Gravimetric Assay of Caffeine in Commercial Tablets Keywords Organic by fuk43069


									                              Chem 127 Lab Experiment #3
       Gravimetric Assay of Caffeine in Commercial Tablets

     Keywords: Organic Chemistry, pharmaceutical analysis, gravimetric analysis

•   To extract caffeine from an appropriate source and through the use of mass
    measurements determine the % caffeine in the tablet.

•   To react the caffeine obtained in the first part of the reaction with salicylic acid and
    obtain the corresponding salt.

•   To determine the % efficiency of the formation of the salt.

When we take a pill or any kind of medication, it is important to know that we are taking
exactly the proscribed amount of the pharmaceutical. The packaging is required to clearly
state the amount of that active ingredient in the tablet and there are stringent checks in
place to ensure this is accurate. Whereas in an ideal case, the amount of the active
ingredient could be determined by simply weighing the pill, this relies on the pill
consisting of the pure material. In reality, this is not the case, many additional materials
are added from stabilizers (to prevent unwanted reactions or degradation of the active
ingredient), colorings (to make the pill look pretty), binders (to hold the pill together) and
in some cases, bulking agents (to increase the mass of the pill – important for drugs
where only a few mg of the active ingredient is present in each tablet). Hence, to
accurately determine the quantity of an active ingredient, all of these extra materials must
be removed.
In this experiment, we will take commercially available caffeine tablets, grind them to a
powder, then through use of filtration to remove insoluble impurities, selective
partitioning between two solvent systems and finally evaporation, obtain the pure
caffeine. Based on the amount of caffeine obtained, we can estimate the % caffeine in the
original pill. In the second half of the lab, we will carry out a simple organic chemistry
reaction and recrystallization by reacting this caffeine with a simple organic acid to
prepare an adduct and we can determine the % efficiency of this reaction (shown in
reaction 1 below.) We have chosen caffeine for this lab as it is readily available, non-
toxic in the doses we are working with and is stable under the laboratory conditions.
Caffeine, more properly known as 1,3,7-trimethyl-1H-purine-2,6(3H,7H)-dione is a
xanthine alkaloid chemical of the empirical formula C8H10N4O2 and the structure shown
on the next page. Pure caffeine melts at 238 ºC but does sublimate (transform directly
from a solid to a gas) at 178 ºC at 1 atmosphere pressure. In nature, caffeine is found in
the leaves of a number of plants where it acts as an insecticide killing and paralyzing
certain insect predators although it is much more well known for its stimulatory effects in

                       Experiment #3 – Gravimetric Caffeine Assay                          3-1

                                        N       N       O

                                   H3C          O

                                   Caffeine (C8H10N4O2)
                                       Fwt 194.19 g
Found and consumed in a wide variety of sources from tea and caffeine, soda and energy
drinks and selected pharmaceuticals. Caffeine is absorbed in the stomach and lower
intestine from where it is then distributed throughout the body upon which it acts as a
central nervous and metabolic stimulant. It is estimated at least 90% of Americans
consume caffeine on a daily basis.
Pure caffeine is a neurotoxic white solid powder which in modern times has been
extracted and purified through soaking coffee beans in water and obtaining the caffeine
from the resulting solution often simply through evaporating off the solvent. Numerous
solvents were used in the past but most have been discontinued for environmental
reasons. Due to the small scale upon which this experiment is carried out in the teaching
lab and the time constraints, we will carry out our extraction from the pill using one of
these solvents, methylene chloride (CH2Cl2).
The complication as mentioned previously is that the pills do not comprise pure caffeine
but rather contain a mixture of undesired materials. However, we can utilize the
partitioning principle to selectively obtain the desired caffeine. First the pill is ground to a
powder then added to boiling water – all soluble materials dissolve but insoluble
materials such as the corn starch do not and can be removed by filtration. Secondly by
mixing the aqueous solution with a non-aqueous solvent such as methylene chloride
which does not mix with the water and forms a separate layer, we can extract the
caffeine. Non-polar materials i.e. the caffeine will preferentially partition into the non-
polar solvent (methylene chloride) and careful separation of the layers allows us to obtain
a solution of pure caffeine in the methylene chloride which can then be evaporated after
drying to obtain the pure caffeine. The separation principle is further illustrated by the
partitioning of the food coloring from the tablet which is a polar material and exclusively
partitions into the aqueous layer. The result after mixing both layers and allowing to
separate is a yellow aqueous layer and a clear organic layer.
Once the layers are formed these can be separated by filtration – since methylene chloride
and water to not mix, by wetting a piece of filter paper with water addition of both layers
to the filter funnel results in the aqueous layer passing through the paper and the organic
layer remaining in the funnel. This can be poured into another container, dried using
sodium sulfate (a drying agent) and the methylene chloride can be evaporated in the hood
leaving behind the pure caffeine.
Based on the mass of caffeine, you can now determine the % weight of caffeine in the
original tablet. How does this compare with what the packaging states?

3-2                     Experiment #3 – Gravimetric Caffeine Assay
In the second part of the reaction, the derivatization reaction, caffeine is reacted with
salicylic acid as shown in Equation 1 to yield the corresponding salt Caffeine Salicylate.
Salicylic acid possesses a closely related structure to that of aspirin and is found naturally
in sources such as willow bark. This reaction is carried out and a % yield obtained
showing the efficiency of this reaction.
                                                                              H      CH3
    N     N       O                      O
                                                                    O       + N      N       O
              N         +                    OH
      N                                                                                  N
                  CH3                                                            N
H3C                                    OH                                                    CH3
          O                                                       OH       H3C       O
Caffeine (C8H10N4O2)        Salicylic Acid (C7H6O3)              Caffeine Salicylate
Fwt 194.19 g                Fwt 138.12 g                         (C15H16N4O5) Fwt 332.31 g

                  Equation 1. The Reaction of Caffeine with Salicylic Acid.
To carry out this reaction, you will have to determine the number of moles of caffeine in
your flask at the start. From this determine how much 1.5 mole equivalents of salicylic
acid would weigh. This is how much salicylic acid you will add.
Why do we add more salicyclic acid than needed? This is done to ensure complete
reaction of all caffeine molecules, the procedure followed removes any unreacted
salicyclic acid leaving behind the pure adduct, caffeine salicylate. In the Organic Lab,
this could be characterized by a number of tests such as IR spectroscopy, melting point
determination, GC-MS but for today, we will assume it is pure and simply determine the
mass of the product.
We now can determine the % yield for this reaction but unlike the copper experiment
where you simply divided final mass by starting mass, the formula weights of the product
and starting material are different. In this case, starting with 1 g of caffeine and ending
with 1 g of product does not reflect a 100% yield. Instead to obtain a % yield, the masses
are converted to number of moles.
   % yield = (# moles of product / # moles of starting material ) x 100
What do we use for starting material since this can be either caffeine or salicylic. We use
the smaller of the two since this is the limiting reagent, in this case the caffeine is our
limiting reagent. This can be illustrated through considering a simple reaction where A
reacts with B to give C. If we have 2 A and 2 B, we obtain 2 C. Simple enough. But if we
have 200 A and 2 B, the maximum we can obtain is still 2 C. We require both
components A and B to make C and B is the limiting reagent. You cannot make more of
a material than what the limiting reagent would allow.
Safety Considerations
   Methylene chloride is mildly harmful and you should avoid spilling this on your skin
   or inhaling the vapors. Methylene chloride is also harmful to the environment so
   dispose of all waste in the appropriate containers.

                         Experiment #3 – Gravimetric Caffeine Assay                          3-3
                               Experimental Procedure
Required Chemicals
       • Methylene Chloride (CH2Cl2)               • Salicylic Acid
       • Anhydrous Sodium Sulfate                  ●    Hexanes

Experimental Procedure: You will work in pairs for this reaction
Troubleshooting Notes:
During the separation steps, it is possible that the layers may form an emulsion and not
clearly separate, a common cause of this is the densities being too similar. If this
happens, you will not see 2 distinct layers but the problem can be rectified by adding
more water to the system. This reduces the density of the aqueous layer and allows
Methylene chloride is relatively volatile possessing a low boiling point and so is easily
lost to evaporation. If you find your organic layer is becoming too small, you may have to
add additional methylene chloride. Be wary of adding too much as it will take
significantly longer to remove at the end of the experiment. You should also avoid
inhaling any of the vapors from the methylene chloride.
Dr Wyllie’s Golden Rule of Separation #45 should be heeded at all times. Never ever
dispose of any layers unless you are absolutely sure it is the correct layer to dispose off.
Once it goes down the sink or into the waste bottle it is gone forever. In addition,
methylene chloride should never be poured down the sink.
A. Extraction of Caffeine
1) Determine the mass of one caffeine tablet after removing it from the packaging.
2) Grind the caffeine tablet to a fine powder using the pestle and mortar
3) Using a hotplate and a small beaker, boil ~ 25 mL of water
4) When the water is boiling, add the powdered tablet and continue to boil for several
5) Allow the mixture to cool to room temperature and filter through filter paper into a
   125 mL Erlenmeyer flask (the solid material left on the paper is predominantly corn
   starch – this can be disposed of into the trash)
6) Add ~ 10 mL of methylene chloride to the flask and stopper the flask.
7) Swirl the flask for several minutes. Record your observations.
8) Take a fresh piece of filter paper, fold and place in the funnel. Wet this thoroughly
   using your water bottle.
9) Pour the solution through this filter paper, only the aqueous layer will go through.
10) Transfer the solution from the funnel into a clean 125 mL Erlenmeyer flask. Stopper
    this flask and keep it safe.

3-4                    Experiment #3 – Gravimetric Caffeine Assay
11) Add another 5 to 10 mL of methylene chloride to the aqueous solution and swirl then
    pour through the same wet paper. Again the aqueous layer will pass through and the
    organic layer will remain in the top. Combine the organic layer with the one from
12) To the organic layer, examine the solution – does it look wet? Add a small quantity of
    sodium sulfate to the flask and swirl – does it look drier? Do you still see water
    droplets around the top of the solution. If so, add more sodium sulfate. Repeat until
    the sodium sulfate retains a granular appearance after swirling.
13) Pour off the methylene chloride into a clean dried, weighed 50 mL Erlenmeyer flask
    ensuring no solid material is transferred. Add ~ 2mL of fresh methylene chloride to
    the solid left behind, swirl and pour this into the Erlenmeyer too.
14) In a tray of warm water heated on a hot plate in the hood, swirl the Erlenmeyer flask
    gently boiling off the methylene chloride. When this is done, you should be left with
    a solid material (caffeine) If you heat this too high, you will sublime the caffeine
    dramatically reducing your yield.
15) Determine the mass of the extracted caffeine by weighing the flask after drying the
B. Derivatization of Caffeine
   1. Based upon the mass of caffeine obtained above, convert this to moles and
      assuming as described earlier that you need 1.5 mole equivalents of salicyclic
      acid, determine the number of moles and hence the mass of salicylic acid required
      for complete reaction.
   2. Add the correct amount of salicylic acid to the Erlenmeyer flask containing your
      caffeine sample followed by 2 -3 mL of methylene chloride.
   3. Warm the flask gently to get all the solid to dissolve. If complete salvation is not
      obtained, add a further 1 mL of methylene chloride.
   4. Allow the solution to cool and add ~ 5 mL of hexanes. Place the solution in an ice
      bath and gently scratch the sides of the Erlenmeyer flask with a glass stirring rod.
      This should induce crystallization and you should see formation of small white
   The adduct is not soluble in hexanes so will precipitate out following hexanes
   addition. Caffeine and unreacted salicylic acid are soluble so selective precipitation
   is observed. The solubility is decreased as temperature decreases so cooling in an ice
   bath maximizes % yield.
   5. After 10 minutes or no further crystal formation is observed, filter the solution
      collecting the crystals on the filter paper. Dispose of the liquid in the halogenated
      waste bottle provided.
   6. Allow the crystals to air dry for approximately 10 minutes then determine the
      mass of the product. Record this and the appearance of the sample in your

                      Experiment #3 – Gravimetric Caffeine Assay                        3-5
Data Analysis / Writing the Report
A. Extraction of Caffeine
         From the mass of caffeine obtained and the initial weight of the pill, what is the %
         weight of caffeine in the pill?
         Examine the packaging of the pill and record how much caffeine should be in the
         pill. How does this compare to the amount you obtained. Why is this higher or

         Calculate the absolute error between the mass of caffeine you obtained and the
         mass as stated by the packaging.

B. Derivatization of Caffeine
      Analysis of the salicylic acid obtained from the derivatization of caffeine is done by
      calculating a % yield for this reaction. Because the formula weight of caffeine and the
      product are different, we have to convert these to moles to determine % yield.
      % yield = (# moles of product / # moles of starting material ) x 100
      What should the expected % yield be? Why is the number you obtained for % yield
      different? How could you improve this?

3-6                      Experiment #3 – Gravimetric Caffeine Assay

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