BIOANALYTICAL CHEMISTRY LABORATORY BSB2442

EXPERIMENT 4:
CHROMATOGRAPHY

Group Morning session (Section B): 9am – 10am

Group member 1. Nurul Syafiqah Binti Nazari
(SB20039)
2. Chen Yoke Ching
(SB20041)
Course BSB2442 Bioanalytical Chemistry
Laboratory
Section B1
Date of assignment 6 December 2021
Date of assignment submission 27 December 2021

Report Outline Person-In-Charge

Introduction Chen Yoke Ching

Methodology Chen Yoke Ching

Results Nurul Syafiqah Binti Nazari

Discussion Nurul Syafiqah Binti Nazari

Conclusion Nurul Syafiqah Binti Nazari

References Chen Yoke Ching

Appendices Nurul Syafiqah Binti Nazari

INTRODUCTION

Chromatography is a preparative or analytical technique used to separate a mixture by

solid or liquid into their components. Preparative chromatography separates the components
and thus purify the components. Analytical chromatography measures the relative proportion
for each of the components detect in the mixture. All types of the chromatography use the
same fundamental principle. Chromatography consists of a mobile phase and a stationary
phase. Mobile phase is a sample dissolved in a solvent and it is pass through stationary phase.
The components in mixture flow through stationary phase with different travel speed. The
travel speed is affected by the size of components, the polarities of the components, the
charge of components and the affinity of components towards stationary phase. The affinity
of components to stationary phase is depending on the two properties of components, which
is adsorption and solubility. The higher the adsorption of components to the stationary phase,
the slower the components to flow through the stationary phase, the longer the retention time.
The higher the solubility of components in mobile phase, the faster they move through the
system, the shorter the retention time. Retention time, which is the time for components to
flow through the system is used to estimate the type of components. The visual output of
chromatography is shown in the chromatogram. Chromatogram is a graph which shows peaks
and patterns on the detected components.

High performance liquid chromatography or high pressure liquid chromatography

(HPLC) is a type of column chromatography. In HPLC, stationary phase which called
column used is silica, alumina or cellulose. The size of stationary phase is in small particle
size to increase the surface area react with the sample components and leads to high
resolution. The mobile phase of sample solution is flowing down the system under the effect
of gravity. A high pressure pump which can generate up to 40MP pressure is applied to the
column. The column is made up by stainless steel so that it can withstand high pressure up to
50MP. The length of column is in the range of 5 to 25cm and the width is about 4.5mm. The
flow rate of mixture solution usually is control in 1 – 3mL/min. The solvent in mobile phase
is select depends on the type of sample. It can be polar or non-polar. The mobile phase is kept
in a solvent reservoir and connected to a high pressure pump. A sample injector is applied to
the high pressure pump to allow sample introduce into the column. The HPLC column was
connected to a detector. The types of detectors used can be UV detector, IR detector,
refractive index detector, mass spectrometer and electrochemical detector. Detector is
connected to the computer to analyse the results. As the sample flows through the HPLC
column, the components in sample move by different velocities, peaks are obtained when the
components reach the detector. A chromatogram was plotted based on the peaks and their
retention time. A standard was used to compare the retention times and estimate the
components in the sample. HPLC also used to find the concentration of component in sample.
A standard which records the retention time and peak of a standard concentration of
component are used to estimate the concentration of component in sample. The higher the
peak, the larger the concentration of the component. The concentration is estimated by
calculating the area of a peak and compare it to the standard.

Gas chromatography (GC) is a type of chromatography used in separation of volatile

compound. A volatile compound is compound which vaporised easily at room temperature.
The components of GC consist of a column, stationary phase, mobile phase, molecular sieve
and detector. The column of GC is arranged in a coil and keep in a chamber with a uniform
temperature to maintain the temperature of column. Types of columns include packed column
and capillary column. The packed column made up of glass or stainless steel. It is 1 – 3m
long and have internal diameter of 2 to 4mm. the capillary column made up of fused quartz. It
is 10 – 100m long and have internal diameter of 0.1 to 1mm. Stationary phase of GC is at the
inner wall of column and made up of silicone grease or wax which withstand high
temperature. The mobile phase used is usually an inert gas or non-reactive gas such as helium
and nitrogen. The mobile phase is kept in a cylinder which connects to a molecular sieve. The
molecular sieve removes unwanted hydrocarbon, water vapour and oxygen in the mobile
phase which may interfere the result. Molecular sieve connected to the column and column
connect to the detector and computer. The gas chromatography is start by first dissolving the
sample in volatile solvent to prepare the mobile phase. Then, the sample injector introduces
the sample into the column. The temperature of the injection region is kept 20°C to 50°C
higher than the column to allow rapid volatilise of sample into vapour. The sample then pass
through the column which has temperature between 150°C to 300°C. The separation of
sample is depending on the reaction between the mobile phase and stationary phase. The less
volatile molecules move with a lower speed and interacts with the stationary phase while the
more volatile molecules pass through the column with a higher velocity. The detector detects
the components elute from the column.

In Gas Chromatography-Mass Spectrometer (GCMS), the sample is separated, the

components exist in the sample is quantified, the unknown peaks are identified by comparing
with the standard. GCMS also used in determine the trace levels of contamination. In GCMS,
the sample is dissolved in a volatile solvent and vaporized into gas state flows into the
column. The components are separate in the column coated with stationary phase based on
their interaction with the stationary phase and the velocities travelled through the column.
The components are eluted with different retention time based on their boiling points and
polarities. The components next go to the detector which is mass spectrometer. The mass
spectrometer separates the ionized and fragmented components based on their mass-to-charge
(m/z) ratio. Then a chromatogram is produced by the computer connected to the mass
spectrometer. The number of peaks in chromatogram indicates the total components exist in
the sample. The chromatogram is used in identifying the components in sample by comparing
to the standard.
OBJECTIVES

1. To determine the standard curve of caffeine.

2. To prepare the sample solution using serial dilution techniques.
3. To learn the techniques to use High Performance Liquid Chromatography (HPLC),
Gas Chromatography (GC) and Gas Chromatography – Mass Spectrometry (GC-MS).
4. To determine the chromatogram of the sample solutions for HPLC, GC and GC-MS.
5. To prepare the replica of HPLC, GC and GC-MS.

METHODOLOGY

Instrumentation: HPLC & GC-MS

Materials:

1. Panadol soluble

2. 1 sachet 3 in 1 coffee

3. Hexane (HPLC Grade)

4. Perfume

5. 2mL HPLC/GC Vials

6. Micropipette (1mL)

7. Micropipette tips

Method:

Part I : Sample Preparation

A) Sample Preparation for HPLC

1. A tablet of Panadol soluble was dissolved into 50mL of water.

2. 1mL of the dissolved Panadol solution was transferred into a vial for HPLC analysis.

3. 1 gram of 3 in 1 coffee was dissolved in 50mL of boiled water and mixed with a stainless-
steel spoon.

4. 1mL of the coffee solution was transferred into a fresh HPLC/GC vial for HPLC analysis
after the coffee solution cool.

B) Sample Preparation for GC-MS

1. 950μL of hexane (HPLC grade) was transferred into 2mL HPLC/GC vial.

2. 50μL of perfume was transferred into the vial.

3. The mixture was mixed by pipetting.

4. A lid was used to close the vial.

Part II: Samples Run and Analysis

A) Samples Analyzed Using HPLC

1. A previous data of serial diluted caffein standard was provided by laboratory staff to
students.

2. A standard curves for caffein was plotted from the data given.

3. The previous HPLC data for Panadol soluble will be provided by laboratory staff. The
amount of caffein in a tablet of Panadol soluble was calculated by comparing to the standard
curve. The result was compared with the original label on the Panadol soluble provided by
the manufacturer.

4. The previous HPLC data for 3 in 1 coffee will be provided by laboratory. The amount of
caffein in the coffee solution was calculated by comparing to the standard curve. The result
was compared with the original label on the 3 in 1 coffee sachet provided by the
manufacturer.
B) Samples Analyzed Using GC

1. The previous data of serial diluted linalool and benzyl acetate standards was provided to
students.

2. A standard curves was plotted from the data given for linalool and benzyl acetate.

3. The previous GC data for perfume was provided by the laboratory staff. The amount of
linalool and benzyl acetate perfume was calculated by comparing to the standard curves.

4. The previous GC-MS data for perfume will be provided by laboratory staff. All the
fragrance compounds in the perfume were identified.
RESULTS
Part A: HPLC Analysis of Panadol and 3 in 1 coffee

Figure 1: HPLC analysis yielded a chromatogram of Panadol soluble.

Peak Retention time (m) Peak area (mAU*s) Peak Area (%)
1 5.465 2.42885e4 100.0000
Table 1: Peak, retention time, peak area, and peak area percentage of Panadol soluble.

From Figure 1, we can conclude that there is no caffeine present in the Panadol soluble. This
is because there is no peak between retention time of 15 min and 16 min in the
chromatogram. The peak between 4 min and 6 min represent another source of compounds.
Therefore, the caffeine standard curve equation cannot be used to estimate the caffeine in
Panadol soluble since caffeine is absent in the Panadol soluble.
Graph 1: Peak Area against caffeine stock concentrations
 Figure 2: HPLC analysis produced a chromatogram of the 3 in 1 coffee.

Peak Retention time (m) Peak area (mAU*s) Peak area (%)
1 2.044 154.07098 25.0802
2 2.552 52.01206 8.4667
3 4.613 14.11189 2.2972
4 5.774 33.54494 5.4605
5 15.734 360.57446 58.6954
Table 2: Peaks, retention times, peak areas, and peak area percentage of 3 in 1 coffee.

The equation y= 48265x + 146.83 was derived from the caffeine calibration curve (Graph 1).
In contrast, with a retention duration of 15.734 minutes and a peak area of 360.57446mAU*s,
the fifth peak in Table 2 showed the presence of caffeine in the 3 in 1 coffee sample.
Substituting y= 360.57446 into the equation y= 48265x + 146.83,

360.57446 = 48265x + 146.83

48265x = 146.83 - 360.57446

x = 0.004mM (concentration in 1000ml)

The value of x indicates the concentration of caffeine in 1000mL of coffee solution.

In 50mL of boiling water, 1 gramme of 3-in-1 coffee was dissolved.

0.004 𝑋 50
Concentration of caffeine in the 3-in-1 coffee sample = 1000

Concentration of caffeine in the 3-in-1 coffee sample = 0.0002mM

The total mass of 3-in-1 coffee powder in a sachet of 3-in-1 coffee = 19g

0.0002 𝑋 19
Total concentration of caffeine in a sachet of 3-in-1 coffee = 1

Total concentration of caffeine in a sachet of 3-in-1 coffee = 0.0038mM

Part B: GC-MS Analysis of perfume

Figure 3: Library Search Research GC-MS analysis of perfume

Spectrum

Figure 4: Chromatogram of perfume by GC-MS

 Table 3:

Peak RT Area Library/ID Ref# CAS# Quality

(%)
.beta.-Pinene
15691 000127-91-3 91
1 5.110 2.28 .beta.-Pinene 15694 000127-91-3 91

Bicyclo[3.1.1]heptane, 6,6-dimethy 15902 018172-67-3 91

l-2-methylene-, (1S)-

D-Limonene
15682 005989-27-5 99
2 5.982 13.43 D-Limonene 15681 005989-27-5 94

Limonene 15667 000138-86-3 93

1,6-Octadien-3-ol, 3,7-dimethyl 26774 000078-70-6 97

3 7.120 9.52
1,6-Octadien-3-ol, 3,7-dimethyl- 26780 000078-70-6 95

1,6-Octadien-3-ol, 3,7-dimethyl- 26781 000078-70-6 80

1,6-Octadien-3-ol, 3,7-dimethyl-, 69339 000144-39-8 90

Propanoate

4 9.417 36.82 1,5-Dimethyl-1-vinyl-4-hexenyl 81047 000078-36-4 90

but yrate

Linalyl acetate 57985 000115-95-7 83

 Isobornyl acetate 57986 000125-12-2 98

Bicyclo[2.2.1]heptan-2-ol, 1,7,7-t 58098 005655-61-8 91

5 9.779 6.64 rimethyl-, acetate, (1S-endo)-

Acetic acid, 1,7,7-trimethyl-bicyc 58077 092618-89-8 91

lo[2.2.1]hept-2-yl ester

6 13.845 31.30 Diethyl Phthalate 78782 000084-66-2 97

Diethyl Phthalate 78784 000084-66-2 97

Diethyl Phthalate 78786 000084-66-2 96

DISCUSSION

HPLC (high-performance liquid chromatography) is a methodology for sorting,

classifying, and quantifying components in a mixture. It is the most popular chromatography
technique used in most laboratories across world. The sample interacts with the mobile
(liquid) and stationary phases, which results in separation (column). The different
components of the sample are segregated depending on their polarities; they will have varied
levels of affinity for the mobile phase, resulting in varying rates of migration through the
column. The merged substances are applied to the stationary phase column's surface, which is
typically a fine adsorbent material such as silica. This must be spread uniformly to avoid air
bubbles interfering with the test results. Glass, wool, or a porous plate is used to inhibit the
column from departing. The mixture separates into bands after the mobile phase has passed
through. Other techniques can be used to collect and analyse these. The approach works since
the components in a mixture are attracted to the stationary phase's adsorbent surface to
variable degrees based on their particular polarity and characterization. A component with a
greater affinity for the stationary phase will move down the column more slowly than one
with a greater affection for the mobile phase. High-performance liquid chromatography
(HPLC), which pumps the sample mixed through the column at a high pressure, is the most
widespread type of polymer chromatography in use today.

HPLC was used to examine Panadol soluble. Panadol's primary component is

paracetamol, which also functions as the active ingredient. The retention time for panadol
was 5.465 minutes, according to Figure 1. The time required for a solute to pass through a
chromatography column is recorded in retention time (RT). The time from injection to
detection is used to determine it. Boiling point, column temperature, carrier gas flowrate, and
column length are all factors that affect retention duration. The peak area for Panadol soluble
was 2.42885e4 mAU*s. The majority of quantitative chromatographic estimations are based
on peak areas. The peak area of 2.42885e4 mAU*s has contribute to 100% of the percentage
of peak area shown in Table 1. The retention time for caffeine is between 15 min and 16 min.
however, in Figure 1, there is no peak in between 15 min and 16 min. Therefore, caffeine is
absent in Panadol soluble sample.
 In addition, HPLC was utilized to assess caffeine in 3-in-1 coffee. The quantity of
the element is proportionally to the peak area. The HPLC data analysis of caffeine in 3 in 1
coffee were tabulated in Table 2. Based on the Graph 1, the equation y= 48265x + 146.83
was derived from the caffeine calibration curve. The graph's regression line was 0.9962,
indicating a striking similarity. In forecasting techniques, regression lines are important. Its
goal is to explain how the peak area and stock concentration are related. The value of R²
indicates how well a linear regression model fits the data. In this case, the R² value is close to
1. Therefore, it indicates a positive linear relationship between the two variables which is
peak area and concentration of caffeine. Graph 1 show the peak area is directly proportional
to the concentration of caffeine which follow the Beer-Lambert Law. In contrast, with a
retention duration of 15.734 minutes and a peak area of 360.57446mAU*s, the fifth peak in
Table 2 showed the presence of caffeine in the 3 in 1 coffee sample. In the fifth peak, the
time it takes for a solute to travel through a chromatography column is the longest. Same
goes to its peak area and the percentage of peak area. The higher the peak indicate the higher
the concentration of caffeine. By substituting y= 360.57446 to y= 48265x + 146.83, the
caffeine concentration was calculated. The obtained x concentration was 0.004mM. By
multiplying 0.004 with 50 and dividing by 1000, the concentration of 1 gramme of 3 in 1
coffee powder dissolved in 50mL hot water was calculated. The concentration was 0.001mM.
Caffeine amount was 0.0038mM in a sachet of 3 in 1 coffee with 19 grammes of total mass.
Based on the standard curve graph, the concentration of caffeine in 3 in 1 coffee should be
between 0.005mM to 0.010mM because the peak area is 360.57446mAU*s, which is between
315.04346mAU*s (for concentration 0.005mM) and 784.03900mAU*s (for concentration
0.010mM).

The analytical technique of gas chromatography–mass spectrometry (GC-MS)

combines the capabilities of gas chromatography and mass spectrometry to determine the
preferences of individual chemicals in a test sample. GC-MS applications include drug
detection, fire investigation, environmental analysis, explosives investigation, and the
identification of unknown components. It can also identify trace elements in substances that
were previously thought to be beyond detection. It, like liquid chromatography–mass
spectrometry, can analyse and detect even very small quantities of a compound. A heated
inlet port, an oven, and a fused silica column (basically a coiled glass tube) coated with a
specific material termed the stationary phase compose up a gas chromatograph (GC). In most
cases, samples are dissolved or diluted in a solvent before being injected into the inlet port.
Other sample preparation techniques, such as solid phase extraction (SPE) and/or
derivatization, may be necessary. In the heated inlet, the liquid sample vaporizes and turns
into a gas. The sample is carried through the column by the mobile phase, which is an inert
gas such as helium. Depending on their chemistry, different chemicals in the sample interact
differently with the stationary phase of the column. As a result, they separate as they go
through the column at various speeds. After that, the separated chemicals depart the column
one by one enter a detector, such as a mass spectrometer (MS). The retention time is the
amount of time it takes for a compound to pass through the column. The GC generates a
chromatogram, a graph in which each segregated substance is indicated by a peak. The
number of peaks in the sample indicates the number of discrete components. The retention
time for each compound is indicated by the position of each peak.

Based on the Figure 3, Figure 4 and Table 3, with a retention time of 5.110 minutes,
the first peak was .beta.-Pinene,.beta.-Pinene, and Bicyclo[3.1.1]heptane, 6,6-dimethyl-2-
methylene-, (1S)-. The proportion of peak area was 2.28%, with a quality score of 91 for
each. At the second peak, followed by D-Limonene, D-Limonene, and Limonene,
respectively. They have a total retention period of 5.982 minutes and a peak area of 13.43%.
The first D-limonene has a quality of 99, the second D-limonene has a quality of 94, and
Limonene has a quality of 93. Three 1,6-Octadien-3-ol, 3,7-dimethyl were found on the third
peak, each with a distinct quality value: the first had a quality value of 97, the second had a
quality value of 95, and the third had a quality value of 80. They had a retention time of 7.120
minutes and a peak area of 9.52%. 1,6-Octadien-3-ol, 3,7-dimethyl-, propanoate, 1,5-
Dimethyl-1-vinyl-4-hexenyl butyrate, and Linalyl acetate were detected on the fourth peak.
They had a retention duration of 9.417 minutes and a peak area of 36.82%. 1,6-Octadien-3-ol,
3,7-dimethyl-, propanoate and 1,5-Dimethyl-1-vinyl-4-hexenyl butyrate both had 90 of
quality value while for Linalyl acetate 83 of quality value. On the fifth peak, Isobornyl
acetate, Bicyclo[2.2.1]heptan-2-ol, 1,7,7-trimethyl-, acetate, (1S-endo)-, and Acetic acid,
1,7,7-trimethyl-bicyclo[2.2.1]hept-2-yl ester were found. They had a retention time was 9.799
minutes, and their percentage of peak area was 6.64%. Their respective quality values were
98, 91, and 91. Three Diethyl Phthalate were discovered in the last peak, which was the sixth
peak. They had a retention time of 13.845 minutes and a peak area of 31.30 percent. Diethyl
Phthalate had a quality rating of 97, whereas the other had a quality score of 96. All the
components identified have quality above 90.
 Table 3 shows the results of the GC-MS perfume analysis. As was shown, the peak with
the longest retention time was the sixth peak, at 13.845 minutes. The longer retention time
indicates the slower the components will be eluted out. The first peak which has retention
time of 5.110m elute fastest among the components in perfume while peak 6th with retention
time of 13.845m elutes the slowest. The fourth peak had the highest proportion of peak area
at 36.82%, which means its concentration is highest among other components in the perfume
essential oil sample. D-Limonene had the highest quality of any ingredient, with a result of
99.

The precautions that should be taken are membrane filters should be used when
handling HPLC because if they are not used, particles can block the column when the sample
is processed through the HPLC column. If there are many particles, the clogging is almost
always irreversible. The second step is to degas the solvent used in the column run. The
apparatus generates pressure in order to flush the solvent through the column. As a result, if
air bubbles are caught in the solvent, the pressure on the HPLC pumps may rise dramatically.
HPLC operates at constant pressure, flow rate, and temperature, as we all know. The presence
of air bubbles can cause pressure and flow rate to be disrupted. In order to keep the mobile
phase (solvents) free of air bubbles, degassing is required. If any particle escapes into the
flow despite taking safeguards, they will be captured by the guard column. A guard column is
a tiny column that is placed in front of the main HPLC column. We also need to eliminate
particle matter and air bubbles, as we've observed earlier. When employing GC-MS analysis,
take precautions such as performing frequent visual inspections and pressure leak checks of
the sampling system plumbing, fittings, and valves, and installing columns according to the
manufacturer's recommendations. Handle glass or fused capillary columns with care and
wear safety glasses to protect your eyes from falling objects while managing, trimming, or
updating them. Switch off and enable heated areas such as the oven, inlet, and detector, as
well as attached equipment, to decrease the temperature before touching them, and turn off
the hydrogen gas supply at its source when modifying columns or maintenance the
instrument.
CONCLUSION

In this experiment, the calibration curve was determined. The calibration curve of
caffeine was obtained with the equation y= 48265x + 146.83. The graph's regression line was
0.9962, indicating almost a fit line. The fifth peak in Table 2 showed the presence of caffeine
in the 3 in 1 coffee sample with a retention duration of 15.734 minutes and a peak area of
360.57446mAU*s.

The techniques to use High Performance Liquid Chromatography (HPLC) which to

ensure certain that all of the buffers are in place and confirm that the pressure is stable, with
no more than 2-3 bar of variation and also, before the real samples, or as part of the same
sequence, run a standard was learned. For Gas Chromatography (GC), the sample is
vaporised before being inserted into the column's head. Elution occurs when an inert gaseous
mobile phase, such as helium, argon, nitrogen, carbon dioxide, or hydrogen, flows through
the system and for Gas Chromatography – Mass Spectrometry (GC-MS), a sample is initially
injected into a gas chromatograph, where it is separated into components based on size and/or
polarity. After that, the components are sent into a mass selective detector. The techniques
were learned through this experiment.

In addition, HPLC was used to examine Panadol soluble and 3 in 1 coffee. The retention
time for the only peak shown in chromatogram of Panadol was 5.465 minutes, which
indicates the absence of caffeine. For 3 in 1 coffee, the retention time was 15.734 minutes.
The retention time for caffeine is between 15 min and 16 min. therefore, the 3 in 1 coffee
shows presence of caffeine.

Besides, the data analysis of GC-MS of perfume were obtained and tabulated in Table 3.
The peak with the longest retention time, at 13.845 minutes, was the sixth peak, as illustrated,
which represent the components in peak 6th will be the last component to elute out. With
36.82 percent of peak area, the fourth peak had the highest proportion of peak area, which
indicates the highest concentration of component among other components in perfume. With
a score of 99, D-Limonene has the greatest quality of any component. This indicate that D-
Limonene had less contamination.
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APPENDICES

Figure 5: The sample preparation of Panadol for HPLC analysis

Figure 6: The sample preparation of 3 in 1 coffee for HPLC analysis

Figure 7: The sample preparation of hexane and perfume for GC-MS analysis