High Performance Liquid Chromatography

Manansala, Dominic H.1, Martin, Marilen2

School of Chemical, Biological, and Materials Engineering and Sciences, Mapua University, Intramuros, 1002
Metro Manila, Philippines

dhmanasala@mymail.mapua.edu.ph1

I. Introduction Further discussing the process

In the study of Biology, more
specifically, but not limited to, in the
field of applied laboratory medicine,
samples are actively collected and
treated. Following this is completing
specific methods and procedures in
order to determine distinct
components found in the mixture. One
of the more common methods used in
experiments similar to this is a
method called “High-Performance
Liquid Chromatography” or HPLC
[ CITATION Hig20 \l 13321 ] which is
basically a more developed technique
derived from column
Chromatography.
Briefly differentiating the two
techniques mentioned, column
chromatography heavily relies on
gravity which allows the solvent to
drip down in the column. On the other
hand, HPLC utilizes a high-pressure
pump instead of gravity which makes
the process quicker relative to column
chromatography.
that occurs during HPLC, the
presence of a high pressure, typically
around 400 atm, forces the solvent to
pass through a column which is filled
by adsorbent. In technical terms, the
immense pressure allows the mobile
phase to proceed to the
aforementioned column known as the
stationary phase [ CITATION Jimst \l
13321 ].
Being an advanced and highly
sensitive, HPLC machines are able to
read and detect various magnitudes of
interaction between the components in
the mixture and the adsorbent present.
The HPLC machine has the ability of
doing so by considering the
differences in chemical and physical
properties. Additionally, the concept
of polarities is also heavily applied.
[ CITATION Ver16 \l 13321 ]
Before proceeding with the
discussion of polarities and property
differences, it is important the two
major types of HPLC namely “normal
phase” and “reverse phase”. The two
types can be distinguished in the
polarities of their mobile and
stationary phases. For the normal-
phase HPLC, the mobile phase is
considered to be non-polar and is then
followed by a polar stationary phase.
The reverse-phase simply has these
two polarities reversed [ CITATION
Lak19 \l 13321 ]. Among the two types,
this experiment utilizes the reverse-
phase type. This is generally the more
common type being that is it relatively
more ideal towards the retention of
organic analytes [ CITATION Mat13 \l 13321
].
For the components in the
mixture found in the sample, each
have their specific interactions with
the beads inside the column which is
affected by the polarity of the
stationary phase and the analyte itself.
Ultimately, this yields different rates
of elution for the distinct components
while flowing out the column.
In displaying the signal peaks
caused by the elution rates, a
chromatogram is produced. The peaks
are caused by the detected retention
times which indicates which
component eluted first up to the last
[ CITATION Chr \l 13321 ]
The success of this experiment
is then guided by these set of
objectives: (1) to understand HPLC as
a chromatographic separation
technique alongside the function of an
HPLC machine. (2) Successfully
complete an HPLC analysis on the
drug sample (3) to determine the
effects of external factors and
parameters (temperature, humidity,
mobile and, stationary phase
composition)
II. Methodology
This experiment was executed
through Labster which is an online
virtual laboratory simulator.
Prior to proceeding with the
actual experiment, the HPLC machine
was calibrated. This is completed by
forcing incremental pressure through
the column beginning with 0.1
mL/min for 10 minutes. After such, a
volume of the subject solvent was
utilized to flush out interferents that
may be contaminate the whole
experiment.
After calibration, the sample
was then prepared accordingly. Using
a mortal and pestle, the drug sample
was powderized to aid in dissolving it
in water.
With the needed preparations
completed, the process of HPLC was
commenced starting by injecting the
prepared drug solution into sample
injector for the HPLC machine to be
able to process the sample following
constant conditions. A chromatogram
of the signal detect will be yielded
accordingly.
For the next half of the
experiment which focuses on the
effects of external factors such as
temperature, the components in the
mobile and stationary phase were
swapped with acetonitrile and C-18
respectively. However, prior to doing
so was the washing and re-
conditioning of the machine by
allowing new variables to be flushed
through.
III. Results and Discussion
After the completion of the
HPLC processes, the following
chromatogram were produced and
analyzed.
Figure 1. Absorbance VS Retention
(at 25 Co)
Observable from Figure 1 are
three peaks with distinct degrees of
absorbance and value for retention (in
minutes).
Being the component with the
lowest value for retention time,
Metformin hydrochloride was
detected first being the peak is found
at around the 5-minute mark.
Followed by Povidone which was
detected by the 9th minute and then
finally at the 16-minute mark is
magnesium stearate. The differences
in retention time and absorbance are
affected by the degree of interactions
between the non-polar silica beads
found in the column.
In dissecting and discussing
each peak that is found on the graph
above, Metformin hydrochloride
(C4H12ClN5), which is highly polar,
yielded the fastest relative retention
time [ CITATION WFa11 \l 13321 ] caused by
the low degree of interaction between
the silica beads which is non-polar,
allowing it to be detected quicker as
soon as it was injected. The strong
absorbance peak of this component is
explained by the occurrence that it
exited the stationary phase
immediately.
Following this component is
Povidone (C6H9NO)n as shown by the
middle, small-sized peak in the graph.
Regarding the position of this peak on
the middle of the graph, it indicates
that its retention time is not the fastest
nor the slowest relative to the drug
components. This is due fact that this
compound is soluble in non-polar as
well as polar solvents [ CITATION JRS20 \l
13321 ] Additionally, it is observable
that it has the weakest absorbance
peak out of the three. These
observations are made possible by the
fact that Povidone is an organic
substance that contains a hydrophobic
or non-polar group and a hydrophilic
or polar group causing a variance in
the interactions in the mobile and
stationary phase.
Lastly for this graph, it is
observed that the largest value for
retention time was detected and
identified as Magnesium stearate
(Mg(C18H35O2)2). This phenomenon is
caused by the structure of the
compound which makes it non-polar
or hydrophobic [ CITATION Mag \l 13321 ].
With that, it tends to actively interact
with the non-polar silica in the
stationary phase. The increase in
degree of interaction yields a
consequence of an increase is the time
of retention inside the column.
Figure2.Chromatogram
comparison (Normal VS Extreme
conditions)
For the next half of the
experiment, a comparative analysis
was done. Between the two
chromatograms shown in Figure 2, the
left chromatogram shows the readings
and detections subjected under normal
conditions (25 Co and 40% humidity).
The chromatogram on the right was
oppositely conditioned being it was
subjected to relatively more intense
conditions (40 Co and 95% humidity).
The motive for this section of
the experiment is to identify how such
external factors such as humidity and
temperature affect the readings for
absorbance and retention times for
certain compounds in a sample.
Initially, it can be observed
between the two chromatograms, that
the retention time and degree of
absorbance for Povidone and
magnesium stearate are similar with
the possible presence of miniscule
discrepancies, which can be securely
disregarded.
However, for the first peak,
which is the identified metformin
hydrochloride, it can be observed that
there seems to be a small yet
quantifiable discrepancy between the
degree of absorbance at normal
conditions compared to the extreme chromatograms in terms of retention
one. time and absorbance. The success in
the completion of the technique
Note that the degree of
allowed the further understanding of
absorbance or the area under the curve
the mechanisms that takes place in an
corresponds to the concentration of
HPLC machine together with the
that specific components in the
polaristic effects and interactions
sample, the slight decrease in the
between molecules.
“height” of the peak indicates that
there is a loss in the concentration at For the next comparative half
an extreme condition [ CITATION Int \l of the experiment, it is determined
13321 ]. This occurrence can be that the change in the environment or
attributed to the possibility of the conditions affects the compound
compound degrading. The increase in possibly through thermal degradation
temperature causes the breakdown of as seen in the decrease in
the components found in the concentration for metformin
compound possibly forming other
hydrochloride [ CITATION Mic18 \l 13321 ]
products [ CITATION Typ \l 13321 ].
Additionally, for the References
comparative analysis of the two given
chromatograms, it indicated that the
components present in the sample are
able to function even with the changes
in environment while noting the small
degradation possible for metformin
hydrochloride.
CONCLUSION
After the completion of the
entirety of the experiment, the
objectives were effectively sufficed.
The theory and application behind
chromatography, more specifically
HPLC, were demonstrated and
applied by identifying the components
present and analyzing the
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