NEPHAR 201

Analytical Chemistry II

Chapter 7
High-Performance Liquid
Chromatography (HPLC)

Assist. Prof. Dr. Usama ALSHANA

1
 Week Topic Reference Material Instructor
1
Introduction Instructor’s lecture notes Alshana
[14/09]
 Principles of Instrumental Analysis, Chapter 6,
2 An introduction to spectrometric
pages 116-142 Alshana
[21/09] methods
 Enstrümantal Analiz- Bölüm 6, sayfa 132-163
 Principles of Instrumental Analysis, Chapter 7,
3 Components of optical
pages 143-191 Alshana
[28/09] instruments
 Enstrümantal Analiz- Bölüm 7, sayfa 164-214
 Principles of Instrumental Analysis, Chapter 9,
4 Atomic absorption and emission pages 206-229, Chapter 10, pages 230-252
Alshana
[05/10] spectrometry  Enstrümantal Analiz- Bölüm 9, sayfa 230-253,
Bölüm 10 sayfa 254-280
 Principles of Instrumental Analysis, Chapter 13,
5 Ultraviolet/Visible molecular
pages 300-328 Alshana
[12/10] absorption spectrometry
 Enstrümantal Analiz- Bölüm 13, sayfa 336-366
 Principles of Instrumental Analysis, Chapter 16,
Omitted 6
[19/10]
Infrared spectrometry pages 380-403 Alshana
 Enstrümantal Analiz- Bölüm 16, sayfa 430-454
Quiz 1 (12.5 %)
7  Principles of Instrumental Analysis, Chapter 26,
Alshana
[26/10] Chromatographic separations pages 674-700
 Enstrümantal Analiz- Bölüm 26, sayfa 762-787
8
[02- MIDTERM EXAM (25 %)
07/11]
9 High-performance liquid
 Principles of Instrumental Analysis, Chapter 28, Alshana
[09/11] chromatography (1)
pages 725-767
10 High-performance liquid
 Enstrümantal Analiz- Bölüm 28, sayfa 816-855 Alshana
[16/11] chromatography (2)
 Principles of Instrumental Analysis, Chapter 27,
pages 701-724, Chapter 29 pages 768-777
11 Gas, supercritical fluid and thin-
 Enstrümantal Analiz- Bölüm 27, sayfa 788-815, Alshana
[23/11] layer chromatography
Bölüm 29 sayfa 856-866, Bölüm 28 sayfa 848-
851
 Principles of Instrumental Analysis, Chapter 30,
12
Capillary electrophoresis pages 778-795 Alshana
[30/11]
 Enstrümantal Analiz- Bölüm 30, sayfa 867-889
13 Quiz 2 (12.5 %)
Alshana
[07/12] Extraction techniques Instructor’s lecture notes
14 Instructor’s lecture notes and from the above given
Revision Alshana
[14/12] materials
15
[21- FINAL EXAM (50 %)
31/12]

2
 Invention of Chromatography by M. Tswett

The Russian-Polish botanist M. Tswett is generally recognized as the first person to establish
the principles of chromatography.
In 1906, Tswett described how he filled a glass tube with chalk powder (CaCO3) and, by
allowing an ether solution of chlorophyll to flow through the chalk, separated the chlorophyll
into layers of different colors. He called this technique “chromatography”.
3
 Comparing Chromatography to the Flow of a River

 Chromatography can be often compared to the flow of a river.

This analogy represents the components of chromatography in the following way:

• River: Separation field
• Leaf and stone: Target components of sample (analytes)
• Standing to watch at the river mouth: Detector
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• Higher degree of separation.
 Refinement of packing material (3 to 10 µm).

• Reduction of analysis time.

 Delivery of eluent by pump
 Demand for special equipment that can withstand high pressures

The arrival of High-Performance

Liquid Chromatography (HPLC)

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 High separation capacity, enabling the batch analysis of
multiple components,

 Superior quantitative capability and reproducibility,

 Moderate analytical conditions,

• Unlike GC, the sample does not need to be vaporized.

 Generally high sensitivity,

 Low sample consumption,

 Easy preparative separation and purification of samples.

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 ⑤
② ③
Detector
④

Column
Pump
⑦

Solvent Sample injection port Waste

reservoir (loop)
(mobile phase) Signal processor
① ⑥

• Other accessories may be added as necessary.

8
 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: 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 to allow air to enter the
reservoir otherwise removal of mobile phase by the
pump will create a vacuum. This prevents mobile Inlet filter

phase from flowing the pump, creating a "vapor

lock" within the pump.

• Besides providing extra protection against particulates entering the pump, the inlet
filter serves to hold the inlet line at the bottom of the reservoir. 9
 Performance Requirements:

1. Constructed of materials inert toward solvents to be used,

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. Syringe pumps
3. Constant pressure pumps
A reciprocating HPLC pump 10
Rheodyne/Valco injectors Autosamplers

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• 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.
• 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.

HPLC injection
port

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 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.
• However, feeding the vial number correctly on auto sampler rack and listing
out the sequence correctly in the computer is very important.

HPLC vials and septa 13

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 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.

• 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.

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Properties of an HPLC column 16
 Stationary phase Mobile phase

High polarity Low polarity

Normal-phase
(hydrophilic) (hydrophobic)

Reversed- Low polarity High polarity

phase (hydrophobic) (hydrophilic)

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 Stationary phase: Low polarity

 Octadecyl (C18) group-bonded silical gel (ODS), etc.

 Mobile phase: High polarity

 Water, methanol, acetonitrile, acetone, tetrahydrofuran etc.

 Salt (or buffer) is sometimes added to adjust the pH or to form

ion pairs.

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 C18 (ODS) type
 C8 (octyl) type
 C4 (butyl) type
 Phenyl type
 Cyano type
 Amino type

CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2

Si -O-Si
CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

C18 (ODS) 19
• 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 a greater retention strength.
Beyond a certain length, however, the retention strength does not change
significantly.
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 C18 (ODS) OH

Weak Polar
Strong
CH3

Non-polar

• In RP-HPLC, strongly hydrophobic substances (i.e., substances with a relatively

low 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 decreasing order of polarity.
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 Stationary Phase
 Silica gel: -Si-OH
 Cyano type: -Si-CH2CH2CH2CN
 Amino type: -Si-CH2CH2CH2NH2
 Diol type: -Si-CH2CH2CH2OCH(OH)-CH2OH
 Mobile Phase
 Main solvents: Aliphatic hydrocarbons (e.g., hexane, cyclohexane),
aromatic hydrocarbons (e.g., toluene), etc.
 Modifier solvents: Alcohols, ethers, etc.

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 SiOH HO
Strong
SiOH
Polar
Very weak

Non-polar

• 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 of polarity. 23
24
 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, that is not possessed by the mobile phase.

Performance of HPLC detectors

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• Absorbance Detector: Is a Z-shaped, flow-through cell for absorbance
measurements on eluents from a chromatographic column.

• 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.

 UV Absorbance Detector with

Monochromator: There are detectors that
consist of a scanning spectrophotometer
with grating optics. Some are limited to UV
radiation; others encompass both UV and
visible radiation. The most powerful uv
spectrophotometric detectors are diode-
array (DAD) instruments.
UV detector cell for HPLC
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 ② Column oven
(thermostatic column
chamber)

① Solvent degasser

• Other necessary HPLC accessories may include:

1. Solvent degasser,
2. Column oven (thermostatic column chamber)

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 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,
2. Sonication
3. Helium purging.

Vacuum filtration Ultrasonic bath

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Why is it necessary to thermostat the column?

Temperature is an important factor in HPLC that may change:

a) Retention time,
b) Resolution,

(a) (b)
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  Isocratic system
Constant eluent composition.

 Gradient system
Varying eluent composition.

• The technique of delivering solution with a constant composition as the eluent

is called “isocratic elution”.

• The technique of varying the eluent composition during a single analysis is

called “gradient elution”.

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 In isocratic mode:

CH3OH/H2O = 60:40
Long analysis time!!

Poor CH3OH/H2O = 80 / 20
resolution

In the analysis of multiple components using HPLC, attempting to clearly separate

every single component results in an extremely long analysis time.
On the other hand, attempting to reduce the analysis time by changing the eluent
composition has an adverse effect on separation among components with relatively
short retention times.
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In gradient mode:

If the eluent composition is changed gradually during analysis...

95%
Concentration of methanol in eluent

30%

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