FACULTY OF APPLIED SCIENCE

UITM CAWANGAN SARAWAK

CHM 260 BASIC INSTRUMENTAL ANALYSIS

LAB REPORT

EXPERIMENT TITLE : EXP 6- HIGH PERFOMANCE LIQUID CHROMATOGRAPHY

LECTERUR : MDM FIRUS MUSFIRAH BINTI POLI
DATE OF SUBMISSION : WEEK 13
GROUP MEMBERS : 1. SHARIFAH BATRISYIA BINTI SYED SHAHIBUL
FADILLAH (2020895276) AS1205C
2. NABILA HUDA BINTI NAZLI (2020464122) AS1205C
3. AINAA ISTIQAMAH BINTI MOHD ROSDI
(2020457062) AS1205C
4. NOR FAIEZA BINTI ABU BAKAR (2020851628)
AS1205A

Declaration of Academic Honesty Theory

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OBJECTIVES
1. To determine the retention time of a standard solution (caffeine).
2. To identify the caffeine peak in a soft drink sample.
3. To determine the amount of caffeine in soft drink sample using the response factor
method.

INTRODUCTION

Technology that are used in chromatography has been greatly improved in recent
years which lead to the common use of High-Performance Liquid Chromatography (HPLC).
HLPC is a laboratory technique that allows for the very efficient separation of small amounts
of components of a mixture. In HPLC instrument, the column contains a tiny particle sized
only about 5µm diameter to ensure a very large surface area which molecules may absorb.
This particle comprises as stationary phase in the chromatography system. Mobile phase
solvent must be forced through the column under a very high pressure due to the tightly
packed tiny particle. To signal when eluents are coming off the column, this HPLC
instrument must be connected to a detector and computer.

Identification of samples is carried out by comparing their retention times. A known

amount of a compound which is a standard, is injected and the retention time is recorded so
the area under the peak can be calculated. A sample containing the same analyte is then
injected under the same experimental parameters. If analyte is the same compound as the
standard, they should give identical retention times. The retion of peak area should give the
amounts of the compound present.

Reverse phase partition chromatography in HPLC employs a polar mobile phase and
a non-polar stationary phase. This is the most frequently used form of HPLC. Other modes of
liquid chromatography include normal phase partition (non-polar mobile phase and polar
stationary phase), adsorption, ion exchange and size exclusion.
CHEMICALS

• Caffeine standard
• Soft drink (coke)
• Distilled Water
• Acetonitrile

INSTRUMENT

• HPLC instrument

PROCEDURE

1. The standard caffeine sample and the coke sample was filtered then was placed in the
autosampler vials.
2. The vials were placed inside the autosampler in number 41 and 42 respectively.
3. The software was set.
4. The mobile phase used was 70% of acetonitrile and 30% of water while the flow rate
of mobile phase used was 1 mL/min.
5. Then, 5 μL of solution B was injected into the HPLC to establish the retention time, t R ,
of caffeine.
6. The peak area of caffeine was measured.
7. 5 μL of the prepared soft drink sample was injected into the HPLC instrument.
8. The peak area corresponding to caffeine was measured by referring to its t R .

Table 6.1 : Instrumental Parameters

Flow rate of mobile phase 1.0 mL/min

Column C18, 150mm x 4.6 ID, 5 μm
Detector UV (254 nm) or PDA
Mobile phase Acetonitrile and water
Ratio of mobile phase 70:30 (Acetonitrile : water)
RESULTS

Name and model of instrument : Agilent Technologies 1260 Infinity

Table 6.2: Retention Time and Peak Area of Caffeine in the Standard and Sample

Compound Retention time, 𝐭 𝐑 (min) Peak area of caffeine

Caffeine standard 0.844 1.14 × 104

Soft drink sample (Run 1) 0.814 4732.65

Soft drink sample (Run 2) 0.815 4714.83

Soft drink sample (Run 3) 0.816 4721.49

Name of soft drink sample : Coke

peak area caffeine standard

Response Factor, RF, caffeine standard = concentration of caffeine standard

1.14 ×104
= 0.1

= 114 000

peak area caffeine in sample

Concentration of caffeine (ppm) in the unknown sample = RF

4721.49
= 114000

= 0.0414
DISCUSSION

Each of the compound were run in 5min time. For the standard solution (caffeine), the retention
time, tR obtained was 0.844min which gave out resolution peak at 1.14 × 104 . This allows the
comparison of the soft drink (Coke) samples 1, 2 and 3. This successful outcome enabled
both objectives number 1 and 2 to be achieved. Apart from that, we were also able to
determine the amount of caffeine in soft drink sample using the response factor method.
Sample 1 acquired the retention time, tR of 0.814min with peak area of 4732.65. Next, sample
2 acquired the retention time, tR of 0.815min with peak area of 4714.83. After that, the retention
time for sample 3 is 0.816min, giving out 4721.49 of peak area of caffeine. In order to calculate
the response factor, sample 3 was chosen to be included in the formula as it comprised the
nearest retention time, tR value with the caffeine standard. Response factor is important in the
determination of the identity and the amount of the unknown in order for good reproducibility
and also to reduce zone broadening which would affect the peak area. Thus, objective number
3 was also successfully achieved with concentration of caffeine in the unknown sample of
0.0414 ppm. Moving on to the components and experimental conditions used in this
experiment. In High Performance Liquid Chromatography (HPLC), there are 5 essentials
which are (in order) sample reservoirs, pumping system, sample injection system, columns,
and detector. The instrument used was Agilent Technologies 1260 Infinity which supported
both guard and analytical columns. The sample injection system used was the autosampler
and its detector supported the UV (254 mm) or PDA detector. Seeing as the mobile phase
used was a mixture of 70:30 acetonitrile-water (polar solvents) with non-polar stationary phase
(soft drink samples and caffeine standard), this partition was a reversed phase partition HPLC,
where the most polarized sample eluted first and produced the highest peak area of caffeine.
From the data above, we know that the highest peak came from sample 1 which was 4732.65
within the shortest retention time of 0.814min, therefore it has the highest amount of caffeine
among the 3 samples being ran. This experiment was carried out with gradient elution as the
ratio of mobile phase was 70:30 acetonitrile-water. Hence, it allowed a more optimized reading
of resolution peak.
QUESTIONS
1. State the types of compounds which are suitable for analysis using HPLC.

Non-volatile (gaseous) and thermally unstable.

2. Differentiate between the HPLC and GC in terms of :

i. Mobile phase
ii. Column

HPLC GC

Mobile phase Solvent reservoirs Carrier gas

- Analytical column - Packed column

Column
- Guard column/pre column - Capillary (open tubular) column

CONCLUSION

In conclusion, the objective of this experiment has been determined. The High - performance
liquid chromatography method is effective for both quantitative and qualitative analysis, and it
is used to evaluate biological and pharmaceutical samples. It provides the best option, most
flexible, and fastest chromatographic analysis technique. The optimal mobile phase
composition for mixture separation is 70:30. (ACN: H2O). A high concentration of organic
solvent composition increases solvent strength, which reduces analysis time. The retention
time of a standard caffeine solution is 0.844 minutes. The caffeine peak area in soft drink
samples 1, 2, and 3 has been identified as follows: Sample 1 has 4732.65, Sample 2 has
4714.83, and Sample 3 has 4721.49. The caffeine content of the soft drink sample was then
calculated by using response factor method. The concentration of caffeine in unknown sample
is 0.0414.
REFFERNCES

Biomedical and pharmacology.(2023).High performance Liquid Chromatography.Retrieved

January 15 2023 from

https://biomedpharmajournal.org/vol15no2/high-performance-liquid-chromatography-recent-
patentsandadvancement/#:~:text=Result%3A%20HPLC%20technique%20is%20important,q
uality%20control%20of%20drug%20components.

Science Direct.(2023).High-Perfomance liquid Chromatography.Retroeved January 15 2023

from https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-
biology/high-performance-liquid-chromatography

Microbe.(2023). HPLC- Definition, Principle, Parts, Types, Uses, Diagram . Retrieved

January 15 2023 from https://microbenotes.com/high-performance-liquid-chromatography-
hplc/