Importance Of Sample Preparation And Analysis Techniques In Mineral And Chemical Analysis Of Beverages

identify the importance of correct sample preparation on the reliability of results 
• compare and contrast the mineral content of various beverages analysed by ICP-AES
• recognise that standard techniques develop over time, yet remain relevant today
• demonstrate correct standard preparation techniques, particularly for ICP-AES analysis
• calculate data using standard curves
• present data effectively using tables
• present data in an oral format to peers

Materials and Methods

1. To determine the concentration of caffeine in beverages by HPLC and UV-Vis spectroscopy
2. To determine the concentration of alcohol in beverages by Gas Chroma trophy and distillation techniques and compare that to the alcohol concentration labeled by the manufacturer
3. To determine minerals concentration in beverages by using ICP-AES

All parts of the Experiment were carried out according to the Laboratory manual.

Results

The Absorbance of Caffeine was measured using the UV-Vis spectrophotometer at 270nm and the calibration curve is shown in figure 1.

The caffeine concentrations of the various samples were calculated from the UV-Vis calibration curve and HPLC chromatograms taking into consideration the dilution factors and the results are summarized in table 1.

 The Caffeine content of Coke, Pepsi, Red bull and Coffee using UV-Vis spectroscopy were found to be 22.760, 23.665, 48.505 and 17.630 mg/100ml respectively and 11.479mg/100ml, 8.885, 35.400 and 48.587mg/100ml respectively using HPLC. The label data for coke, Pepsi and red bull indicated 9.6, 10.7, and 30mg/100ml respectively while the recommended value from the NUTTAB were 10, 10, and 24mg/ 100ml. there was no label data available for coffee while the recommended value from the Australian tables was 31mg/100ml.

For coke and Pepsi, the results obtained using HPLC were closer to both the Label and the NUTTAB values.  The results using UV-Vis spectroscopy were very high indicating massive errors in the experiment. A possible reason for the high concentrations could be due to improper dilutions in the sample preparation step.

For red bull, the HPLC results were slightly higher than the Label data. Both the label data value and the Experimental value were higher than the NUTTAB value indicating that the caffeine content of the Red bull did not comply with Australian standards. The UV-Vis results were twice as high as the experimental results using HPLC indicating possible errors in the dilution factor calculations. For Coffee, no caffeine was detected using HPLC. The results using UV-Vis spectroscopy were lower than the NUTTAB value for decaffeinated coffee implying the coffee complied with the Australian standards. There was no label data available for caffeine in coffee.

In all samples, the HPLC method gave lower values than the UV-Vis spectroscopy method.  A possible reason for errors in the UV-vis data could be the presence of compounds other than caffeine in the samples that absorbs at the wavelength of analysis (270nm). In Uv-Vis, the absorbance measured is from all the sample components that absorb at the given wavelength while in HPLC, the components are first separated and then the absorbance of each component is measured as it leaves the column. Thus, the absorbance measured with HPLC is strictly that of caffeine.

 The experimental values obtained using HPLC were closer to both the literature values for most of the samples and thus, HPLC is the most suitable method for determination of caffeine in beverages.  

Results

The results obtained for ethanol using the GC and the distillation methods are shown in table 2. Quantification of the GC results was done using both the standard curve and the relative response factor (RRF) techniques. Also presented in the table are the Ethanol content calculated from the Label data and the recommended value from the Australian Tables.

 The ethanol content of beer by Gas Chromatography was found to be 5.9% using the standard curve technique and 6.64% using the Relative Response factor technique.  The distillation method gave 3.83% whereas the label data and the NUTTAB values were 4.9% and 4.0% respectively.  From the results, Wine has almost thrice as much ethanol as beer. The GC results were higher than both the label and the NUTTAB values whereas the distillation method results were lower than both reference values.  However, the distillation method results were closer to the NUTTAB values as compared to the GC method results. Thus, the distillation method gives better results than the GC method for the determination of ethanol in beverages.

Caffeine in Beverages by UV-Vis Spectroscopy and HPLC

The ethanol content of red-wine by GC was found to be 17.5% using the standard curve technique and 20.3% using the RRF technique. The label data value was 13.5% while the Australian Tables value was 9.6%. The results given by the standard curve technique were more close to the label and NUTTAB values than the RRF technique results.  Based on these results, the standard curve technique gave better results than the RRF technique.

For both samples, the GC method gave higher values than both the Label and Australian Table values. A possible reason for this could be due to the fact that the samples used were not properly diluted and thus affected the response of the chromatograms. Gas chromatography works well with very dilute samples. Concentrated samples result into thick tailing peaks and that are not very reproducible. One way of improving the resolution of the chromatogram, is by using very dilute samples and standard of low concentration.  A possible source of error in the distillation method could be the use of wrong temperatures. The density conversion tables are given at 20 ⁰C yet the experimental densities were measured at room temperature.

Based on the label data, both samples had higher ethanol content than the recommended values from the Australian Tables. However, the experimental value for the beer obtained using distillation was within the recommended range.

The concentrations of sodium, potassium and Calcium were calculated, taking into consideration the dilution factors and the results are summarized in tables 3-6

The sodium and potassium in coke was not detectable. The label data indicated a concentration of 4 and 3mg/100ml respectively while the recommended NUTTAB values were 11 and 1 mg/100ml respectively. Based on the experimental data, coke meets the Australian standards for Sodium and Potassium. Calcium concentration of coke was 11.29mg/100ml which was higher than both the label and the NUTTAB values.

Sodium, Potassium and Calcium concentration of red-bull were 7.8, 238.24 and 3.32 mg/100ml. Na & Ca values were lower than both the Label and NUTTAB values and thus, red-bull complies with the Australian standards.  The Potassium value was unusually high indicating major non-compliance with the standards. The recommended K value for energy drinks is 0mg.

Sodium and Calcium in red wine was 8.7 and 9.69mg/100 ml respectively while Potassium was not detected. Sodium and Potassium were within the recommended NUTTAB values thus the wine complies with the Australian standards. However, the Ca was above the NUTTAB recommended value. There was no label data available for all the three minerals.

The sodium and Calcium in Coffee was 3.35 and 12.87mg/100ml respectively. Potassium was not detected in coffee. All the minerals were within the NUTTAB recommendations thus the coffee complies with the Australian standards.

Conclusion

All the objectives of the experiment were successfully achieved.  It was concluded that HPLC was the best method for caffeine analysis as compared to UV-vis spectroscopy because HPLC results were comparable to the Label and NUTTAB values.  The caffeine content of the analyzed samples increased in the order Pepsi <Coke<Red Bull<Coffee.

Red wine was found to contain almost thrice as much ethanol as Beer.  Both Beer and Red wine had higher levels of ethanol than the recommended NUTTAB values when analyzed with GC. However, the Distillation method results were within the recommended value. Thus, Distillation gave more reliable results than the GC method for Ethanol analysis in beverages.

The mineral content of the beverages were successfully determined. Coke met the Australian standards for Sodium and Potassium but the Calcium level was higher than the recommended.  Red-bull complied with standards for Sodium and Calcium but the potassium exceeded the recommended value. Red-wine satisfied the recommended values for Potassium and Sodium but the Calcium was beyond the recommended value.  Coffee satisfied the recommended values for all the three minerals.

References

Laboratory Manual.

NUTTAB. (2010). NUTTAB 2010 Online searchable database: Retrieved from https://www.foodstandards.gov.au/science/monitoringnutrients/nutrientables/nuttab/Pages/default.aspx