Caffeine: a Drug that has a Place in our Life

  INTRODUCTION       -       METHODS and MATERIALS       -       RESULTS       -       DISCUSSION

                                                                                                                                                                       A) Meaning of the Results

                                                                                                                                                                       B) Sources of Errors

                                                                                                                                                                       C) Integrative Discussion

1- Introduction

Caffeine is a legal drug which is taken everyday by millions of people all around the world.  It is the most common drug, it is more common than any medicine and this since few thousand years(1). It is found in several plant leaves like tea, coffee and mate, also in guarana paste and cola nuts(2). Many popular drinks and food contain this drug in different amounts.  Among them is certainly coffee, but also tea, soft drinks, energy drinks and chocolate. We can ask ourselves how these beverages differ in caffeine concentration.

Caffeine is a conjugated organic molecule.  It is an alkaloid, which are naturally-occurring compounds found in plants.  They are organic compounds composed of carbon, hydrogen, nitrogen and usually oxygen. Alkaloids exhibit basic properties.  Many of them, such as caffeine, are valuable as medicines because of their physiological effects(3).

In its pure state, caffeine is a crystalline white powder and has a bitter taste. It is easily soluble in chloroform, i.e. one gram in 5.5 ml, and partially soluble in water, i.e. one gram in 46 ml.  Its molecular formula is C₈H₁₀N₄O₂ and it has the following structure(4).

 Figure 1.1 - Caffeine Structure

Caffeine is also a drug and has physiological effects, which are looked for by consumers.  Its main effect, which is the main reason of consumption, is the catalysis of the human body metabolism. Because caffeine concentrations that affect human beings are contained in different beverages drank everyday, we will attempt to find the caffeine concentration in a cup of coffee, of tea, and of cocoa, and in energy drink and in Coca Cola. Then, these concentrations will be related to the biological effects of caffeine.

Previous determinations of caffeine content in beverages have been done using high-performance liquid chromatography(5). This requires an expensive high technology equipment. Chromatography is a technique that separates all the component of a sample.  It works by passing the sample in high pressure solvent through a steel tube packed with sorbents. The components of the sample are separated because of the different interactions of the components with the solvent and the tube, which are mainly due to the different polarities of the components(6).

The concentration we expect to obtain are regrouped in the following table.  They are the value reported in a The World of Caffeine, and will be regarded as being the theoretical values for the concentration.

                 Table 1.1 – Theoretical Concentrations of Caffeine

In this experiment, the concentration of caffeine will be found with an ultraviolet spectrometer that is relating the absorbance to the concentration.  Compounds containing multiple bonds, as does caffeine, usually absorb a part of electromagnetic radiation, in the ultraviolet of visible regions, that passes through it.  The quantity absorbed depends on the wavelength of radiation and the structure of the compound.  The absorption of radiation is caused by the absorption of the energy by electrons in the molecule that are excited to higher energy orbitals.  An absorption spectrum, also called a wavescan, is necessary to determine the wavelength of maximum absorption.  This is the wavelength reported in books and it is also the one used when determining concentration of unknown sample. Compounds that contain conjugated multiple bonds, i.e. compound having a single bond in between two multiple bonds, have maximum absorption at wavelength longer than 200 nm.  “When a molecule absorbs light at its longest wavelength, an electron is excited from its highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO).”  The size of the gap between these two energy levels is what changes the wavelength of maximum absorption. A smaller gap will occur in conjugated molecules, which requires less energy for the electron movement and thus a greater wavelength.  The absorption of a certain sample is also determined by the concentration of the absorbing molecule in the sample since more molecules will absorb a greater amount of light(7).

“The Beer-Lambert Law, which states that A=abc, where A is the absorbance, a is the absorptivity constant for [a given compound], b is the path length of the light through the sample, and c is the concentration,”(8) can be use to determine the concentration of an unknown compound in a sample, provided that a and b are known.  A calibration curve can be made from known concentration samples by plotting the absorbance as a function of concentration to obtain the value of ab.  The absorption spectrum of the sample is use in comparison with the one for the pure sample containing only the compound to verify if the nature of the compounds is likely to be the same in the two cases.   

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2- Method and Materials

       The first step in this experiment was to analyze pure caffeine. To use this pure substance in the UV spectrometer, the only thing that had to be done was to dilute it in chloroform, which was chosen as a solvent. Caffeine was diluted at different known concentrations.  These solutions were used to build a calibration curve to analyze our results. However, this step is explained below in this part of our laboratory report.  Here, it is important to find the absorbance spectrum of pure caffeine to compare it to the other spectrums from the extracted substances. Before placing a diluted solution of pure caffeine into the spectrometer, a reference scan was made using only chloroform. Then, the diluted solution was inserted into the spectrometer for a scan ranging between the maximum wavelengths possible on the machine, 200 nm and 900 nm. Then, a graph was plotted with the data obtained and a bell shaped curved was expected.  If the absorbance was greater than three, it meant that the concentration of the solution was too high and it had to be diluted more.  These steps were repeated until the reading on the graph plotted was analyzable, that is smaller than three. Then the wavelength, at which caffeine absorbs the most UV rays, was determined.  This maximum wavelength of absorption of pure caffeine was the wavelength used afterwards to determine absorption of the different solution made from the beverages. Moreover, knowing between which wavelengths was situated the bell shaped curve permitted to shorten the range of analysis in the machine for the wavescan of the beverages.

Secondly, the caffeine was extracted from the first substance to be tested, the Red Bull.  To do that, 5 mL of Red Bull were poured into a 30 mL separatory funnel.  Then, 10 mL of chloroform were added.  The two substances were mixed by shaking the separatory funnel carefully for a few repetitions. The top stopper was then opened to let out any gas producted. These two important steps were repeated about 25 times.  Then, the bottom layer of liquid, which was the chloroform containing caffeine, was pooled in a graduated cylinder of 50 mL or 100 mL.  After, the same operation was done twice, using 15 mL of chloroform and then 10 mL of chloroform. After, three extractions, a small amount of the third one done was put in the UV spectrometer to verify the remaining content of caffeine. This was done by measuring the absorbance at wavelength of 275 nm, the maximum absorption wavelength of pure caffeine we determined previously.  If this absorbance was small enough (lower than 0.05), no caffeine or a very small amount of it absorbed UV light, and thus there was almost no caffeine present anymore in this last extraction which meant that we could presume that there were no more in the sample of the beverage. If the absorbance was too high (more than 0.05), then other extractions were performed until the absorbance of the last extraction was small enough. After most or all of the caffeine was extracted from the Red Bull.  The volume of solution was recorded.  The solution of caffeine and chloroform was then kept in a Teflon capped glass bottle for the UV analysis that would be done later. The next important step was then to extract caffeine from the other chosen substances containing this psychoactive drug, i.e. coffee, tea, cocoa and Coca Cola. The same process as explained above was performed with all the substances. Of course, the different substances did not need the same number of extractions. In fact, coffee, cocoa and Coca Cola needed five extractions while tea required three. There is only one difference between the extraction of Red Bull and the others; for the four last substances, the volume of chloroform used by extraction was of 15 mL for the first and 10 mL for the others, which is different from the way the caffeine was extracted from the Red Bull.

Before analyzing the extracted solutions of caffeine and chloroform, the calibration curve had to be built as a reference for the analysis. This trend line was constructed with five solutions of known concentration of pure caffeine that were evaluated with the spectrometer. Each solution was placed in the machine to measure its absorbance at the maximum wavelength, 275 nm. Then, using Excel, the linear function was made.  One important point has to be noted; this curve had to be made each day that some extracted substances were evaluated in the spectrometer since a calibration curve can differ each day since the external condition of experiment can differ. Thus, a trend line was built only in the two last days since the extracted solutions of caffeine and chloroform were analyzed on these days.

The analysis of these substances followed the same process as the evaluation of UV absorption of pure caffeine. A sample of each substance was placed in the spectrometer for a scan from 225 nm to 350 nm, the range between which the bell shaped curve of caffeine absorption of UV light was situated. Then, using Excel, the graph was plotted from the data obtained. If the absorbance obtained was greater than three, the substance was diluted until the bell curve was observed. The absorbance at 275 nm was recorded three times to insure accuracy.  The data and plot were then saved and kept for the analysis of the results.  We had control over the volume of the chloroform-caffeine solution whereas we had no control over the absorbance of our samples.  Consequently, the volume of solution collected can be called our independent variable and the absorbance our dependant variable.

              Safety precautions were observed during the lab since pure caffeine is extremely toxic¹ and so is chloroform.  These two compounds must be handled with hood ventilation systems, gloves and goggles.  Since the gloves were made of plastics, they had to be changed often and every time chloroform was spilled on them.  Precautions not to inhale chloroform and caffeine also had to be taken.  Also, we only made used of glass ware since chloroform dissolves plastic.

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3- Results

            Table 3.1- Caffeine calibration curve determined on March 15, 2004

            Table 3.2- Caffeine calibration curve determined on March 22, 2004

Table 3.4- Wavescan of various substances at λ=275 nm

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4- Discussion

A) Meaning of the results

Interestingly enough, the results show that the substance with the highest concentration of caffeine is cocoa, with 2979 ± 5 ppm.  Then, the results  for the other substances show what we expected, Red Bull being the substance with the second highest concentration of caffeine (881 ± 2 ppm), followed by coffee (459 ± 1 ppm), tea (114 ± 1 ppm) and Coca Cola (76 ± 5 ppm) respectively. 

The uncertainties on the concentrations are fairly small.  They represent only between 0.17% and 0.88% in all cases except for Coca-Cola, which has an uncertainty of 6.58% of the concentration.  This uncertainty is big enough to give us doubts about the reliability of the result.  Furthermore, the values obtained for the concentrations are within uncertainty for all beverages except Coca-Cola.  Trial 1 is off by one ppm, which represents a small percentage of the value and is not significant enough to discard the value obtained for this trial.  Still we have to regard the concentration of Coca-Cola as less reliable than the other ones.

The following table shows the comparison between the theoretical concentrations of caffeine in the substance we tested and the experimental results we got.

Table 4.1 – Comparison table between expected and experimental data

We can see that the experimental concentrations for coffee and tea are within the theoretical range.  The caffeine concentration of Coca cola, even though it is not very close to the theoretical value, could be said to be in an acceptable range, taking into account that its uncertainty is bigger and that the theoretical values are only approximations.  The experimental result for Red Bull also seems to be quite far off if we consider the fact that it is more than twice the theoretical value.  Note that Red Bull is a new kind of drink on the market and that not many data are available concerning its caffeine content.  Our theoretical concentration for Red Bull was taken form internet and it was not confirmed by other sources.  However, because of the small uncertainties for our data and because we do not know the procedure that was used to get the theoretical values, we can be confident in our results and we can assume they are at least as reliable as the theoretical ones; therefore, we cannot really know which set of results (theoretical or experimental) are off.

 Also, the theoretical results are all in the form of ranges, which are not precise at all, or are approximations, which suggest imprecision.  The brands used to perform the test might also be different from those used in the determination of the theoretical values.  Also, the process used to make the coffee, tea and hot chocolate might result in different concentrations from time to time.  This might be the reason for having ranges and approximations, but it also partly explains the results that differ from the theoretical.  As for the experimental concentration of caffeine in cocoa, it is really far off if we consider the theoretical values; it might even seem to be non sense since it is more than 50 times the theoretical value.

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 B) Sources of errors and suggested improvements in experimental design

For cocoa, we first thought that compounds like theobromine or theophylline might have dissolved into chloroform and because they have a similar structure to caffeine, they would have absorbed at a wavelenght similar to that of caffeine.  We discarded this hypothesis since both of these compounds are too polar to dissolve in chloroform.  The difference in the results could be explained by the amount of cocoa powder that was dissolved in water before we performed the extraction.  Not all the powder was dissolved into the water and we had a considerable residue at the bottom of the bottle.  This residue might have contained an excess of caffeine which could have dissolved in chloroform and have increased the concentration of caffeine in our sample.  However, because we do not know the procedure followed to obtain the theoretical concentration, we suppose that they might have taken out the residue and therefore, this might partly explain why the theoretical concentration is much lower than the one we obtained.  In other words, if they tested a cocoa solution in which only a small amount of powder was present, it could have all dissolved completely and their caffeine concentration would thus be lower than ours.  If cocoa powder is to be tested in further years, it might be a good idea to filter the solution before performing the extraction.  This way, the students will avoid having excess caffeine dissolved in their chloroform.  The use of hot chocolate from a coffee machine or from a hot chocolate envelop could also reduce the concentration.

            Also, the uncertainty on other results may have been caused by the imprecision on the calibration curve.  The determination of the calibration curve is a crucial point in the experiment since a falsen calibration curve would cause all our final concentrations of caffeine to not represent reality.  The very first step of the determination of the calibration curve was to weight out 0.100 g of pure caffeine.  A slight imprecision in this initial step would have repercussion on all other solutions that were made from the initial solution.  Another problem encountered with the calibration curve is that the Beer-Lambert law supposed that the curve must be linear and go through (0,0).   The Beer-Lambert law being A=abc, where A is the absorbance, a is the absoptivity constant of the compound, b the path lenght of light through the sample and c the concentration of the compound.  Our data give a pretty good linearity since our coefficients of linearity (R²) are good for both calibration curves.  But they do not go to zero.  After doing some research, we can say that Beer-Lambert’s law applies linearity up to absorbance 0.3.  We worked with concentrations of caffeine that were too high and the absorbances we got for those concentrations are above the critical point of absorbance.  The procedure followed for the calibration curve could be improved for further experiments by taking smaller concentrations to determine the calibration curve and to test the substances.  This way, the absorbances would be lower and it would assure the students a better corelation with Beer-Lambert’s law.

            The calibration curve may not be the only place where uncertainty was introduced.  As we discussed above in the explanation of the caffeine concentration in cocoa, the presence of other compounds dissolved in the chloroform layer as to be taken into account.  Many compounds compose the initial substances and some acids with a long non-polar end, often present in food, might have dissolved in chloroform.  It would be likely that we would notice the presence of those acids on the wavescan since they would probably not absorb at the same maximal wavelenght as caffeine does.  The presence of those acids might explain the results difference, in the case of Red Bull for example. In order to minimize the chance of having those acids present in the chloroform layer, a new experimental design could include the addition of a base to every substances tested in order to neutralize those acids.  With the addition of a base such as Na₂CO₃, those acids would become ionic compounds and would certainly not dissolve in chloroform, being too polar.

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 C) Integrative discussion

          Why does caffeine “kick in” in humans? The answer is not only complicated but also uncertain. Following Weinberg and Bealer’s work, The World of Caffeine, there are three theories explaining the mechanism of action of the drug. However, two have been proven to be wrong for normal conditions and the last one is not confirmed yet. The first theory states that caffeine is, with its relatives in the family of methylxanthines, an inotropic agent, a compound increasing “the force of cardiac muscles”(9). This agent acts by augmenting the entry of calcium ions in the cells, the molecules responsible for muscle contractions. Because the cardiac muscles are stronger, an increase in cardiac output and the body metabolism is speeded up. However, this theory is not considered as the main explanation for caffeine mechanism of action since only a toxic dose of caffeine (around ten to a hundred times greater concentration than in coffee) will have this particular effect.

   The second theory claims that caffeine increases the level of a certain hormone produced by the body, cyclic adenosine monophosphate (cAMP). This compound has an enhancing effect on the use of glycogen, one of the body’s storage of energy. Therefore, caffeine would favor the burning of this important source of energy and would then give the possibility to the body to use energy more easily. Yet, the same problem as in the first theory is encountered here; the concentration of caffeine in the body needs to be extremely high for the mechanism to work. Thus, very toxic doses are required to show an effect that would be related to the increased level of cyclic adenosine monophosphate.

            The main and most recent theory on caffeine mechanism of action states that this legal drug acts in the brain on adenosine, a neuromodulator that slows down the body metabolism. In fact, some of the effects of this inhibitor are mood-depressing, sleep-inducing, blood pressure-inducing and heartbeat-slowing effects. Caffeine plays a part in the system by blocking adenosine uptake at the receptor sites in the brain cells. Caffeine stops a compound that slows down the body and therefore speeds up the metabolism. Moreover, some findings would show that the effects of the well-known drug could be more completely explained by the fact that caffeine increases the dopamine uptake by blocking the way to its inhibitor, adenosine. Thus, the last theory is not only more complete than the two first but it also works at normal doses of the drug, which is different from the calcium uptake and the cAMP increase theories. Still, the theory needs more confirmative results to be fully accepted.

            Caffeine has a main biological effect, as explained above. However, this effect has also several other side-effects that have strong impacts on the human body. As it was said, there are a lot of these but four are more important than the others: the effects on the cardiovascular system, on physical activity, the dependence and the toxicity of caffeine.

            As most people think, caffeine would be dangerous for the heart and vascular system because it increases the body metabolism and thus the blood pumping. In fact, this thought is not actually true at normal caffeine concentration in the blood, which is created by ingesting a normal quantity of the drug-containing beverages. This fact is proven by several studies; caffeine does not have long-term effect on the heart. Thus, the effects on the cardiovascular system are only on a short-term basis. Indeed, the rule that has to be followed with the ingestion of caffeine is that lower doses slow and higher doses speeds the heartbeat. And why does people that take caffeine regularly are said to not have more risk of heart problems than people who does not ingest the drug? Because the drug creates a tolerance. Therefore, people ingesting caffeine regularly are tolerant to the drug and the dose taken is not effective enough to have the unwanted effects, the increased heart beat. Furthermore, an interesting study shows that caffeine has a positive effect on systolic and diastolic pressure in the heart, associated with respectively the contraction and the relaxation of the cardiac muscles. The drug would lower these blood pressures, which is a good effect on the body that lessens the risk of cardiovascular problems. In fact, the study showed that people ingesting caffeine regularly tend to have a lower blood pressure than people not taking caffeine regularly. On the whole, in all the studies about caffeine performed, the results are trustable, mostly positive and related to the usual consumption of caffeine.

              Since the International Olympic Committee (IOC) put caffeine in its restriction list in 1962, the drug has been considered as a physical activity enhancer. Scientific thinking and studies have been done since to evaluate whether caffeine benefits physical exercise or not. Three assertions would explain the positive answer to this question. The first is based on the fact that caffeine favors lypolysis, the catabolism of fat in adipose tissues, following the studies of Dr. John Williams, a specialist of caffeine. Moreover, in the same trend of idea, caffeine, as it was explained above, blocks the effects of adenosine, which is an inhibitor of lypolysis. The second assertion states that caffeine renders the burning of glycogen more efficient, following the second theory on caffeine’s mechanism of action. The third claim is more about perception of the individuals affected by the drug than a physiological. Since caffeine speeds up the body metabolism, they feel less fatigued and can then do more exercise than they would if they had no concentration of caffeine in the body. However, these assertions can be shown to be true only at high doses of the drug ingested such as 900 mg of it, which is as much as about nine cup of coffee in a short time. Furthermore, as the several studies on the subject showed, caffeine seems to be a positive enhancer in physical activity axed on endurance. Thus, caffeine would improve the athletic capacity of the body in lower intensity work done for a longer time.

            Caffeine is a psychoactive drug having relatively strong effects on the brain. Since it is psychoactive, it is most likely to be addictive. However, to explain the addictive property of caffeine more precisely, the effects of the drug must follow at least one of the two definitions of addiction: the physical dependence and the clinical dependence. A physical dependence is developed in an individual consuming a certain substance when withdrawal symptoms are diagnosed and a tolerance to the drug is developed on the person. This first definition is thus followed by the addiction on caffeine since a regular user of the drug that stops his consumption will have several symptoms encountered in every similar case. These symptoms are sleepiness, work difficulty, irritability, decreased sociability, flulike symptoms, increased depression, anxiety and impaired psychomotor performance. Moreover, the affected individual can also easily develop a tolerance to caffeine. A drug will cause a clinical dependence syndrome when it has a positive effect on the consumer that will encourage him to continue taking it. This is also the case for caffeine since it gives reinforcing effects such as euphoria, energy or self-confidence, which are consequences on the consumer’s mind that will encourage him to continue ingesting caffeine. Furthermore, the drugs that create the physical and clinical dependence syndromes are usually considered as abusive drugs. Therefore, caffeine would be a dangerous drug as are cocaine and heroin. However, the effects of addiction on caffeine are much milder than those on the strong drugs and are not as lethal.

            The possibility of lethality in caffeine consumption brings an interesting possible effect of caffeine: the toxicity of the drug. Of course, several cases of caffeine intoxication exist and thus the drug is toxic to a certain point as every substance is. To which point is caffeine toxic? Following the cases existing, the dose of caffeine ingested needs to be much higher than the normal ones. Therefore, a lot of cups of coffee have to be drunk before the apparition of any symptoms of intoxication on caffeine. These are nervousness, anxiety, insomnia, gastrointestinal disturbances, irregular heartbeat, tremors, psychomotor agitation, urination, headaches, diarrhea and irregular breathing. One of the good known example cases for caffeine intoxication is found in psychiatric hospital where schizophrenics consumed jars of about 250 milliliters full of instant coffee with a spoon. The intoxication in the patients caused an increase in schizophrenic symptoms. When there is a possibility of toxicity, there is also a possibility of lethality. In fact, it has been calculated that the LD-50 of caffeine (the lethal dose for fifty percent of the population) is 10 grams. Thus, a person that drinks a hundred cup of coffee (about 100 mg per cup) has a great chance to die of this consumption.

            On the whole, caffeine has one main effect on the brain by blocking the adenosine. This effect catalyses the body metabolism and engenders several other effects. The most important impacts on the body are observed on the cardiovascular system and on the athletic capacities, and they can create dependence and toxicity in the body. For each of the impact, an important concentration is needed to create the effect, which is often more than the normal consumption of the drug.                       

If we compare the results we got with the biological effects of caffeine, some very interesting observations can be made.   First, according to our results for the concentration of caffeine in coffee and according to the mass of caffeine that is needed to die (lethal dose), 87 cups of coffee would be a lethal dose.  With the results we got for each substance and using the same reasoning, we obtain that the lethal dose for the other substances would be the following: 351 of tea, 528 cups of Coca-cola, 46 cups of Red Bull and 13 cups of cocoa.  For the first four substances, the results make sense if we compare them to what we observe in every day life; however, for cocoa, the lethal dose got from our results does not really make sense if we consider that, according to common sense, drinking 13 cups of cocoa would cause death.  This shows once again that our result for the concentration of caffeine in cocoa is probably not accurate and a quite far off.  Also, if we compare the concentrations we got with the biological effects they should cause, the cocoa result does not really make sense.  For example, our results show that cocoa contains more caffeine than all the other substances we tested; therefore, according to these results, cocoa would create more dependence than coffee and Red Bull and it would give more energy and affect more the metabolism than these same two substances.  Obviously, this is again not what we usually observe and therefore, this makes us question our result for cocoa. 

We suggest that future research could be directed toward the difference of caffeine content among the same kind of beverages, that is, between different coffee brands.  Also, different kinds of coffee could be analysed.  For example, the difference in caffeine content between espresso, cappuccino and moka could be analysed.  Finally, different coffees made from the same coffee beans and the same bag of coffee could be analysed to see if the concentration of caffeine can vary from one coffee to the other, even though the beans from which the coffee was made are the same.

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9 Idem as 1 p. 224

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