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Practical 5: Analysis of Vitamin C using HPLC


1. To learn the operational principles of a high performance liquid chromatography(HPLC)

and its apparatus
2. To quantify the unknown concentration of ascorbic acid using HPLC method and analyse
the one with fruit juice.


Vitamin C is an important physiological antioxidant which helps to protect cells from

damage caused by the free radicals. Free radicals are highly reactive uncharged molecules having
an unpaired valence electron. They are very unstable and react quickly with other compounds to
gain stability. Attacked molecule may become a free radical itself once it loses its electron and
begins a chain reaction that can cascade and causes the disruption of a living cell (National
Institute of Health, 2011). The body can handle free radicals in normal condition but absence of
antioxidant or excessive free-radical production may lead to diseases. Antioxidant like vitamins
C and E are very stable and can neutralizes free radicals by donating one electron to the highly
reactive molecules. Vitamin C or also known as ascorbic acid is water-soluble and its presence
can be analysed by conducting high performance liquid chromatography (HPLC).

HPLC is a form of column chromatography used to separate, identify and quantify

compounds based on their retention time in the stationary phase inside the column. A sample
mixture or analyte is pumped in a solvent known as the mobile phase at a high pressure through a
column with chromatographic packing material which conducts the stationary phase. The sample
is carried by a moving carrier gas stream of helium or nitrogen. Analyte retention time varies
depending on the strength of its interactions with the stationary phase primarily due to different
polarities in the analytes, the eluent strength of the mobile phase and the flow rate (Hanai, 1999).
The principle is that analytes which has the least amount of interaction with the stationary phase
or has the most amount of interaction with the mobile phase will exit the column faster (The
Linde Group, 2016). A detector produces the separated compound bands as they elute from the
high pressure column which information is sent to a computer that will then generate a
chromatogram. The mobile phase exits the detector and is either sent to a waste or collected.

Methods and Materials


1 ml of autopipette, 10 ml volumetric flasks, 0.45 µm filter


Ascorbic acid standard 1 mg/ml, unknown ascorbic acid solution, fruit juice, ultra-pure water


1. A series of standards which containing a variable concentration of analyte (see table) was
prepared by one practical group due to time constraints. The standards were diluted with
ultra-pure water inside volumetric flasks.

Standard Ascorbic acid Total volume ( Ascorbic acid Peak area

solution standard 1mg/ ml ¿ concentration
ml (ppm)
1 0.2ml 10 20
2 0.4ml 10 40
3 0.6ml 10 60
4 0.8ml 10 80
5 1.0ml 10 100

2. The samples had to be diluted by all practical groups. 0.5ml of each sample was
transferred into different volumetric flasks and each volumetric flask was topped up to 10
ml .
3. A 0.45 µm filter which prior to injection was used to filter the samples and standard
solution. Lecturer or lab staff was referred to on how to use and wash the filter.
4. 20 µl of each standard and unknown solution were injected into the HPLC by using a
Hamilton syringes. Lab staff was referred to on operating and injection procedures.
5. The peak height was measured for each standard. The data was used to prepare a
calibration graph and the concentration of analyte in the samples were calculated.


Standard Ascorbic acid Total volume ( Ascorbic acid Peak area

solution standard 1mg/ ml ml ¿ concentration (ppm)
1 0.2 10 20 650375
2 0.4 10 40 805978
3 0.6 10 60 1039690
4 0.8 10 80 1664862
5 1.0 10 100 1916072
Table 1.1 Ascorbic Acid Concentration and Peak Area of Ascorbic Acid Standard

Unknown Ascorbic acid Total volume (ml) Ascorbic acid Peak area
sample standard 1mg/ ml concentration (ppm)
1 0.5 10 50 1232298
Table 1.2 Ascorbic Acid Concentration and Peak Area of Unknown Sample

Peak Area with Ascorbic Acid Concentration


f(x) = 16951.39 x + 198312
Peak Area (µV*sec)

R² = 0.95



10 20 30 40 50 60 70 80 90 100 110
Ascorbic Acid Concentration (ppm)

Peak Area Linear (Peak Area)

The standard curve of peak area of 5 standard solutions against their ascorbic acid concentration
is shown on the graph above. The R2 value obtained from the graph above is 0.9494. However,
the conclusion that our result obtained is not very accurate since the R2 value we obtained is
quite far from 0,99, which is theoretical value. Therefore, we deduced that some experimental
errors may be did during the experiment and cause the value not very accurate.


y = 16951x + 198312, whereby y = peak area (µV*sec), x = ascorbic acid concentration (ppm)

To determine the ascorbic acid concentration (mg/mL) of an unknown sample given its peak area

Peak area = 1232298

1232298 = 16951x + 198312
1232298 – 198312 = 16951x
1033986 = 16951x
x = 60.99ppm
= 60.99 x dilution factor
= 60.99 x 20
= 1219.8 mg/mL

High performance liquid chromatography (HPLC), is a technique in analytical chemistry

used to separate, identify and quantify each component in as mixture. It relies on pumps to pass a
pressurized liquid solvent containing the sample mixture through a column filled with a solid
adsorbent material. Each component in the sample interacts slightly differently with the
adsorbent material, causing different flow rates for the different components and leading to the
separation of the components as they flow out the column (Aburjai, Alzweiri and Al-Hiari,
2011). HPLC has been used for manufacturing, legal, research and medical purposes. The
components of the sample mixture are separated from each other due to their different degrees of
interaction with the adsorbent particles. The pressurized liquid is typically a mixture of solvents
and is referred to as a “mobile phase”. Its composition and temperature play a major role in the
separation process by influencing the interactions taking place between sample components and
adsorbent. These interactions are physical in nature, such as hydrophobic, dipole-dipole and
ionic, most often a combination. Aqueous solvents, organic solvents, and mixtures of these types
of solvents are usually used as the mobile phase in HPLC.

Reversed phase HPLC(RP-HPLC) has a non-polar stationary phase and an aqueous,

moderately polar mobile phase. RP-HPLC operates on the principle of hydrophobic interactions,
which originates from the high symmetry in the dipolar water structure and allows the
measurement of these interactive forces. The binding of the analyte to the stationary phase is
proportional to the contact surface area around the non-polar segment of the analyte molecule
upon association with the ligand ton the stationary phase. The energy released in this process is
proportional to the surface tension of the eluent and to the hydrophobic surface of the analyte and
the ligand respectively. The retention can be decreased by adding a less polar solvent into the
mobile phase to reduce the surface tension of water. Gradient elution uses this effect by
automatically reducing the polarity and the surface tension of the aqueous mobile phase during
the course of the analysis. The polarity of the mobile phase altered by varying the ratio of
methanol and water. With the high ratio of methanol, the polarity of the mobile phase will be
reduced. In the other hand, the polarity of the mobile phase will be increased with the high ration
of water. There is because methanol is less polar whereas water is more polar.
The retention time of ascorbic acid will be altered by varying the methanol concentration.
With such stationary phases as RP-HPLC, retention time is longer for molecules which are less
polar, while polar molecules elute more readily. The retention time can be increased by adding
more water to the mobile phases thereby making the affinity of the hydrophobic analyte for the
hydrophobic stationary phase stronger relative to the now more hydrophilic mobile phase. In the
other hand, the retention time can be decreased by adding more organic solvent to the eluent.
Therefore, the retention time of ascorbic acid will decrease if the methanol concentration is
increased due to the decrease in polarity of mobile phase.

The importance of controlling mobile phase pH when analyzing ionizable compounds by

RP-HPLC is often recognized and easily understood, however it is often equally important to
control pH when working with field samples of non-ionizable compounds due to the presence of
ioniable impuritities. Usually, acid modifiers are used to adjust pH. It is also common to employ
strong or weak acids alone to control pH at low values, for commonly used trifluoroacetic and
acetic acids. When acetic acids are used, this method of pH control does not provide a buffered
mobile phase and may not be as effective for all types of samples, especially basic ones.
However, it has become popular for adjusting the pH of the mildly ionizable compounds such as
peptides and proteins. The mobile phase is prepared by using acetic acids to adjust the pH. By
pH adjustment, the ion-pair effect may occur and there may be some influence on the analysis
results (SHIMADZU (Shimadzu Corporation), 2016).


Objectives of this lab have been achieved. Concentration of ascorbic acid was quantified and
principle of HPLC were determined and understood. The ascorbic acid concentration of an
unknown sample given has a peak area of 1232298 is 1219.8 mg/mL. Therefore, the HPLC
technique were deeply understood from this experiment.

Aburjai, T., Alzweiri, M. and Al-Hiari, Y. (2011). Temperature and Pressure Behaviours of
Methanol, Acetonitrile/Water Mixtures on Chromatographic Systems. American Journal of
Analytical Chemistry, 02(08), pp.934-937.

Hanai, T. (1999). HPLC- A practical guide. Cambridge: Royal Society of Chemistry.

National Institute of Health, (2011). Vitamin C. [online] Available at: [Accessed 17 Oct. 2016].

SHIMADZU (Shimadzu Corporation). (2016). Preparing the Mobile Phases : SHIMADZU

(Shimadzu Corporation). [online] Available at: [Accessed 19 Oct. 2016].

The Linde Group, (2016). High performance liquid chromatography (HPLC). [online]
Available at:
hy.html [Accessed 17 Oct. 2016].