UNIVERSITI TEKNOLOGI MARA

KOTA KINABALU SABAH

CHM260

BASIC INSTRUMENTAL ANALYSIS

LABORATORY REPORT

EXPERIMENT 3

High Performance Liquid Chromatography (HPLC)

GROUP : AS1206A1

GROUP MEMBER:

NADIA MAJITOL
: 2012199581
STEPHANIE FREDIRICK
: 2012566461
LECTURER
: Julenah Ag Nuddin

DATE
: 16th SEPTEMBER 2015
AIM

The aim of this experiment is to study how HPLC works in order to analyse the unknown present in a
mixture. Students will also be exposed on how the separation of mixtures occurs whereby; it can be
determined by obtaining the retention time in standard caffeine. After that, the retention time of caffeine
will be compared the retention time in several type of soft drink. This experiment also enable student to
understand the retention factor, number of theoretical plate, resolution, selectivity factor, area and peak of
the HPLC.

INTRODUCTION

High Performance Liquid Chromatography, or HPLC, is the most common analytical separation tool and is
used in many aspects of drug manufacture and research. HPLC is used

for qualitative and quantitative analysis of unknown mixtures which determining what is there, and how
much. HPLS is also used for the separation of mixtures for later analysis which is preparative HPLC. In
chromatography a small volume of a mixture of chemicals is passed through a column using a solvent and
different molecules exit the column at different times, this is called separation. (Terill R., n.d.)

The separation of a compound involves its physical interaction with a stationary phase

and a mobile phase. In chromatography a tube is filled with stationary phase (typically surface-modified
silica particles or silica “gel”)phase(solvent)andis passeda mobil through the system. In HPLC the
stationary phase is extremely small. A standard particle size

for column chromatography is 60 microns, while that for HPLC is typically 5 microns, or the size of a
speck of dust. Solvent flow through such dense material requires a high pressure, so in HPLC the stationary
phase is packed in a stainless steel tube, and solvent is pumped through the system under high pressure, up
to several thousand pounds per square inch (psi).

This pressure results in a flow rate of several ml’s (Knauer,per nminute.d.).

Why the stationary phase in HPLC is so tiny? A small, high-surface-area stationary phase maximizes the
interaction between the substance to be separated and the stationary phase, which results in better
separation. The primary parts of an HPLC are a solvent PUMP, an injector, the column, and a
detector/recorder: Each of these components are connected in a series to each other by steel tubing. The
pump controls the flow of solvent through the system. Upon leaving the pump, solvent enters the injector,
then passes through the column, and finally through the optical unit of a detector. The injector allows a
convenient and controlled introduction of sample directly onto the column, while the detector indicates
when
a particular analyte (also called a band) elutes (or leaves the column). If desired, the separated components
can be individually collected for later analysis. The two important things to know about each analyte
appearing as a peak in the chromatogram are 1 the retention time and 2 the peak area. An example of a
chromatogram of diet Pepsi (using much different conditions than we will use) is below:

OBJECTIVES

To determine the retention time of a standard solution (caffeine).

To identify the caffeine peak in a soft drink sample.

To determine the amount of caffeine on soft drink sample using the response factor method.

REVIEW OF LITERATURE

Many beverages such as soft drinks, coffee and tea contain the mild stimulant caffeine
(C8H10N4O2). The caffeine content varies widely from about 100 μg/mL (100 ppm) to over 1000 μg/mL in
Thecertaincaffeineseparationtypesiseasily achievedof coffe using HPLC. Liquid chromatography can be
divided into several types, including normal-

phase (e.g., a silica or alumina column), reversed-phase and ion exchange. Reversed-phase partition
chromatography uses a non-polar organic coating on a silica structure for the stationary phase. The non-
polar coating is commonly formed by reacting an organochlorosilane with the OH groups on the silica
surface.

With normal-phase HPLC, the most commonly used solvents are hexane, isopropanol or THF, whereas a
more polar mobile phase such as methanol/water or acetonitrile/water is commonly used for reversed-phase
partition chromatography. In this experiment, the caffeine separation will be done using a non-polar
C18column and a methanol/water mobile phase. A series of caffeine standards that bracket the unknown
sample concentration will be measured to construct a calibration curve. A comparison of the caffeine peak
area in the soft drink
sample compared to those for the standards permits a quantitative determination of the
caffeine content.

A study conducted by Kurina Baksh (2013) by using HPLC to calculate the amount of
caffeine present in several soft drink which are Redbull and Sting. She analysed alongside a
caffeine standard stock solution of concentration0.205 mg/mL by using a HPLC-UV/Vis
Detector at the wavelength of 254 nm, a Pecoreversed-phase C18 column, a mobile phase
80:20 (v/v) of water: methanol and a pump flow rate of 1mL/min. For quantitative purposes,
2
serial dilution of the caffeine standard gave a R value of 0.999 and the retention time of 2.7

± 0.2 minutes. With respect to a 12 oz can and using peak height, she found out that Red Bull
had a caffeine content of 88.76 mg and Sting 42.60 mg. With respect to a 12 oz can and using
peak area, it was found that Red Bull had a caffeine content of 63.91 mg, Full and Sting
17.75 mg. It was concluded that Red Bull had the higher amount of caffeine than Sting.
Below are the peaks for standard caffeine, Redbull and Sting.
Peah height (mAU) Versus Retention time (minutes) of Caffeine Standard 0.10 mg/mL
Peah height (mAU) Versus Retention time (minutes) of Redbull
Peah height (mAU) Versus Retention time (minutes) of Sting
APPARATUS

Beaker

Burette

Glass rod

Volumetric flask 10 mL

Dropper

CHEMICALS

Caffeine standard

Soft drink (Coke and Pepsi)

Distilled water

Acetonitrile

PROCEDURE

A. Experimental

Caffeine Standard Solutions

Stock solution, 1.0 mg/mL (Solution A)

A 10 mg caffeine was accurately weighed into 10 mL volumetric flask. It was then dissolved in
distilled water. The volume was made up with distilled water.

Flow rate of mobile phase

1.0 mL/min

Column
C18. 150 mm x 4.6 ID, 5 µm

Detector
UV (at 254 nm) 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)

To operate this instrument, the system was firstly switched on. The PC was not switched on.
The button ON was switched according to the labelled hardware number.

Running the software:

The method was created by clicking to instrument 1 at Class VP. To login window, the
username was System and Password was 2001. Next, the pump icon was clicked on, Low
pressure gradient mode was selected and the flow was set up to 0.2 ml/min. for the pressure
limitation, maximum was 2845 psi and 0 psi for minimum. A: Buffer solution, B: Methanol
(MeOH), C: ACN (Acetonitrile) and D: H2O. CTO was clicked, 10 AS vp menu. The oven
temperature was set to 28°C, Tmax was 85°C. The SCL to AV was checked, Trigger type
Expernal and Power On Event 1. After that in the status log menu, download, ok and apply
buttons were clicked. LC Setup Assistant icon was clicked and PDA image was clicked. In
general menu, the wavelength was started with 190 nm, and end at 800 nm. Library menu
was not change and in the purity menu, the wavelength was setup to 190 nm to 800 nm. In
D/A output menu, Channel 1. In the spectrum menu, nothing need to be changed. In the
spectrum menu, an appropriate data was inserted in the Active and Wavelength column. In the
ratio menu, an appropriate wavelength was added to Channel 1. After that, File, Method and
Save as was clicked. A description of title was added. The LC Setup Assistant was clicked
and the pump was also clicked whereby injection process was proceed. Before the injection
process begin, Control was set to Simple run and the sample ID, Data file were filled. Next,
the start button was clicked. When filling the syringe with sample, the maximum volume was
set to 20 µm and all samples were quickly injected into the port. The result was shown with a
several peaks, depending upon sample composition.
Working solution, 0.1 mg/mL (solution B)

1 mL of solution was pipetted into a 10 mL volumetric flask. The volume was made up with
distilled water.

Sample preparation

10 mL of soft drink sample (Coke and Pepsi) was poured into a small beaker. A magnetic
stirrer was used to remove any carbonation (sonnicate if necessary). 2-5 mL aliquot was
filtered through a 0.45 µm pore diameter membrane filter to remove particulate matter. 10 µL
solution B was injected into HPLC instrument. The peak area corresponding to caffeine was
measured referring to its tg.

B. Operation of the HPLC

Instruments: 1. HPLC (UV DETECTOR) –PERKIN ELMER SERIES 200

2. HPLC (PDA DETECTOR) –SHIMADZU VP SERIES

Sample preparation

A syringe and a filter (SRP 15 (0.45 µm) were used to transfer sample (10 –20 pL) into a vial.
To prepare a mobile phase, the mobile phase was filtered. For methanol, water, buffer and
acetonitrile (ACN), a membrane filter was used. An ultrasonic cleaner (Model UC 05) was
used. For the sample injection, single run acquisition was clicked and the syringe as well as
the load was inserted. After that, sample was injected and turned down. Control was selected
and extents run to few minutes. Report was selected and the Method custom report was
viewed. The result was printed. To clear the process, LC setup assistant was clicked. For
mobile phase B, C and D, washed with 100% ACN. For buffer, it was washed with water and
100% water was clicked.
DATA

Name and model of instrument:

Compound
Retention time, tr (min)
Peak area of Caffeine

Caffeine standard
1.494
97.6366%

Soft drink sample (Coke: Run 1)

1.493
55.6450 %

Soft drink sample (Pepsi: Run 2)

1.491
58.3264%

Name of soft drink sample: Coke and Pepsi

CALCULATIONS

Caffeine Standard

Capacity
Selectivity

Resolution, Rs

Number of theoretical
factor, α

plates

[
]

[
]
[
]

]
= 3.11

= 0.47

= 1.792
[

]
=

Coke

Capacity
Selectivity
Resolution, Rs

Number of theoretical

factor, α

plates

[
]

[
]
[
]

]
= 2.48

= 1.04

= 0.292
[

=
Pepsi

Capacity
Selectivity

Resolution, Rs

Number of
theoretical

factor, α

plates

[
]

[
]
[
]

]
= 2.96

= 0.40

=
= 3.095
[

=
Response Factor

539.49 ppm

561.16 ppm
DISCUSSION

In this experiment, HPLC was run only once for caffeine standard, three run for Coke and Pepsi. The
objective of this experiment is to identify amount of caffeine on soft drink sample using the response
factor method. According to the data obtained from HPLC, the retention time for caffeine is around
1.494 min, and for Coke and Pepsi, the retention time were 1.493 and 1.491 respectively. The
response factor of caffeine standard is concentration of caffeine (ppm) in coke is 5.85924. The
concentration of caffeine in Coke is obtained by dividing the area of caffeine in sample over RF, the
concentration of caffeine in coke is 539.49 ppm and 561.16 ppm for Pepsi. Amongst the three run
conducted by HPLC on Coke and Pepsi, the first run for both Coke and Pepsi were chosen because it
shows the nearest retention time of caffeine as in the retention time of caffeine in standard caffeine.

The capacity factor,dmeasure thek’migrationwasratescalculateofanalytesin column. The HPLC result

for standard caffeine shows the value of 0.47 capacity factor. According to this value, it indicates that
the separation is poor. As in Coke and Pepsi, the k’ were 1.04 and 0.40 respectively, which also
indicates poor separation.

As for the selectivity factor which is a measure of the relative migration rates of species A and B
with a stationary phase material, caffeine standard, Coke and Pepsi shows readings as much as 1.792,
0.292 and 3.095 respectively. Moving on, for the column efficiency, the numbers of theoretical plates
were calculated for caffeine standard, Coke and Pepsi which each shows an average number of
3 3 3
theoretical plates of 3.58 x 10 , 2.92 x 10 and 3.41 x 10 respectively. Resolution, Rs is the measure
of the separation of two chromatographic peaks. The Rs for caffeine standard, Coke and Pepsi is
3.11, 2.48 and 2.96 respectively.

In conducting this experiment, there were several precautionary steps that should be considered. The
first precautionary is to avoid any alteration in mobile phase composition which can lead to wide
fluctuation, noise and disturbances in the analytic recordings. Moreover, the presence of minute
particles in the mobile phase can block the column leading to interruption of the process and the
presence of air bubbles in the mobile phase can vary the pressure applied by the pump on mobile
phase.

For the maintenance of instrument, make sure that the waste containers are kept empty after using
the HPLC. It is recommendable to test for a column that has not been used recently or is old, to make
sure that it is working properly and has not contaminated. To ensure a smooth process during HPLC,
it is important to flush the system before running the
samples in order to insure that the solvent used for the previous sample does not interfere
with the current samples. The mobile phase flow rate is important and can range from 1-10
mL/min, through 1 mL/min is a good place to start with most experiments. It is also
important to monitor pressure when adjusting the flow rate, as the pressure should not exceed
400 bar. (Operating Manual, n.d.)
PRE-LAB QUESTION

Define ‘normal’ phase partition and ‘reve

Normal Phase

In normal phase, the stationary phase is polar and the mobile phase is less polar. The least polar compound
elutes first because in a relative sense, it is the most soluble in the mobile phase.

Reversed phase

In reversed phase, the stationary phase is non-polar and the mobile phase is polar. The most polar component
appear first.

What is meant by? Why‘responseisfactor importantfactor’inthe determination of the identity and the
amount of an unknown?

Response factor is a measure of the relative mass spectral response of an analyte compared to its internal
standard. Response factor is important to identify the slope of the line between the response for a given
standard and the origin. The average response factor for each analyte will then be used to calculate the
concentration of unknown.

QUESTION

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

Compounds which are suitable for analysis using HPLC are compounds that are organic, inorganic,
biological samples, synthetic or natural polymers and thermal labile compound.

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

(a) Mobile phase

HPLC

GC

Mobile phase is a liquid

Mobile phase or carrier is a gas

(b) Column
HPLC

GC

1. Analytical column
1.
Packed column

2. Guard column
2.
Capillary column
CONCLUSION

As a conclusion, the retention time of a standard solution (caffeine) was able to be

determined by using HPLC which were 1.494 min. The caffeine peak in a soft drink sample
of Coke and Pepsi were able to be identified which were 593.00757 mAU at 1.493 minute
retention time and 622.93848 mAU at 1.491 minute retention time respectively. The amounts
of caffeine on soft drink sample by using the response factor method were determined. In
coke, the concentration of caffeine is 539.49 ppm and 561.16 ppm in Pepsi.
REFERENCES

th
Terill R. (n.d.). CHEM 55 Lab. HPLC. Retrieved 14 September, 2015 from
http://www.chemistry.sjsu.edu/rterrill/ped/55/Lab/HPLC%20analysis%20from%20155. pdf

Knauer. (n.d.). Application Note. Quantitative analysis of Caffeine using the KNAUER HPLC
th
Educational system. Retrieved 13 September, 2015 from http://www.knauer.net

Operating Manual.(n.d.). High Performance Liquid Chromatography, Agilent 1100;VWD,

DAD, FLD, and RID. Retrieved 123th September, 2015 from
http://share.psu.ac.th/system/assets/media/files/000/007/356/original_Operating_Manua
l_HPLC_1100.pdf?1306827231

Kurina Baksh. (2013). A Quantitative Study to Determine the Amount of Caffeine Present in
Three Popular Brands of Energy Drinks (Red Bull®, Full Throttle® and Sting®) using
th
Reversed-Phase High Performance Liquid Chromatography. Retrieved 15 September, 2015
from http://www.academia.edu
APPENDICES