Determination of caffeine in beverages by hplc

Determination of caffeine in tea by HPLC
Preparation of standard solutions
You will require standard solutions of Caffeine in Methanol: Concentration Caffeine µg / ml Mobile Phase
Preparation of tea sample
1. Grind approximately 7g of the tea sample.
Carry out the rest of the procedure in duplicate.
+/- 0.001g of the sample in duplicate into a 500 ml round bottomed flask.
3. Add 4.5g magnesium oxide and a few anti-bumping granules.
4. Add 300ml deionised water and weigh the flask to the nearest 0.1g.
5. Mix the contents and set up the flask for reflux.
6. Bring the flask to the boil and simmer for 20 minutes.
7. Allow to cool to room temperature.
8. Add enough deionised water to bring the flask contents back to the weight recorded at step 4.
9. Allow the contents to settle, then filter approximately 3ml of the flask contents through a 0.2µ syringe HPLC Conditions
Flow rate:
Injection volume:
Samples should be bracketed with standards (i.e.
run four standards, a maximum of eight samples,then run four standards again).
Using HPLC sample vials
Samples for analysis by HPLC are placed in small glass vials which are then sealed using a cap which isfixed in place using a special crimping tool.
Standard solutions: Carefully transfer about 4 ml of each of the solution to a sample vial. Place a cap on
each tube and seal using the crimping tool. Label vial with a marker pen.
Beverage solutions: using a plastic syringe, draw up about 5 ml of your beverage solution. Place a 0.2µ
filter on the end of the syringe and push about 4 ml of the solution into a sample vial (you have to press quite
hard to force liquid through the filter – but do not press to hard!). Seal the tube with the crimping tool. Repeat
with the other sample. Label vials with a marker pen.
Analysis of standards and beverage sample
Assistance will be provided with the HPLC – if in doubt ask.
(1) Check the temperature of the column heater is set to 45oC and that mobile phrase is flowing
through the column.

(2) Place samples in autosampler
Place your vials in the first open positions on the autosampler carousel. (If you need to move the carouselaround, press and hold the arrowed button on the autosampler control panel). Check that positions 95 and96 contain sample vials filled with filtered deionised water.
(3) Set up the program
On the computer screen you will see a window similar to the one shown below. You will need to change orcheck certain values:• Change "Data name" to any sample name you chose.
• Check "Repeat" = 1, "Vials" = (however many you have), and "From number" = 1 (change any values if When you are satisfied, click the blue button and the analysis will begin. The software will then report that it
is “CONDITIONING THE SYSTEM” and, a few minutes later, the run will begin and a graph showing the
progress of the chromatogram will appear on the screen.
When the first sample has been analysed you should have a chromatogram similar to the one shown
The system will then automatically begin to analyse the next sample,and then each sample in turn until all your samples have beenanalysed.
Construction of calibration curve / determination of caffeine content of beverage

This can be done either by hand or automatically using the software provided.
If calibration data is provided, go to step 3 (plot graph) below.
(1) By hand
1. Print out each of your three chromatograms.
2. Measure the peak height of each of the caffeine peaks for your standards.
3. Plot a graph of caffeine concentration against peak height.
4. Measure the peak height for your beverage sample.
5. Obtain the caffeine concentration of your beverage samples and the “unknown caffeine” solution using (2) Using the software provided
You will be instructed in the use of the software if you construct your curve using the supplied software.
The Eurochem 2000 HPLC system
Basic principle of operation
High-performance Liquid Chromatography (HPLC) is a versatile form of chromatography, used with a widevariety of stationary and mobile phases, to separate individual compounds of a particular class of moleculeson the basis of size, polarity, solubility or absorption characteristics.
At its most simple a HPLC system pumps a solvent (called the mobile phase) through a stainless steel
column containing an absorbent material (called the stationary phase). A small amount of a sample
containing mixture of substances is injected before the column. Different substances adhere to the
stationary phase to different extents and so take varying amounts of time to pass through the column. After
passing through the column the various components of the mixture are detected, often by UV absorption.
The mobile phase (and any substances in it) then leave the system and are collected in a waste bottle.
Most modern HPLC systems are computer controlled and have automatic sample injection.
Components of the system
(See over page for a schematic diagram of the system.) Solvent (Mobile Phase) bottles
There are four bottles each of which can contain a different mobile phase. For this experiment we are only
using bottle A, which contains a 70 : 30 (v/v) mixture of Methanol and Water.
The Pump
Pumps mobile phase through the system at a controlled rate (1.0 ml per minute in this experiment).
As mobile phase enters the system the degasser removes air bubbles by applying a vacuum.
(If there are any air bubble in the mobile phase they may affect flow rate and detector response.)
Mixing chamber
When more mixtures of more than one different mobile phase are being used the mixing chamber ensures
that they are well-mixed before moving through the system.
(As noted above, we are only using a single mobile phase for this experiment.)
The autosampler allows us to automatically inject over 90 samples without human intervention. For each
sample the autosampler needle is washed with water, then draws 20 µl of sample into the system, the
needle is then washed with water again. When that sample has been analysed, the cycle repeats itself until
all the sample have been analysed.
The Column
The heart of the HPLC system. It contains the stationary phase which separates the various components of
a mixture.
UV Detector
This measures the UV absorption of the liquid passing through it. When a sample passes through the
detector a sharp peak in absorption occurs. For the caffeine experiment we are measuring the absorption at
254 nm.
After passing through the detector, the liquid is collected in a waste bottle.
The computer contains the controlling software and is used to store chromatograms, methods and other
Interface Box
This is a piece of hardware which allows the various components of the system to "talk to each other"
Schematic diagram of system
JG10: Determination of caffeine in beverages by UV
Preparation of standard curve
Dichloromethane and solutions in Dichloromethane present possible long-term hazards. They
must only be handled in the fume cupboard and nitrile gloves must be worn.
Stoppered glass cuvettes must be used for the UV determination.
Do not place Dichloromethane-filled cuvettes on the wooden bench
Place any waste Dichloromethane in the "Chlorinated solvents waste bottle."
You are provided with the following standard solutions of Caffeine in Dichloromethane: Concentration mg per 100 ml Dichloromethane
Concentration mg per 1 ml Dichloromethane
Determine the absorption of the one of these solutions at 277 nm, using Dichloromethane as the blank.
Check to see if this is the maximum. If it is not, locate the maximum and use this wavelength for yoursubsequent determinations.
Extraction of Caffeine from beverages
Dichloromethane and solutions in Dichloromethane present possible long-term hazards. They
must only be handled in the fume cupboard and nitrile gloves must be worn.
Place any waste Dichloromethane in the "Chlorinated solvents waste bottle."
1. Where necessary, remove carbonation by pouring the beverage from one beaker to another until 2. Pipette 10 ml of sample into a 100 ml separating funnel, add 5 ml Oxidising Solution and mix.
3. After exactly five minutes add 10 ml Reducing Solution and mix.
4. Add 1 ml dilute Phosphoric Acid solution and mix.
5. Add 1 ml Sodium Hydroxide solution and mix.
6. Extract with 50 ml Dichloromethane.
7. After separation, drain the lower layer through a 7 cm filter paper into a 100 ml volumetric flask.
8. Add 2-3 ml of Dichloromethane to the separating funnel and drain through the paper to rinse the 9. Wash the paper with a further 2–3 ml of Dichloromethane.
10. Re-extract the aqueous solution with 40 ml Dichloromethane, and wash funnel and paper as before.
11. Add to the first extraction aliquot and dilute to the mark with Dichloromethane.
12. Scan for maximum absorption using UV spectrophotometer and supplied software.
13. Measure the absorbance of the standards at the determined maximum.
14. Measure absorbance of sample at the determined maximum.
5g Sodium Sulphate and 5g KSCN in water, dilute to 100ml.

15g concentrated Orthophosphoric Acid + 85ml water.



Carbonated beverage “A”
Carbonated beverage “B”
Carbonated beverage “A”
Carbonated beverage “B”
Carbonated beverage “A”
Carbonated beverage “B”


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