1.00 × 10᎑4 M vanillin and o-vanillin are shown in Figure 2. Because the solution of vanillin in water was colorless and the vanilla extract was yellow, probably due to the presence of other absorbing components, the students decided to analyze the sample with CE and RP-HPLC and not with UV–vis.
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Wavelength / nm
Results and Discussion
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o-vanillin
Safety glasses should be worn all the times in the lab. Goggles and gloves should be worn while preparing the BGEs and standard solutions from concentrated reagents. Methanol is flammable and methanolic solutions must be prepared in a fume hood. Acetic acid is corrosive and SDS, vanillin, and Tris are harmful by inhalation and are irritating to eyes, skin and respiratory system. The hazards of the prepared solutions are minimal at the low concentrations used. The risk descriptions of all substances are given in the Supplemental Material.W
Micellar Electrokinetic Capillary Chromatography CE is a separation method (7, 8) in which all components are separated before they are identified and quantified. Because vanillin is a neutral component, the micellar electrokinetic capillary chromatography mode (MECC) (9, 10) was used with sodium dodecylsulphate (SDS) as surfactant. Electropherograms for the separation of 6-s pressure injections of a mixture of 6.00 × 10᎑4 M of vanillin and o-vanillin and diluted (500×) Baukje vanilla extract are shown in Figure 3. The isomers vanillin and o-vanillin can easily be separated and vanillin can be determined in vanilla extract without any pretreatment. For the determination of vanillin in vanilla extract, calibration graphs have been set up, plotting the measured peak areas of standard solutions of vanillin versus the concentration of vanillin over the range 1.0–10.0 × 10᎑4 M. For all determinations the sample solutions were measured twice and the average value was used for the calculation of the concentration of vanillin. The results of the vanillin determination, the relative standard deviation, and the regression coefficients of the calibration graphs are given in Table 1.
vanillin
1.0
A EOF marker vanillin
B EOF marker 5
10
Time / min Figure 3. Measured electropherograms for the separations of 6-s pressure injections of (A) a mixture of 6.00 x 10᎑4 M of vanillin and o-vanillin and (B) 500x diluted Baukje vanilla extract. The migration time of the electroosmotic flow (EOF) is marked.
Table 1. Results for the Determination of the Vanillin in Baukje Vanilla Extract from Student Group 1 Method
[vanillin]det/M
RSD (%)
Regression coefficienta
HPLC1
0.400
0.62
0.99990
HPLC2
0.450
1.43
0.99940
MECC1
0.442
0.26
0.99998
a
The regression coefficients were calculated from the the calibration data.
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In the Laboratory Table 2. Concentration of Vanillin in Baukje Vanilla Extract Determined by Various Student Groups
a
[vanillin]a/M
Student Group
HPLC 1
HPLC 2
MECC 1
MECC2
1
0.400 (0.62)
0.450 (1.43)
0.442 (0.26)
---
2
0.412 (0.53)
0.421 (1.08)
0.432 (1.95)
0.437 (1.36)
3
0.403 (0.96)
---
0.404 (1.46)
---
RSD values (%) are in parentheses.
High Performance Liquid Chromatography
Conclusions
The students also used reversed-phase high-performance liquid chromatography (RP-HPLC) (11) for the determination of vanillin, at a wavelength of 280 nm. For the determination of vanillin in Baukje vanilla extract, calibration graphs have been set up, plotting the measured peak area or peak height of standard solutions of vanillin versus the concentration of vanillin over the range 1.0–10.0 × 10᎑5 M. For all determinations the sample solutions were measured twice and the average value was used for the calculation of the concentration of vanillin. The results of the vanillin determination, the relative standard deviation, and the regression coefficients of the calibration graphs are given in Table 1. The chromatograms are shown for a mixture of 10.0 × 10᎑5 M vanillin and o-vanillin and diluted (5000×) Baukje vanilla extract in Figure 4. A good agreement between MECC and HPLC values was obtained.
The concentrations of vanillin in Baukje vanilla extract determined by various groups of students with the separation techniques RP-HPLC and MECC are shown in Table 2. It can be concluded that the concentration of vanillin can easily be determined in vanilla extract. By applying two analytical methods it can be verified whether the students worked accurately. The results obtained with RP-HPLC and MECC are similar and no internal standard is needed although different wavelengths are used in HPLC and MECC. For MECC, higher concentrations are needed for standards and sample because of the very short optical path length. The differences between the measured concentrations for vanillin are larger than could be expected from the RSD values. This may be caused by errors made by diluting the Baukje vanilla extract, which has a very high viscosity. W
Supplemental Material
Notes for the instructors and student data from the experiments are available in this issue of JCE Online. Relative Absorbance at UV Detector
vanillin
Literature Cited o-vanillin
A
vanillin
B
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Time / s Figure 4. Measured chromatograms for (A) a mixture 10.0 x 10᎑5 M vanillin and o-vanillin and (B) 5000x diluted Baukje vanilla extract. Measuring conditions: flow rate 0.7 mL/min; wave length detector 280 nm.
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1. Beckers, J. L. J. Chem. Educ. 2004, 81, 90–93. 2. Hocking, M. B. J. Chem. Educ. 1997, 74, 1055–1059. 3. Lampman, G. M.; Andrews, J.; Bratz, W.; Hanssen, O.; Kelley, K.; Perry, D.; Ridgeway, A. J. Chem. Educ. 1977, 54, 776– 778. 4. Lampman, G. M.; Sharpe, S. D. J. Chem. Educ. 1983, 60, 503–504. 5. Fowler, R. G. J. Chem. Educ. 1992, 69, A43–A46. 6. Ainscough, E. W.; Brodie, A. M. J. Chem. Educ. 1990, 67, 1070–1071. 7. Foret, F.; Krivankova, L.; Bocek, P. Capillary Zone Electrophoresis; Electrophoresis Library, VCH: Weinheim, Germany, 1993. 8. Kuhn, R.; Hoffstetter-Kuhn, S. Capillary Electrophoresis: Principles and Practice; Springer Verlag: Berlin, Germany, 1993. 9. Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 111–113. 10. Vindevogel, J.; Sandra, P. Introduction to Micellar Electrokinetic Chromatography; Huthig: Heidelberg, Germany, 1992. 11. Harvey, D. Modern Analytical Chemistry; McGraw-Hill: New York, 2000.
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