Extraction of Caffeine

Extraction of Caffeine from Coffee by liquid/liquid extraction, tested for purity with Thin Layer Chromatography

Worked with Jaeone

  1. Introduction

Coffee has become a major part of modern society. It has been consumed since the 15th century. Due to its property of causing sleeplessness and increase in concentration when absorbed it is consumed largely by students or businessmen who are required to focus for a long time. However, the trend is diverging in the recent years. Scientists discovered that caffeine is the substance in coffee that contributes to the decreasing of the drowsiness. Thus, the demand for caffeine than raw coffee has increased significantly. In this experiment, caffeine will be extracted from coffee to be tested for purity which can be further used for other benefits.

A process in which an organic compound is separated from a mixture of compounds is called extraction. Liquid/liquid extraction in specific is a method of extraction through using solvent that can dissolve only certain compounds, leaving the rest undissolved. In this process, the solvent that has dissolved the specific compound is called the extract. An important point in liquid/liquid extraction process is that the two solutions, the original mixture and the solvent, has to have a different polarity so that they are immiscible with each other. Immiscible by definition is that two solutions cannot mix and maintain homogeneity. Thus an immiscible solution to the original mixture that is capable of dissolving the desired compound while leaving the unwanted compounds is ideal in separating a compound from the mixture. In this experiment, dichloro-methane (also called methylene chloride) was used. Since dichloro-methane is non-polar compared to water and since like dissolves like, it can dissolve more caffeine compared to water while being immiscible with water. However, a problem with using dichloro-methane in this experiment is that it can also dissolve tannin, a non-polar polyphenolic compound that is also present in coffee, thus making hard to isolate caffeine only from coffee. However, Tannin can be converted to salt by deprotonating its hydroxyl (–OH) group to (-O) group thus making the molecule highly polar. To solve the problem, sodium carbonate was used to react with tannin in order to produce phenolic anion, a salt which does not dissolve in dichloro-methane due to high difference in polarity. Furthermore, even though it has been assumed that dichloro-methane and water are highly immiscible, there may be a slight chance of water being dissolved through a phenomenon called emulsion. Emulsion is a process in which immiscible solutions mix due to droplets of either of the solutions getting trapped in the other solution’s bubble. To purify caffeine as much as possible, anhydrous sodium sulfate is added. Anhydrous compounds are substances that lack water of crystallization, thus absorbing water to create crystals of itself. Thus, when enough of anhydrous sodium sulfate is added to the solution, the salt crystallizes by absorbing the water in the solution, leaving the solution free of water, isolating caffeine.

To test for the purity of the caffeine that has been extracted, chromatography was used. Chromatography is a method of separating mixture. It uses two substances that vary in their polarity in which one dissolves the mixture, called mobile phase and one that does not, called stationary phase. To be more precise, stationary phase also builds the structure for the mobile phase to travel. In performing a chromatography, mobile phase is allowed to travel through the stationary phase. As it travels, due to difference in polarity for all compounds in the mixture, the distance that each compounds travels will vary. Thin layer chromatography, or TLC for short, is a branch of chromatography where a layer of absorbance on a glass plate is the stationary phase and the liquid that is being absorbed by the absorbance on the glass plate is the mobile phase. In this experiment, a non-polar absorbance, silica coating (SiO2) and a polar mixture of 5% acetic acid and 95% ethyl acetate was used. Rf value is a mathematical way of representing the result of TLC. It is the value of the distance that the substance traveled divided by the total distance that the mobile phase traveled. Thin Layer Chromatography was used for the testing of the result of the extraction since it allows the comparisons of the distance that pure caffeine and the extracted material travels thus making it easier to distinguish if the extracted material is made of pure caffeine or is a mixture including other impurities.

  1. Materials
    1. Brewing of Coffee
      1. Roasted and grinded coffee bean
      2. 500mL Beaker
  • 500mL Erlenmeyer flask with side arm
  1. Erlenmeyer flask stopper
  2. Filter paper
  3. Pump
  • Rubber cable
  • Glass cylinder funnel
  1. Glass support base with sand core permeable top
  2. Metal clamp
    1. filtration_vacuum-filtration-appartus
  1. Liquid/liquid extraction
    1. 6g sodium carbonate
    2. 75ml methylene chloride
  • Spoonful anhydrous sodium sulfate
  1. 250mL Beaker
  2. Separating funnel
  3. Plastic stopper
  1. Thin Layer Chromatography
    1. TLC plate coated with SiO
    2. Ethanol
  • Acetic Acid
  1. Ethyl Acetate
  2. Hexane
  3. Pure Caffeine
  • 10mL Beaker
  • Hot Plate
  1. Glass capillary
  2. Glass chamber
  3. Solid Iodine
  1. Procedure
    1. Brewing of Coffee
      1. Set the vacuum filtration apparatus by connecting the sidearm of the Erlenmeyer Flask to the pump by the rubber pipe, applying the stopper, connecting it with the glass base, setting a filter paper on the base, placing the glass cylinder on the set and holding it firm with the metal clamp like the Figure 1.1.
      2. In 500mL beaker, put all the roasted coffee powder and pour water to make 300mL of coffee. Stir it thoroughly to brew as concentrated as possible.
  • Pour the coffee into the glass cylinder and turn the pump on. If in the process of filtering, coffee bubble erupts and is about to go above the level of sidearm in the Erlenmeyer Flask, turn the pump off to slow the filtering process. Turn the pump back on when the bubble subsides.
  1. Liquid/liquid Extraction
    1. To the 300mL of coffee solution in the Erlenmeyer Flask, add 6g of Sodium Carbonate (NaCO3). Swirl the solution until all sodium carbonate is dissolved
    2. Divide the 300mL of coffee to two separation funnel for each group, 150mL of coffee each.
  • To each separation funnel, add 36.5mL of dichloro-methane. Close the funnel entirely and swirl it vigorously for 1 or 2 minutes. Do not swirl it excessively or emulsion will form. In the process of doing so, open the tab to allow the gas to escape. Repeat the process several times
  1. Allow the mixture to settle and separate into two layers, the dark top layer and a clear dichloro-methane bottom layer. A middle layer can exist, but it should not be significant.
  2. Carefully open the tab to pour out the bottom layer into a clean beaker. Pour out the middle layer into another beaker and the bottom layer into a separate beaker.
  3. Pour the middle layer into the same separation funnel and repeat process iii. by adding only as much amount of dichloro-methane as the solution itself. By this process, middle layer may not exist. If it does not, collect the bottom layer with the beaker used to collect the bottom layer in process v. and the top layer with the beaker used to collect the top layer. The top layers can be discarded.
  • To the dichloro-methane solution, add a spoonful of anhydrous sodium sulfate and swirl it vigorously. The salt will form crystals. When crystallization is no more observed, filter out the crystals of hydrous sodium sulfate using a filter paper and a funnel.
  • Let the dichloro-methane evaporate, leaving pure caffeine. Dry the solid and weigh the mass.
  1. Thin Layer Chromatography
    1. Measure out about the same amount of pure caffeine as the extracted caffeine.
    2. Dissolve it in 8mL of ethanol solution in a 10mL beaker.
  • Use a capillary tube to let it dissolve. You may use the hot plate to heat the solution to speed the dissolving process.
  1. Using the glass cutter, cut TLC plates, into rectangular pieces that will fit in the glass chamber.
  2. On the coated side of the TLC plates, drip the ethanol-caffeine solution into a small dot. Let it dry and drip it again. Repeat the process three to four times. Dry the solution and mark the position it has been dripped.
  3. Make a stock solution of 5% acetic acid, 95% ethyl acetate.
  • Pour a little amount of stock solution into the glass chamber and put the TLC plate in so that the dripped point is not below the solvent level but has enough distance to travel.
  • When the stock solution has traveled till the very end and is about to reach the end, take out the TLC plate and mark the starting point, and the ending point of the traveled solvent.
  1. To a dry glass chamber, add 20 to 30 beads of solid iodine.
  2. Place the TLC plate in the iodine glass chamber and wait until the plate has turned yellowish shows other dots above the originally marked points.
  3. Take out the TLC plate and quickly mark the newly made dots. Calculate the Rf value of the substance.
  • Repeat from process ii. to process xi. with the extracted caffeine. Also record the data.
  • Compare the Rf of the two solutions to check what you have extracted is caffeine.
  1. Data
    1. Qualitative Observation
      1. Liquid/liquid Extraction Process

The aqueous layer of coffee rested on top of the organic layer of dichloro-methane and caffeine. The top layer was dark brown and the bottom layer was transparent with a yellowish hue. The middle layer was a lighter brown than the top layer and had an oily texture to it.

When anhydrous sodium sulfate was added, the powder of sodium sulfate started to form spherical, white and non-transparent crystals. As the swirling continued for a longer time, the crystals broke down to smaller units of still spherical and solid shape.

The extracted caffeine was yellowish white compared to the pure caffeine which is purely white.  It was in powder form, stuck to the bottom of the beaker.

  1. Thin Layer Chromatography

The chromatography results of the pure caffeine contained only a single dot. However, the result of the extracted substance had two define dots with different Rf values.

  1. Thin Layer Chromatography Results
    1. Chromatography of pure caffeine
      • Distance from the origin to the dot
        • 5mm
      • Distance from the origin to the end point of the solvent travel
        • 5mm
  1. Chromatography of the extract
    • Distance from the origin to the first dot
      • 0mm
    • Distance from the origin to the second dot
      • 0mm
    • Distance from the origin to the end point of the solvent travel
      • 0mm
  1. Analysis
    1. Analysis of the Qualitative data

Caffeine has a pure white color. Thus, looking at the fact that dichloro-methane solution and the final, dry extract had a yellowish color shows that it would have contained impurities.

The fact that anhydrous sodium sulfate broke down to smaller units show how reaction of absorbing of water has gone to completion. This is because if reaction did not go to completion, and the solution still had water, the anhydrous sodium sulfate would keep forming bigger crystal. However, since it did not, it can be safely stated that the reaction has completed.

Chromatography result shows that the extracted substance had one more dot compare to the chromatographic result of the pure caffeine. This implies that the extracted substance contained impurity other than caffeine.

  1. Analysis of the Chromatographic Results and Calculation of Rf Values
    1. Calculation of Rf value
      1. Rf calculation
    2. Rf value of pure caffeine
      1. Rf value of pure caffeine
    3. Rf value of the Extracted Caffeine
      1. Rf value of extracted caffeine

Looking at the evaluation of the Rf values, it can be inferred that the second dot was caffeine and the first dot, impurity.

  1. Discussion

It can be easily inferred that this experiment has failed to isolate caffeine only. Firstly, caffeine is a single compound that does not decompose due to chromatography. Thus, in chromatography, pure caffeine should result in only one dot. However, the chromatographic result of the extracted substance turned out to have two dots. This shows that the experiment has failed to isolate caffeine only. This is further shown through the fact that the chromatography result of the extracted substance did not exactly match the chromatography result of the pure caffeine. As analyzed above, the first dot that appeared in TLC can be interpreted as the impurity since it does not match any of the Rf value. On the other hand, the second dot, whose Rf value matches the one of pure caffeine can be interpreted as the extracted caffeine.

To discuss about the impurity that has been measured, there seems to be two possibilities; water and tannin. Of course, impurities in beaker and several human mistakes would have created this mistake too. However, to be only reasonable and sound reason for mistake was discussed. Firstly, the fact that the reaction between anhydrous sodium sulfate and water has gone to completion, dealt with in analysis section, shows that it couldn’t have been water molecule left in dichloro-methane. On the other hand, tannin seems to be a likely candidate for the impurity. As explained in the introduction, tannin, is another stimulant commonly found in coffee. It is non-polar like caffeine, even more so than caffeine due to its high molecular mass, making itself easy to be dissolved in dichloro-methane in the process of liquid/liquid extraction. Furthermore, even though it has been tried to eliminate as much tannin as possible by deprotonating them through dissolving carbonate ion, if carbonate ion dissolved was not enough to eradicate all tannin, some tannin would have taken part in the reaction, getting dissolved in dichloro-methane and showing its appearance in chromatography. To explain the chromatographic results, the impurity, assumed to be tannin, has traveled less than caffeine along the non-polar stationary phase. It can be safely said that tannin is more so non-polar even compared to caffeine due to its polyphenolic structure and high molecular mass. This makes tannin more stable with non-polar stationary phase than caffeine is. On the other hand, caffeine is more stable with polar mobile phase. Thus, caffeine will travel further than tannin with polar mobile phase due to the principle of like-dissolves-like making tannin not travel further than caffeine.

This mistake as discussed above should have been created due to the lack of sodium carbonate in the coffee solution leaving non-polar tannin behind. Thus, to improve the experiment, more sodium carbonate should have been dissolved in the solution, even past its saturation point to be filtered. This would have completely deprotonated tannin leaving only caffeine to be the non-polar molecule in the coffee solution. This would have definitely isolated caffeine.

Work Cited

Nam, Y. E; Hill, M. P; Randall, J; Organic Chemistry with Vernier; Vernier Software and Technology, 2012

Ramalakshmi, K; Raghavan, B; Caffeine in Coffee: Its Removal. Why and How?. Crit. Rev. Food Sci. Nutr., 2010, 39, 441-456

Extraction of Caffeine from Tea. http://vlab.amrita.edu/?sub=3&brch=64&sim=169&cnt=1 (accessed April 18, 2016)

For the Actual Document click Extraction of Caffeine

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