EXPERIMENT

Isolating caffeine from ground coffee using different extraction techniques

KEY LEARNING CONCEPTS

•   To understand two phase extractions using aqueous and organic solvents.

•   To describe how functional groups of organic molecules affect their polarity and therefore their solubility in solvents used for extraction.

•   To compare and contrast multiple methods of extractions for potential use on an industrial scale.

•   To develop communication skills through the generation of an executive summary.

AIMS OF THE EXPERIMENT

•   To isolate caffeine from commercially available ground coffee using Soxhlet extraction and high pressure/temperature extraction with a coffee espresso machine.

•   To evaluate and compare the extraction techniques used to isolate caffeine with respect to yield, cost and environmental impact.

Note:

You will require a basic scientific calculator as well as your Student Notes and Report Template. It is a good idea to bring these to all practical classes.

READING

• Chemistry Human Activity, Chemical Reactivity, Mahaffy, Bucat, Tasker, Kotz, Treichel, Weaver and McMurry 2nd  ed. 2015:

Intermolecular Forces: Section 6.3, pages 165-175

Solid and Liquid State: Section 11:10, pages 433-436

• Online guides – see LMS “Laboratory Information”:

• See CHEM 1000 Lab Manual Techniques and instrumentation:

o Top-loading balances: page 149;

o Laboratory equipment and glassware: page 151;

o Filtration: page 153;

CHEMCAL PRELAB MODULE

There is a ChemCAL Prelab module, which may be accessed from the LMS and will provide some exercises related to this experiment. This is not compulsory.

INTRODUCTION

Isolation of natural products, namely organic compounds found in plants or animal sources, is a         profitable global industry due to the fact that many naturally occurring products are challenging and often expensive to synthesise.

Natural products can be extracted from natural plant materials (e.g. seeds, roots and leaves) by a       process known as Soxhlet extraction. In this process, the natural source material is continuously         washed with a given solvent that is constantly being cycled through processes of boiling and                recondensation. In each cycle, the organic compounds that are soluble in the solvent are extracted    and become more concentrated in the solvent phase. However, issues surrounding the cost of            heating large volumes of solvent, alongside safety and environmental concerns, mean that the use of this technique on a large scale is declining in industry.

Recent innovations in product extraction have led to development of techniques such as pressurised hot water extraction. Water is forced through a natural material at high temperature and pressures  (in excess of 9 bar). This allows water to be heated beyond its boiling point to facilitate a more           efficient extraction. In this experiment, a household espresso machine will be used to simulate these conditions.

Solubility and Extraction

Extraction processes depend on the relative strength of intermolecular attractive forces between the species in solution to the solvents used. Commonly a mixture of polar and nonpolar solvents are         used, forming two immiscible layers.  Polar organic molecules and inorganic salts preferentially            dissolve (are attracted more strongly) into the more polar solvent.  Large organic molecules which      are predominantly nonpolar in nature preferentially dissolve in the organic or nonpolar solvent.

As the number of polar substituents and those that can participate in hydrogen bonding increase, the solubility of an organic molecule in polar solvents increases.  A change in pH can also increase water  solubility of organic molecules with acidic or basic substituents that react with OH-  or H+ to form         charged groups on the structure.

The extraction solvents used in this experiment are ethyl acetate, acetone and water (see Fig 3.1)

Major constituents in coffee beans: Roasted coffee beans contain many hundreds of compounds responsible for the taste, colour and aroma, and caffeine is a relatively minor component.

Fig 3.2a. caffeine structure

It is surprising to note that the caffeine molecule is insoluble in water at room temperature.  In the presence of acid, the nitrogen becomes protonated to form a positively charge species which is      water soluble.

Fig 3.4

Thin Layer Chromatography

Before the properties and/or structure of a compound can be examined, it must be pure. One very useful technique for establishing purity is thin layer chromatography (this being classed as one type of liquid-solid chromatography).

In the thin layer chromatography experiment, the sample to be analysed (which will frequently           consist of a mixture of the compound of interest and one or more impurities) is first adsorbed onto   an inert substance called the stationary phase (in this experiment a thin layer of2silica gel – SiO2 ·xH2O – on an aluminium backing) which has some polar groups. The stationary phase is then brought into  contact with a less polar organic solvent (mobile phase). As the solvent moves past the point on the  TLC plate where the mixture has been applied, the components of the mixture move different            distances depending on their polarity.

Polar organic molecules, having a higher proportion of oxygen- and nitrogen-containing functional     groups, are attracted more to the polar stationary phase and don’t shift far from the point at which   they were applied. Less polar compounds containing a higher proportion of carbon and hydrogen are less attracted to the polar stationary phase and move further from the point of origin than more        polar compounds.

Developing communication skills in the business/industrial context.

Government and private companies, large and small, consult with experts in various fields to assess the risks and benefits of new ventures, investments and innovations (a cost-benefit analysis).           Consultants are expected to produce a detailed report, but for ease of review of the findings, these reports begin with an Executive Summary.  This is a brief summary of the methodology used to        assess the proposal, the major findings and a recommendation from the expert, weighing up all       factors.  As a scientist, with expertise in chemistry, you may be expected to act as a consultant; or,  after obtaining your degree, you may work for a company or create your own company to provide  these consultation services.  In this experiment, you are going to have a taste of this type of              professional engagement and reporting style.

A pharmaceutical company wishes to extract caffeine from ground coffee for use in their line of caffeine containing tablets. They have enlisted the help of The University of Melbourne Chemistry Department to determine in which technique they should invest. They have requested that a comparison of two extraction techniques (Soxhlet and high temperature/pressure) be undertaken in order to determine which technique is the most suitable with respect to yield by percentage mass of ground coffee, cost of chemicals and processes, and environmental impact.

Your task:

i) To perform a Soxhlet extraction using a small amount of an organic solvent and

ii) To use a high pressure/temperature extraction with water as the solvent (the use of an espresso machine mimics these conditions well).

iii) You will determine the percent by mass of caffeine in ground coffee using each of these extractions and the purity of the extractions via thin layer chromatography (TLC).

iv) You will summarise your percent by mass of caffeine and purity results and recommend a method for the executives of the company in the form of an executive summary.

EXPERIMENT REPORT

There is a complete report template for students to complete and submit.  All your results and         answers can be entered there and the report format is in the template - you do not need to copy or write any supplementary report.

Print it and have it ready for entering data.

You can complete any parts of your report before your Lab session that you feel confident to do. Practice the calculations.  Prepare well and have questions ready to ask your demonstrator. It is highly recommended that you aim to complete all your report during your lab session while  you are able to question your demonstrator.

SAFETY

Safety Warning:

Ethyl acetate and acetone are volatile and flammable liquids.  Keep away from

Risk Assessment

Before you undertake this experiment, you must read through the experimental procedure,             including the Risk Assessment sheet. For On-Campus classes: Please sign the declaration at the      end of these notes to acknowledge that you have read and understood the information on the     Risk Assessment sheet. Your demonstrator will check and COLLECT this declaration at the start of class.

EXPERIMENTAL PROCEDURE

This is a group experiment – half the group will perform Method A and half the group will perform Method B – students will then share results.  For both Method A and Method B, students will work in pairs.

The flow chart providing guidelines for the Soxhlet extraction (Method A) and extraction using espresso machine (Method B) is given below in Figure 3.5.

Figure 3.5 The flow chart for the Soxhlet extraction (Method A) and extraction using espresso machine (Method B)

REFERENCES

1.  Figure 3.3 adapted from http://www.chem.science.unideb.hu/Pharm/Notes5.pdf (2018).

Part A: Extraction of Caffeine from Ground Coffee

Using a top-loading balance, weigh approximately 1.0 ̶ 1.2 g of coffee into a Soxhlet thimble. You   will need to gently tap the thimble to fit in more coffee. (It is easier to stand your thimble in a small beaker while weighing). Place the thimble into the Soxhlet apparatus.

Record the exact amount weighed into Table 1

The following Steps 2 to 6 of Method A MUST be conducted in a fumehood.

1.  Place 3 to 4 boiling chips in a CLEAN and DRY 50 mL round bottom flask and add 25 mL of acetone. Attach the Soxhlet apparatus to the neck of the round bottom flask

(see diagram on right). Your demonstrator will show you the set up for the

Soxhlet apparatus and refer to page 159.

2.  The water condensers have already been arranged in the fumehood and held gently in place by a clamp. Slide the top of the Soxhlet apparatus over the      bottom of the reflux condenser, then place the base of the round bottom       flask over the steam-bath.

(NOTE: Do not tighten the grip of the clamp as this can lead to breakage of

the water condenser)

3.  Allow the acetone to reach boiling and complete 15 cycles of extraction (one Soxhlet cycle is completed once the solvent from syphon arm has returned   to the round bottom flask).

This normally takes 10 ̶ 15 minutes. You should obtain a brown/yellow solution at this time.

4.  After you have completed 15 cycles and obtained a coloured solution, disassemble the Soxhlet apparatus and place the extractor in the tub provided in the fumehood (including the thimble  with coffee grounds).

5.  Evaporate acetone to dryness, inside the round bottom on a steam bath in the fumehood, leaving brown solid residue at the bottom offlask. This normally takes about 5 minutes.

6.  Remove the round bottom flask from steam bath and allow the flask to cool for 3 to 4 minutes at your laboratory space before continuing onto the next step.

7.  Add 10 mL of 2 M NaOH solution, via zippette, into the round bottom flask and swirl thoroughly to mix for 2 ̶ 3 minutes.

8.  Transfer the solution mixture gently from the round bottom flask into the separating funnel.

NOTE: Avoid disturbing any brown residue and boiling chips as much as possible. Particularly avoid transfer of any boiling chips into the separating funnel as they may block the tap.

It is IMPORTANT to release the pressure in separating funnel in a fumehood.

9.    Extract caffeine into ethyl acetate: Add 15 mL ethyl acetate, via zippette, to the separating funnel. (Note: your demonstrator will explain how to use the separating funnel).

Drain and discard the LOWER aqueous layer into a 250 mL beaker, closing the tap, as the meniscus separating the two layers just reaches it (see Figure 3.6).

Figure 3.6 Separating funnel.

10. Add 5 mL of 2M NaOH to the UPPER layer (ethyl acetate) in separating funnel and repeat the    extraction procedure in Step 10. Drain and discard the LOWER aqueous layer into the same 250 mL beaker.

11.Repeat Step 11 with another 5 mL of 2 M NaOH solution.

12. Drain the UPPER layer of ethyl acetate into a CLEAN and DRY 50 mL conical flask.

13. To remove any water dissolved in the ethyl acetate layer before boiling off:

a)  Add approximately one spatula full of magnesium sulphate (MgSO4 ) powder to the ethyl     acetate in the conical flask (from Step 13). When the magnesium sulphate no longer clumps and is suspended in solution when swirled, it is a good indication your compound is dry.

b)  Add 2 to 3 boiling chips to a CLEAN, DRY and LABELLED 50 mL beaker. Weigh the beaker using an analytical balance and position it underneath the glass filter funnel in fumehood.

• Record the mass into Table 1 in your report.

c)  Place a piece of fluted filter paper into the filter funnel (see Figure 3.6).

Figure 3.7 Method for fluting filter paper

d)  Pour the MgSO4/ethyl acetate mixture through the filter paper into the above PRE-WEIGHED

50 mL beaker. This is to remove the magnesium sulphate from the ethyl acetate by gravity filtration.

14. Place the beaker on the steam bath to evaporate all of the ethyl acetate (about 15 mL) to dryness. A white residue should appear at the bottom of beaker.

15. Remove the beaker from the steam bath with a wooden/plastic peg and allow it to cool to room temperature.

• Weigh the beaker, with the boiling chips still inside, using an analytical balance and record the mass to determine the % by mass of caffeine obtained.

• This caffeine sample is used to perform thin layer chromatography in Part B.

1.  Using a top-loading balance, weigh approximately 15 – 16 g of coffee into an espresso group head, then pack the sample mixture down using the coffee tamper provided.

• Record the exact amount weighed in Table 2 in your report.

Figure 3.8 Example of group head packed with sample mixture

2.  Attach the group head with your coffee sample and turn the group head handle to the right until the group head locks into place.

3. Place a 250 mL beaker under the spout of the group head (Figure 3.8) and turn the dial to the       LEFT for extraction. This extraction should take approximately 30 seconds and you should collect

approximately 50 – 60 mL of a brown coffee extract. This is to be shared by a number of groups – usually, it is not necessary to perform another extraction.

4.  Empty the coffee extract into the 100 mL measuring cylinder and allow the bubbles to settle.

• Record the exact volume of coffee extract in Table 2 in your report.

5.  Measure 10 mL of coffee extract using a 10 mL measuring cylinder and transfer into the separating funnel with the valve closed.

6.  Add 5 mL of 2 M NaOH solution, via zippette, to the coffee extract in separating funnel and swirl to thoroughly mix.

It is IMPORTANT to release the pressure in separating funnel in a fumehood.

7.  Add 15 mL ethyl acetate, via zippette, to the separating funnel funnel (Note: your demonstrator will explain how to use the separating funnel).

Drain and discard the LOWER aqueous layer into a 250 mL beaker, closing the valve, as the meniscus separating the two layers just reaches it (see Figure 3.6).

8.  Add 5 mL of 2 M NaOH to the UPPER layer (ethyl acetate) in separating funnel and repeat the extraction procedure used in Step 7.

Drain and discard the LOWER aqueous layer into a 250 mL beaker,

9.   Repeat Step 8 with another 5 mL of 2 M NaOH solution.

10. Drain the UPPER layer of ethyl acetate (about 15 mL) into a CLEAN and DRY 50 mL conical flask.

11. To remove any water dissolved in the ethyl acetate layer before boiling off:

a)  Add approximately one spatula full of magnesium sulphate (MgSO4 ) powder to the ethyl     acetate in the conical flask (from Step 10). When the magnesium sulphate no longer clumps and is suspended in solution when swirled, it is a good indication your compound is dry.

b)  Add 2 to 3 boiling chips to a CLEAN, DRY and LABELLED 50 mL beaker.

c)  Weigh the above beaker using an analytical balance and position it underneath the glass filter funnel.

• Record the mass into Table 2 in your report.

d)  Place a piece of fluted filter paper into the filter funnel (see Figure 3.7).

e)  Pour the MgSO4/ethyl acetate mixture through the filter paper into the above PRE-WEIGHED

50 mL beaker to remove the magnesium sulphate from the ethyl acetate.

12. Place the beaker on the steam bath to evaporate all of the ethyl acetate (about 15 mL) to dryness. A white residue should appear at the bottom of beaker.

13. Remove the beaker from the steam bath with a wooden/plastic peg and allow it to cool to room temperature.

• Weigh the beaker, with boiling chips still inside, using an analytical balance and record the mass to determine the % by mass of caffeine obtained.

• This caffeine sample is used to perform thin layer chromatography in Part B.

Part B: Thin Layer Chromatography (TLC)

1.  Take the TLC plate provided and be careful not to touch the surface with your fingers. Lightly draw a pencil line 1.5 cm from the bottom edge using a ruler. Then evenly mark this line with three small “dots” as shown in Figure 3.8. These marks will offer a guide when spotting your samples:

A.   Caffeine obtained from Method A

B.   Caffeine obtained from Method B

C.   Caffeine reference standard (provided)