Environmental & Analytical Chemistry II Analysis of vitamin supplements Expt 2407a

Experiment 2407
Analysis of vitamin supplements.
You will be working in pairs for this practical.
The experiment will be conducted in the Physical Chemistry lab – Johnson Building room 118.
Labs start at 1:00PM. See the practical roster on MyUni for further information.

Safety Information
This practical involves working with 6M Hydrochloric Acid. Contact with HCL causes burns. Always
wear gloves when handling solutions containing HCL and use extreme care. 5% Acetic Acid is also
used extensively. Avoid skin and eye contact. Other reagents may be considered as mildly toxic.
Avoid skin contact and exercise good lab practises.

Preliminary Questions
Your answers must be approved by a demonstrator BEFORE YOU BEGIN!
1. During the experiment you are required to dissolve part of an iron tablet and make the resulting
solution up to 50mL in a volumetric flask. You are told that some of the iron tablet will not dissolve,
but it is insignificant compared to the 50mL volume. What would happen if the filler material was
significant? Hint: consider what will happen when you make the solution up to the mark.
2. Why is it necessary to cool the iron tablet solution before making it up to the 50mL mark in a
volumetric flask?
3. When preparing the replicate solutions of ‘complexed’ iron tablet you will be using 50mL
volumetric flasks instead of the 100mL flasks. As such you must scale down the volume of each
complexing reagent by 50%. Calculate the new volume required of hydroxylamine, acetate buffer
and phenanthroline solutions.
4. To determine the riboflavin content of a vitamin supplement you will use the method of ‘standard
additions’. What advantage does standard additions have over a direct calibration?
5. You are required to make up a series of standard addition flasks to determine the riboflavin
content of a vitamin supplement. Calculate the required volume of 100ppm riboflavin standard
required for each flask.
6. What attribute of a ‘standard additions’ calibration graph is used to determine the unknown
concentration?

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Environmental & Analytical Chemistry II Analysis of vitamin supplements Expt 2407a

Introduction
This practical contains two distinct parts – the determination of iron and the determination of
riboflavin. Each part can be undertaken in parallel; that is if you are waiting for something to
happen for one part you may continue with the other.

While this practical may seem long and daunting it is actually quite easy. Most solutions are just
simple dilutions which can be undertaken in very little time. The data acquisition on the instruments
is also very fast; taking only ~5-10 minutes for each part.

If you get stuck, or are unsure as to how to proceed, please speak to a demonstrator. They are
here to help you and will guide you through everything you need to do.

Introduction – determination of iron

In this procedure, the iron in a supplement tablet is dissolved in acid, reduced to Fe 2+ with
hydroquinone, and complexed with o-phenanthroline to form the intensely coloured iron(II) tris-
phenanthroline complex.

The absorbance of the complex is measured at the λmax of 508 nm using a UV-Vis
spectrophotometer.

By analysing a number of standards you can construct a calibration curve of iron content vs
absorbance. Unknown solutions can then be analysed and concentrations calculated using the line
of best fit.

The method is based on an approved method of the Association of Analytical Chemists.

You have been supplied with the following reagents:
 Hydroxylamine NH2OH: Aqueous solution containing 10 g/L. This has been stored in an
amber bottle to prevent air / light oxidation.
 Sodium acetate: 1 M sodium acetate in 1M acetic acid solution (buffer).
 o-Phenanthroline: 2.5 g in 1000mL of water. Stored in an amber bottle.
 Standard Fe (0.04 mg Fe/mL): 0.281 g of reagent-grade Fe(NH4)(SO4)2.12H2O in water
in a 1 L volumetric flask containing 1 ml of 98% H2SO4.

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Environmental & Analytical Chemistry II Analysis of vitamin supplements Expt 2407a

Introduction – determination of vitamin B2 (riboflavin)

In this procedure you will be using fluorescence spectroscopy to determine the concentration of vitamin B2 (riboflavin)
in a vitamin supplement. Riboflavin is ideally detected by fluorescence as it has a strong emission at around ~500-
530nm when excited by 374nm light. In contrast riboflavin has a broad UV-Vis absorbance spectrum which may have
interferences due to other components of the vitamin supplement. This analysis will be undertaken using the method
of standard additions.

Fluorescence spectroscopy:
The fluorescence spectrophotometer operates on a different principle to the regular UV-VIS spectrophotometers you
have used in the past (or even just a short time ago for the iron section). Firstly light is introduced into the sample at a
given wavelength. This is called the excitation wavelength as it promotes the analyte of interest into its higher energy
‘excited’ state. The spectrophotometer then measures the light emitted from the sample at another given wavelength
which is greater than that of the excitation wavelength. This is called the ‘emission wavelength’ as it indicates the
wavelength of light which is being emitted by the analyte as it decays from the excited state into a lower energy state.
There are also two other parameters which control the spectral resolution – these are called the excitation and
emission slit widths. The larger the number of each parameter means that more light is either incident on, or captured
from the analyte respectively. This comes with a tradeoff – the larger the value the less precision. In simplest terms
the slit widths are similar to the error in each parameter; for example a slit width of 5nm means that the light passing
through can only be resolved to ±2.5nm.

The fluorimeter is able to scan across a range of excitation or emission wavelengths and measure the intensity of light
fluoresced by the sample (in arbitrary units). You will be using the fluorimeter in emission mode as you are only
interested in the intensity of light emitted by the fluorescence of riboflavin.

What are standard additions?

One of the biggest issues in analytical chemistry is a phenomenon called ‘matrix effects’. A matrix effect is something
that causes a change in a measured value due to some chemical or other external influence. For example consider a
turbid water sample analysis using UV-Vis spectroscopy. Assume you calibrate the UV-Vis using a series of standard
solutions using clean water; obtaining a perfect linear relationship. You then place the unknown solution into the UV-
Vis and record the absorbance. The absorbance measured, and thus concentration calculated, is not valid as the
turbidity in the water has scattered some of the incident light rather than absorbed it. This did not happen in the
standards as the water was clean.

The method of standard additions aims to eliminate matrix effects. This is achieved by adding a fixed amount of
sample (or ‘spike’) to each standard solution. Consider the previous turbid water example; by adding a fixed amount of
turbid water to each standard; each respective standard is now equally turbid. The recorded absorbance for each
standard solution is influenced by the turbidity equally; and thus the result is now valid.

Preparing standard addition solutions is almost as easy as a direct calibration. Firstly you must decide on a range of
concentrations which will encompass your unknown. For example assume 0, 1, 2, 3, 4ppm. You then add the correct
volume of stock solution to each flask but DO NOT make it up to the mark. Next the ‘spike’ of unknown solution must
be added. The spike is chosen by an educated guess; ideally you want to add enough unknown so that when each
standard addition flask is made up to the mark it will have a concentration similar to your standards (i.e. around 1-
4ppm). In reality it can even sit outside your standard range by a significant amount; so this part isn’t as hard as it
seems. Once the spike is added the flasks are made up to the mark. Measurement in the instrument is the same as
direct calibration.

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Environmental & Analytical Chemistry II Analysis of vitamin supplements Expt 2407a

Analysis of standard addition results:

At this point it is not clear on how this can be used to extract the unknown concentration as there is a ‘calibration’ but
no unknown sample has been run. Luckily by a trick of mathematics the unknown can be easily extracted from the
calibration curve itself.

Since we are using fluorescence in this practical let us assume that intensity emitted is linearly proportional to
concentration (in fact it isn’t; however this holds well enough for low concentrations). Note if you were performing an
experiment by measuring absorbance this example is still valid, just replace intensity with ABS.
𝐼𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 = 𝑘 × 𝐶
Now consider our standard addition flasks – there are actually two concentrations of riboflavin present. The first is due
to the addition of standard, the second is due to the addition of the ‘unknown’. Since the unknown has been added in
equal concentration to each flask we then have:
𝐼𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 = 𝑘 × (𝐶𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 + 𝐶𝑜𝑛𝑠𝑡𝑎𝑛𝑡𝑢𝑛𝑘𝑛𝑜𝑤𝑛 )
Therefore this takes the form of a function like:
𝐹(𝑥) = 𝑘 × (𝑥 + 𝐴)
Where A is some constant value. For those maths aficionados you might recognise that if we add a constant A to each
x value this has the effect of ‘translating’ the graph on the x-axis by that given value. For example consider a simple
function y=3x. When y=0, x=0; and the function has a slope of 3. If we now substitute x = (x+1) then you will notice that
the function still has a slope of 3; however when y= 0, x = -1. By adding the constant A=1 we have offset the graph on
the x axis by –A. If this doesn’t quite make sense it’s worth 5 minutes of your time trying it out on Graphpad/Excel. You
can then graphically see what is happening. Remember that x has been substituted with (x+A) in brackets – this is
important as if you forget them your just changing the y-intercept!
Since a constant ‘spike’ of unknown solution has been added to each flask (including the zero) this relationship proves
useful. If the concentration of riboflavin standard added vs intensity is plotted out; the negative of the x-intercept will
be equal to the unknown ‘spike’ concentration.
Remember that the ‘spike’ concentration will not be equal to the original concentration of the unknown as it was diluted
in the standard addition flasks. For example if you put 1mL of spike into a 100mL flask you have diluted the unknown
by 100 times!

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Environmental & Analytical Chemistry II Analysis of vitamin supplements Expt 2407a

Experimental
You have been supplied with two types of ‘over the counter’ vitamin supplement tablets. Your task is to determine the
iron and riboflavin content of each respective supplement and relate the result to the manufacturers claims (on the
label).

Disposal of solutions:
All solutions and reagents used in this practical are to be poured into the waste container on the bench.

Determination of iron:

Preparation of standard solutions:

Begin by preparing 5x clean 100mL volumetric flasks. These will contain your standard solutions. Pipette 10, 5, 2, 1
and 0mL of 0.04mg/mL Fe standard solution into the 5 respective flasks. Make sure you label the concentration on
each flask, not the volume of iron standard (you will need to calculate these). In the following order (using measuring
cylinders) add 2mL of hydroxylamine solution, 10mL of sodium acetate buffer and 10mL of phenanthroline solution to
each flask. Make up the flasks to the mark and mix thoroughly using the swirl method. If you are unfamiliar with this
method, speak to a demonstrator. Allow the solutions to stand for at least 10-15 minutes to allow the complex to form.

Preparing the iron supplement tablet:

Choose an iron supplement tablet and weigh it on the 0.1mg analytical balance. Do not forget to record the mass of
the chosen tablet to 5 SF in your lab book.

The active ingredient in the supplied iron supplement is ferrous succinate. This is quite soluble in the stomach
compared to ferric phosphate which is used in some other variants. Begin by preparing a clean mortar and pestle.
Take your chosen iron tablet and grind using the pestle. Once the tablet is finely ground weigh ¼ of the tablets original
mass into a 100mL beaker. Add 25mL of 6M HCL to the beaker using a measuring cylinder. Place the beaker onto the
steam bath and allow to heat for about 25 minutes to allow the iron to dissolve. Since the tablet contains some
insoluble filler material (chalk) the solution will be a muddy brown colour.

When dissolved allow the solution to cool in the fume cupboard next to the steam bath. Once the solution is at room
temperature transfer the contents of the beaker quantitatively to a clean 100mL volumetric flask. Make sure you rinse
the beaker with deionised water several times; transferring the rinsings into the flask. Mix thoroughly using the swirl
method.

Preparing the replicate unknown solutions:

It is useful to perform replicates on an experiment to determine the reproducibility of a result. In this experiment you
will be complexing the iron tablet solution three times (in 3 separate flasks). Each flask should theoretically contain the
same iron content, however some variation may exist.

Begin by preparing 3x clean 50mL volumetric flasks. Label them replicate A, B and C. Pipette 2mL of iron tablet
solution into each flask. You must now add the complexing reagents (in order) to each flask using the reduced
volumes calculated in the preliminary questions. Make up to the mark with water and mix thoroughly using the swirl
method. Allow 10-15 minutes for the complex to form.

Collecting data on the UV-Vis spectrophotometer

Once your solutions have been prepared you may use the Metertek UV-Vis spectrophotometer to determine the
absorbance of each respective solution. You have been supplied with 10mm plastic UV-Vis cells which are stored in
grey cell holders. Whenever the cells are not in use (or being filled) they should be left inside the holder. This avoids
the chance they may be knocked over. Fill each cell with the solution of interest to a level of about 8mm from the top.
Overfilling is not required and will most likely result in spillage. Do not forget to label the cell holder so you can identify
the cell contents. Never label the cell itself!

Take the cells (in the holders) to the UV-Vis equipment. Using the instructions provided obtain absorbance data at
508nm for each solution. Print out two copies and then check your results with a demonstrator.

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Environmental & Analytical Chemistry II Analysis of vitamin supplements Expt 2407a

Determination of riboflavin:

You have been supplied with a sample of tablets which contain approximately 100mg of riboflavin (vitamin B2). Your
task is to determine the concentration of riboflavin by standard additions and fluorescence spectroscopy. All
solutions are made up using 5% acetic acid; do not use water!
Disposal of solutions:
All solutions and reagents used in this practical are to be poured into the waste container on the bench.

Preparation of the vitamin tablet solution:

Begin by weighing one vitamin tablet on an analytical grade balance (0.1mg precision). Note the weight of the tablet in
your lab book to 5 significant figures.

Return to the bench and grind the same tablet in a clean and dry mortar and pestle until fine. Return to the balance
and weigh out 1/20th of the mass of the ground tablet into a clean and dry 100mL beaker. Avoid any tablet chunks
such as the tablet coating; only weigh the fine powder. Write the exact mass you weighed into your lab book as this
will be required in further calculations.

Add ~50mL of 5% acetic acid into the beaker, using the beakers markings as a rough guide. The exact volume in this
step is unimportant provided you do not exceed ~75mL. Cover the beaker with alfoil to prevent evaporation and then
leave on the bench. Swirl gently every 2-3 minutes until approximately 10 minutes has elapsed. This gives time for the
riboflavin to dissolve into the acetic acid.

Quantitatively transfer the contents of the beaker, including any undissolved solids into a 100mL volumetric flask.
Use 5% acetic acid to rinse out the beaker and collect all the rinsings into the 100mL flask. Be careful not to rinse too
much at a time – you do not want to overfill the 100mL flask!! Once transferred make the flask up to the mark with 5%
acetic acid. Mix thoroughly using the swirl method. Note that some insoluble filler material may still be floating around.
In terms of volume the amount of material is insignificant. If the material causes light scattering in the instrument the
effect will be cancelled out as there is a constant amount of ‘unknown’ in the standards.

Preparation of the standard addition flasks:

Begin by labelling 5 clean, dry 50mL volumetric flasks “0ppm, 1ppm, 2ppm, 3ppm, 4ppm”. Note that the concentration
written on the flask is the concentration due to the added riboflavin standard, not the total concentration of riboflavin
in the flask.

Pipette the required volume of 100ppm riboflavin standard solution into 4 flasks (not the 1st flask as it is zero!). Do not
make the flasks up to the mark yet!

Decant about 25mL of the vitamin tablet solution into a small, clean and dry beaker. Let the beaker sit for about a
minute so that any particulate sinks to the bottom. From the beaker pipette 1mL of solution into each of the 5 flasks
(including the zero). Make up each flask to the mark using 5% acetic acid and mix thoroughly using the swirl method.

Preparing the cells for use in the fluorimeter:

On the bench use a transfer pipette to fill 5x clean and dry plastic cuvettes with each respective standard addition
solution. Fill to a maximum of 2/3 full. If the cuvettes are dirty they must be washed using deionised water and then
thoroughly rinsed with the solution of interest. The slightest contamination due to water or foreign material in these
cells will cause a significant change in the measured value on the fluorimeter. Wipe each cell externally using a
kimwipe to ensure no fingerprints or dirt is present.

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Environmental & Analytical Chemistry II Analysis of vitamin supplements Expt 2407a

Determining the emission wavelength:

To save time the excitation wavelength of riboflavin has been supplied (374nm). This was determined by taking an
absorption spectra on a UV-VIS spectrometer and choosing an absorbance maxima. You must however determine an
appropriate emission wavelength for riboflavin before continuing.

Before continuing speak to a demonstrator who will explain the fluorimeter software.

Take the 5 cells over to the fluorimeter and place the 4ppm standard addition solution into the instrument. Load the
‘SCAN’ software, select the emission option, select the accumulation scan option and set the instrument to the
following settings depending on which instrument you are using:

Excitation wavelength: 374nm

Emission slit: LS50b fluorimeter: 3.0nm - LS55 fluorimeter: 2.5nm
Excitation slit: LS50b fluorimeter: 3.0nm - LS55 fluorimeter: 2.5nm
Emission scan range: 400nm to 650nm
Number of scans: 3

Click on the traffic light button to begin the scan. Once the scan begins you will need to periodically click on the ‘resize
Y axis button’ which looks like two vertical arrows. The spectra will not rescale unless you click this button. Once
complete you should observe a peak around ~500 to ~530nm. Remember this spectra is showing the intensity of light
fluoresced, or emitted, at each given wavelength (in arbitrary intensity units). Use the cursor tool to determine the
exact emission wavelength maxima and write the value down in your lab book.

Collecting data on the fluorimeter:

For this section it will help to have a spreadsheet open in which to record the values. Each fluorimeter computer has
Microsoft Excel installed for this purpose.

Remove the cell (if any) from the fluorimeter and replace with the first solution (the 0ppm standard addition solution).
Load the ‘READ’ software and set the instrument to the following settings:

Excitation wavelength: 374nm

Emission slit: LS50b fluorimeter: 3.0nm - LS55 fluorimeter: 2.5nm
Excitation slit: LS50b fluorimeter: 3.0nm - LS55 fluorimeter: 2.5nm
Emission wavelength: The value you determined earlier
Integration Time: 10 seconds
Select ‘do not save’ data option

Press the traffic light button and watch the readout. Every 10 seconds the readout will update with a new value (the
integration time dictates the time period). Record 3 values displayed into your spreadsheet (i.e. record a value, wait 10
seconds, record a value etc). Once complete press the traffic light button to stop recording.

Repeat the above for the remaining 4 standard addition solutions, taking 3 readings for each. Once complete remove
all solutions from the fluorimeter.

Make sure you save your spreadsheet to either the G drive or a flash drive. You will need these values for the
analysis!

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Environmental & Analytical Chemistry II Analysis of vitamin supplements Expt 2407a

Cleanup (before analysis)

All solutions are to be poured into the waste container on the bench. Rinse all glassware thoroughly with deionised
water in the sink and place neatly back in the locker.

Analysis of results - Iron

Begin by plotting the concentration vs absorbance for the Fe standard solutions in Graphpad Prism. Obtain a linear fit
which specifies the standard error for both the slope and intercept. Also obtain the 95% confidence interval for the
slope and intercept.

Calculate the nominal (or average) iron content in milligrams for each replicate (A,B,C) individually. This is done by
using the equation of best fit. Remember you dissolved and diluted part of the iron tablet – these steps must be taken
into account when calculating the mass of Fe.

Using a different approach you can also calculate the 95% confidence limit in the result. Calculate the average
absorbance from the 3 replicates (A,B,C). Using this calculated absorbance, use the line of best fit to calculate the
nominal (average) iron content in the tablet. Furthermore use the 95% confidence limit data to calculate the upper and
lower limits for the tablet. Your result should be in the form: Nominal iron content = xxx mg, Upper 95% iron content =
yyy mg, Lower 95% iron content = zzz mg.

One other useful statistic available from the calibration is the detection limit. The detection limit is defined as the
minimum concentration that can be reliably measured using this technique. Conceptually the detection limit is defined
as the concentration of the blank + 3 times the standard error in the blank. Hint: What was the concentration of your
blank solution? What axis is it intercepting at this concentration? Graphpad may have already inadvertently performed
this calculation.

Finally comment on your result (including confidence intervals, error etc) and compare it to the manufacturers claim on
the label. Did your mass of Fe in the tablet agree with the label? Is it greater or less than the manufacturers claim?
Does the label value fall within the 95% confidence interval?

Analysis of results - Riboflavin

Begin by averaging your 3 readings for each standard addition solution. Notice each reading is slightly different
(usually no more than 1-2%). This is due to the fact fluorescence readings are inherently noisy; that is the measured
intensity fluctuates due to a number of external factors.

Using Graphpad Prism plot standard addition concentration (independent variable) vs averaged fluorescent intensity
(dependant). Apply a linear fit using ‘Linear Fit’; do not use the Chemistry II linear fit equation as it misses out some
details. Obtain the concentration of the unknown ‘spike’ and then calculate the concentration of the unknown solution.
From the concentration of the unknown solution calculate the amount (in mg) of riboflavin in the tablet you analysed.
Hint: remember your dilutions; and remember you only used part of a vitamin supplement tablet! Does your calculated
value agree with the label?
If time permits you will also notice that Graphpad has given you the 95% confidence interval for the X-intercept.
Using this information calculate the 95% confidence limit for the mass of riboflavin in the vitamin supplement. Hint: it’s
a lot easier than the CI for iron, just plug a couple more numbers into a few dilution calculations! Does the 95% CL
encompass the manufacturers claim?

Questions
These additional questions should be answered in your practical report.
1. Suggest a reason why the iron or riboflavin content in each tablet may be slightly higher than the value on the
label? Hint: Consider the manufacturing process – is each tablet identical? Are riboflavin and iron stable?
2. The method of standard additions has many advantages, yet it is not used as frequently as direct calibration.
What is one of the biggest disadvantages of standard additions compared to a direct calibration?

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Environmental & Analytical Chemistry II Analysis of vitamin supplements Expt 2407a

References
1. From Harris Expt 19
2. R.C. Atkins, J. Chem. Ed. 1975, 52, 550.

Acknowledgements
Matthew Bull – Added fluorescence component and revised Fe – 2/2018
Matthew Bull – Revised 7/2012
A/Prof Gerald Laurence – Original Edition

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