royal society of chemistry analytical division - ne region ... east region...

Schools' Analyst Irn Bru Practical...............................................

© David Crowther, Sheffield Hallam University, February 2003.

Royal Society of Chemistry

Analytical Division - NE region

Schools' Analyst competition heat 2003

Name…………….....................………………………

School………….......................……………………….



Health and Safety

This is a practical exercise, so normal rules for safety in the laboratory apply.

Wear laboratory coats and safety spectacles at all times.

Do NOT eat or drink in the laboratory.

Always use the pipette fillers provided, and handle glassware carefully to avoid breakages

and cuts.

Keep long hair under control.

IF IN DOUBT ABOUT ANYTHING THEN ASK A DEMONSTRATOR.

......................................................................................................................................

Nomenclature:

You will find that volumes are given as litres (L) or millilitres (mL). Remember that 1 L

= 1 dm3

and 1 mL = 1 cm3.

There are answer sheets at the back of each of these instruction booklets for your

individual use. You may keep this instruction bookelt after the exercise.

Each team must complete one answer booklet, attach graphs to it and submit it at

the end of the exercise.



The Problem

"Made in Scotland from girders"

the adverts used to proclaim. Now the soft drink Irn-Bru, a Glaswegian delicacy, has gone

techno:

http://www.irn-bru.co.uk/irnbru.asp

What exactly is in it though, and are there likely to any effects on health if large amounts

are consumed?

Tha label indicates ferric ammonium citrate at 0.002% plus a preservative (E211, sodium

benzoate) and caffeine at unspecified concentrations. You will check that the contents do

seem to fit the label description, and measure the concentrations of the unquantified

ingredients. You will also recommend a maximum allowable consumption in order to

stay within guidelines for the ingestion of caffeine.

There are three experiments for you to do. Each will take about 2 hours if you work

efficiently:

i) Measure the amount of iron present in the drink. The method that you will use for

this is called colorimetric analysis.

ii) Extract some of the organic components of the drink and identify them by thin

layer chromatography.

iii) Extract the caffeine and determine its concentration by uv spectrophotometry.

You need to plan your work carefully to allow completion of the exercise. You

should allow at least 30mins at the end for calculations and completion of the

questions.

What is the best way to organise the work within your team? Show this as a

diagram below:



Experiment 1 Determination of iron by colorimetry

In this experiment you will determine the concentration of iron by complexing with

1,10-phenanthroline. The complex between ferrous ion and 1,10-phenanthroline is

red-orange in colour, and its concentration can be measured by colorimetry. In

order to determine the total iron content you must first reduce the ferric form to the

ferrous form with hydroxylamine, then determine with 1,10-phenanthroline.

Equipment

Uv/visible spectrophotometer (colorimeter)

Plastic 1 cm pathlength cells

Pipettes

Pipette fillers

Pasteur pipettes

Volumetric flasks (15 x 100mL plus others)

Storage bottles (if insufficient volumetric flasks)

Distilled water

Stock iron solution (ferrous ammonium sulphate, 2.00 x 10-3

mol L-1

)

1,10-phenanthroline solution (10.0 x 10-3

mol L-1

in water)

Hydroxylammonium chloride solution (1.00 x 10-2

mol L-1

in water)

Procedure - colorimetry

1 Prepare a series of standard solutions of the ferrous ammonium sulphate stock

solution to cover the range 1.00 to 4.00 x 10-5

mol L-1

iron. Four solutions will be

sufficient. Choose dilutions that are evenly spaced and volumes that are easy to make up

with the glassware available.

2 In two 100 mL flasks make 2-fold and 4-fold dilutions of Irn-Bru (i.e. pipette 25.0

mL IrnBru into one flask and 50.0 mL into the other), add 50.0 mL of the hydroxyl

ammonium chloride solution to each and add distilled water to the mark for the 4-fold

dilution. Label them clearly as"2-fold dilution" and "4-fold dilution".

3a) Pipette 5.00 mL of the 1,10-phenanthroline solution into a clean 100mL

volumetric flask, add 10 mL hydroxylammonium chloride solution and make up to the

mark with water. Label this as "0 x 10-6

mol L-1

Fe".

b) Repeat the process in four other flasks to get a set of calibration solutions from

your diluted iron standards in step1. Take a clean 100mL volumetric flask and pipette in

25.0mL of one the standard solutions, then 10 mL hydroxylammonium chloride solution

then 5.00 mL of the 1,10-phenanthroline solution and make up to 100 mL with water.

Label each flask with the standard concentration.

(NB if there is insufficient volumetric glassware you can pour your samples into

labelled storage bottles, rinse the 100mL volumetric flasks and then reuse them)



c) Repeat the process with each of the diluted Irn-Bru samples from step 2 (i.e. each

flask should have 25.0 mL of one of the diluted IrnBru's, 10 mL hydroxylammonium

chloride solution, 5.00 mL of 1,10-phenanthroline solution and be made up to 100 mL

with water). Label each flask with the iron concentration or IrnBru dilution.

d) Also make up two flasks (one from each of the diluted Irn-Bru solutions) without

the 1,10-phenanthroline reagent. Label these as "2-fold blank" and "4-fold blank".

You should have 9 samples from this step for absorbance readings.

4 ASK FOR A DEMONSTRATION OF THE USE OF THE

SPECTROPHOTOMETER

5 Set the spectrophotometer to 515 nm wavelength then zero it with distilled water

in the cell(s).

6 Read and record on the result sheets the absorbance of each of the mixtures from

step 4. Rinse the cell between each reading.

7 Plot a calibration graph using the absorbances read from each of your standard

solutions.

8 Correct for the background absorbance of the colouring material in the drink by

subtracting the appropriate "blank" reading from each of the two sample readings (see

result sheet).

9 Read off the concentration of iron in each of the diluted IrnBru samples.

10 Correct for dilutions then report the concentration of iron in the original IrnBru

drink on the result sheet.

11 Carry out the further calculations on the result sheet.



Experiment 2 Extraction and identification of organic components

In this experiment you will perform a solvent extraction to remove some of the

organic compounds from the Irn-Bru, then separate and identify them by thin layer

chromatography.

Equipment

100 mL separating funnel and stopper

50 mL measuring cylinder

10mL measuring cylinder

Retort stand with ring or clamp

4 mol L-1

sodium chloride

100 mL beaker

Dichloromethane

Methanol

2 mol L-1

hydrochloric acid

Thin layer chromatography plate (silica gel with fluorescent indicator)

4 spotting tubes

Chromatography tank with ethyl acetate solvent

Standard solution of caffeine (200 mg L-1

) in methanol

Standard solution of benzoic acid (100 mg L-1

) in methanol

Standard solution of citric acid (2000 mg L-1

) in methanol

Ultraviolet plate viewer

Procedure

We are doing qualitative analysis here, so very accurate volume measurements are not

required. This means that you can use measuring cylinders for the volumes (in this

experiment only).

1 Place 40 mL of Irn-Bru into the separating funnel then add 5 mL of 2 mol L-1

hydrochloric acid and 5 mL of 4 mol L-1

sodium chloride solution.

2 Add 2 mL dichloromethane (CARE: organic solvent), stopper the funnel and

invert a few times.

CARE: Hold the funnel upright (stopper at top) and loosen the stopper to

relieve gas pressure; dichloromethane is very volatile and pressure will build up inside

the funnel.

Repeat the process a few times, relieving pressure after each, then shake the flask

vigorously for about 20 secs, place on the stand and allow the layers to separate.

3 Run off the lower (dichloromethane) layer into the beaker, being careful not to

allow any of the upper Irn-Bru layer to come out.



4 Use a pencil to lightly mark an origin line about 1cm from the end of a TLC plate.

Now use spotting tubes to place evenly spaced spots of your extract, the benzoic

acid, the caffeine and the citric acid on the line. Write in pencil on the other end of the

plate (away from the line and spots) what each spot is. You will probably need to make

several applications to each spot, allowing time for drying between each application, to

get enough on. Allow the spots to dry then place the TLC plate in the developing tank.

Remove when the solvent is about 3/4 the way up the plate, and immediately mark the

position of the solvent front with a pencil.

5 When the plate is dry, examine under ultraviolet light in a plate viewer and mark

the position of each spot with a pencil.

6 Remove the plate from the viewer, measure the disance from the origin to each of

the spots and to the solvent front, then calculate the Rf value for each spot (this is the

distance travelled by the spot divided by the distance travelled by the solvent front).

If your plate is overloaded or underloaded then try again, changing the amount of

material spotted onto the plate.

7 Write your results in the table and draw a diagram of your TLC plate in the answer

section, labelling it clearly.

8 Is there evidence of all three compounds in the Irn-Bru extract? Explain your

answer.



Experiment 3 Determination of caffeine by solid phase extraction and uv

spectrophotometry

In this experiment you will extract caffeine from Ir-Bru and measure how much is

present in the drink. To do this you will pass the drink through a solid phase

extraction cartridge which will retain the caffeine then, after washing the cartridge

you will elute the caffeine and measure its concentration by uv spectrophotometry.

Equipment

0.1 & 10mL pipettes (1 of each)

Pipette fillers

Small beaker (25 or 50 mL)

50 mL volumetric flask

Pasteur pipette

C18 solid phase extraction (SPE) cartridge

Vacuum filtration assembly (SPE manifold or 50mL Buchner flask with syringe needle

through stopper)

Dilute ammonium hydroxide (1 mol L-1

)

Dilute hydrochloric acid solution (1 mol L-1

)

Methanol

Caffeine solutions 5.00, 10.0, 15.0, 20.0 mg L-1

in 1 mol L-1

hydrochloric acid

Ultraviolet scanning spectrophotometer with fused silica UV cell

Procedure

1 Pipette a 5.00 mL sample of Irn-Bru into a clean small beaker and add 5 mL of 1

mol L-1

ammonium hydroxide.

2 Condition the SPE cartridge by fitting it to the funnel and flushing through 5 mL

of methanol followed by 5 mL of 1 mol L-1

ammonium hydroxide. Try throughout the

experiment to get a flow rate of 1-2 drops per second.

Turn off the vacuum when the liquid level gets down to the top of the packing and

disassemble the apparatus. Discard the liquid in the collector and reassemble.

3 Load some of the alkaline Irn-Bru sample from step 1 and reconnect the vacuum

line. Add more when the level nears the top of the packing.

4 After the Irn-Bru, pass 5 mL of 1 mol L-1

ammonium hydroxide through the

cartridge to wash the packing (the orange dye will probably come off here). Disconnect

the vacuum line when the liquid gets to the top of the packing.

5 Discard the eluate in the flask into the sink, rinse the flask and empty out as much

water as possible.



6 Reassemble the system and pass through 10.0 mL of methanol, being careful to

collect the eluate. Let this pass through until no more liquid is pulled from the cartridge.

7 Disconnect from the vacuum line, remove the bung and transfer the liquid to a

50.0 mL volumetric flask. Make up to the mark with 1 mol L-1

hydrochloric acid, stopper

the flask and label it.

8 ASK FOR A DEMONSTRATION OF THE USE OF THE SPECTROMETER

YOU MUST USE THE SILICA CELLS FOR THIS EXPERIMENT, NOT PLASTIC

ONES. Care - they are expensive and will smash if dropped!!

9 Set the spectrophotometer wavelength to 274 nm then zero it with distilled water

in the cell(s). You only need to have the cells 3/4 full. Use a Pasteur pipette for filling the

cells.

10 Remove the sample cell, discard the contents, refill with the lowest concentration

of the supplied set of standards and record the absorbance on the result sheet.

Repeat for the rest of the standard solutions, from low to high concentration.

Rinse the cell and Pasteur pipette with water before continuing.

11 Fill the cell with your diluted extract from step 7 and record its absorbance at 274

nm.

12 Rinse the cell, replace the sample with water, set the instrument to scan from 240

nm to 340 nm and store a baseline.

13 Rinse the cell, replace the sample with the highest concentration standard and

record its spectrum from 240 to 340 nm

Label the spectrum.

14 Rinse the cell, replace the sample with the extract from step 7 and record its

spectrum from 240 to 340 nm

Label the spectrum.

15 Plot a calibration graph of the standard absorbances at 274 nm against

concentration.

16 Read off the concentration of the extract and record on the result sheet.

17 Compare the spectra of the standard solutions with that of the extract. Is there

evidence of other compounds being present?

Write down your reasoning on the answer sheet and attach your spectra.



Background

UV / visible spectrophotometry

1,10-phenanthroline forms a complex with ferrous ion more readily than with ferric ion,

and the complex with the ferrous form is more intensely coloured. The intensity of the

colour can be used to measure how much of the complex is present. This is the structure of

the complex:

Caffeine, although forming a colourless solution, absorbs light in the ultraviolet (you will

record its absorbance spectrum in this region), and its concentration in solution can be

measured most easily by the absorbance at 274nm.

Caffeine:

N

N

N

N

O

H3C

CH3

CH3

O

Light itself can be split into the spectrum of colours that we see in a rainbow; different

colours signify different wavelengths and, therefore, different energies. We call a beam of

light of one colour monochromatic.

Light will be absorbed by an atom, ion or molecule when the energy of one quantum of a

particular wavelength of light matches the energy required to cause an electron in an outer

orbital to jump to a higher energy level.



Each absorption band is caused by the transition between a given pair of energy levels;

because the energy level differences vary with different electronic structures, absorption

spectra can often be used to help identify the analyte atom, ion or molecule.

The technique of spectrophotometry relies on the absorption of light by the analyte; the

intensity of a beam of light is measured in the absence then presence of analyte and the

decrease in transmitted intensity is used to determine the analyte concentration.

I I0

light detector

mono-chromatic

The Beer-Lambert law expresses the relationship between absorption and concentration:

A = bc

where A = absorbance, = molar absorptivity (L mol-1

cm-1

), c = concentration (mol L-1

)

and b = optical pathlength (the distance that light travels through the sample, in cm). If this

relationship is valid, then a graph of absobance against concentration for a solution will be

a straight line which passes though the origin.

This is the basic equation of spectrophotometry. The spectrophotometer (or colorimeter)

can only measure the intensity of light, however, so we need an additional relationship

linking absorbance to I and I0. This is A = log (I0/I).

Thin Layer Chromatography (TLC).

In thin layer chromatography a glass or plastic sheet is covered by a uniform thin layer

of adsorbent material. Sample analyte, dissolved in a suitable medium, is applied to

the thin layer plate, dried and the plate placed in a tank containing solvent mobile

phase.

The mobile phase moves up the plate carrying the dissolved sample with it. The

components of the sample will be retarded relative to mobile phase by interaction with

the stationary adsorbent phase, which in this case is silica gel (an acidic polar

adsorbent). In general polar adsorbents retain polar compounds whilst non-polar

adsorbents retain non-polar compounds (the "like likes like" principle).

When the mobile phase almost reaches the top of the plate it is removed and dried. The

spots can be visualised by placing the dried playte under an ultraviolet lamp; the silica

gel has a fluorescent compound impregnated in it, and the spots appear as dark patches



against a greenish background.The position of each spot is marked and the Rf value for

each compound is calculated from the ratio:

Rf = distance moved by analyte spot

distance moved by solvent front

The Rf value is used to identify the analyte. This is an example of development

chromatography.

Solvent-solvent extraction

Solvent-solvent extraction, as used in experiment 2, is a classical method of removing

organic compounds from aqueous solution. It relies on the use of two immiscible (non-

mixing) solvents, one very polar (e.g. water) and one much less polar (e.g. an alkane or, as

here, the haloalkane dichloromethane). If the substance that we want can exist in both

ionised and neutral forms (as acids and bases can) then by adjustment of the pH we can

make the substance dissolve in one or other of the two solvents. For instance, if we make

the water solution acid, then organic acids will be protonated to their neutral form. These

are more soluble in the less polar solvent, and so the organic acid will move from water to

the less polar solvent. On the other hand, if we make the solution alkaline, then the acids

will be ionised and stay in the water, while bases will be neutral and accumulate in the

organic layer.

Solid phase extraction

Solid phase extraction, as used in experiment 3, is a modern version of solvent-solvent

extraction.The device that you have used here has a very thin layer of an 18-carbon alkane

(the less polar solvent) covalently bonded to a solid suport (silica), over which the more

ploar solvents are passed. The less polar solvent is "locked" in place and has a large surface

area for the substance to interact with, which improves the efficiency of extraction (and also

means that you don't have to shake it!). This method reduces the amount of organic solvent

that we have to use, so is more environmentally-friendly.



Results

Experiment 1 Determination of iron by colorimetry

What is the iron-containing compound added to Irn Bru?:

What is the concentration specified on the bottle?:

Absorbance of standard solutions:

Standard iron

concentration:

Absorbance:

Absorbance of Irn-Bru samples:

Sample: 2-fold dilution 4-fold dilution

Absorbance with

phenthroline

Absorbance without

phenanthroline

(blank)

Difference

(corrected

absorbance)

Attach calibration graph

Readings from graph:

(keep to a realistic number of significant figures)

Concentration of iron in 4-fold dilution: mol L-1

Concentration of iron in 2-fold dilution: mol L-1



Calculations:

Explain each step of your calculation in words, write the relevant numbers in an equation

and include units at each stage

Correct for the dilutions of each sample:

Convert the molar concentrations to mass concentrations (g L-1

) of iron:

(relative molar mass Fe = 55.85, i.e. one mole of iron weighs 55.85 g):

Explain your calculation.

Now convert to mg L-1

(or parts per million, ppm). Remember that there are 1000 mg in a

g:

Final answers:

The concentration of iron in Irn Bru is: mol L-1

or g L-1

or

mg L-1



Experiment 2 Extraction and identification of organic

components

Draw a diagram of your TLC results here:

Distance moved by solvent front =

Spot

identity:

Distance

moved:

Rf value calculations:

Explain the calculation in words and show the equation for each sample:

Conclusions from TLC data:



Experiment 3 Determination of caffeine by solid phase extraction and uv

spectrophotometry

Standard solutions of caffeine

Concentration:

Absorbance:

Absorbance of Irn-Bru extract =

(attach calibration graph)

Concentration of caffeine in extract (from graph): mg L-1

(keep to a realistic number of significant figures)

Calculations:

Explain each step of your calculation in words, write the relevant numbers in an equation

and include units at each stage

Correct for dilution of the sample:

(the extract was in 50.0mL which contained all the caffeine from 5.0mL of Irn-Bru, so

you should multiply the value from the graph by an appropriate factor (what will it be??)

to get the concentration of caffeine in the original Irn-Bru sample)

Convert the caffeine concentration from mg L-1

to g L-1

(remember that there are 1000 mg

in one g):

Convert the caffeine concentration from g L-1

to mol L-1

(the relative molar mass of

caffeine is 194.2, i.e. one mole of caffeine weighs 194.2 g). Explain your calculation in

words and show the numbers clearly:



Deductions

Compare the uv spectrum of pure caffeine with that of your extract. Is there evidence of

other compounds being present in this extract ?

Attach your spectra (label each clearly!) and explain your answer.



Questions

1 What are the oxidation states of iron in:

i) the ferrous form (e.g. in FeCl2 ):

ii) the ferric form (e.g. in FeCl3)

2 Draw the structure of benzoic acid (C6H5COOH) in both its neutral and ionised

forms:

3 Why did we need the "blank" IrnBru samples (without phenanthroline reagent) in

the first experiment?

4 How many significant figures did you express your answers to, and why did you

think this number to be "realistic"?



5 The following symptoms may be exhibited when amounts of caffeine greater than

250mg have been ingested (from Desk Reference to the Diagnostic Criteria from DSM-

3-R (American Psychiatric Association, 1987): restlessness, nervousness, excitement,

insomnia, flushed face, diuresis, gastrointestinal disturbance, muscle twitching, rambling

flow of thought and speech, tachycardia or cardiac arrhythmia, periods of inexhaustibility,

psychomotor agitation.

How much Irn-Bru would you have to drink to exhibit such symptoms? (show

your working):

royal society of chemistry analytical division - ne region ... east region...

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