Caffeine Extraction from Tea

This is one of the most popular, and fun, labs in the TOPS Chemistry Program. It could be used as a cool introduction to chemistry labs at the beginning of the school year or as a fun wrap-up at the end of the course.  The techniques are simple and straight forward and only really require students to follow directions and proper safety protocols, i.e. don’t touch a hot hotplate, wear safety goggles, etc.

A couple of years ago we were on the verge of not being able to provide it any longer because we ran out of the extraction solvent that had been used for years, 1,1,1-trichloroethane.  Turns out it’s an ozone-depleting chlorinated hydrocarbon regulated by the Montreal Protocol being rapidly phased out.  So, while our multi-year supply started to dwindle, we scrambled trying to find a suitable substitute.  The challenge was finding a solvent that not only worked, but that could be used in a high school science classroom.  As many of you know, the solvent of choice for these types of extractions in a college lab is methylene chloride (dichloromethane), however, it can’t be used in a high school setting.

CAUTION

Hexane and isopropanol are EXTREMELY FLAMMABLE.  Do not use near open flame!  Hot plates can cause burns, be careful.  Caffeine is poisonous in large quantities, exercise caution when handling it and wash your hands when you leave the laboratory area.  Goggles are mandatory whenever you are in the lab area and laboratory work is being done.  See the MSDS sheets for additional health and safety concerns.

PURPOSE
To extract and measure the caffeine in a tea bag.

PROCEDURE
1. Put on your goggles.  Check that your lab station has all supplies as listed above.

2. Find the mass of a tea bag with tea.  Record the mass.  Copy the mass of the empty tea bag from the chalkboard.

3. Place 10 mL of a 1 M Na₂CO₃ solution in a 50 mL beaker.  Add tea bag, cover with foil until solution boils, then remove foil.  Boil for 5 minutes.  Note:  Add distilled water from wash bottle as needed to maintain 10 mL level.  Keep the bag off the bottom of the beaker to avoid burning a hole in the bag.

4. Remove beaker from hot plate carefully, using tongs; let air cool for a few minutes; then cool beaker in ice bath; squeeze out excess fluid from tea bag being careful not to break bag.  Discard the tea bag.  At this point you should have no more than 10 mL of solution.  If you have more, boil off some of the water to concentrate the solution.  (Make sure 50 mL beaker is dry on the outside before putting it back on the hotplate).  Cool the extract (liquid) for one minute in ice bath.

5. Pour the cooled extract into a 15 mL centrifuge tube.

6. Now, using the dispensette, pump 3 mL of hexane/isopropanol into your centrifuge tube and screw  on the cap.  Gently shake the tube for 10 seconds then relieve pressure by partially unscrewing the cap.  If tube is shaken vigorously and opened quickly, contents may spew out of vial.  Retighten the cap and shake gently for at least 30 seconds.  Relieve pressure in tube every ten seconds by partially unscrewing the cap.

7. Dry the outside of the tube and tighten the cap.  Place tube in centrifuge. Note the centrifuge number for your tube. Centrifuge the mixture for three to four minutes, being sure to balance the centrifuge with another tube of approximately equal volume.

8. Carefully remove the clear top layer of liquid with a glass Pasteur pipet and place it in your numbered glass vial.  Avoid putting the pipet tip into the lower aqueous phase – you want the top layer (or organic phase).  Squeeze the bulb of the pipet before putting the tip of the pipet into the liquid.  Put the pipet tip just below the surface of the top layer in the centrifuge tube and release the bulb slowly.  This will be a mixture of your product and hexane/isopropanol.  Repeat this process until most of the top layer has been transferred into your group’s numbered vial.

9. Repeat the extraction process (See steps 5-8) two more times using 3 mL of fresh hexane/isopropanol each time.  After each subsequent extraction be sure to pipet the clear top layer into your vial.  Avoid taking any of lower aqueous layer.  Screw the cap on your vial and note and record the number of the vial.

10. After the third extraction is completed, discard remaining lower dark layer into a waste container provided by your teacher.  Save your vial with its contents for the next part of the lab.

—STOP-HERE-IF-YOU-DO-NOT-HAVE-TIME-TO-COMPLETE-THE-LAB-TODAY—

11. Add a small amount (pea-sized) of Na₂SO₄ crystals to your vial of solvent and caffeine.  Screw cap on vial and swirl gently.  The crystals will clump.  Continue to add small amounts of crystals to the vial and swirl it until the addition of new crystals does not produce more clumping.  You should observe individual crystals of Na₂SO₄ in your vial as you swirl it.

12. Using the stirring rod, decant the fluid portion of your vial into the 100 mL beaker.  Avoid getting any crystals of Na₂SO₄ into the beaker.

13. After the decanting process is completed the hexane/isopropanol mixture must be carefully and slowly evaporated leaving only the crude caffeine behind.  To evaporate the hexane/isopropanol mixture, place the 100 mL beaker into a sand bath heated to 90°C in the fume hood.  Gently swirl the beaker in the sand bath.  Just as the solvent has evaporated, remove the beaker.  The residue can be slightly moist.  The crude caffeine will coat the bottom of the beaker.

Caution: DO NOT BOIL THE BEAKER DRY!

14.  Assemble the sublimation apparatus as shown in Figure 1.  The outside of the inner 50 mL beaker must be very clean and dry.  Do not add ice until after the beaker is placed on the hotplate.

15. Place the sublimation apparatus on a hot plate and begin heating.  Fill the small beaker with chips of ice.  Be careful not to let ice or water fall into the larger beaker.

Figure 1  SUBLIMATION APPARATUS

16. Continue heating until the sublimation process is complete.  When heated, the pure caffeine will sublime and then be deposited on the outside of the 50 mL inner beaker.

17. Once sublimation is complete, remove the apparatus from the hot plate with tongs and allow to cool.  Remove the inner beaker very cautiously and carefully pour off the ice water making certain no water comes into contact with the sublimed caffeine crystals.

18. Dry the inside of the 50 mL beaker with Kimwipes and find the mass of this beaker with the caffeine on its bottom.  Record this mass.  Make careful observation about the physical properties of caffeine.  Scrape the caffeine from the beaker and turn the product in to the instructor.

19. Carefully clean and dry the beaker you used.  Find the mass of your clean, dry 50 mL beaker and record.

20. Find the difference in masses and record as the mass of caffeine obtained.

Factors Affecting Enzyme Activity (Amylase)

In this lab we study the activity of enzymes, a very important group of proteins.  They speed up nearly all the important biochemical reactions within a cell (which would otherwise take an exceedingly long amount of time) and thus are an indispensable part of life on earth.  Specifically, we’re looking at how certain external factors (temperature, pH, etc.) can affect the activity rate of enzymes.

For this activity we’re using the catalytic enzyme amylase, which breaks down starch (a polysaccharide made up of amylose and amylopectin) into maltose (a disaccharide) and dextrin.  Humans produce amylase in the salivary glands and pancreas to aid in the digestion of starchy foods, the most common carbohydrate we consume.

How do we use this information to set up a test (assay) for the evidence of enzyme activity, i.e. production of maltose?  Well, we know that maltose is a reducing sugar (you all remember redox reactions, OIL RIG and all that, right?)  that reacts with and reduces the pale yellow colored alkaline 3,5-dinitrosalicylic acid (DNS) to the orange-red colored, 3-amino, 5-nitrosalicylic acid after being heated for 5 minutes.  Essentially, the intensity of the color is proportional to the concentration of maltose present in the solution (per Beer’s Law).  Thus, increased amylase activity produces more maltose, which reduces more DNS, which then turns the solution a darker orange-red.

Once we’ve done that, we can use a spectrophotometer to do a colorimetric analysis of samples and figure out how much maltose was produced in different conditions.  A higher absorbance will correlate with the darker color (higher optical density).  We use a wavelength of 540nm, which is the lambda max for the orange-red color of the “reduced” DNS.  The resulting numbers can then be graphed to see the pattern of enzyme activity in changing external conditions.

Introducing the spectrophotometer into the lab is a good way to show students the difference between qualitative and quantitative analysis.

Now that we figured out a test for the presence of maltose, and hence the activity of amylase, what do we use as the substrate?  As you know, we use amylopectin (from corn).  But why don’t we just use a starch solution with potato or corn starch instead?  Remember that starch is made up of amylopectin and amylose, both polysaccharides.  Amylose is a linear, helical (more tightly packed) polymer of glucose while amylopectin is a highly branched polymer of glucose.  Those branches in amylopectin provide more surface area for the amylase to break it down into maltose units.  So, using amylopectin will yield a slightly higher production of maltose during the 10 minutes allotted for the reaction than using either starch or amylose.

Which brings me to the next question, what’s the purpose of the tartrate solution?  Remember, maltose is a reducing sugar  What do you think will happen if it reacts with dissolved oxygen in the solution?  We add the tartrate to prevent oxidation of the maltose and stabilize the color.

Before we get to the lab procedures, let me mention a few things to keep in mind before actually running the lab with your students.

1.- Set up the water bath at least 2 hours before the start of the lab.  If it’s the first period of the day, it might be better to set the temperature at about 6 and leave it on over night, then in the morning raise the temperature to the highest setting.  Remember, use only distilled water!

2.- Make sure the Brinkmann Dispensettes are pumping correctly and primed before the students use them.  There should be no air bubbles in the glass cylinder or discharge tube.  You’ll have to go over proper operation with your classes.  Point out that the piston must be raised slowly and evenly, then gently pushed down.

3.- It would be very helpful to have students practice using the micropipettes before doing the lab.  The accuracy of the data obtained by each group is affected by how well they measure and dispense the different volumes required.  So, a little practice beforehand will help tremendously.  Have students pay close attention to where the three “stops” are located, they should be able to feel where each one is.  Also, make sure they keep track of the upper and lower limits for each micropipette. They should never go below 100uL! They will jam the micropipette if they do this.  The 50uL of amylase needed for test tube 2 in Part B will be measured using a different micropipette set aside just for this task.

4.- Timing matters!  We’re measuring the activity of an enzyme under different conditions during a set amount of time (10 minutes).  It’s important that students understand when the timing should start, i.e. when enzyme and substrate combine.  It seems an obvious point, but many students don’t get it at first.  They need to be aware of when, for the part they’re working on, they’re mixing the enzyme and substrate and be ready to keep track of the reaction time.  Something that can be asked to confirm understanding of the process behind the lab procedures is whether it matters if you add the reagents with the dispensette before adding the reagents with the micropipettes.  Of course for Part A and D it doesn’t matter, but it will for Part B and C.

Part A:  Measuring the Amount of Maltose Present in a Sample.

The data obtained from this part of the lab will be used to create a standard curve pairing an absorbance reading with known amount of maltose.