NEAR EAST UNIVERSITY

Faculty of Pharmacy
Department of Analytical Chemistry

Analytical Chemistry Lab. II

Assist. Prof. Dr. Usama Alshana

2020-2021
TRNC

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 High-Performance Liquid
EXPERIMENT 5
Chromatography (HPLC)
Determination of Preservatives in Medicines

5.1 Aim

The aims of this experiment are to make students familiar with:

 the principles of HPLC

 basic analytical procedures in HPLC analysis (e.g., preparation of mobile phases,
standard aqueous solutions and samples, plotting calibration graphs, etc.)
 interpreting the results of HPLC analysis
 determinating methylparaben in cough syrup by HPLC

5.2 Safety Precautions

Lab. coats, safety glasses and enclosed footwear must be worn at all times in the laboratory.
Methanol used in this experiment should be handled with extra care; Ingestion of as little as one
to four ounces can cause irreversible injury to the nervous system, blindness or even death.
Methanol can cause poisoning, systemic acidosis, optic nerve damage and central nervous
system effects. Methanol can also degrease the skin, which may cause dermatitis. Wear rubber
gloves when handling it.

5.3 Introduction

Fundamentally, chromatography is a technique used to separate the components contained in a

sample. High-performance liquid chromatography (HPLC) is a type of chromatography that,
because of its wide application range and quantitative accuracy, is regarded as an indispensable
analytical technique, particularly in the fields of organic chemistry and pharmaceutical analysis.

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It is also widely used for quantitative and qualitative analysis of organic and inorganic
compounds as well as a preparation technique for the isolation and purification of target
components contained in mixtures. There are various reasons behind the popularity of HPLC in
many fields (Figure 5.1).

Figure 5.1: Reasons behind the popularity of HPLC

In order to increase the separation capability of column chromatography, in addition to

increasing the surface area of the stationary phase so that the interaction efficiency is increased,
it is also necessary to homogenize the separation field as much as possible so that dispersion in
the mobile and stationary phases is minimized. The most effective way of achieving this is to
refine the packing material.

Refining the packing material, however, causes resistance to the delivery of the eluent to
increase. This is similar to the way that water drains easily through sand, which has relatively
large particles, whereas it does not drain easily through clay-rich soil, which has relatively fine
particles.

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Depending on gravity and capillary action would cause analysis to take a very long time to be
completed, and the idea of delivering the eluent forcibly using a high-pressure pump was
proposed. This was the start of high-performance (or pressure) liquid chromatography (HPLC).

HPLC Instrumentation

An HPLC instrument differs from a column chromatograph in that it is subject to the following
performance requirements.

1. Solvent Reservoir(s)

A solvent reservoir is where the mobile phase is placed and pumped from. The mobile phase can
be a pure solvent or a mixture of two, three or even four miscible solvents.

2. Delivery Pump

A solvent delivery pump that can maintain a constant, non-pulsating flow of solvent at a high
pressure against the resistance of the column is required.

3. Sample Injection Unit

There is a high level of pressure between the pump and the column; a device that can inject
specific amounts of sample under such conditions is required.

4. Column

The technology for filling the column evenly with refined packing material is required. Also, a
material that can withstand high pressures, such as stainless steel, is required for the housing.

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5. Detector

Higher degrees of separation have increased the need for high-sensitivity detection, and levels of
sensitivity and stability that can respond to this need are required in the detector.

A block diagram of an HPLC instrument is shown in Figure 5.2.

Figure 5.2: A block diagram of an HPLC instrument

Solvent Reservoirs

The requirements for a solvent reservoir are:

1) The reservoir and its attachment to the pump should be made of materials that will not
react with or contaminate the mobile phase. Such materials include Teflon, glass, or
stainless steel.
2) The vessel should have a cap to prevent particulate matter from contaminating the
mobile phase.
3) Caps have another hole than the main hole of the tubing that allows air to enter the
reservoir otherwise removal of mobile phase by the pump will create a vacuum.
Preventing the mobile phase from flowing to the pump creates a "vapor lock" within
the pump and can damage it.

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 4) Besides providing extra protection against particulates entering the pump, the inlet
filter serves to hold the inlet line at the bottom of the reservoir (Figure 5.3).

Figure 5.3: Solvent reservoir and inlet filter in HPLC

Solvent Degasser

Why is degassing of HPLC mobile phase necessary?

Bubble formation on mixing of solvents can lead to a number of problems in HPLC analysis
which can be prevented by degassing of mobile phase. These include:

1) Unstable and noisy baselines,

2) Air bubbles passing through detectors lead to spurious (fake) peaks,
3) Excessive pressure can develop which can lead to eventual pump failure.

Degassing techniques

Commonly used degassing practices for HPLC mobile phase are:

1) Vacuum filtration (solvent filtration) (Figure 5.4),

2) Sonication,
3) Helium purging.

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 Figure 5.4: Solvent filtration and degassing system

Solvent Delivery Pump(s)

Performance Requirements:

1) Constructed of materials that are inert toward solvents to be used in the mobile phase,
2) Deliver high volumes (flow rates) of solvent (up to 5 mL/min),
3) Deliver precise and accurate flow (<0.5% variation),
4) Deliver high pressure (up to 400 atm),
5) Deliver pulse-free flow,
6) Have low pump-head volume,
7) Be reliable.

Types of HPLC pumps:

1) Reciprocating pumps
2) Displacement pumps
3) Pneumatic pumps

Reciprocating pumps

Reciprocating pumps are currently used in about 90% of HPLC instrument. In this type of
pumps, a process consisting of (1) aspiration, (2) compression and (3) discharge of the mobile
phase is repeated. The operating principle is shown in Figure 5.5.

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 Figure 5.5: A reciprocating HPLC pump

Advantages of reciprocating pumps:

 They have small internal volume (35-400 L). Therefore, they have low dead volume.
 They offer high output pressure (up to 10,000 psi).
 Their ready adaptability to gradient elution.
 They provide constant flow rates that are independent of column back-pressure and
solvent viscosity.

Sample Injection Port

Sample injection in HPLC can be classified as manual and automatic.

Manual injection

Injection is done through specially designed 6-port rotary injection valve. The sample is
introduced at atmospheric pressure by a syringe into a constant volume loop. In the LOAD
position the loop is not in the path of the mobile phase. By rotating to the INJECT position the
sample in the loop is moved by the mobile phase stream into the column (Error! Reference
source not found.).

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It is important to allow some sample to flow into waste from the loop so as to ensure there are no
air bubbles in the loop and previously used sample is completely washed out to prevent memory
effects.

Automatic injections

 Advanced HPLC systems are equipped with an auto injector along with an auto
sampler.
 Automatic injection improves laboratory productivity and also eliminates personal
errors.
 The software programs filling of the loop (generally from 0 to 100 µL) and delivery of
the sample to the column.
 The computer also controls the sequence of samples for injection from vials kept in
numbered positions of the auto sampler (Figure 5.6). However, feeding the vial
number correctly on auto sampler rack and listing out the sequence correctly in the
computer is very important.

Figure 5.6: Manual and automatic HPLC injectors

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 HPLC Columns

Columns for HPLC are of two types: guard and analytical columns (Figure 5.7).

Figure 5.7: Guard and analytical HPLC columns

Guard column

A guard column is introduced before the analytical column to:

1) Increase the life of the analytical column by removing not only particulate matter and
contaminants from the solvents but also sample components that bind irreversibly to
the stationary phase.
2) The guard column serves to saturate the mobile phase with the stationary phase so that
losses of this solvent from the analytical column are minimized.
3) The composition of the guard-column packing is similar to that of the analytical
column; the particle size is usually larger. When the guard column has become
contaminated, it is repacked or discarded and replaced with a new one.

Analytical column

The HPLC analytical column is the heart of an HPLC instrument where separation of the
components of the sample is achieved. They can be categorized into two types: Normal-phase

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and reversed-phase columns. Partition chromatography can be performed in one of these two
modes: normal phase and reversed phase. The combination of a stationary phase with a high
polarity and a mobile phase with a low polarity is called “normal phase” and the opposite
combination is called “reversed phase”. Accordingly, if a normal-phase column is applied, the
method is referred to as normal-phase HPLC and when a reversed-phase one is used, it is called
reversed-phase HPLC (Table 5.1).

Table 5.1: Polarity of stationary and mobile phase in normal- and reversed-phase HPLC
Stationary Phase Mobile Phase
Normal-phase Polar (hydrophilic) Non-polar (hydrophobic)
e.g., silica, alumina e.g., hexane, cyclohexane, toluene
Reversed- Non-polar (hydrophobic) Polar (hydrophilic)
phase e.g., C4, C8, C18 e.g., Water, methanol, acetonitrile,
tetrahydrofuran

Reversed-phase HPLC (RP-HPLC)

The term “reversed phase” gives the impression that this technique is somewhat uncommon.
However, in fact, most people that use HPLC perform separation with reversed phase
chromatography, and it can fairly be described as a standard separation mode.

Compounds with a low polarity, such as those composed of aliphatic chains without localized
electrons, are used. However, to be used as packing material for HPLC, the substance used must
be chemically stable and capable of withstanding high pressures, so it is not true to say that any
substance with a low polarity is sufficient.

The most commonly used substance is produced by chemically bonding an octadecyl group (-
C18H37) to the surface of silica gel. This type of packing material is commonly known as “ODS”,
and an “ODS column”, into which ODS is packed, is almost synonymous with a “column for
reversed-phase chromatography”.

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If a stationary phase produced by chemically bonding an aliphatic chain to silica gel is used, the
length of the aliphatic chain influences the retention strength for the solute. It is said that, in
general, longer chains have greater retention strength. Beyond a certain length, however, the
retention strength does not change significantly.

The most commonly used solvents are water, methanol, and acetonitrile. Water is the solvent
with the highest polarity, and by mixing it with methanol or acetonitrile, which have lower
polarities, the overall polarity of the solution can be adjusted. Salts and acids are also added
sometimes in order, for example, to adjust the pH value or form ion pairs.

In reversed-phase HPLC, strongly hydrophobic substances (i.e., substances with a relatively low
polarity) are strongly retained by the stationary phase, and therefore have relatively long
retention times. Therefore, in a chromatogram containing multiple peaks, the substances are
eluted, broadly speaking, in descending order of polarity. An example of the effect of polarity on
the retention time in RP-HPLC is shown in Figure 5.8.

Figure 5.8: Effect of polarity on the retention time in RP-HPLC

Normal-phase HPLC (NP-HPLC)

In NP-HPLC, hydrophilic substances (i.e., substances with a relatively high polarity) are strongly
retained by the stationary phase, and thus have relatively long retention times. Therefore, in a
chromatogram containing multiple peaks, the substances are generally eluted in increasing order

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of polarity. An example of the effect of polarity on the retention time in RP-HPLC is shown in
Figure 5.9.

Figure 5.9: Effect of polarity on the retention time in NP-HPLC

HPLC Detectors

Types of detectors: HPLC detectors are of two basic types:

1) Bulk property detectors which respond to a mobile phase bulk property, such as
refractive index, dielectric constant, or density.
2) Solute property detectors which respond to some property of solutes, such as UV
absorbance or fluorescence, which is not possessed by the mobile phase.

HPLC Absorbance Detectors

Absorbance detector is a Z-shaped, flow-through cell for absorbance measurements on eluents

from a chromatographic column (Figure 5.10Error! Reference source not found.). Many
absorbance detectors are double-beam devices in which one beam passes through the eluent cell
and the other through a filter to reduce its intensity.

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UV absorbance detector with monochromator: There are detectors that consist of a scanning
spectrophotometer with grating optics. Some are limited to UV radiation while others encompass
both UV and visible radiation. The most powerful UV spectrophotometric detectors are diode-
array detectors (DAD).

Figure 5.10: UV detector cell for HPLC

HPLC Data Processors

The main components and features of an HPLC data processor (i.e., software) are:
Methods, data acquisition, integration, quantification, peak Identification, calibration,
automation, evaluating system suitability, and spectra library.

HPLC Waste

Waste Disposal Procedure

Use proper personal protective equipment and dispose chemicals in an appropriate organic waste
container.

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1. Label Waste

Affix a hazardous waste tag on all waste containers as soon as the first drop of waste is added to
the container

2. Store Waste

Store hazardous waste in closed containers in a designated location.

Waste must be under the control of the person generating and disposing of it

3. Dispose of Waste

Dispose of regularly generated chemical waste within 90 days by calling responsible authorities.

5.3.1 Parabens

Parabens are a class of widely used preservatives in cosmetic and pharmaceutical products.
Chemically, they are a series of para-hydroxybenzoates or esters of para-hydroxybenzoic acid
(also known as 4-hydroxybenzoic acid). Parabens are effective preservatives in many types of
formulas. These compounds, and their salts, are used primarily for their bactericidal and
fungicidal properties. They can be found in shampoos, commercial moisturizers, shaving gels,
personal lubricants, topical/parenteral pharmaceuticals, spray tanning solution, makeup and
toothpaste. They are also used as food additives.

Their efficacy as preservatives, in combination with their low cost, the long history of their use,
and the inefficacy of some natural alternatives like grapefruit seed extract (GSE), probably
explains why parabens are so commonplace.

In this experiment, four parabens, namely, methyl-, ethyl-, propyl- and butylparaben, which are
the most commonly used ones, are separated by HPLC. In addition, methylparaben will be

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quantitatively determined in a cough syrup. The chemical structures of the studied parabens are
given in Figure 5.11.

Figure 5.11: Chemical structures of the studied parabens

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5.4 Experimental

5.4.1 Instrument

An Agilent Technologies 1200 SERIES Gradient High-Performance Liquid Chromatograph

(HPLC) with a Diode Array Detector (DAD).

5.4.2 Chemicals and apparatus

You will be provided with the following solutions and equipment:

 Cough medicine or a similar syrup

 DI water
 Methanol
 Methanol/water (mobile phase)
 Methyl-, ethyl-, propyl- and butylparaben (pure standards)
 Methylparaben-containing syrup (real sample)
 Solvent filtration system

5.4.3 Procedure

A. Preparation of parabens stock and standard solutions

1) Weigh 10.0 mg of each paraben using an electronic balance.

2) Separately transfer this mass to 10-mL volumetric flasks.
3) Add 5.0 mL of methanol to dissolve the solid and then complete the volume to the
mark with DI water. Each of these stock solutions contains 1000 µg of parabens in
each mL (or 1000 ).
4) Prepare a 100.0 intermediate stock solution containing the four parabens in a
10-mL volumetric flask by transferring 1.0 mL of each stock solution and then
completing the volume to the mark with methanol (Solution A).

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 5) Prepare a 2.0 mixed standard solution containing the four parabens by
transferring 200.0 µL of Solution A into a 10-mL volumetric flask and then complete
the volume to the mark with DI water.

B. Separation of parabens

Inject 20.0 µL of this solution into the HPLC instrument using the separation conditions shown
in Table 5.2.

Table 5.2: HPLC experimental conditions

Column ACE-C18. 3 mm ID x 12.5 cm (5 µm)
Mobile phase flow rate 1.0
( )
Column temperature (°C) 20
Detector/wavelength UV. 258 nm (BW 4). Reference 360 nm (100 BW)
Injection volume (µL) 20
Mobile phase Methanol:H2O 60:40 (%, v/v)

C. Constructing a calibration graph for methylparaben

1) Prepare methylparaben standard solutions containing 2.0, 4.0, 6.0, 8.0 and 10.0
in DI water.
2) Inject 20.0 µL of each solution into the HPLC instrument using the separation
conditions shown in Table 5.2. Record the peak area you get for each standard in Table
8 of your Post-Lab. Report.
3) Plot a calibration graph of peak area (y-axis) versus methylparaben concentration in
(x-axis).

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D. Determination of methylparaben in the cough syrup

1) Transfer 50.0 µL of the syrup into a 10.0 mL volumetric flask and complete the
volume to the mark with DI water (Solution B).
2) Vortex the solution for 10 s and inject 20.0 µL of this solution into the HPLC
instrument using the separation conditions shown in Table 5.2.
3) Measure the peak area of the peak corresponding to methylparaben. Record the peak
area you get in Table 9 of your Post-Lab. Report.
4) Calculate the concentration of methylparaben in Solution B using the linear equation
you obtained from the calibration graph (in Step C.3) with the standards.
5) Hence, calculate the concentration of methylparaben in the original syrup (in )
by multiplying with the dilution factor (DF), i.e., 200 times. Record your results in
Table 9 of your Post-Lab. Report.

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References

[1] Skoog D. A., West D. M and Holler F. J., “Fundamentals of Analytical Chemistry”, 7 th
Edition, Saunders College Publishing, Orlando, Florida, 1996.
[2] John R. Dean, “Atomic Absorption and Plasma Spectroscopy”, 2nd Edition, ACOL series,
John Wiley & Sons, Ltd, UK, 1997.
[3] Peter A. S., Brian C., “Chromatographic Separations (Analytical Chemistry by Open
Learning)”, Wiley-Blackwell, 1987.
[4] M. Tevfik Orbey, Nilgün G. Göğer, Nusret Ertaş, Şebnem Yılmaz, et al., “Analitik Kimya
Pratikleri”, Gazi Üniversitesi, 2012.
[5] Usama Alshana, “NEPHAR202 Analytical Chemistry II Laboratory Manual”, Near East
University, 2018.
[6] Hayati Çelik, “NEPHAR202 Analytical Chemistry II Laboratory Manual”, Near East
University, 2014.
[7] http://www.chemguide.co.uk/analysis/chromatography/paper.html (24/09/2016)
[8] Quanfei Zhu, Chao Ma, Huaixia Chen, Yaqi Wu, Jianlin Huang, “A molecular imprint-
coated stirrer bar for selective extraction of caffeine, theobromine and theophylline”,
Microchimica Acta (2014) 181:303–311.
[9] Snyder L. R., Kirkland J. J., “Introduction to Modern Liquid Chromatography”, John Wiley
& Sons, New York, 1974.

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