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?