CHM 260 LABORATORY REPORT

Experiment Number: 6

Title: High Performance Liquid Chromatography

(HPLC)

Name: Nor Syafikah binti Mohd Nasir

Student no.: 2015440114

Date of Report Submission: 15th December 2017

Lab Partner’s Name: Aqilah binti Anuar

Lyana Fasihah binti Ahmad Sobri

Nur Atiqah Farzana binti Zaini

Lecturer’s Name: Miss Hanani binti Yazid

Experiment 6: High Performance Liquid Chromatography (HPLC)

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

High Performance Liquid Chromatography is a type of separation analysis that uses

liquid mobile phase and liquid stationary phase. The separation occurs based on the
differences in the polarity of the analytes. The analyte that interacts most in the
stationary phase will elute later than the analyte that interact least in the stationary
phase. Reverse phase chromatography is used in which the stationary phase is non-
polar while the mobile phase is the polar mixture. The changes in the mobile phase
polarity will affect the interaction between the analyte and the stationary phase and
also affect the efficiency of the separation. Changes in the mobile phase composition
can be done either by isocratic elution whereas the composition in the mobile phase
is constant throughout analysis or by gradient elution in which the composition of the
mobile phase is change during separation either continuously or in step in order to
separate wide range of polarity of the analytes.

APPARATUS

Beaker

Burette

Glass rod

Volumetric flask 10mL

Dropper
CHEMICALS

Caffeine standard

Soft drink (Cola)

Distilled water

Acetonitrile

PROCEDURES

A. Experimental
1. Caffeine Standard Solutions
i. Stock solution, 1.0mg/mL (Solution A)
a) Weigh 10mg of caffeine accurately and pour into 10mL
volumetric flask.
b) The caffeine was dissolved in distilled water.
c) The volume was made up with distilled water.
ii. Working Solution. 0.1mg/mL (Solution B)
a) 1mL of solution A was pipetted into a 10mL volumetric flask.
b) The volume was made up with distilled water.
2. Sample Preparation
i. 10mL of Cola sample was poured into a small beaker.
ii. Sonnicate was used to remove any carbonation.
iii. 2-5mL aliquot was filtered through 0.45µm pore diameter
membrane filter to remove particulate matter.
3. 10µL of solution B was injected into the HPLC to establish the retention
time, tR, of caffeine.
4. The peak area of caffeine was measured.
5. 10µL of the prepared soft drink sample was injected into the HPLC
instrument.
6. The peak area corresponding to caffeine was measured by referring to its
tR.
B. Operation of the HPLC

Flow rate of the mobile phase 1.0mL/min

Column C18, 150mm x 4.6 ID, 5µm
Detector UV (at 245nm) or PDA
Mobile phase Acetonitrile and DI water
Ratio of mobile phase 70:30 (Acetonitrile : water)

Operating Instructions

Starting the instrument:

LC-10 AT (liquid chromatography)
DGU -14A (degasser)
CTO- 10AS (column/oven)
SPD – M10A (diode array detector)
SCL -10A (system controller)

1. To operate this instrument, the system was switched on first before the
PC.
2. The button was on according to the labelled hardware number.

Running the software:

1. To create method, go to class VP, instrument 1 was clicked.
2. Login window was on,
Username: System
Password: 2001
Login was clicked
3. Pump icon was click on,
Pump window was on
Select mode: Low pressure gradient
Set up for flow: 0.2ml/min
Pressure limit, max : 285psi
 Min: 0 psi
A: Buffer solution
B: Methanol (MeOH)
C: ACN (Acetonitrile)
D: H2O
4. CTO – 10AS vp menu was clicked. Oven temperature: 28°C, T max: 85°C.
5. SCL-10AV was clicked: Trigger type External
Power on ; Event 1
6. Go to status log menu: Do not change anything.
7. Go to time program menu: Do not change anything.
Download was clicked.
OK was clicked, then Apply was clicked.
8. LC Setup assistant icon was selected : Click PDA image.
9. In general menu, the wavelength was started : 190nm, end wavelength :
800nm.
10. In library menu: Do not change anything.
11. In purity menu: from 190 nm to 800 nm.
12. In D/A output menu: Channel 1……….nm.
13. In spectrum menu: Do not change anything.
14. In multi menu: the appropriate data was inserted.
15. In ratio menu,
Channel 1
Wavelength (nm): ________
16. Click File > Method > Save as…….
Description: (title)
17. LC Setup assistant was clicked.
18. Turn on pump was clicked. At this moment, injection was proceeded.
19. Before injection taking place
Click Control: Simple run
Sample ID: ……………
Data file: …………..
Start was clicked
20. When the syringe was filled with the sample, the maximum volume should
be 20mL and it must quickly inject all samples into the port.
21. Results will show a chromatogram with a several peaks, depending upon
sample composition.

Sample preparation:
1. A syringe and a filter were used to transfer sample (10-20mL) into a vial.
Type of filter: SRP 15 (0.45mm).
2. The mobile phase was filtered to prepare mobile phase. For methanol,
water, buffer and acetonitrile (ACN) , use membrane filter.
3. Ultrasonic cleaner was used.
4. Sample injection.
Single run acquisition was clicked. The syringe was inserted and load.
The sample was injected and turn down.
5. Control was selected > Extent run _____min.
6. Report was selected, > view > Method custom report.
7. Results were printed.
8. For clean up process, LC set up assistant was clicked.
For mobile phase B, C and D, wash with ACN. 100% ACN was
clicked.
For buffer, wash with water. 100% water was clicked.
9. The instrument was switched off.
QUESTIONS

1. State the types of compound which are suitable for analysis using HPLC.

The types of compound which are suitable for analysis using HPLC are
compound that is non-volatile and thermally unstable such as organic,
inorganic, biological samples, synthetic or natural polymers.

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

i. Mobile phase
The mobile phase of chromatography equipment is the substance
that moves the sample through the machine. In HPLC the mobile
phase is a liquid made up of an organic solvent, ultrapure water
and other ingredients to ensure its compatibility with the sample.
GC uses gas for its mobile phase. Gases used include helium,
nitrogen, argon or hydrogen, depending on what is being
analyzed.

ii. Column
As samples travel over chromatography columns, the sample and
mobile phase interact with the column contents causing the
components of the sample to elute at different time. HPLC
columns are typically four-to-six inch-long metal or glass tubes
tightly packed with silica or differing carbon chain lengths. GC
systems have coiled capillary columns with interior walls coated
with various materials depending on the lab's needs. Stretched
out, GC columns can reach lengths of 100 feet.

CALCULATION

Response Factor, RF, caffeine standard = Peak area caffeine standard

Concentration of caffeine standard

Concentration of caffeine (ppm) = Peak area caffeine in sample

RF
(Conversion factor: 1mg/mL = 1000 ppm)

RF = 1479720
0.1 mg/mL
= 14797200 mg/mL

Concentration of caffeine (ppm) in Coke sample:

= 2237029
14797200
= 0.1512 mg x 1000 ppm
mL 1mg/mL
= 0.1512 ppm

DISCUSSION

In this experiment, HPLC is used to determine the retention time of caffeine and the
caffeine peak in a soft drink sample. Although gas chromatography has a higher
sensitivity and speed, it cannot be utilised in this case as both compounds are not
sufficiently volatile and may degrade at high temperatures. HPLC has the ability to
separate, identify and quantitate the compounds that are present in any sample that
can be dissolved in a liquid B. Specialized component is required for an HPLC
separation because of the high pressures and low tolerances under which the
separation occurs. If the results are to be reproducible, then the conditions of the
separation must also be reproducible. Thus HPLC equipment must be of high
quality; it is therefore expensive. There are some components of the HPLC
instrument used in this experiment.
Firstly is the solvent. The mobile phase, or solvent, in HPLC is usually a mixture of
polar and non-polar liquid components whose respective concentrations are varied
depending on the composition of the sample. As the solvent is passed through a
very narrow bore column, any contaminants could at worst plug the column, or at the
very least add variability to the retention times during repeated different trials.
Therefore HPLC solvent must be kept free of dissolved gases, which could come out
of solution mid-separation, and particulates.
Secondly is the injector which is the syringe injection through self-sealing
elastomeric septum. This experiment used a glass Hamilton syringe that designed to
withstand pressure up to 1500 ps.
Next component is the column. Type of analytical and guard column used in this
experiment. Guard column used to prevent deactivation of the analytical column by
adsorption and to minimize band broadening. The function of the guard column is to
increase the lifetime of a HPLC column. Also, prevents clogging of the analytical
column while extending its lifetime and preserving its performance. Thus, guard
column is sacrificed to protect the more expensive analytical column. The column
used in the lab should have 5µL to 20µL in inner diameter.
Besides, another component used in HPLC instrument is detector. The HPLC
detector, located at the end of the column, must register the presence of various
components of the sample, but must not detect the solvent. For that reason there is
no universal detector that works for all separations. A common HPLC detector that
used in this experiment is a UV absorption detector, as most medium to large
molecules absorb UV radiation.
Besides, another component used in HPLC instrument is detector. The HPLC
detector, located at the end of the column, must register the presence of various
components of the sample, but must not detect the solvent. For that reason there is
no universal detector that works for all separations. A common HPLC detector that
used in this experiment is a UV absorption detector, as most medium to large
molecules absorb UV radiation.
After that, some techniques are used in this experiment to make sure the results are
to be reproducible and for good separation. This includes the degassing system
technique. The purpose of degassing the solvent is to remove dissolved gases and
dust from the liquid. Dissolved gases can lead to irreproducible flow rates and band
spreading. Also, both bubbles and dust will interfere with the performance of most
detectors. In this experiment we have degassed the solvent using filtration through a
membrane filter.
If the stationary phase is more polar than the mobile phase, the separation is
deemed normal phase. If the stationary phase is less polar than the mobile phase,
the separation is reverse phase. In reverse phase HPLC the retention time of a
compound increases with decreasing polarity of the particular species. The key to an
effective and efficient separation is to determine the appropriate ratio between polar
and non-polar components in the mobile phase. The goal is for all the compounds to
elute in as short a time as possible, while still allowing for the resolution of individual
peaks.
If the composition of the mobile phase remains constant throughout the HPLC
separation, the separation is deemed an isocratic elution. Often the only way to elute
all of the compounds in the sample in a reasonable amount of time, while still
maintaining peak resolution, is to change the ratio of polar to non-polar compounds
in the mobile phase during the sample run. Known as gradient chromatography, this
is the technique of choice when a sample contains components of a wide range of
polarities. For a reverse phase gradient, the solvent starts out relatively polar and
slowly becomes more non-polar. The gradient elution offers the most complete
separation of the peaks, without taking an inordinate amount of time. A sample
containing compounds of a wide range of polarities can be separated by a gradient
elution in a shorter time period without a loss of resolution in the earlier peaks or
excessive broadening of later peaks. However, gradient elution requires more
complex and expensive equipment and it is more difficult to maintain a constant flow
rate while there are constant changes in mobile phase composition. Gradient elution,
especially at high speeds, brings out the limitations of lower quality experimental
apparatus, making the results obtained less reproducible in equipment already prone
to variation. If the flow rate or mobile phase composition fluctuates, the results will
not be reproducible.
Based on the data obtained, the retention time of caffeine standard is 0.908 min and
its peak area is 1,479,720 µV/sec. Then, the soft drink sample which coke cola was
injected to the HPLC three times to see if the retention time of the soft drink sample
were same to the caffeine standard. The retention time for those three injection were
0.903, 0.906 and 0.873 min. The chromatogram that has the retention time value
nearest with the standard caffeine’s retention time was chosen to calculate the
amount of caffeine in the soft drink. The area peak of caffeine for three sample of
soft drink were 2, 082, 175 µV/sec, 2, 237, 029 µV/sec and 2, 164, 372 µV/sec. The
results showed that there were caffeine in the coca cola sample because it has
nearest retention time with the caffeine standard. Also, for better result, the
chromatogram with the lowest retention time is chosen because short and well
defined separation time produced good result. Then, the concentration of caffeine in
Coke sample was calculated using response factor. The result obtained was
0.1512ppm.
During the experiment was conducted, there were several precautions that must be
taken in order to minimize error of the data. Firstly, shake all the solution adequately
to ensure dissolution for 5min before injection into chromatograph. Then, make sure
the column is not clogged, it maybe cause by the precipitation of proteins in the
column caused by removal of stabilizing agents during fractionation. Beside that, the
syringe was rinse and clean before and after each sample was injected to prevent
any contaminant and other solution that will affect the process. Lastly, avoid
formation of bubbles during the experiment because it will affect the reading in the
column while the measurement was taken.

CONCLUSION
At the end of the experiment, we were able to determine the retention time of a
standard caffeine solution which is 0.908min. Besides, we were identified the
caffeine peak in a soft drink. The three peak for the coca cola sample were 2, 082,
175 µV/sec, 2, 237, 029 µV/sec and 2, 164, 372 µV/sec. Lastly, the amount of
caffeine in the Cola soft drink was determined using response factor method. There
was 0.1512 ppm of caffeine found in the Cola drink.

REFERENCES
1. Ariffin Z., 2016, Basic Instrumental Analysis Laboratory Experiments for
An Introductory Course in Instrumental Analysis, High Performance Liquid
Chromatography, Experiment 6.
2. Skoog, D.; Holler, F.; Crouch, S. Principles of Instrumental Analysis, New
York, (2007), retrieved on 11 December 2017, 4.10pm.