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ABSTRACT High performance liquid chromatography (HPLC) technique is used to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying and purifying the individual components of the mixture. HPLC is also considered an instrumentation technique of analytical chemistry, instead of a gravitimetric technique. Benzoic acid and caffeine standard is prepared with different concentration. Then, both sample and standard were tested in the HPLC. The mixture of compound that we need to separate is caffeine and benzoic acid with the soft drinks sample. In this experiment, presence of caffeine and benzoic acid in soft drink sample is identified and the amount of caffeine in soft drink sample was determined. The sample was degassed by placing it in a vacuum flask before filtered through the filter paper. Compound differentially retained in the stationary phase reach the detector at different times to produces a set of peaks along the time line. Each component of the mixture reaches the detector at the different time and produces a signal at the characteristic time called the retention time. The area under a peak is related to the amount of the component present the mixture. In this experiment, serial dilution also will be prepared to be as standard caffeine and to determined if caffeine is present in the soda sample by use retention time. Other than that, by using the concentration to peak area relationship, the concentration of caffeine in the soda sample can be determined. The peak of caffeine will appeared after 2 second and by measure caffeine peaks of the standards, the amount of caffeine in a sample can be determined. The area is getting bigger when the concentration is increasing. This shows that the higher concentration of caffeine will make a bigger effect.


CONTENTS Title Page Abstract Table of Contents List of Tables List of Figures List of Symbols


1.0 Introduction 2.0 Methodology 2.1 Procedures 2.2 Analysis of data 3.0 Results & Discussion 4.0 Conclusions

1 3 5 5 6 9

References Appendices


LIST OF TABLES Table No. 3.1 Title Retention time of caffeine in standard Page 6

LIST OF FIGURES Figure No. 2.1 3.1 Title How the HPLC actually works Standard curve for peak area vs. concentration Page 3 7


m L mL ppm v.s

micrometer micro liter milliliter part per million peak area (microvolume x second)


INTRODUCTION A soft drink is a beverage that typically contains water (often, but not always carbonated

water), usually a sweetener, and usually a flavoring agent. The sweetener may be sugar, highfructose corn syrup, fruit juice, sugar substitutes or a combination of these. Soft drinks are called "soft" in contrast to "hard drinks" (alcoholic beverages). Widely sold soft drink flavors are cola, cherry, lemon-lime, root beer, orange, grape, vanilla, ginger ale, fruit punch, and sparkling lemonade. Soft drinks may be served chilled or at room temperature. They are rarely heated. Soft drinks are made by mixing dry ingredients and fresh ingredients like lemons, oranges, etc. with water. Production of soft drinks can be done at factories or at home. Soft drinks can be made at home by mixing either syrup or dry ingredients with carbonated water. The main ingredients in this soft drink are usually caffeine and benzoic acid. Caffeine is a natural component of chocolate, coffee and tea, and is added to colas and energy drinks. It is found in varying quantities in the seeds, leaves, and fruit of some plants, where it acts as a natural pesticide that paralyzes and kills certain insects feeding on the plants. It is most commonly consumed by humans in infusions extracted from the seed of the coffee plant and the leaves of the tea bush, as well as from various foods and drinks. Beverages containing caffeine enjoy great popularity among the teenagers specially. (Wikipedia, 2011) On the other hand, benzoic acid occurs naturally in various berries notably cranberries, cinnamon, plums, currants, cloves, etc. It has long been used to inhibit microbial growth in many products including non-alcoholic beverages, jams and emulsified sauces. The salt of the benzoate is more stable than the acid form and more soluble in water making the benzoates a favourable choice for the soft drinks industry. Benzoic acid is very effective against moulds, yeasts and bacteria. It is particularly well suited for use in soft drinks, such as carbonated, still and juice beverages because it works best between pH levels of 24. (UNESDA, 2010) The composition of the drink therefore has an effect on its efficiency and suitability for use. The major cause of benzene in soft drinks is the decarboxylation of benzoic acid in the presence of ascorbic acid (vitamin C) or erythorbic acid (a diastereomer of ascorbic acid). Benzoic acid is often added to drinks as a preservative in the form of its salts sodium benzoate,

potassium benzoate, or calcium benzoate. Other factors that affect the formation of benzene are heat and light. Storing soft drinks in warm condition speed up the formation of benzene. In order to check or identify if there is a presence of caffeine or benzoic acid in the soft drink, we used the high performance liquid chromatography (HPLC) system. Compounds are separated by injecting a plug of the sample mixture onto the column. The different components in the mixture pass through the column at different rates due to differences in their partitioning behavior between the mobile liquid phase and the stationary phase. Thus a mixture component will separate with one another. (Lindsay, 1997) HPLC typically utilizes different types of stationary phases, a pump that moves the mobile phase and analyte through the column, and a detector that provides a characteristic retention time for the analyte. The detector may also provide other characteristic information (i.e. UV/Vis spectroscopic data for analyte if so equipped). Analyte retention time varies depending on the strength of its interactions with the stationary phase, the ratio/composition of solvent used and the flow rate of the mobile phase. With HPLC, a pump (rather than gravity) provides the higher pressure required to propel the mobile phase and analyte through the densely packed column. The increased density arises from smaller particle sizes. This allows for a better separation on columns of shorter length when compared to ordinary column chromatography. (Lindsay, 1997)

1.1 Objective: 1.1.1 - To Identify the present of Benzoic acid/ Caffeine in soft drink sample 1.1.2 - To determine amount of caffeine in soft drink sample


METHODOLOGY The equipment required for this experiment is an isocratic HPLC system with UV

detector, C18 column, vacuum and 0.45m filter paper. In addition, the following equipment along with the specified technical data is also required:

Additional Equipment: A 0.45 m filter syringe A 100 L syringe A 60 mL syringe A Volumetric flask

Other than that, the experiment also used the chemical substances which is caffeine 1000ppm standard (stock solution), methanol (HPLC grade), double distilled water that filtered with 0.45m filter paper and the soft drink sample.

The HPLC works as shown in Figure 2.1;

Figure 2.1: How the HPLC actually works

A reservoir holds the solvent thats called the mobile phase, because it moves. A highpressure pump is used to generate and meter a specified flow rate of mobile phase. An injector is able to introduce by injecting the sample into the continuously flowing mobile phase stream that carries the sample into the HPLC column. The column contains the chromatographic packing material needed to effect the separation. This packing material is called the stationary phase because it is held in place by the column hardware. A detector is needed to see the separated compound bands as they elute from the HPLC column. Most compounds have no color, so it is impossible to see them with normal eyes. The mobile phase exits the detector and can be sent to waste, or collected, as desired. When the mobile phase contains a separated compound band, HPLC provides the ability to collect this fraction of the elute containing that purified compound for further study. This is called preparative chromatography. Before the solvent are prepared to test, it must go through the degasification process first to make sure no more carbon dioxide is left in the soft drink sample. It disturbs the result of the experiment if there is still carbon dioxide gas in the sample.




The following steps were carried out during the experiment.


Preparation of Benzoic acid/ caffeine standards -The standard caffeine samples of 20 ppm, 40 ppm, 60 ppm, 80 ppm and 100 ppm was prepared by diluting portions of the 1000 ppm solution with distilled water.


Preparation of soda samples -The soft drink sample was obtained. -The sample was degassed by placing it in a vacuum flask and connecting the flask to a vacuum pump or water aspirator. It will left under vacuum until no more bubbles appeared in the soda sample. (If no vacuum is available, allow the soda to stand open overnight.) -The degassed soda was filtered through filter paper.


Analysis of data


The standard benzoic acid /caffeine retention time was used to identify the benzoic acid/caffeine peak and their retention time was recorded.


By using all information, the presence of benzoic acid/caffeine in the soda sample was determined.


The different concentration of standards peaks area was recorded and a standard calibration curve (concentration vs. peak area) was plotted.


The caffeine peak in the soda sample chromatograph was measured and standard calibration curve (concentration vs. peak area) was used to determine the concentration of Benzoic acid/ caffeine in the soda sample.

Note: All raw data must be record in table form.



RESULT AND DISCUSSION In this experiment, High Performance Liquid Chromatography (HPLC) was used to

determine caffeine in soft drink which was Coca-cola as the sample (Harris, 2005). It was also used to find the amount of a chemical compound within a mixture of other chemicals. Before the sample was run in the HPLC, standard of caffeine must be prepared. 1mL, 2mL, 3mL, 4mL and 5mL (see calculation in Appendix) of caffeine stock solution was taken out from 1000ppm caffeine to dilute 20ppm, 40ppm, 60ppm, 80ppm, and 100ppm of caffeine standard respectively in 50mL volumetric flask. Then, double distilled water was poured in the flask and the flask was shacked until the mixture is well mixed. To run the HPLC, Standard Caffeine sample was used to identify the Caffeine peak and the retention time of the caffeine was being recorded. The peak was increased from 20ppm to 100ppm. The retention time was used to determine whether caffeine was present in the Cocacola sample or not. The amount of the caffeine in the sample was determined and the caffeine peak of the standards was measured (see Table 3.1), and a standard curve was constructed (see Figure 3.1). The caffeine in the Coca-cola sample chromatograph was measured and the concentration to peak area relationship was used to determine the concentration of Caffeine in the Coca-cola sample. Table 3.1: Retention time of caffeine in standard Concentration /ppm 20 40 60 80 100 Retention Time [min] 1.122 1.980 2.068 2.090 2.105 87919.30 110641.08 156997.94 170023.59 216500.43 Peak area / v.s


Peak area / v.s vs Concentration, ppm

250000 200000 Peak area, v.s 150000 100000 50000 0 0 20 40 60 80 100 120 Concentration, ppm Peak area / v.s Linear (Peak area / v.s) y = 1582.7x + 53453 R = 0.9763

Figure 3.1: Standard curve for peak area vs. concentration Based on the result obtained, caffeine retention time is 2 minutes. The peak area for 20ppm is 87919.30 v.s with time 1.122 minutes, 40ppm is 110641.08 v.s with time 1.980 minutes, 60ppm is 156997.94 v.s with time 2.068 minutes, 80ppm is 170023.59 v.s with time 2.090 minutes, and 100ppm is 216500.43 v.s with time 2.105 minutes. As expected from the experiment, the highest peak area for this experiment is at 100ppm with 216500.43 v.s. This shows that the result obtained obeys the theory that states that the higher the concentration, the higher the peak area whereas a higher concentration gives a greater effect towards the area (Fernndez, 2000). The data also shows a positive result when there is absence of caffeine in the sample. Graph for this experiment is not a straight line and accurate because only got R 2 0.9763. Although the comparison of the calculated and literature values of the analyte concentrations yielded large percent errors, the standard addition plots yielded R2 values close to 1, which implies that the method of standard addition was successful (Leacock, et al., 2011). All the objectives in this experiment were achieved as there was presence of caffeine in soft drink sample and the amount of caffeine in soft drink sample can be determined. As stated by Barone and Roberts (1996), caffeine is a pharmacological active substance and depending on the dose, can be a mild central nervous system stimulant. It is noted that

caffeine is not food but a drug working through nervous system. Excessive amount should be avoided since caffeine consumed in large amounts has adverse health effects. In particular, people suffering from high blood pressure should be advised to avoid use of caffeine containing beverages since caffeine is known to increase the blood pressure. In addition those with coronary heart disease should avoid such beverages as caffeine disrupts normal heart rhythm.



CONCLUSION AND RECOMMENDATIONS As the conclusion, the experiment has met the objectives which are to identify the present

of Benzoic acid/ Caffeine and determine the amount of caffeine in soft drink sample. The present of Benzoic acid/ Caffeine was identified in the soft drink sample by using HPLC. A standard of caffeine was prepared from caffeine stock solution to identify the caffeine peak and the retention time. The retention time was used to determine whether caffeine was present in the soft drink sample.The amount of the caffeine in the sample was determined and the caffeine peak of the standards was measured as shown in Table 3.1 and a standard curve was constructed in Figure 3.1. Based on the result, the caffeine retention time is 2 minutes. A comparison of the caffeine peak area in the soft drink sample compared to standard curve allows determination the amount of caffeine. The result also shows that the result obtained obeyed the theory as the higher the concentration the higher the peak area.The graph for this experiment is not a straight line and accurate because only got R2 0.9763 the nearest the value of R2 to 1 is more accurate. All the objectives in this experiment were achieved as there was presence of caffeine in soft drink sample and the amount of caffeine in soft drink sample can be determined. In order to get more accurate value, there are several recommendations to improve the result obtained. Firstly, try to reduce as much of contamination as the procedures involve dealing with the analysis of sample that contamination could affect the result obtained. Next, make sure that avoid any errors while handling HPLC such as bubbles present in the syringe that injected into HPLC.



Barone JJ, Roberts HR. (1996). Caffeine Consumption Food Chemical Toxicology, Volume 34, p119, Coca-Cola Company, Atlanta Harris, D.C., (2005). Quantitative Chemical Analysis; 5th Edition, W.H. Freeman and Company, New York Leacock, R. E., Stankus, J. J., Davis, J.M. (2011). Journal of Chemical Education. Simultaneous Determination of Caffeine and Vitamin B6 in Energy Drinks by High-Performance Liquid Chromatography. Volume 88, pp.2 Lindsay, S. (1997). High performance liquid chromatography. [Online]. Available from: http://www.osti.gov/energycitations/product.biblio.jsp?osti_id=7013902 October 2012]. P. L. Fernndez, M. J. Martn, A. G. Gonzlez and F. Pablos. (2000). Analyst, Volume 125, pp.421-425, RSC Publishing Union of European Soft Drink Association (UNESDA), (2010). Qualitative and quantitative control of benzoic acid and caffeine in soft drinks. United States: UNESDA Publications. Wikipedia (2011). Benzene in soft drinks. [Online]. Available from: [Accessed by 26

http://en.wikipedia.org/wiki/Soft_drink [Accessed by 27 October 2012].


APPENDIX Sample of calculation:

1) 20 ppm M1 = 1000 ppm V1 = ? mL M2 = 20 ppm V2 = 50 mL

M1V1 = M2V2 (1000)(V1) = (20)(50) V1 = 1 mL #