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Bioanalytical Chemistry
Practical 2021-
Dosage of caffeine by spectroscopy
Detection of caffeine in tea leaves and sodas
Objectives
In this laboratory experiment, we will have 3 sessions:
Session 1:
Identification of caffein in soda and tea leaves extract by TLC
Calibration of pipet
Measure the pH value of a soda
Session 2
Measure the concentration of an unknown caffeine solution by UV spectroscopy
Session 3
Measure the concentration of an unknown caffein solution by HPLC-DAD
Observe the UPLC MSMS and GCMS system
Background
Caffeine (figure 1) is a derivative of xanthine: 1,3,7 trimethyxanthine. Related
compounds are theophylline (1,3-dimethylxanthine ) and theobromine (3,7-
dimethylxanthine).
The major sources for caffeine are the seeds of the coffee plant, cola nuts, Mate which is
used as tea in Paraguay and tea leaves ( camellia sinensis ); Three types of tea are
commercially available from the tea leaves (green, oolong and black) which differ only
in their processing methods. To obtain green tea, fresh leaves are sun-dried or steamed
to destroy the natural enzymes that causes fermentation. If the leaves are allowed to
fermentate an oxidation process occurs which gives oolong and with a longer
fermentation process black tea. Theobromine is found mainly in cacao.
Caffeine has a tertiary amine but is a very weak base with a pKa value of about 0.
(Beware the literature is confusing, this pKa value corresponds to the equilibrium BH
ļ³ B + H
; in the literature a value around 14 is often cited corresponding to the
equilibrium B ļ³BH
+ OH
). Caffeine ā a polar compound- is soluble in water (about
20g/L at 25Ā°C), and the solubility increases with the temperature and is also very
soluble in polar non protic solvent like dichloromethane CH 2 Cl 2 with a partition
coefficient dichloromethane/water of about 8-10. It is less soluble in ethylacetate. Tea
leaves contain also cellulose, proteins and amino acids, tannins, pigments and saponins.
Cellulose
Although cellulose is very polar it does not dissolve in water due to its high molecular
weight.
Proteins and pigments
Proteins and pigments are very soluble in water and do not extract into the organic
phase. The content of pigments varies between the kind of tea and the level of oxidation.
Tannins
Tannins are polyphenolic compounds with molecular weight of up to 20 000. Tea
tannins are soluble in water and give the typical bitter taste of tea. Tea tannins belong to
a subgroup named hydrolysable tannins. The core structure is D- glucose to which
several units of gallic acid are attached via ester bonds. Note the structure of gallic acid.
The presence of soluble tannins in tea leaves complicates the isolation of caffeine as low
molecular weight tannins are soluble in methylene chloride. However, we can take
advantage of the hydrolysis of ester bonds in hydrolysable tannins. When tea leaves are
boiled in the presence of a base, such as CaCO 3, the ester bond is cleaved to give glucose
and a calcium salt of gallic acid which are not extracted into methylene chloride.
Saponins
Saponins are amphiphilic glycosides MW 1200-1300(found also in Ginseng and other
plants) who have soap-like properties. So they tend to give emulsions when shaken in
Each practical session will be separated into sub-groups. Each individual values
obtained during a practical session will be collected and available to everybody. From
these data everybody will calculate the mean value and the standard deviation.
Overview of the Procedure:
Dilution for the calibration curve
Experimental section
Starting materials
Caffeine stock solution: 500 mg/L
Tea bags
Caffeine
standard
Tea soda
extracti
on
n
Boiling
CaCO
solvent
extractio
nn
TLC pH
Remova
l
CO
2
no
mg/L
Stock
solution
5 mg/L
mg /L
mg/L
mg/L
mg/L
X 10
X 10
X 10
X 2
X 2
X 5
2 mg/L 1 mg/L
Soda: Pepsi or Coca Cola
Thin Layer Plate 5x 10 cm silica gel with fluorescent indicator 254 nm
Dichloromethane CH 2 Cl 2
Ethylacetate
Calcium carbonate CaCO 3
UV cells Brand
pH meter
spectrophotometer
micropipette
Part 1: Identification
Caffeine extraction from tea
Place the tea bag inside a 100 mL beaker (leave the label outside)
Add 0.8-1 g of CaCO3 and 50 mL of water
Bring the solution to a gentle boil
Allow the solution to cool down
Squeeze gently the tea bag (be careful not to puncture the bag) and remove the bag
Take 2 ml of this extract in HPLC vial. This solution will be analyzed in Part 3.
We are ready to spot the TLC plate
Spotting the TLC plate
Take gently a plate by its sides (do not put your fingers on the plate)
Draw lightly with a pencil (not a pen) a line at about 1- 1.5 cm from the bottom (on the
silica side)
Mark very lightly 5 spots with a pencil 1 cm apart (see below, do not write the number
on the plate)
Spot with a micropipette (as instructed)
caffeine 50 mg/mL solution Spot 1
tea spot 2
tea extract: with ethylacetate spot 3
tea extract with dichloromethane spot 4
soda without extraction spot 5
bottom
1 5
Cuvette 1: Blank: distilled water
Cuvettes 2-7: calibration solutions arranged in increasing concentration
Cuvette 8: unknown concentration
First: calibrate the instrument (ask for the instructor)
Then read and record the absorbance of all your cuvettes. Be sure that the wavelength is
set up at 275 nm.
Put the cuvettes back exactly the way they were.
After you finish clean carefully the cells
Make the calibration curve:
By hand on millimetric paper (provided) (and then with excel if you wish)
Calculate the unknown concentration
Additional note: Not part of the practical
This extraction procedure would permit to get pure caffeine as follows. After extracting
the tea water twice with dichloromethane, Extract 1 and extract 2 would be combined
and the solvent dried and evaporated. Caffeine would then be obtained pure by
sublimation (goes from solid to gas) at a relatively low temperature and the pure
caffeine would be collected on a cold spot (centrifuge tube filled with ice) as shown
Part 3: Quantification by HPLC-DAD system
Sample preparation
Standard solutions (from 0,5 to 10 mg/L) and tea extract were prepared in previous
session.
Set up method
See instructorās guide and note in your report
Put your vials in the sample manager of HPLC system
Run analysis
Data analysis
Build calibration curve and calculate the concentration of caffein in tea extract. Report
this result in your report.
Observation of LCMSMS and GCMS system
See instructorās guide and note in your report
Report
1. One single report for a subgroup. Be sure to give
a. the last name and first name of each member of the subgroup
b. the date of the practical
2. The practical report consists of 2 parts
a. A prelab report which should be prepared before coming. Based on
the answers the instructor may give a note during the session.
b. A lab report, which should be short and be completed upon
leaving.
LAB Report: Questions
1. TLC :
a. Draw a schematic figure of the TLC plate after chromatography
with the spots observed
b. What is the Rf value of caffeine standard?
c. Do you see a spot corresponding to caffeine in every lane? What is
the Rf values?
d. Do you see any difference between lanes 2, 3, 4? Why?
e. Comments
2. Quantitation of caffeine by spectroscopy
a. Values for the pipette calibration: mean value, relative standard
deviation and relative accuracy of the pipette.
b. With this pipette what is the uncertainty in the concentration of the
50 mg/L solution
c. Plot the calibration curve on the millimetric paper (do not forget
the units). Is the curve linear?
d. Give the concentration of your unknown solution (be sure to report
the name of that unknown).
e. Using excel or the tables used in the Prelab report give the values a
and b in Absorbance = a + b. conc. Calculate the coefficient r
2
f. Plot the residual A obs
-A
estimated
y ā y
) = f(conc): Comment.
g. What is concentration of the stock solution in mol/L (M)
h. Calculate the molar absorption coefficient of caffeine in your
experimental conditions.
3. pH of soda
a. give the pH value you obtained
b. Give the mean value and the standard deviation of all pH values
obtained during the session
4. Calibration the pipets
a. report the mean, SD, RSD of 10 time repetition
5. HPLC and LCMSMS, GCMS
b. If we extract with dichloromethane we have 2 phases. Which phase will
the lower phase? Why?
c. If we extract with ethylacetate which phase will be the lower phase? Why?
- Uncertainty in measuring volumes.
a. We are using automatic pipettes and we have to be sure that the volume
dispensed is the one indicated on the pipette ( 0.5 , 1 mL, ā¦ ). A pipette is
calibrated by weighing. Last year a group reported the following values
(in g) for a 500 Ī¼L pipette (0.4935, 0.4931, 0.930, 0.4993, 0.4898, 0.4976,
0.4971, 0.4974, 0.4952, 0.4972, 0.4970. Calculate the mean value, the
standard deviation and the relative accuracy of the pipette.
b. If we use that pipette to prepare 5 mL of the 50 mg/L solution by diluting
10 times the stock solution what is the relative uncertainty of the diluted
(50 mg/mL) solution.
4. Thin layer chromatography:
a. Watch: https://www.youtube.com/watch?v=rgkRf3LD5OU
b. Is the chromatographic system used in our experiment Normal phase or
Reversed phase?
c. In our chromatographic system which of xanthine or caffeine would have
a higher Rf value? Why?
d. We are detecting caffeine by UV. Suggest a reagent that would give a
colored spot to detect caffeine.
e. We are using 100% ethyl acetate as the mobile phase. Some authors use a
(19/1 ethylacetate/methanol vol/vol) mixture. Would the Rf value of
caffeine increase or decrease if methanol is added to the mobile phase.
5. Spectroscopy:
a. Give the theoretical relationship between absorbance and concentration
b. What are the units for Absorbance and the molar absorption coefficient?
c. From the literature what are the wavelengths of maximum absorption of
caffeine in the UV range? Why do we have to use the one around 275 nm?
6. Quantification:
