This document describes a series of experiments using analytical techniques such as HPLC, GLC, and ICP-AES to identify components in samples from perfumes, sports drinks, and sweeteners. The results indicate significant differences in the concentrations of sweeteners and metal ions between branded and generic products, highlighting that homemade drinks can have comparable or superior nutrient profiles. The findings suggest that branded perfumes may have longer-lasting scents compared to high street variations.

1. 1
Using the analytical techniques HPLC, GLC and ICP-AES to identify components in samples
given from various perfumes, sports drinks and sweeteners by Molly Winterbottom
Abstract
Carrying out various analytical techniques can allow the identification of various
components of a sample; this sample can be contained in a mixture of components all with
different structures or compositions and from everyday life since analytical techniques can
be used for a wide range of applications in many industries like environmental science and
many more.
1. Introduction
The 3 analytical techniques we used are reversed-phase high performance liquid
chromatography (HPLC), Gas-liquid chromatography (GLC) and inductively couples plasma—
atomic emission spectroscopy (ICP-AES). HPLC is a very common technique used for
separation of a mixture of components, it is used to identify, purify and quantify these
individual components that are in the mixture1
. HPLC can be used to separate compounds in
a mixture that have been dissolved in a solution and determine how much of each
compound is in that mixture. This separation is all based on the molecule’s hydrophobicity2
.
We want to use this technique to identify the 3 different types of sweeteners in branded
and generic diet soft drinks since the sample will be dissolve in a solution and therefore will
be in a mixture. GLC is an analytical technique used to separate and analyse compounds that
are volatile but without decomposing them. We use this technique when identifying
between the 2 perfumes due to their ability to be vaporized. ICP-AES is a spectral method
that can be used to determine a samples elemental concentration. This is done using
wavelengths, once the analyte is burned using a flame ionisation detector the colour that is
emitted will indicate the concentration of that element present in the sample3
. We use this
technique when investigating the elements inside homemade and branded sports drinks to
identify the individual metal element concentrations.
In these series of experiments, we want to identify the difference between a designer
perfume and a high street brand perfume using GLC; the difference between 3 types of
sweeteners in branded and generic diet soft drinks using reversed-phase HPLC and lastly to
identify the metal ions in a solution using ICP-AES comparing homemade sports drinks with
branded sports drinks. We want to make these comparisons to see whether there is a
significance difference between cheaper/homemade Vs more expensive and branded.
2. Methods
The first experiment carried out in this series was the identification and analysis of the 3
different types of sweeteners (Aspartame, Saccharin and acesulfame K) in the branded and
generic diet soft drink using HPLC. In this experiment we needed to compare the retention
times for each sweetener present using a chromatogram. To start off we needed to produce
the chromatograms by using our samples provided, the HPLC data system was set to have
our samples injected into the HPLC injector port in a certain order starting with the Branded
diet drink (degassed) and then the generic diet drink (degassed). Then inject our sweeteners

2. 2
starting with 50 mg L-1
of Saccharin, then 25 mg L-1
of Acesulfame K, after which our
standards needed to be injected for Aspartame. By using 5 standards for Aspartame (100-
500 mg L-1
) it will allow for further analysis of the branded and generic diet drinks to find
their concentrations of aspartame later on. In a HPLC the injector port has a Rheodyne (6-
point injection) valve that allows a certain amount of sample to enter the column by getting
rid of excess as waste and by mixing the sample with the mobile phase, in this case the
mobile phase used was HPLC eluent: 20% acetonitrile / 80% buffer (10 mM ammonium
acetate, 0.1% trifluoroacetic acid, pH 2.6) and wavelength set to around 255nmas this
wavelength is present in the absorbance of all 3 sweeteners allowing a cross section
between the 3 as seen below.
Figure 1 – UV spectra of the 3 sweeteners showing a “cross-over” at a wavelength around
255nm
Figure 2 – Column specifications that were used during the HPLC experiment

3. 3
Shown above in Figure 2 is the column that our sample, mixed with our mobile phase,
enters once the correct volume is acquired and all the waste is removed from the Rheodyne
valve. This glass column contains the stationary phase that is non-polar and will retain most
organic analytes, once it was out of the column the samples were separated into their
components which could then pass through a detector and a chromatogram was produced
where we were able to identify which sweeteners were present in each diet drink (which
can be seen in results and discussion section under experiment 1).
The second experiment was the comparison of the 2 perfumes using GLC and a ‘smell test’.
To provide a suitable sample the syringe was rinsed using ethanol roughly 3 times taking up
a full syringe of 1 microlitre. Before injecting the perfume samples, they needed to be
diluted using a 20-fold dilution (1:20).
Then by the use of one of the diluted perfumes samples the syringe was rinsed and the
syringe was filled to its full capacity of 1 microlitre. Once the sample was in the syringe it
was taken to the injection port, whilst taking care around it since the temperature of the
port was 250 degrees Celsius. Then inserting the syringe into the port and injecting 0.2
microlitres of the sample into the GLC injector, where it then moved into the
injector/column oven (starting temperature of 60 degrees Celsius) and entered the coiled
column for separation. Once the sample reached the flame ionisation detector the
chromatogram was produced for each perfume. The mobile phase, in this case nitrogen gas,
then mixed with the samples injected 1 at a time and carried it to the liquid stationary
phase, inside the glass capillary column. The oven temperature rose from low to high with a
gradual increase of 10 degrees per minute and finished with a temperature of 220 degrees
Celsius with a hold time of 4 minutes.
Whilst the chromatogram was being produced, a clean tissue was taken and labelled “aged
designer” and had a syringe full (1 microlitre) of the designer perfume placed on it and left
on a clear area of a bench. This was repeated with the high street perfume in order to
conduct the ‘smell test’. After 30 minutes these steps were repeated in order to produce
the “fresh” perfume sample and therefore the previous samples were used as the “aged”
samples. Immediately after the new “fresh” samples were produced, the samples were
smell tested and using the fragrance wheel, the descriptions of the scents were recorded in
a table. (The result can be viewed under experiment 2 in results and discussion)
The last practical done was using ICP-AES. To start off, dilutions of the soft drink samples
needed to be done using a pipette, filler and volumetric flask making sure to label each glass
volumetric pipette with the dilution carried out. The dilutions made were 100.0 mL of a 1 in
10 dilution. Suitable disposal of the pipettes was then carried out making sure to rinse them
with deionised water and place them tip-down in the pipette boot to be washed. Then by
taking our dilutions to the ICP-AES we were able to produce concentrations of our elements
detected by using an internal standard calibration graph produced by the technical staff on
the instrument’s software. The results were collected for the drink samples and can be seen
in the results and discussions section under experiment 3.

4. 4
3. Results and discussion
Experiment 1 – HPLC
This table above shows the retention time (in minutes) and peak areas of each peak present
in all the samples and standards in this experiment. We can establish a presence of
Acesulfame K and aspartame standard 2 in the branded drink and in the generic drink there
are similar retention times that correspond to saccharin and aspartame standard 1.

5. 5
Branded Diet Drink
Generic Diet Drink
Acesulfame K
Saccharin

6. 6
From the 2 blue circles shown above we can clearly establish that the branded diet drink
contains Acesulfame K sweetener, and the generic diet drink contains the sweetener
saccharin due to the appearance of the exact same peaks with near enough the same
retention time. The retention times of both Saccharin and Acesulfame K varies as multiple
peaks are present in acesulfame K, whereas only 1 peak is present in Saccharin.
Figure 3 - The Peak area of Aspartame against the concentration of Aspartame
Our 2 sample peak areas with unknown concentrations: Branded = 138535.04 uV*sec Peak
area; Generic = 83887.44 uV*sec peak area
By using the table above (table 2) we can use the peak areas of the branded and generic diet
drinks to find the concentration of aspartame in the drinks.
Figure 4 – calibration curve determining the concentrations of our unknown diet drinks
A) the unknown branded B) the unknown generic
A
B

7. 7
So, from figure 3 we can see that the calibration curve determines the concentration of
branded diet drink to be around 150 mg L-1
and the generic diet drink concentration to be
around 95 mg L-1
The acceptable daily intake for aspartame is 50 mg per kg of body weight then for a person
who is 100kg would be 100 x 50 = 5000mg
With a graph concentration of Aspartame in a generic drink of 95 mg L-1
Would result in a volume of 5000/95 = 52.6 litres
Experiment 2 – GLC
Table 2 – Quantitative data taken from the perfume samples by GLC
Figure 5 – quantified data from table 3 showing the amount of limonene present in each
perfume A) designer perfume B) High street perfume
Using the data from table 3 and plotting a calibration graph we can now find out the
concentration of limonene in the designer and high street perfumes. The line labelled A in
Figure 5 indicates that the concentration of limonene in the designer brand perfume is
A
B

8. 8
around 50-55 mg L-1
and the limonene concentration for the high street brand perfume
shown by line B is around 25 mg L-1
. However, due to these perfumes being diluted in a 20-
fold dilution we would need to multiply the concentrations found by 20. Therefore, the
designer perfume would have a concentration of 1000-1100 mg L-1
and the High street
perfume would have a concentration of 500 mg L-1
The ‘smell test’
Figure 6 – The fragrance wheel used during the ‘smell test’ experiment to describe the
fresh and aged perfume sample
In the ‘smell test’ section of this experiment the fragrance wheel shown above in figure 6
allowed each participant to describe what they were smelling in a word or two. Whilst the 2
groups of 11 partners described what they smelt the descriptions were recorded on the
table below (table 4) and later on colour coded showing a clear difference between the
designer perfume and the high street’s fresh and aged samples.
Fragrance wheel (Michael Edwards, 1983)

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Table 3 – ‘Smell test’ results in a qualitative table colour coded
Key – FLORAL = RED
SOFT/WEAK FLORAL = ORANGE
CITRUS = GREEN
WOODY = BROWN
WATER/FRESH = BLUE
WATER, FLORAL = PURPLE
CITRUS, FLORAL = PINK
Taking into account that this ‘smell test’ is more subjective we do see a difference between
the designer and high street brand perfumes. These differences include how long the
fragrances lasted over 30 minutes and how strong the scent was fresh compared to aged. In
the chemistry 2 group we can see that the majority of participants though there was a floral
scent present in the fresh designer perfume which led to a soft floral scent after the 30
minutes and in some cases, seen in group 1, the floral scent was described as still strong
more frequently than in group 2.
By using the chromatogram produced by the GLC we can now start picking out more
accurate chemical differences by comparing both the perfumes retention times and their
peak areas.

10. 10
GLC chromatogram of designer perfume
GLC chromatogram of High Street Perfume
Between the 2 samples the first peak established has the same retention time of 0.72 mins
(indicated by the blue circle) so to start there would not be a significant difference between
the 2 smells although they do have a different peak area. Going further down the list of peaks
we notice that there are 16 more peaks established in the first GLC sample possibly suggesting
that it will overall have a longer retention time of the smell of the perfume than the other
sample. We can also see this cluster of peaks shown in the designer perfume that isn’t as
dominant in the high street brand so quite a lot of the peaks that are different are mainly in
this area of the chromatogram. We can also see that there is roughly a 3 min difference in the
retention time of the last peak in both the samples. The sample with the most peaks has a
16.559 min retention time of the last peak whereas the other sample has a retention time of
13.992 mins for its last peak (shown by the green circles) suggesting that the first sample with
the longer retention time will have the perfumes fragrance for longer.

11. 11
Experiment 3 – ICP-AES
Table 4 – ICP Short Report of the elements ran through the ICP-AES
In this experiment we use a 1 in 10 dilution of 100 ml this would mean that our samples
injected was 10 ml of the sample mixed with 90 ml of water to dilute the drink therefore we
have 10-fold dilution.
In order to calculate the undiluted concentration, we need to multiple by the inverse of our
dilution factor. Therefore, we need to take our final concentration and multiply it by our
dilution factor.
CALCIUM
So, starting with Calcium it has a calculated concentration of 2.39 in the homemade drink
and 3.53 in the branded sports drink, therefore our undiluted concentrations would be 2.39
x 10 = 23.9 for the homemade sports drink and 3.53 x 10 = 35.3 for the branded sports
drink.
POTASSIUM
Potassium has a calculated concentration of 24.46 in the homemade drink and 23.57 in the
branded drink. So, the undiluted concentration of the homemade drink would be 24.46 x 10
= 244.6 and for the branded drink it will be 23.57 x 10 = 235.7
MAGNESIUM
Magnesium has a calculated concentration of 1.24 in the homemade drink and a value of
1.74 in the branded drink. So, the undiluted concentration of the homemade drink would
be 1.24 x 10 = 12.4 and the branded drink would have an undiluted concentration of 1.74 x
10 = 17.4
SODIUM
Sodium has a calculated concentration of 94.89 for the homemade drink and for the
branded drink it had a calculated concentration of 100.07. So, for the homemade drink we
can calculate the undiluted concentration, this will give us 94.89 x 10 = 948.9 and for the
branded drink the undiluted concentration would be 100.07 x 10 = 1000.7

12. 12
By looking at the concentration values for the metals in both the homemade and branded
sports drinks we can establish that this specific homemade drink contains 8.9 more
potassium which could suggest that this person needs more of this electrolyte possibly to
help with muscle contractions during exercise4
4. Conclusion
By comparing branded to non-branded products, we can see that especially in the sports
drinks the difference in concentrations of metallic elements is not as significant as expected,
in some ways it would be better to make homemade sports drinks filled with the
electrolytes your specific body needs to enable your body’s full ability to function and
recover after exercise. On the other hand, comparing the 2 different perfume brands we can
establish that the fragrance will last longer and maybe stronger with the branded perfume
than the high street perfume.
1 Sanjay Kumar D and D.R. Harish Kumar IMPORTANCE OF RP-HPLC IN ANALYTICAL METHOD DEVELOPMENT: A REVIEW Department of
Pharmaceutical Analysis, Krupanidhi College of Pharmacy, Sarjapura Main Road, Carmelaram post, Bangalore-560 035, Karnataka, India
https://ijpsr.com/bft-article/importance-of-rp-hplc-in-analytical-method-development-a-review/?view=fulltext
2 Aguilar MI. (2004) Reversed-Phase High-Performance Liquid Chromatography. In: Aguilar MI. (eds) HPLC of Peptides and
Proteins. Methods in Molecular Biology™, vol 251. Springer, Totowa, NJ
3
Andrew R. Barron, in Physical Methods in Chemistry and Nano Science, 2019 version 1.11
https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Book%3A_Physical_Methods_in_Chemistry_and_Nano_Science_(Barron)/
01%3A_Elemental_Analysis/1.05%3A_ICP-AES_Analysis_of_Nanoparticles
4
https://medlineplus.gov/potassium.html