Preparing The Caffeine Extract Biology Essay
Getting the Most Caffeine Out of Tea
AIMA NABILA BINTI ABDUL BASID
MAISARAH BINTI AHMAD
NUR ALIFAH SHAIRAH BINTI JOHAN ARIFIN
NUR FATIHAH BINTI ZAINAL ABDIM
NURUL MAHFUZAH BINTI BAHARUDDIN
Group : 4 D
Date : 19TH April 2013
Lecturer : Dr. Nurhuda Manshoor
To separate the components of a mixture by using Thin Layer Chromatography (TLC) instrument.
To separate the components of a mixture by using High Pressure Liquid Chromatography (HPLC) instrument.
To calculate the concentration of caffeine in the sample.
In this laboratory session, we wanted to learn about the chromatography technique by applying the skills required in handling the equipment needed in the chromatography technique.
Chromatography is a technique used to separate each component present in a mixture of compound. In this experiment, we did two types of chromatography techniques which are Thin Layer Chromatography (TLC) and the liquid chromatography (High Performance Liquid Chromatography). Each type of technique used different type of apparatus but apply the same principle which is by separating the components in the mixture of compound.
Thin layer chromatography uses a TLC plate. The principle in using the TLC plate is simple in way that the plate will be dissolved in the solvent until the solvent had reached the frontline. In the TLC plate technique, we used UV light to view our caffeine component in the sample by observing and comparing which spot having the same level as the standard spot.
Another chromatography technique we applied was the liquid chromatography (High Performance Liquid Chromatography) to test for the concentration of caffeine with different times of extraction. Initial assumption was the longer the time of extraction, the higher the concentration of caffeine extracted. Thus, we ran the machine to test the initial assumption and compared the result with the standard calibration curve we plotted earlier. The calibration curve plotted was between the peak areas against the concentration. From the standard, we can compare our peak area of sample with the standard curve. From there, we can extrapolate to determine the concentration of caffeine sample and plot the graph of concentration of caffeine against time elapsed to reach peaks.
Therefore, from the TLC plate test, we were able to determine the retention time while from HPLC test we can obtain the concentration of caffeine sample in the teabag.
The longer the time of extraction, the higher the concentration of caffeine extracted.
On TLC plate, the spot having the same level with spot produced by standard caffeine indicates the presence of caffeine component in sample.
Thin layer chromatography (TLC) plate
Preparing the caffeine extract
5 ml of methanol was drawn into the syringe and then was squirted into the waste container.
2 ml of tea was drawn into the syringe.
The syringe filter was attached and 1 ml of filtered tea was injected into a 10 ml graduated cylinder.
4 ml of methanol was added into the 10 ml graduated cylinder using pipette.
All the apparatus was cleaned after use.
Thin layer chromatography
A 5 x 10 cm thin layer chromatography (TLC) plate was prepared.
A baseline measured 1 cm from the base of the TLC plate was drawn in order to apply the sample.
A frontline that is measured 1 cm from the top of the TLC plate was drawn.
A 1-cm band of sample was applied on the baseline of the TLC plate.
A band of caffeine standard was applied onto the TLC plate, separated by 1 cm from the sample band.
The solvent was allowed to completely evaporate.
An elutant was prepared with ratio of chloroform to methanol is 90: 10 v/v.
The elutant was poured into the separation chamber without exceeding 1 cm in height.
The TLC plate was placed into the separation chamber and was closed with a cover glass.
The plate was removed from the chamber when the solvent touches the frontline.
The plate was dried and the chromatogram was visualized in a UV cabinet.
High Performance Liquid Chromatography
200 µl syringe was filled with the sample and the sample was squirted out on a paper towel.
The syringe was filled with 20 µl the sample and it was injected into the HPLC system.
The chromatogram was labeled.
The results were recorded and analysed.
RESULTS AND DISCUSSION
Standard Calibration Curve
Concentration of standard caffeine (ppm)
Retention time, Rf (min)
Peak area (mAU)
STANDARD CALIBRATION CURVE
y = 19.608x + 213.91
R2 = 0.9998
y = 2.6155x + 27.612
The graph shows the relationship between the concentration of caffeine and peak area (mAU). There is positive relationship between two variables. As the concentration of the caffeine sample increases, the peak area of the standard caffeine also increases.
This calibration curve is plotted in order to enable us to find the concentration of the caffeine samples of unknown concentrations. Therefore, based on various time to steep in TLC which are 5s, 10s, 20s and 25s, we can find the concentration of the caffeine in the sample by referring to the standard calibration curve.
Thin layer Chromatography (TLC)
Extraction period of tea samples (steep time) (sec)
Distance traveled by the substance, (cm)
Distance traveled by the solvent, (cm)
Retention time, Rf
Thin Layer Chromatography (TLC) is an easy, fast, and cheap technique used in pharmaceutical analysis to identify and separate components in a mixture.
For this experiment, we want to identify whether the five tea samples that were extracted at different extraction period contain caffeine and if their concentrations vary with time or not.
Chromatographic separation works based on the fact that different substances are separated differently between two phases, a mobile phase and a stationary phase. In this experiment, the mobile phase is the eluent containing 90:10 v/v of chloroform: methanol, while the stationary phase is the TLC plate coated with a thin layer of solid adsorbent silica.
To determine whether there is caffeine in the tea samples, we compared the Rf of sample mixture with the standard caffeine. At the baseline of TLC plate, we spotted the standard caffeine and the tea samples using glass capillary. Then, the plate is placed in separation chamber containing the eluent. Eluent is a solvent used to enhance the pulling action of the substances in the mixture up to the top. By using the capillary action, the solvent will be slowly rising up the TLC plate until it reached frontline of the plate. The solid will adsorb some fraction of each component in the sample mixture.
Each component of the tea sample was attracted differently with the stationary phase that already washed along with the mobile phase according to the polarity of the mixture and the stationary phase. Caffeine is a polar molecule because it is soluble in water and the molecular structure of caffeine showed that it is a hydrogen bond acceptor from water.
The mobile phase contains chloroform mostly, which is a non-polar solvent with a low dielectric constant of 4.8.
The concept of the separation is "like attracts like" which means if the stationary phase is non-polar, so non-polar components of the tea sample will be more adsorbed to the stationary phase. Likewise, it goes to the mobile phase that washing over the stationary phase. If the mobile phase is a non-polar solvent, it will attract and carry along the non-polar components easily and vice versa.
As the capillary action move further, it allows the mobile phase to separate the mixture of tea samples at different rates. Besides that because of the different polarities between mixture with mobile phase and stationary phase, hence different components travel at different speed.
As the mobile phase rise up along the TLC plate with the tea sample band and standard caffeine band, the mixture of the tea sample will also rise up together and spread out. As we applied the band, we should make sure that we applied enough sample on the TLC paper. Because if we applied too little sample, the spot formed maybe too faint to see as it moves further up the TLC plate. When the mobile phase already moved up until frontline, remove the plate from the separation chamber and dry the plate. After that, we can compare the chromatogram between the standard caffeine and the sample in a UV cabinet.
From the spot formed, we can determine whether caffeine is present in the tea sample or not. From the spot, we can calculate the Rf value of caffeine. The Rf value of a given component is the same under the same experimental condition. Rf value is unitless.
The equation to calculate Rf value is:
Rf = distance traveled by the substance
distance traveled by the solvent
C:\Users\Toshiba\Downloads\Pharmacy Sem 4\PHC470\Chromatogram TLC.PNG
From the result obtained, we can see that different extraction period of tea samples yielded slightly different Rf value. With the same experimental condition, the Rf value should be the same for all tea samples, but maybe because of the different extraction period of tea samples made the concentration of caffeine in every tea samples slightly vary from each other. But basically, the Rf value of the tea samples are close to each other between 0.654 to 0.800.
High Performance Liquid Chromatography (HPLC)
Extraction period of tea samples (steep time) (sec)
Retention time (min)
Peak area of Caffeine (mAU.s)
Concentration of caffeine (diluted)
Actual concentration of caffeine (ppm)
In this experiment, the caffeine separation is done using a non-polar column and a combination of 40% of methanol and 60% of water as mobile phase. A series of caffeine standards with known sample concentration which are 25, 50 and 100 ppm was measured to construct a calibration curve.
HPLC is accomplished by injection of 20 µl of caffeine’s sample into a moving stream of the mobile phase that passes through a column packed with particles of a stationary phase. A component retained in the column is determined by its partitioning between the liquid mobile phase and the stationary phases.
There are two main ways to interpret a chromatogram which are:
Determination of the peak height of a chromatogram peak.
Determination of the peak area.
Therefore, we decided to interpret the chromatogram by determining the peak area. A comparison between the caffeine’s peak areas to those for the caffeine standards allows a quantitative determination of the caffeine content.
Before injection of the samples, they are already diluted to total volume of 5 ml. Therefore, based on dilution factor of 5, the actual caffeine concentration is calculated using formula as follows:
Actual caffeine concentration = Concentration of caffeine x
= Concentration of caffeine x Dilution factor of 5
For 5 Sec,
Actual concentration of caffeine = 13.81678 ppm x 5
= 69.0839 ppm
For 10 Sec,
Actual concentration of caffeine = 22.87096 ppm x 5
= 114.3548 ppm
For 15 Sec,
Actual concentration of caffeine = 30.69817 ppm x 5
= 153.49085 ppm
For 20 Sec,
Actual concentration of caffeine = 48.56685 ppm x 5
= 242.83425 ppm
For 25 Sec,
Actual concentration of caffeine = 37.0651 ppm x 5
= 185.32550 ppm
From the above graph, it shows that the concentration of caffeine being extracted from the sample increases, as elapsed time for the sample of tea to steep becomes longer. At one point, the concentration of caffeine being extracted slowly decrease after it reached particular time of maximum amount of caffeine to be extracted from the tea. It is noted as the extraction time reaches 20 seconds, the curve showed a maximum concentration of caffeine.
There is an error occurred during this experiment in which at 25 seconds, we observed that the concentration of caffeine starts to decrease. Theoretically, the concentration of caffeine being extracted should remain constant after maximum concentration has been achieved. The reason is mostly likely due to the systemic error during preparing of caffeine extract from tea sample.
Keep HPLC solvent away from open flame because it is flammable.
Dispose of all liquid waste in the appropriate waste container.
The TLC plate must not be immersed exceeding the baseline when we first put the plate in the container containing the solvent.
The separation chamber should be closed with a cover glass during experiment to prevent the eluent from being evaporated.
In pharmaceutical industry, there is always need to test for the content or purity of a pharmaceutical product. Hence, there are varieties of techniques available to separate each component contained in a sample such as using traditional TLC or advanced HPLC method.
From the experiment, the concentration of caffeine in each sample is:
Extraction period of
tea samples (sec)
Actual concentration of caffeine (ppm)
Thus, it can be concluded that as the extraction time increases, the concentration of caffeine increases too, but not to exceed the maximum amount of caffeine extractable from the tea.
Volume of liquid in the pipette is 4 ml.
Height of peak shown in the diagram is 2.8 cm.
Yes, chemical mixtures can be separated. It is because it is a mixture, not a compound. A mixture can be separated either by means of physical or chemical methods.
Example of physical method is filtration by using a filter paper.
Example of chemical method is chromatography technique by which it separates the mixture using chemical interaction between the components of mixture and the solvent. This applied in the one of the chromatography technique which is the liquid chromatography.
Line graph :
y = 0.135x - 0.15
R² = 0.995
Concentration will reach 0.6 mg/L at 5.56 minute.