Experiment 10 Overview

EXPERIMENT 10
Caffeine in Soft Drinks

PRE-LAB REQUIREMENTS
PRE-LAB REQUIREMENTS FOR EXPERIMENT 10 ARE…...

1. Read the entire experiment, contained in the following pages

2. Read the Experimental resources:

Become familiar with the techniques used in the experiment by working through the
following resources:

Techniques:
• Working in a Fume Hood Video – Complete the Fume Hood Quiz
• Extraction
• Purification (Vacuum Sublimation and Melting Point Analysis)
• Rotary Evaporators

Virtual Lab:
• Complete the Virtual Lab Experiment 10: Extraction of Caffeine from Cola
• Bring the Feedback Form, for the Evaluation questions, to the lab
• No late submissions will be accepted

3. Complete the Experimental Design Form (Pre-lab)

Complete the Pre-lab portion of the Experimental Design Form:
As you design your procedure and complete the EDF (in pen):
• Use the Experimental Overview and Virtual Lab Tutorial to assist with
designing your procedure
• As you read through the Experimental Overview, choose glassware,
equipment and chemicals for each step from the Laboratory Stockroom

4. Library Research Assignment:

Use CRC Handbook of Chemistry and Physics to find the literature values for:
• melting point of caffeine
• boiling point and density of dichloromethane
Cite these values in your Introduction and write them on your EDF (before the lab)

5. Prepare for the In-lab Quiz:

The pre-lab quiz will be based on the following areas of this experiment:
• Techniques
• Calculations
• Chemical theory
If you have prepared well for the experiment, the quiz should be straight forward

There is a lot of work for this experiment. Do no leave it until the last minute or you will be overwhelmed
 Experiment 10 Overview

EXPERIMENT #10 LABORATORY LEARNING GOALS

A. Basic laboratory skills
A1. Adhere to instructions on laboratory safety, recognize hazardous situations, and act appropriately. (Refer to A1.b in the
General Laboratory LGs.)
A1.a: General Safety: Cautiously handle dichloromethane, a carcinogenic organic solvent. Do not release pressure from
the separatory funnel while pointing it at another student.
A2. Perform common laboratory procedures correctly (new and old). (Refer to A2.b & c in the General Lab LGs.)
A2.a: New laboratory procedures
• Perform a Liquid-Liquid Extraction (as outlined in the lab manual)
• Remove residual water from the dichloromethane layer (Gravity filtration)
• Use dichloromethane’s physical properties to isolate crude caffeine
• Carefully evaporate volatile liquids in a fume hood
• Purify solids using Vacuum Sublimation (as outlined in the lab manual)
• Perform Melting Point Analysis
o Use proper technique to measure the melting range of your crude and purified samples
A3. Maintain an up-to-date laboratory record that is of sufficient detail that others could repeat any of your experiments if
necessary. (Refer to A3 in the General Laboratory LGs.)
A3.b: Accurately record data and observations in an organized way
• Prepare data tables and record experimental results/observations in the flowchart
A3.c: Measure (and report) uncertain quantities with appropriate precision
• Consider the precision of the experiment with respect to the significant figures afforded by the
procedure
A4. Apply appropriate methods of analysis to data and convert raw data into meaningful units.
• Calculate the amount of caffeine in the cola sample (original vs. lab sample volumes)
• Compare caffeine levels between cola brands
• Compare literature melting points to measured values
A5. Perform logical troubleshooting of laboratory procedures.
• Recognize which layer in the separatory funnel contains the desired compound
• Identify procedures to prevent and/or deal with an emulsion or aid layer separation
• Evaporate the dichloromethane at a rate which effectively removes only dichloromethane
• Follow instructions to ensure uniform reading of the melting point range
B. Communication (Refer to B1 & B2 in the General Laboratory LGs.)
B3. Access (and properly cite) relevant information from the library and other resources.
• Use ChemFinder to locate literature values of physical properties
• Find alternative methods for extracting caffeine
C. Responsibility (Refer to C2 in the General Laboratory LGs.)
C1. Effectively prepare in advance for laboratory work.
• Complete the online techniques on Extraction, Melting Point Analysis, and Vacuum Sublimation
D. Context
D1. Use the scientific method.
D2. Describe what your data does and does not say.
• Interpret the data: Consider the limitations of the experiment (hint: partition coefficients)
E. Integration and application of knowledge/experience
E1. Apply critical thinking in the laboratory.
• Describe why each step of the experiment is needed to extract caffeine from soft drinks
• Describe the purpose and value of multiple, small dichloromethane extractions vs. one large extraction
• Explain the importance of recording the temperature range when performing melting point analysis
• Describe the purpose and value of melting point analysis
E2. Recognize whether results and conclusions "make sense". Questions to consider:
- Are your measured levels of caffeine comparable to values provided in the lab manual?
- How do melting points compare between different student samples and literature values?
- Do colas thought to have more caffeine demonstrate higher levels than others?
 Experiment 10 Overview

EXPERIMENT 10
Caffeine in Soft Drinks

AIM:
To isolate and purify caffeine contained in a cola beverage.

OBJECTIVES:
❑ Design an experimental procedure to extract crude caffeine from a cola drink
❑ Determine the most effective method of extraction through team collaboration
❑ Calculate the amount of crude caffeine extracted
❑ Perform vacuum sublimation to purify the crude caffeine
❑ Determine the purity of the caffeine through melting point analysis

INTRODUCTION:
Caffeine (C8H10O2N4) is a compound that belongs to a large class of substances called alkaloids. Alkaloids
are usually defined as nitrogenous plant products having a marked physiological action when
administered to mammals. Many alkaloids are extremely difficult to synthesize, hence plants are still
important commercial sources of them. Caffeine is present
in tea leaves and in coffee to the extent of about 4%. Tea O CH3
contains two other alkaloids, theobromine and
theophylline, which are similar to caffeine in chemical CH3 C N
structure. Caffeine stimulates the heart and respiratory N C
system, but theophylline and theobromine relax the smooth
C H
C C
muscles. Tea thus relaxes some physiological systems while N N
stimulating others. By way of contrast, coffee contains no O
relaxing alkaloids and thus has a more stimulating effect. CH3 CAFFEINE
Cola products contain varying amounts of caffeine, allegedly
to improve their taste. However, Roland Griffith of Johns Hopkins University recently conducted a study
proving that most cola-drinking adults cannot tell whether the beverage contains caffeine or not.
[Challenge: can you find this article in the scientific literature?] The levels of caffeine listed in Table 1
below have been specified by the British Columbia Alcohol and Drug Commission.

Table 1: Caffeine Levels in Various Food Products

Average cup of regular coffee 90-150 mg
Average cup of instant coffee 60- 80 mg
Average cup of tea 30- 70 mg
Bottle of cola 30- 45 mg
Average chocolate bar 30 mg
Stay-Awake pill 100 mg
Cold relief tablet 30 mg
 Experiment 10 Overview

The Theory Behind the Experiment

LAB PROJECT OVERVIEW
You are working in an analytical laboratory. Your team is given a project to determine the amount of
caffeine, and its purity, present in a Cola product, by isolating the crude caffeine and then purifying it. You
must establish the most efficient method to isolate caffeine, using the Manufacturer’s caffeine content
as a reference (will be provided in the lab).
For your convenience, the cola products have been heated by the technical staff to remove carbon
dioxide. During the heating process, the volume of the cola was reduced to exactly half of its initial value.
Keep this in mind when reporting the amount of caffeine present in your cola product.
When isolating and purifying caffeine from cola, the challenge is to find a method to separate this single
compound in an efficient way (without undue losses) from the host of other compounds present in cola.
Isolation and Purification
This is achieved by:
• selective extractions. In the lab project summary, you will be provided with a generalized
procedure for extracting caffeine from an aqueous sample. Adapt this procedure to meet the
goals of your project, to determine the most effective extraction method.
• removing unwanted substances
• subliming the crude caffeine
These techniques take advantage of the fact that, unlike some other materials found in cola, caffeine is:
• soluble in water and dichloromethane (DCM) at room temperature
• caffeine is basic and does not react with 10% ammonium hydroxide as do the acids present in
cola. These unwanted acids remain in an aqueous layer, during separation, in the form of
ammonium salts
Once the crude product is isolated, it must be identified as caffeine.

Identification and Purity

This is achieved by:
• Melting point analysis can be used for identification. and also provides a check on the purity of
the final sublimed product.

SAFETY CONCERNS
In this experiment you will use Dichloromethane (DCM), which is a carcinogen (cancer-causing agent)
Because of this:
• all work where DCM is being used will be carried out in the fume hood
• all round bottom flasks containing DCM, that are removed from the fume hood, must have a
glass stopper in them.
Before coming to your Experiment 10 lab session make sure you watch the Working in a Fume Hood
Video for correct fume hood operations and complete the fume hood quiz. If you do not complete the
fume hood quiz, with 100% score, BEFORE coming to the lab, you will be seen as a safety hazard and
asked to leave. No make up lab will be given.

Remember DCM is a carcinogen so avoid touching it or breathing in its fumes.

 Experiment 10 Overview

The Experiment
LAB PROJECT SUMMARY

Perform an experiment that allows you to quantitatively achieve the following tasks:
1. Extract crude caffeine from a cola drink.
2. Determine the mass and melting point of the crude caffeine isolated.
3. Purify the crude caffeine by vacuum sublimation.
4. Determine the melting point of the purified caffeine.
Once you have obtained all this data in the lab, you will share your results with your team.
Using the combined data, discuss with your team the validity of the results and sources of error.

EXPERIMENTAL PROCEDURE
Extraction of the Caffeine
Points to consider Procedure
• What is the purpose of this part of the You will be given a 50 mL sample of cola.
procedure? Extract the caffeine using a separatory funnel.
• From what other compounds are you trying to Watch the video on using a separatory funnel
separate the caffeine?
• Caffeine is more soluble in DCM than in water, Add 10-15 mL of ammonium hydroxide to the cola
but what about the principal impurity, and swirl to mix the liquids. Include in your
phosphoric acid? procedure, an explanation of why ammonium
hydroxide is added.
• Which layer is the organic layer? Consider
which layer should have the greater relative
volume and whether water or DCM has the
greater density. Note that DCM is a carcinogen
• Which phase (aqueous or organic) would you (cancer-causing agent) so avoid
like the impurities to end up in? touching it or breathing its fumes.
• How would you make phosphoric acid less
soluble in an organic solvent (and therefore
more soluble in water)? Keep in mind that a
weak acid's salt is usually more soluble in
water than the acid itself. Add a portion of dichloromethane to the mixture
in the separatory funnel.
• What method of extraction will give the more Note that you are given 30 mL of DCM for all the
product? separations you are going to perform.
o 1 rinse with 30 mL of DCM or 2 rinses
To ensure more effective extraction of caffeine,
with 2 lots of 15 mL DCM etc
split the 30 mL of DCM into smaller portions and
• Complete the section in the Partition
repeat the extraction process with each portion.
Coefficient calculations in the Virtual Lab to
Combine all the organic extracts together into one
answer this question and help you devise your
beaker.
procedure.
Make sure that you understand which compound is going into which phase before proceeding.
 Experiment 10 Overview

Drying the Organic Extract

Points to consider Procedure
Any residual water present in your collected Weigh a clean dry 100 mL round bottom flask
organic extract must be eliminated, so as not to (with plastic holder) on the analytical balance, it
interfere in the subsequent solvent-removal step. will eventually contain your caffeine product.
How can this be done? Note: The yield will be so small that it will be
During extraction, the DCM and the aqueous cola impossible to quantitatively transfer the product
sample form two immiscible layers when mixed. into another vessel for weighing.
Can this idea of immiscible layers be used to Figure 1, shows hot to fold a sheet of filter-paper
separate any residual water? to create a “fluted” filter-paper.
If the filter paper is soaked in DCM, will any
residual water pass through the filter paper? Figure 2 shows how to set up the equipment
needed for this procedure.
Fluted filter-paper greatly increases the efficiency
of filtration, because the liquid only has to pass Once your fluted filter paper is in the dry funnel,
through one layer of paper at all points and runs “wet” the filter paper completely using a dropper
freely down the side of the paper inside of the full of DCM. Carefully pour your collected organic
filtration funnel. extract onto the filter paper and let gravity pull it
through and into your pre-weighed round bottom
flask.

Once your organic extract is in the round bottom flask, you must place a glass stopper in the flask to
stop any of the DCM fumes escaping in to the lab. DO NOT take the flask out of the fume hood without
a glass stopper in it. Your TA will remove the stopper before the solvent removal process.
Note: This technique can only be applied to organic liquids that are immiscible with water and so can
be used to dry a DCM solution containing only small amounts of caffeine; but not an ethanol solution
as ethanol is miscible with water.

How to Flute Filter Paper

To flute, fold the paper in half, then into quarters (1). Fold each quarter in half again (2 to 3) so that the
filter is now divided into eighths. Fold the “outside” eighths (4) and the “inside” eighths in half again (5
to 6). Finally, open up the folded paper as shown in (7) and seat the filter in the glass funnel.
Figure 1: Folding fluted filter paper. Figure 2: Gravity Filtration
 Experiment 10 Overview

Solvent Removal: Isolation of the Product

Points to consider Procedure
From the above “drying” step, you should be left The final isolation step will be removing the DCM
with an extract that consists solely of caffeine from the round bottom flask. This will be done
dissolved in DCM. using a rotary evaporator (rotovap). A TA will
DCM is a very volatile solvent. Why is this operate the rotovap, but you should understand
important in this step? why it is used and what will happen to your sample
How can you increase a solvent's rate of in this step.
evaporation?
Technique: Rotary Evaporators
What is the boiling point of DCM?
Once the solvent has been removed, you will be
left with crude caffeine.
Quantitatively weigh your sample. Make sure the
round bottom flask is at room temperature!
Use this mass of crude caffeine to compare the amounts of caffeine present in your team’s samples.

Determination of the Melting Point of Caffeine

Points to consider Procedure

How do your results compare with the literature Put about 3 mm of your dry solid sample into a
value? melting point capillary tube. Technique: “Filling a
Is your own sample relatively pure (within ± 2 ºC capillary tube”
of the literature value)? Use your observations to Prepare another capillary tube with pure caffeine
comment on the purity of your crude caffeine. obtained from your TA.
Place both tubes into the melting point apparatus
Remember, a broad melting point range indicates at the same time and conduct a melting point
the presence of impurities. analysis. Technique: Performing a Melting Point
Observe and record the melting point range of
both the crude and pure samples.
DO NOT put used capillary tubes in the garbage. Place in “used capillary tubes” beaker.

Purification of Caffeine
To confirm that your extracted product is caffeine, you must perform a melting point analysis on a purified
sample of your product. This purification of your crude product is achieved by vacuum sublimation, a
simple physical process used to purify solids. In sublimation, a volatile solid is separated by evaporation
from less and more volatile impurities. The sublimation process used in this lab results in a very low yield
of pure product. Therefore, for you to have enough of pure product to be able to perform a melting point
analysis, you will need to combine your crude sample with other students in your TA section prior to
sublimation.
Remember that the validity of your hypothesis will be determined using the mass of your crude caffeine
and not the pure caffeine. Here we are simply verifying that the extracted product is indeed caffeine.

Points to consider Procedure

As the solid evaporates, pure caffeine is deposited In your procedure state you will use this technique
onto the outside of the cold-finger via for purification. Briefly explain why this apparatus
condensation. This material is the sublimate. has to be dry. Technique: Vacuum Sublimation
 Experiment 10 Overview

Points to consider Procedure

Are the crude product and the sublimate exactly Inspect the sublimate.
the same colour?
How does this melting point compare to the Conduct a melting point analysis on the purified
literature value and the value you have obtained caffeine and record the value.
for the pure caffeine earlier in the experiment?
Does this identify your product as caffeine?
What does this tell you about the relative purities
of your “crude” sample and your group's sublimed Do not turn off the pump BEFORE the vacuum is
sample? broken, as water sucking back into the hot flaks,
could cause an explosion and SERIOUS INJURIES.
Note: If your sample is wet during sublimation, or
the technique is not performed correctly, this can
sometimes increase the temperature range and
drop the MP compared to your crude sample.

CLEAN UP
Glassware Rinse glassware with tap water, return it to your kit, follow the clean-up sheets
Separatory funnel Rinse with tap water and replace in ring clamp with stopcock open and cap on
Round bottom flask Rinse the flask and place in front of your kit in the holder
Funnel Rinse and return to “used funnel” bin
Used capillary tubes In the “used capillary tubes” beaker
Chemical disposal
Caffeine Pour down sink with plenty of water

PRE-LAB CHECKLIST
Make sure to do the following before you do the experiment:
❑ Read through the all components of the experiment
❑ Watch the Working in a Fume Hood Video – Complete the Fume Hood Quiz
❑ Formulate a research question and hypothesis and chose a research article relevant to your experiment
Complete the Virtual Lab Experiment 10: Extraction of Caffeine from Cola (Evaluation question 1 only)
❑ and bring the feedback form (printed or screenshot) to your Experiment 10 lab session.
❑ Prepare for the In-lab quiz. You can be tested on techniques, calculations and experimental overview.
Complete IN PEN the pre-lab part of the EDF listed below,
• Research question and Hypothesis
❑ • Glassware / equipment and reagents needed
• Procedure - Remember, keep it concise. If you want to bring in additional notes for your own
reference, that is ok, but do not include them in your EDF.
The more you prepare for the wet lab the easier your experiment will seem. Familiarize yourself with:
• your own procedure so you are aware of the order the experimental steps are performed
• any techniques in the experiment
• prepare tables to record data collected during the experiment
 Experiment 10 Overview

SUBMISSION DEADLINES
Task Submit Submit (where and when)
Prepare for lab:
• Read experimental overview
• Read online material
• Find research article
• Watch Working in a fume
hood video
Submit before the start of your Experiment 10
lab session*
Complete Fume Hood Quiz Quiz Submit in Canvas
If this quiz is not completed with 100% score, you
will not be allowed to perform the experiment
Bring to lab
Complete before the start of your Experiment
Virtual Lab Feedback Form (screenshot
or printout)
10 lab session*
Complete before the start of your Experiment
Complete pre-lab portion of EDF 10 lab session*
Submit with your lab report
This quiz will be a closed book quiz held in the first 10 minutes of
Prepare for the In-lab Quiz
the lab session
Complete before the end of your Experiment
Complete in-lab portion of EDF 10 lab session*
Submit with your lab report
Submit your Experiment 10 lab report
AM labs submit by 11 am** one-week after lab
session
Complete Lab Report Lab Report
PM labs submit by 4 pm** one-week after lab
session
Submit to box outside B370
*No late submissions **Late penalties apply (2 marks deducted per hour after the deadline)