BE184P Instrumentation in Biological Engineering 2

EXERCISE 2.1 CHROMATOGRAPHIC ANALYSIS - HPLC

Objectives:

At the end of the virtual simulation, students should be able to…

1. Understand the different compartments of an HPLC machine and functions
2. Understand the principles of HPLC separation
3. Understand the lipophilic interaction between the analyte and the mobile and stationary phase
4. Understand the different changes in parameters (such as the column, mobile phase, temperature
etc.) and its effects on analyte separation and concentration measurements

Introduction:

Chromatography is essentially a physical method of separation in which the components of a mixture are
separated by their distribution between two phases – the stationary phase and the mobile phase.
Chromatography is a method by which a mixture is separated by distributing its components between two
phases. The stationary phase remains fixed in place while the mobile phase carries the components of the
mixture through the medium being used. The stationary phase acts as a constraint on many of the
components in a mixture, slowing them down to move slower than the mobile phase. The movement of
the components in the mobile phase is controlled by the significance of their interactions with the mobile
and/or stationary phases. Because of the differences in factors such as the solubility of certain
components in the mobile phase and the strength of their affinities for the stationary phase, some
components will move faster than others, thus facilitating the separation of the components within that
mixture.

High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a
sample mixture or analyte in a solvent (mobile phase) at high pressure through a column with
chromatographic packing material (stationary phase). HPLC has the ability to separate, and identify
compounds that are present in any sample that can be dissolved in a liquid in trace concentrations as low
as parts per trillion. Because of this versatility, HPLC is used in a variety of industrial and scientific
applications, such as pharmaceutical, environmental, forensics, and chemicals.

In performing HPLC which involves separation of polar components, a reversed-phase HPLC is used
wherein the stationary phase is lipophilic and the mobile phase is polar. If the samples to be separated
are nonpolar, then the stationary phase used is polar and the mobile phase nonpolar.

Instructions: Access the Labster link provided in Blackboard. Answer the guide questions for Exercise 2.1.
You may need to print screen or copy images for some of the questions in the report form.
BE184P Instrumentation in Biological Engineering 2

Name: Celestra, Den Mark O. Student No.2019102287 Program/Year: BE/3 Date:3/17/21

EXERCISE 2.1: CHROMATOGRAPHIC TECHNIQUES – HPLC

REPORT FORM

I. ABSTRACT: Minimum of 250 words. (Give a brief description/summary of the virtual experiments
conducted.) (5%)

Chromatography is a widely used separation technique that can be applied in various industries such as
pharmaceutical, food, chemical, and biological. This type of technique utilizes a column that is filled with
a specific bead depending on the sample that is being separated. The separation process using
chromatography depends on its various kinds such as adsorption chromatography, partition
chromatography, ion exchange chromatography, and size exclusion chromatography. High Performance
Liquid Chromatography (HPLC) is a package device that applies the discipline of chromatography while
integrating high operational pressure to flush the sample into the column. In this report, HPLC was utilized
to separate the contents of metformin hydrochloride and investigate the effects of extreme conditions
when it is stored. The data that was gathered suggests that the combination of C-18/silica column with
acetonitrile/hexane as mobile phase with temperature above 40°C provides faster rate of separation
without compromising the peaks. After determining the peak differences, statistical analysis was
done using linear regression using the regression formula y=21.536x which reflected that the
concentration of metformin hydrochloride under normal condition was 70.3 g/L, while 68.1 g/L
if under extreme condition. The concentration gathered were then converted into their original
amount considering that the samples came from the tables diluted and 10mL of liquid. As a
conclusion, It was determined in this report using HPLC that storing the medicine under extreme
conditions will decrease the metformin hydrochloride concentration by 3.1%. It is then advised
that the medicine be stored at its optimal condition with 40°C and 95% humidity.

II. Data Gathered:

A. In the simulation, a standard curve was created. This is done by subjecting standard sample with
different concentrations the absorbance was generated for each. Fill in the table with the
data/information gathered in Labster.

Sample Concentration Absorbance Units (AU)

Metformin hydrochloride (g/L)
1 12.5 258.8
BE184P Instrumentation in Biological Engineering 2

2 25 552.5
3 50 1100
4 75 1575
5 100 2170

Insert/paste here the plot of the Standard Curve:

If the unknown metformin tablet exposed at high temperature had an absorbance of 2000AU, what is its
concentration in g/L? The sample was prepared by dissolving it and with a final volume of 10mL.

The sample will range from 91 g/L to 95 g/L

B. What happens to the separation of the three components: metformin hydrochloride, povidone and
magnesium stearate, if parameters such as column type, mobile phase and temperature? Insert an image
of the results on the following chromatographic parameters and briefly describe each.
BE184P Instrumentation in Biological Engineering 2

a. Column: C18 Mobile phase: acetonitrile Temperature at 25°C

There were three peaks or three compounds that were eluted from the sample. The first elution which
signifies the first peak was during the 4th minute, the second peak started at the 8th minute, and the third
elution started during the 13th minute.

b. Column: C18 Mobile phase: acetonitrile Temperature at above 40°C

BE184P Instrumentation in Biological Engineering 2

3 peaks were generated from the chromatograph wherein the first effluent was at the 3rd minute, the
second effluent was eluted at the 5th minute, while the last compound was eluted at the 8th minute. It can
be observed from the graph that the temperature played a role in how fast the effluents were eluted in
the chromatography process.

c. Column: silica Mobile phase: hexane Temperature at 25°C

The three peaks that can be seen from the graph equates to the number of effluents that was eluted from
the chromatography process. It can be observed that all three compounds took approximately 3 minutes
before they were completely eluted from the column.

d. Column: silica Mobile phase: hexane Temperature at above 40°C

BE184P Instrumentation in Biological Engineering 2

It can be observed that the temperature above 40°C allowed the effluent to be eluted in a faster rate
compared to the 25°C sample. It can be observed that all three compounds were eluted for a minute
starting on the third minute.

Would you expect that components will be separated using silica and methanol? Why or why not?

No, because silica and methanol for column and mobile phase respectively did not produce a good quality
peak. Based on the figure below, the results of chromatography did not produce a distinct peak which a
good determinant that the combination of silica and methanol are not an efficient tools for separation.

Would you expect that components will be separated using C18 and methanol? Why or why not?

It can be determined with the visibility of the three peaks that the components were able to be separated.
The definition of the peaks is due to the polarity difference of the stationary and mobile phase.
BE184P Instrumentation in Biological Engineering 2

C. Comparison of Chromatogram

Insert here the image of the comparison of chromatograms. Briefly describe if there are any differences
in the two chromatograms. If there is a difference, briefly explain what may have brought about this
difference.
BE184P Instrumentation in Biological Engineering 2

The low peak of the second chromatogram was visible because it is the sample that has been stored under
extreme condition.

Concept Analysis:

1. Identify the labelled parts of the HPLC and briefly describe its function.
BE184P Instrumentation in Biological Engineering 2

Label Part Function

A Carries the mobile phase solution that allows it to
Mobile phase reservoir be pumped into the column.
B Detects or identifies the component of the sample
Detector that has been eluted from the chromatography
column.
C Pump Allows the mobilization of the sample into the
system by generating high pressure.
D Manual Injector Where the sample is administered.

E Column oven Where the separation process happens. Typically

filled with beads specific for each experiment.

2. If samples are solid, briefly describe its sample preparation for HPLC analysis.

Solid samples are first crushed using mortar and pestle. The powdered sample are then dissolved into a
solvent and filtered using a 0.5um filter to get rid of the lumps that was not dissolve properly.

Sample preparation of solid states that a polar solid sample should be dissolved in a polar solvent while a
non-polar solid sample has to be dissolved using a non-polar solvent.
BE184P Instrumentation in Biological Engineering 2

3. Briefly describe the effects of temperature on the separation of components in HPLC.

Based on the data that was gathered it can be determined that the temperature is a great factor in
separating compounds from a sample. It was reflected that the higher the temperature, the lower the
amount of time needed to separate compounds from the sample. Aside from the time, the viscosity can
also be affected with temperature rise as temperature is inversely proportional to viscosity. Moreover, the
solubility of the compound is also accommodated with the rise of temperature as solubility is directly
proportional to temperature.

4. Will there be expected components separation if the stationary phase and mobile phase have the same
nature of polarity? Why or why not?

No, because the polarity of both mobile and stationary will allow the mobile phase to only adsorb to the
stationary phase which will lead to the inability to separate the components of the sample.

5. Briefly describe the differences between the reverse phase and normal phase HPLC.

Reverse-phase HPLC is when the stationary phase is non-polar while the mobile phase is polar. This
means that the polar compounds of the sample are eluted first before the non-polar compounds. Normal-
phase HPLC is when the stationary phase is polar coupled with non-polar (or moderately polar) mobile
phase. This means that the non-polar compounds are eluted first before the polar compounds.

References:

[1] Belanger, J. M., Paré, J. J., & Sigouin, M. (1997). High performance liquid chromatography (HPLC):
principles and applications. In Techniques and Instrumentation in Analytical Chemistry (Vol. 18, pp. 37-
59). Elsevier.

[2] Cazes, J. (1999). Basic Principles of HPLC and HPLC System Troubleshooting.

[3] Corradini, D., Eksteen, E., Eksteen, R., Schoenmakers, P., & Miller, N. (1998). Handbook of HPLC.
CRC Press.