College of Pharmacy and Nursing
Department of Pharmacy
Spring 2010/ 2011 Semester
Advanced Instrumental Analysis (CHEM451)
Code CHEM451
Title Advanced Instrumental Analysis
Credits 2
Pre-requisite -CHEM260
Description The course deals with the principles and applications
of modern analytical instruments. Emphasis is placed
upon the theoretical basis of each type of instrument,
its optimal area of application, its sensitivity, its
precision, and its limitations.
Teaching Strategies
Lecture tutorial
Learning outcomes
The main goals of this course are to lay the ground
work for an understanding of the use of
instrumentation together with chemical data, to
convey the theoretical principles upon which
methods such as chromatography and, spectroscopy
techniques are based, and to cultivate relevant
laboratory skills and data taking and data analysis
methods. In more detail, you should:
1. Show familiarity with the principles of
chromatography and electrophoresis.
2. Be able to enumerate the data domains
characterizing an instrumental measurement.
3. Be able to apply the classical theory of wave
motion to the propagation of electromagnetic
radiation, and show of treatment is appropriate.
4. Demonstrate an understanding of how the
quantum mechanical theory of electromagnetic
radiation leads to the generation of spectra.
5. Be able to give a definition of emission,
absorption, luminescence, and scattering methods.
6. Be able to describe the roles and types of
components found in typical optical instruments,
including lasers, monochromators, and
interferometers.
7. Show knowledge of the specifics of infrared
absorption, ultraviolet absorption, and luminescence
methods for the spectroscopic study of atoms and
molecules.
8. Be able to explain the principles of mass
spectrometry and its applications to the study of
atoms and molecules.
9. Be able to give an account of how the interaction
between nuclear magnetic moments and external
magnetic fields leads to the phenomenon of nuclear
magnetic resonance, and of how the phenomena of
chemical shift and spin spin coupling arise.
1- Introduction to Instrumentation
Course outline
2 -UV-Visible spectrometry
3 -Infra red spectroscopy
4 -Nuclear Magnetic Resonance
5 -Mass Spectrometry
6 -Chromatography
References
1 -Practical Pharmaceutical Chemistry, 4th ed part1&11, A.H. Beckett, J.B.Stenlake
2-Silverstein, Spectroscopy
Lecturer: Dr. Afaf Mohammed
Academic Activity Section/s Day & Time Building & Room
Theory
1
Sat 10:00-10:50 15-15C
Tutorial
1
Mon 11:00-1:50 15-15C
Lecture and Lab Plan
Week
1 Chromatography
2 Column C & TLC
3 Gas Chromatography and HPLC
4 Introduction to Instrumentation
5 Interaction of matter with radiation
6 UV-Visible spectrometry
7 Calculation of λmax
8 Infra red spectroscopy
9 Determination of the structure using IR Spectrum
10 Semester Break
11 Nuclear Magnetic Resonance
12 Proton magnetic resonance
13 13C Magnetic resonance
14 How to use Nuclear magnetic resonance for structure elucidation
15 Mass Spectrometry
16 �ٌReview of the course
17 Final Exam 40%
Summary of Assessment
Assessment Theory
1st Continuing assessment 10% week8
Mid Semester assessment 20% week 12
2nd Continuing assessment 10% week 13
Open book Exam and oral exam
20%
Final assessment 40% week17 or18
Prepared by
Dr. Afaf Mohammed
Course Coordinator and instructor
Approval
Professor Dr. Nafsiah Binti Shamsudin, RN
Dean of college of Pharmacy and Nursing
Advanced Instrumental Analysis
Course Description:
The course deals with the principles and applications of modern
analytical instruments. Emphasis is placed upon the theoretical basis
of each type of instrument, its optimal area of application, its
sensitivity, its precision, and its limitations.
CONTENTS
AN INTRODUCTION TO CHROMATOGRAPHY
A General Description of Chromatography
The Rate Theory of Chromatography
Separations on Columns
Qualitative and Quantitative Analysis by Chromatography
LIQUID CHROMATOGRAPHY
Column Chromatography
Thin Layer Chromatography
Electrophoresis and Ion exchange chromatography
GAS-LIQUID CHROMATOGRAPHY
Principles of Gas-Liquid Chromatography
Apparatus
Applications or Gas-Liquid Chromatography
Gas-Solid Chromatography
Examples of Applications of Gas Chromatography
HPLC High Performance Liquid Chromatography
Principles of HPLC
Advantageous of HPLC over gas-chromatography
Apparatus
Limitations of HPLC
ELECTROMAGNETIC RADIATION AND ITS INTERACTIONS
WITH MATTER
Properties of Electromagnetic Radiation
The Interaction of Radiation with Matter
Emission of Radiation
AN INTRODUCTION TO ABSORPTION SPECTROSCOPY
Terms Employed in Absorption Spectroscopy
Quantitative Aspects of Absorption Measurements
APPLICATIONS OF ULTRAVIOLET AND VISIBLE
ABSORPTION MEASUREMENTS
Absorbing Species
Some Typical Instruments
Application of Absorption Measurement to Qualitative Analysis
Quantitative Analysis by Absorption Measurements
INFRARED ABSORPTION SPECTROSCOPY
Theory of Infrared Absorption
Infrared Instrument Components
Some Typical Instruments
Sample Handling Techniques
Qualitative Applications of Infrared Absorption
Quantitative Applications
Infrared Fourier Transform Spectroscopy
NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
Theory of Nuclear Magnetic Resonance
Experimental Methods of NMR Spectroscopy
Environmental Effects on Proton NMR Spectra
Applications of Proton NMR
Application of Proton NMR to Quantitative Analysis
Fourier Transform NMR13C Nuclear Magnetic Resonance Spectroscopy
Applications of carbon 13 NMR
MASS SPECTROMETRY
The Mass Spectrometer
Mass Spectra
Qualitative Applications of Mass Spectrometry
Quantitative Applications of Mass Spectrometry
Chromatography: -
Chromatography basically involves the separation of mixtures due to
differences in the distribution coefficient (equilibrium distribution) of
sample components between 2 different phases. One of these phases is a
mobile phase and the other is a stationary phase.
In adsorption chromatography (liquid-solid chromatography) a bulk
liquid phase (usually a mixture of compounds in solution) is brought into
contact with a finely divided solid adsorbent and selective adsorption of
the components of the mixture occurs on the surface of the solid.
In partition chromatography (liquid-liquid chromatography) one phase is
a liquid adsorbed on the surface of a solid (stationary liquid phase) and
the other is a mobile liquid phase, separation depends largely upon
multiple partitions between the two liquid phases. Paper chromatography
is an important example of partition chromatography in which filter paper
serves as a support for the immobile phase (water).
In thin layer chromatography, thin layers of adsorbents (alumina, silica
gel, etc., generally mixed with a binding agent such as calcium sulphate
to facilitate adhesion to glass) are supported on glass plates.
The technique, apart from its obvious advantages, is in many ways
similar to paper chromatography but the range of separation is large,
extending from few micrograms to 75—100 mg or even more.
Ion exchange chromatography is a special example of liquid-solid
chromatography in which strong ionic attractions replace the relatively
weak polar adsorption forces. Ion exchange resins are used; Separation in
Ion-exchange Chromatography is based on the competition of different
ionic compounds of the sample for the active sites on the ion-exchange
resin (column-packing), these consist of organic macromolecules
containing ionizable groups.
A supplementary technique is known as paper electrophoresis
(or iono- phoresis): this utilizes the different speeds with which organic
ions move under the influence of an electric field towards the anode or
cathode. Electrophoresis can be carried out on strips of moistened filter
paper between the ends of which a potential difference is applied.
1. Adsorption Chromatography:-
Solutes having different adsorption coefficients towards a certain solid
can be separated by liquid-solid chromatography. This involve the
preparation of a cylindrical column of the solid adsorbent (stationary
phase) - hence name Column Chromatography - and the addition of a
concentrated solution of the mixture (liquid phase) at the top of the
column. As the solution penetrates into the column, the solutes are
adsorbed. When the solution has just completely adsorbed on the column,
fresh solvent is added at the top. The solvent flows down the column and
redissolve the solute in amounts determined approximately by the usual
adsorption law and carries them to lower sections of the column.
As more solvent percolates through the column, the cycle of adsorption
and desorption continues and the solutes gradually move down the
column in concentrated bands this is called development of the
chromatogram (the banded column of adsorbent is termed a
chromatogram). With solutes possessing different adsorption coefficients,
the least tightly held material tends to move ahead more rapidly. If the
coefficients are sufficiently different or the column is long enough, the
faster moving will form a separate band below the slower moving one.
A + B + C OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOO OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOO OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOO OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO
Sample (A+B+C)
Column
Solid Particles(packing material- stationary phase)
Eluant (eluate)
DIAGRAM OF SIMPLE LIQUID COLUMN CHROMATOGRAPHY
A
B
C
Solvent(mobile or moving phase)
Fig 1. Column Chromatography.
At the lower end, the solutes are forced out of the column and can be
collected separately in successive fractions.
If the components of the mixture are colored, their positions on the
column are readily apparent as a series of colored bands. Sometimes the
positions of the bands can be detected by their fluorescence in ultra-violet
light, or by the use of an indicator or marking substance, it is usually
more convenient to continue the addition of the development solvent so
the components pass in turn out of the column with the solvent and are
collected separately. This is called elution. The components may be
identified in the various portions of e1uate by chemical or physical
methods.
An effective adsorbent should have a high but Selective adsorption power
and large surface area; it must be chemically inert, preferably white, and
readily available, it should be finely divided to give high surface area per
unit weight.
The following substances, listed in order of decreasing absorption
strength for polar molecules, are commonly used:
- Activated alumina,
- Charcoal,
- Magnesium silicate,
- Silica gel,
- Magnesia,
- Calcium carbonate,
- Sucrose, and
- Starch.
The solvents in increasing order of eluting ability towards alumina are:
- Saturated hydrocarbons,
- Aromatic hydrocarbons,
- Ethers,
- Halogenated hydrocarbons,
- Ketones, and
- Alcohols.
Mixtures of these solvents are often used, e.g., petroleum ether-benzene,
benzene-ether, and ether-methanol.
2. Paper chromatography:
Paper chromatography is a partition chromatography in which the
stationary phase is the absorbed water always present in filter paper
(ca. 22%), the support the paper itself, and the moving phase a solvent
previously saturated with water. The method is not invariably a partition
process, adsorption phenomena may be involved.
It may be involved that filter paper is made of highly purified cellulose
(C6 H10O5)n ,which is a polyhydroxy compound of high molecular weight.
A drop of a solution of the mixture, or drops of the individual
components of the mixture, is app1ied to the paper by means of a
capillary tube, and the paper dried. The paper is then placed in a suitable
container so that it can be irrigated with an organic liquid or mixture of
liquids (downwards by gravity - descending technique, or upwards by
capillarity- ascending technique) without losses by evaporation. Then the
solvent travels the required distance, the paper is removed from the
container, the position of the solvent front noted, and the paper dried. If
the spot are not colored, the location of the spots is determined by
spraying the paper with a chemical reagent which produces insoluble
colored derivatives with the solutes (e.g., ninhydrin for amino acids)
Sometimes the solutes exhibit fluorescence in ultra-violet light and their
presence can be detected in this way. The movement of any substance
relative to the solvent front in a given chromatographic system is constant
and characteristic of the substance. The constant is called Rf value and is
defined as:
Fig.2 Paper chromatogram
In Fig. 1 the upper line contains the point of application of the drop and
the lower line the position of the solvent front,
3. Thin Layer Chromatography:
Paper chromatography is limited to separations on cellulose since other
media, such as silica gel and alumina, cannot be conveniently and simply
made into suitable sheets. This limitation can be removed by making thin
layers of these substances supported on glass plates.
It is usual to employ solid layers which adhere to the glass plate,
generally by virtue of a binding agent, such as calcium sulphate, which is
incorporated with them. A particular advantage is that corrosive reagents,
which attack paper, can be used for spraying. The prepared thin layer on
glass is often called a chromaplate The sample ‘spots’ tend to remain
more compact than in paper chromatography so that smaller amounts of
substances can be separated and identified, On the other hand, much
larger quantities of mixtures can be separated by suitably increasing the
size of plates and the thickness of the layers. The time of separation is
also reduced. For routine separations, a thickness of not less than 0.25
mm. is common and for laboratory demonstration of the technique a glass
plate 20x5 cm. is a convenient size.
Chromatoplates are prepared by applying a uniform layer of an
appropriate material in the form of a thin aqueous paste to clean glass
plates. The usual sorbents include silica gel, alumina, kieselguhr and
cellulose powder. Silica gel, largely used because it can serve as a
medium for the separation of polar compounds (by partition) and non-
polar compounds (by adsorption). Since the layers are relatively delicate,
the ‘spot’ origin is generally marked by a special metallic tool (‘scriber’)
supplied with a frame in which ‘holes’ are placed at regular and equal
distances. The mixture may be applied from a fine capillary tube.
Development is carried out by the ascending technique in small glass jars.
The choice of solvent will depend upon the nature of the substance to be
separated and the sorbent. It is desirable to match the polarity of the
solvent to that of the substance being separated. Visualization of the spots
is usually made with appropriate spraying reagents (table 1). Sometimes
an inorganic fluorescent indicator is incorporated in the adsorbent. The
whole plate is then fluorescent under a UV lamp and dark spots appear
where a migrating compound has quenched the fluorescence.
Table 1:
Reagents Colour of Spots Examples
- Iodine vapours. - Yellow, brown - Digitoxin, Digoxin, Lanatoside.
- Mercury (II) chloride and potassium permanganate.
- White spots on grey ground, yellow spots on violet ground.
- Barbitone.
- Nitric acid. - Red. - Ajmaline.
- Vanillin/H2SO4 - Red. - Volatile Oils.
- Ninhydrin/n-butanol - Yellow to violet, purple
- Amino acids, aliphatic amines.
- p-Dimethylamino benz aldehyde.
- Yellow to violet. - Aromatic amines.
- Dragendorff-Reagent:
(Pot. Tetraiodo bismo-tate (III).
- Yellow - red - N- Heterocycls, Alkaloids: Atropine, Opium
e.g., Silica Gel GF254 contains 13 % w/w of calcium sulphate and 1.5
w/w of a fluorescent indicator having a maximum intensity at
254 nm (B.P-88).
precoated thin layer chromatographic
Fig.3
They are of flexible solvent-resistant polyester and are of sufficient
thickness to be self-supporting and also flexible coated with silica gel,
the adsorbent layer contains a small amount of a polyvinyl alcohol binder
which results in a coating that is highly porous and allows the solvent to
penetrate quickly. Sheets of the precoated material can be activated, if
necessary, cut to the desired size with a cutting board or a pair of scissors
(Fig:2), and used just as normal thin layer glass plate. After the separation
has been made, the sheet can be stored in a note book, etc. The
performance of the thin layer chromatographic sheets is similar to that of
thin layer glass plates (utilizing silica gel as the adsorbent material) on
which the migration rates of solvents are slightly slower.
4. Ion Exchange Chromatography:-
Ion exchange chromatography may be regarded as a special example of
liquid-solid chromatography wherein strong ionic attractions replace
relatively weak polar adsorption forces. Ion exchange materials are
usually described as cation exchange resins or as anion exchange resins.
These resins are supplied in the form of small beads and are usually made
by the co-polymerization of such compounds as styrene and
divinylbenzen: suitable groups, upon which the exchange principle
depends, can be introduced either before or after condensation.
Styrene (Vinylbenzen)
Cation exchange resins are those in which the resin matrix contains
sulphonic acid groupings (-(SO3) H+), the hydrogen ions being capable of
exchange. These are known as strongly acidic cation exchangers. Resins
containing carboxylic acid groups (-(COO)-H+) are known as weakly
acidic cation exchangers.
Anion exchange resins are resins with similar structures except that the
resin matrix contains ions which are capable of exchanging with anions in
the surrounding medium. These may contain quaternary ammonium
groups (-(NMe3) +X- (where X = Cl or Br), and are strongly basic anion
exchangers. There are also weakly basic anion exchangers which contain
amino groups (such as -HMe2) instead of the quaternary ammonium
groups.
Some applications include:-
(a) Conversion of a sodium salt of a carboxylic acid into the free acid:
(R-SO3-)H+ + (R-COO-Na +) (R-SO3
-) Na+ + (R-COO-H+) (Solution) (Solution)
(b) Conversion of a salt of a weak base into the free base:
(R-NMe3+OH- + (R’NH3)+ Cl- (R-NMe3
+)Cl- + (R’NH3) + OH- (R’NH2 + H2O). (Solution) (Solution)
(c) Removal of acids from mixtures of acids and neutral compounds.
(a) Removal of bases from mixtures of bases and neutral compounds.
The resins may be regenerated with appropriate reagents and used again.
5. Paper Electrophoresis:-
Electrophoresis is a technique which is closely associated with
chromatography and is often used in conjunction with it. Separa-tions
depend upon differences in electrical properties of the components of a
various organic ions move under the influence of an electric field,
towards the anode or cathode; form the basis of separations of mixtures.
The separation is carried out in a supporting medium; only filter paper
will be considered here. The pit of the electrolyte is important and is
usually controlled by a buffer solution.
6. Gas Chromatography: -
Gas chromatography (GC) is similar to other forms of chromatography
except that the mobile phase and the sample are in the vapor state. It can
be applied on any mixture of compounds, all the components of which
should be volatile at the temperature used (up to 350°C).
In Gas chromatography, the stationary phase is packed in a small
diameter tube (1 /16-1/4 in.) of moderate length (4-15 ft). The tube is
usually of stainless steel and bent into a helix or “U’ shaped to fit the
instrument. The moving phase, an inert gas such as helium, argon,
nitrogen, or sometimes hydrogen, is allowed to flow through the column,
which is enclosed in a thermostatically controlled oven. The temperature
at which a separation is carried out is usually a few degrees below the
boiling point of the major component of the mixture. The sample is
injected with a micro-syringe through a rubber septum into the column,
then components of the sample flow through the column at varying rates
and is detected as they emerge by a detector which transmits a signal to a
recorder (Fig.4).
Fig.4: Scheme of a gas chromatograph
Figure 4 is a schematic diagram that illustrates what occurs when a
solution of solutes A and B is injected into the column.
Fig.5 A schematic diagram of a gas chromatogram
Gas chromatography is frequently divided into gas-solid chromatography
(GSC) and gas-liquid chromatography (GLS). The distinction is made on
the basis of the stationary phase.
Stationary phase in GSC: The main application of GSC is in the analysis
of gases such as CO, CO2, O2, N2 and light organic gases. In this case, the
stationary phase required is a solid, finely divided, and with adsorptive
power. Only three solid adsorbents are in Common use. These are:
a) Molecular sieves (4A, 5A, …..), synthetic inorganic materials similar
to the natural zeolites (hydrated aluminosilicates) but with very regular
crystal structures and pore sizes.
b) Silica gel.
c) Activated charcoal.
Most gas analysis can be conducted at room temperature.
Stationary phase in GLC: In GLC, the stationary phase is a liquid or
low-melting solid, which is coated on an inert, finely particulate support.
The most common supports are: Chromosorb W, Chromosorb P,
Chromosorb T, and glass beads.
The liquids or low- melting solids being used as the stationary phase must
be non-volatile under the conditions of the experiment. Stationary liquids
are usually classified on the basis of their polarity. The most common of
these liquids are presented in table 2.
Table 2:Some stationary liquids in GLC columns:
Liquid (or low-melting solid) Max. Operating temperature (°C)
a) Non-polar liquids
- Silicone gum rubber (SE-30)
- Silicone gum rubber (OV-1)
- Apiezon L hydrocarbon grease
(Paraffin oil, squalane 300)
- 250- Higher than 250
- 250
b) Polar liquids:
- Polyethylene glycol 400
(PEG 400, Macrogol 400)
- Carbowax 1500 & 1540
(PEG 1500 & 1540
- Carbowax 20M
(PEG 20,000)
- Polyesters
- 100
- 150
- 250
- 225
(e.g. Neopentyl glycol succinate)
Operating temperatures:
The choice of a temperature at which a GC separation is carried out is the
most important factor to be considered. If the operating temperature is
higher than the required, column will separate less effectively, retention
times are reduced and the peaks are closer together and poorly resolved,
At too high temperatures, the stationary liquid itself can be eluted, a
phenomenon known as “bleeding”. On the other hand, if the temperature
is low than the required, fewer poorly shaped peaks result with distinct
tailings.
A good initial choice is a temperature few degrees lower than the boiling
point of the major component or components of a mixture.
Detectors:
The detector indicates the presence and measures the amounts of the
components of a mixture in the effluent of a GC column. Thermal
conductivity detectors, flame ionization detectors, and electron capture
detectors are the most used ones.
Gas Chromatography of some non-volatile compounds:
Some non-volatile compounds can be converted into volatile derivatives
and, therefore, can be gas chromatographed. Fatty acids such as stearic,
palmitic, arachidic, etc. are not themselves volatile, but their methyl
esters are so. Consequently, for gas chromatography of these acids, they
should be converted to their corresponding methyl esters by treatment
with anhydrous methanol in presence of a suitable catalyst such as boron
trifluoride (BF3) or boron trifluoride ethyl ether. (F3B-O (C2H5)2).
Similarly, hydroxy compounds such as sugars, flavonoids, glycosides,
etc. can be gas chromatographed as their trimethylsiloxy derivatives.
Gas chromatographic data:
Retention times:
The retention time (tR) is the time in minutes or seconds elapsing between
the time of the injection of the sample and the time of the maximum
detection of a given substance emerging under the same conditions
(temperature and gas flow) from a chromatographic column. Similar to
the Rf value, the tR value can serve as a first approximation to the
identification of substances.
Relative retention:
Drugs may be identified by means of their relative retention, determined
by the equation:
in which t2 is the retention time of the desired drug, t1 is the same for a
reference standard determined with the same column and temperature,
and ta is the retention time for an inert component (Fig. 6).
Resolution:
Resolution (R) is a rneasure of efficiency of the separation of two
components in a mixture. It is determined by the equation:
where w is the width of the peak base obtained by extrapolating the
relatively straight sides of the peak to the base line (Fig.5).
Fig.5 Determination of “Relative retention” as well as “Resolution”
Area Measurement:
For quantitative analysis, the area of the peak provides accurate
information about the amount of the corresponding component.
7. High Performance Liquid Chromatography :
Fig. 6 High Performance Liquid Chromatography Instrument
High performance or high pressure liquid chromatography (HPLC), a so
much sophisticated form of column chromatography, was developed
recently as a result of further advances in column technology. Fine
column packing particles (3 to 50 µm) are used and therefore require high
pressure pumping systems capable of delivering the moving liquid phase
at pressures up to 5000 pounds per square inch (psi) to achieve
appropriate flow rates. It is often necessary to use samples less than 20µg
with such packing, sensitive detectors and data handling systems are used
to analyze the co1mn effluent.
There are many compounds cannot be handled effectively by GC, either
because they are insufficiently volatile and cannot pass through the
column, or because they are thermally unstable and decompose under the
conditions of separation. It has been reported that only 20% of known
organic compounds can be handled satisfactorily by GC without prior
chemical modification of the sample. Pressurized LC on the other hand is
not limited by sample volatility or thermal stability. Thus HPLC is ideal
for the separation of macromolecules and ionic species of biomedical
interest, labile natural products, and a wide variety of other high
molecular-weight compounds.
Liquid chromatography also enjoys certain other advantages with respect
to GC. Very difficult separations are often more readily achieved by
liquid than by GC. The reasons for this include:
a) Chromatographic separation is the result of specific interactions
between sample molecules, the stationary and moving phases. These
interactions are essentially absent in the moving gas phase of GC, but are
present in the liquid phase of LC, thus providing an additional variable
for controlling and improving separation.
b) Furthermore, a greater variety of stationary phases has been found
useful in LC, which again allows a wider variation of these selective
interactions, and greater possibilities for separation.
c) Finally, chromatographic separation is generally enhanced as the
temperature is lowered, because, intermolecular interactions then become
more effective. This favors procedures such as LC which are usually
performed at room temperature.
Liquid chromatography also offers a number of unique detectors which
have so far found little or no application in GC.
- Colorimeters combined with color-forming reactions of separated
sample components.
- UV absorption and fluorescence detectors.
- Radiometric detectors.
- Conductivity detectors.
- Polar graphic detectors.
- Refractive index detectors.
A final advantage of HPLC versus GC is in the relative ease of sample
recovery. Separated fraction: are easily collected in LC, simply by
placing an open vessel at the end of column. Recovery is quantitative and
separated sample components are readily isolated. The recovery of
separated sample components in GC is also possible but is generally less
convenient and quantitative.
A modern liquid chromatograph (Fig.7) consists basically of a solvent
reservoir equipped with degassing system, high-pressure pump and
pressure gauge, analytical column, sample introduction system,
thermostated oven (optional), detector and recorder.
The stationary phase is packed in stainless steel or glass tubing The
length varies from 25 to 125 cm, either straight or U-shaped while the
internal diameter varies from 1-4 mm. Generally, HPLC is conducted at
room temperature, but if precise control of temperature is required then
the analytical co1umn should be enclosed in a thermostated oven.
Column packing in HPLC:
Packing for HPLC column with very fine particle usually ranges from 3
to 50 µm, these can be classified according to the following criteria:
a) Rigid solids or hard gels or soft gels.
b) Spherical or irregular.
c) Porous or super-facially porous (pellicle beads).
The type of material to be packed into a column is determined to a large
extent by the HPLC method which will be used.
Fig.7 Scheme of High Performance Liquid Chromatograph
Q1 Explain
a- In TLC the adsorbent is mixed with calcium sulphateb- The pressure gauge in HPLC is very importantc- Temperature higher than required is not good in GC analysis.d- UV spectrum and IR spectrum appears as bands not a single peak.e- Monochromators are essential part of the spectrometer.f- IR spectrum is more useful than UV spectrum.
Q2 How would you detect a nonfluorescent substance on TLC plate.
Q3 The most usable detectors in GC are------------?
Q4 How do you choose the temperature in GC?
Q5 What are the limitations for GC?
Q6 What are the advantageous of HPLC over GC?
Q7 TLC is preferred over paper chromatography, Explain.
Q8 What are the different of interaction between the stationary phase and sample in chromatography?