Instrumental analysis

PHC 321

Lecture (9)
Chromatography
 Questions

A. Discuss the mechanism of the following:

• Hollow cathode lamp
• Chopper

B. Compounds A & B was determined by TLC using hexan: ethyl acetate: ammonia
(4:5:1, v/v/v) as mobile phase, the Rf was found to be 0.35 & 0.67 respectively. Answer
the following questions:
• Discuss the principle of TLC.
• How to calculate the Rf value?
• Which compound has more affinity to the stationary phase?
 Practice questions

If you are supplied with three metals Ca0, Na0 and K0, discuss each of the following:

1- How can you differentiate between these metals qualitatively?

Resonance wavelength of Flame emission or atomic emission

2-What is the main precaution to be taken if each of these metals will be analyzed separately by
flame emission spectroscopy?

The main precaution is keeping the flame temperature regular as the number of excited atoms
are greatly affected by temperature, where an increase in temp by 10°K is accompanied by 4%
increase in the excited atoms.
It must be sufficient to cause atomization only and not ionization.
3- What is the main difference in the instrument if each of these metals will be
analyzed separately by atomic absorption spectroscopy? And explain how you can
eliminate interferences due to flame emission.

The difference is the presence of hallow cathode lamp as external light source
The Chopper will eliminate the flame interference, when the disc rotates at a
constant high speed, when the mirrored quarter is in front of the lamp, the radiations
are reflected. When the opened quarter is in front of the lamp, the radiations pass to
the sample and is absorbed.
 Interactive teaching methods and activities
• https://www.youtube.com/watch?v=lVu2BFlqHfs
• https://www.youtube.com/watch?v=ZN7euA1fS4Y
• https://www.youtube.com/watch?v=eCj0cRtJvJg
• https://www.youtube.com/watch?v=YRFSN9q523M
• https://www.youtube.com/watch?v=lboUQfX8cI4
• https://www.youtube.com/watch?v=VOSkyj1dtbc
• https://www.youtube.com/watch?v=8_7cdfNO7OY

● https://www.flippity.net/fc.php?k=1koJbA9FHr
RES2ERtA4_EHd-igNJUjJz7Qays0M8Ket0
 Learning outcomes

At the end of the lecture the students will acquire knowledge about:

▪ Chromatography and its classification

▪ The principle of HPLC separation and components of instrument used
 Classification of Chromatographic Methods

I. According to the nature of mobile phase:

Liquid chromatography Gas chromatography

II. According to the stationary phase:

Normal phase chromatography Reversed phase chromatography

III. According to the technique used

Planner chromatography Columnar chromatography

IV. According to the mechanism of separation

 IV. According to the mechanism of separation

▪ Adsorption chromatography

▪ Partition chromatography

▪ Ion exchange chromatography

▪ Size exclusion chromatography

▪ Affinity chromatography
1- Adsorption Chromatography
▪ It is the oldest type of chromatography
▪ The stationary phases (the adsorbent) is solid containing the
active site to which the substance to be separated (adsorbate)
adhere and the mobile phase is a fluid (liquid or gas)
▪ The strength of the interaction between the adsorbate and
adsorbent is governed by the number interacting sites on the
surface.
▪ The efficiency of the separation depends on:
1. The solubility of the molecule in the mobile phase.
2. Binding strength to the stationary phase

Physical adsorption Chemical adsorption

occurs through weak physical forces Occurs through formation of
like Van der Waals and dipole dipole hydrogen bond
interactions
 2- Partition Chromatography

▪ The stationary phase is liquid and mobile phase is liquid or gas.

▪ The liquid of the stationary phase is wetting a solid support like

cellulose or silica particles or alumina spread in a form on thin
layer on a sheet or packed in a column

▪ The substances to be separated distribute themselves between

the two phases according to their solubility depending on the
analyte partition coefficient between the two phases.

▪ Component which is highly soluble in the stationary phase

travels more slowly than that of lower solubility.
3- Ion exchange chromatography
It is used for the separation of ionized molecules (+/-)

The stationary phase is a bead polymer (resin), its surface is composed of a large
number of ionizable functional groups and the mobile phase is buffered solution.

• Cations (+ve) are separated on stationary phase called cation exchange resin
(-ve) which contains negatively charged groups (-SO3-H+) covalently bonded to
the stationary phase.

• Anions (-ve) are separated on stationary phase called anion exchange resin
(+ve) which contains positively charged groups (-R3N+Cl-) covalently bonded to
the stationary phase.

The stronger the charge on the ion, the greater is the retention time in the column.
Ion exchange columns
Anionic exchanger column
 4- Size exclusion chromatography
• The stationary phase is a material containing pores with
network structure whose dimensions are different in size.
• Substances are separated according to their molecular weight
(size).
• Usually, the analyte of the small molecule goes through the pore of
the stationary phase particles (strong retention), while the analytes
of large molecules just move between the particles taking shorter
pathway.
• The larger size of the analyte molecule, the faster is its elution
from the column
• When mobile phase is aqueous, the technique called gel filtration
while when it is an organic solvent the technique is rather called gel
permeation
 5- Affinity Chromatography
• This technique is highly specific and usually used to purify biomolecules
such as enzymes, antibodies, and recombinant proteins.
• It can also help to remove harmful substances such as pathogens.

• A particular ligand is chemically immobilized or “coupled” to a solid

support so that when a complex mixture is passed over the column, those
molecules having specific binding affinity to the ligand become bound.
The other sample components are washed away, the bound molecule is
stripped from the support, resulting in its purification from the original
sample.

• For example, a protein in a solution can be purified by passing it through a

column having another molecule attached to it (the ligand) that has an
affinity for the protein.
• When they bind, this will allow the unreactive, non-bound solution to pass
through the column. The target molecule is then eluted from the ligand so
that the protein can be removed from that surface.
 Technique

Planner Columnar

Paper HPLC/UPLC

TLC/HPTLC GC
High Performance (pressure) Liquid Chromatography (HPLC)
Chromatogram

Where:
tR = retention time
tM = void time
Wb = baseline width of the peak in time units
Retention time tR

tR
Retention volume Rv
the volume of mobile phase that passes through the column from the time of
injection to the time when the peak maximum of the injected compound elutes

Rv= tR x F (mobile phase flow rate)

Advantages of HPLC:
• Shorten the time required for separation.
• Applicable for very dilute samples.
• Easy automation of instrument operation and data analysis
• High accuracy and resolution.

Disadvantages of HPLC:
1. HPLC has high cost.
2. High quality components are needed.
3.The solvents and columns used in HPLC are expensive.
4. Regular maintenance and calibration is needed which add extra cost.
5. Sophisticated software is required for data analysis.
Instrumentation

As shown in the schematic diagram in Figure above, HPLC instrumentation

includes a sample reservoir, pump, injector, column, detector and integrator
or display system. The heart of the system is the column where separation
occurs.
1. Solvent Reservoir and degassing system
Mobile phase contents are contained in a glass reservoir.
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.

Degassing system is used to remove O2 and N2 gases which

interfere with the chromatographic separation .

Requirements of mobile phase:

• High purity
• Low viscosity
• Low reactivity (inert) to avoid chemical interaction with
the separated components or stationary phase.
• Immiscible with the stationary phase.
Types of chromatographic elution
Isocratic elution:
In this type of elution, mobile phase is either single solvent or mixture of solvents
without change in its composition.
The flow of the mobile phase is continued until the analysed mixture is completely
separated to its components.

Gradient elution:
The composition of the mobile phase is changed during the separation process.
It can be used for more complex samples to hasten the elution of the analytes of
strong affinity to the stationary phase.
2. Pump:
A pump aspirates the mobile phase from the solvent reservoir and
forces it through the system’s column and detector.

3. Sample Injector:
The injector is used to introduce the sample (mixture to be
separated) into the stream of solvent passes from the pump to the
column.

The analyst can manually load the sample into the injector using
a syringe (manual injector) or the samples are loaded in vials and
placed in an autosampler tray (can hold up to 100 samples)
4. Columns
Columns are usually made of polished stainless steel, are
between 50 and 300 mm long and have an internal diameter of
between 2 and 5 mm. They are commonly filled with a
stationary phase with a particle size of 3–10 µm.

Columns may be either analytical column or guard column.

Ideally the temperature of the mobile phase and the column
should be kept constant during an analysis.
Precolumn (guard column):
-Usually proceeds the separation column.
-It protects the main column from plugging and contamination by
samples and mobile phase. Thus, prolonging the life of the analytical
column.
-They are easy to replace, and it generates less charge than
replacing the main column.
-It has the same packing material as the separation column.
Temperature Control in HPLC
Why is it needed?
1- Reproducibility
Retention in HPLC is temperature-dependent
If temperature varies, then it is difficult to assign “peaks” to specific compounds in
the chromatogram and the peak areas/heights may vary

2- Solubility
Certain chemical compounds may have low solubility in the HPLC mobile phase.
If they are injected into the flow stream they may precipitate, or other difficulties may
arise

3- Stability
Certain chemical compounds, especially biological compounds such as enzymes or
proteins, may not be stable at room temperature or higher
 What is UPLC ??
Ultra performance liquid chromatography
Differences between HPLC and UHPLC

1. Particle sizes – In HPLC particle sizes of the stationary phase are typically in the order of 3-5 µm,
whilst UHPLC is characterized by particles of 2 µm or less.

2. Column dimensions – As with particle sizes there is a corresponding reduction in column

dimensions with UHPLC. A typical HPLC column has an internal diameter of 4.6 mm and a length of
250 mm, whilst a UHPLC column has internal diameters of 2.1 mm or less and is much shorter, 100
mm for example.

3. Flow rates – UHPLC runs at much lower flow rates than HPLC, for example 0.2 – 0.7 ml/min against
1-2 ml/min respectively.

4. Backpressure – With the smaller particles and reduced column diameter then this manifests itself
into higher backpressures in UHPLC compared to HPLC. HPLC instruments typically operate at
maximum pressures of 400-600 bar, whilst UHPLC instruments can operate at up to 1500 bar.
Advantages of UPLC:

▪ Decrease in consumption of mobile phase volume by at least 80% compared

to HPLC.
▪ Decreased run time and cost of operation.
▪ Lower injection volume is required.
▪ Reduction in band broadening thereby increasing the sensitivity.
▪ The higher column temperature minimizes the mobile phase viscosity resulting
in the high diffusion coefficient without significant loss in efficiency and
increase in column back pressure.
Disadvantages of UPLC:
▪ Higher back pressures compared to conventional HPLC which decreases
the life of the columns.

▪ The particles of less than 2 μm are mostly non-regenerable therefore, have

a narrow use.
 References:

• J. Mendham, R.C. Denney, J. D. Barnes, M.J.K. Thomas, Vogel's

Quantitative Chemical Analysis (6th edition, 2010), Prentice Hall, ISBN
0582226287.

• Donald L. Pavia, Gary M. Lampman, George S. Kriz, James A. Vyvyan,

Introduction to Spectroscopy (4th edition, 2012), Brooks Cole, ISBN
0495114782