INSTRUMENTAL METHODS OF ANALYSIS

Presentation by:

R.Pushpalatha

Dept. of Chemical Engineering

SSN College of Engineering

What is Chromatography?

Elution : always (100%) dilution

What is Chromatography?

samplein

eluentin

CaCO3

(adsorption)

column

eluantout

detector

chromatogram(mass spect. IR

spect. etc)

Chromatography

The most powerful tool for separating & measuring the components of a complex mixture.

• The separation of a mixture by distribution of its components between a mobile and stationary phase over time

– mobile phase = solvent– stationary phase = column packing material

• A solute equilibrates between a mobile and a stationary phase.

� The more it interacts with the stationary phase, the slower it is moved along a column.

Uses for Chromatography

Chromatography is used by scientists to:

• Analyze – examine a mixture, its components, and their relations to one another

• Identify – determine the identity of a mixture or components based on known components

• Purify – separate components in order to isolate one of interest for further study

• Quantify – determine the amount of the mixture and/or the components present in the sample

Uses for Chromatography

Real-life examples of uses for chromatography:

• Pharmaceutical Company – determine amount of each chemical found in new product

• Hospital – detect blood or alcohol levels in a patient’s blood stream

• Law Enforcement – to compare a sample found at a crime scene to samples from suspects

• Environmental Agency – determine the level of pollutants in the water supply

• Manufacturing Plant – to purify a chemical needed to make a product

Types

• Gas Chromatography• Liquid Column Chromatography• High Performance Liquid Chromatography

(HPLC)• Thin Layer Chromatography (TLC)• Paper Chromatography• Electrophoresis• Affinity Chromatography• Immuno chromatography

Gas ChromatographyUsed to determine the chemical composition of unknown substances, such as the different compounds in gasoline shown by each separate peak in the graph below.

Paper ChromatographyCan be used to separate the components of inks, dyes, plant compounds (chlorophyll), make-up, and many other substances

Liquid ChromatographyUsed to identify unknown plant pigments & other compounds.

Thin-Layer ChromatographyUses thin plastic or glass trays to identify the composition of pigments, chemicals, and other unknown substances.

Examples of Chromatography

• Liquid Chromatography – separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase)

• Gas Chromatography – separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase)

• Paper Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase)

• Thin-Layer Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel

(stationary phase)

Types of Chromatography

Chromatography

Chromatogram - Detector signal vs. retention time or volume

time or volume

Det

ecto

r Si

gnal

1 2

Milestones in Chromatography

• 1903 Tswett - plant pigments separated on chalk columns

• 1931 Lederer & Kuhn - LC of carotenoids

• 1938 TLC and ion exchange

• 1950 reverse phase LC

• 1954 Martin & Synge (Nobel Prize)

• 1959 Gel permeation

• 1965 instrumental LC (Waters)

Separation Mechanism

Instrumentation

What is Chromatography?

Factors Governing Retention Time

• Retention time of a solute : time taken by the solute to reach the detector from the moment of its injection into the column.

• Depends upon

� Nature of the stationary phase

� Composition of the mobile phase

� Column length (dia too)

� Mobile phase flow rate

Partition Ratio, K

• Partition ratio/ coefficient or Distribution ratio / coeficient : K

• How the solute distributes between the stationary phase & mobile phase

• tR = tm ( 1+K (Vs/Vm))

• K = Cs / Cm

• If K= 1, the solute is equally distributed

• If KA = KB, no separation occurs

• If KB > KA, then A elutes first

Column Capacity Factor, k’

• Column Capacity Factor /Column Retention Factor

• Also called mass distribution ratio

• A normalised quantity used to describe the migration rates of the solute through the column

• Ratio of the total amount of solute present in the stationary phase to that in the mobile phase

• K’ =(CsVs) / (CmVm) = K /(Vm/Vs) = K / β

• β is Phase ratio

• tR = tm ( 1+k’) or k’ = (tR – tm) / tm

• Value of k’ should be between 1 & 5

Retention Volume

• Retention volume (VR) : Volume of the mobile phase (in ml) required to elute a solute from the point of its introduction at the head of the column to its exit from the column.

• VR = tR x F = V0 ( 1 + K(Vs/Vm)) = V0 + KVs

• V0 = Vm : void volume or dead volume:tm x F

Separation of Peaks

� For pairs of bands

� Efficiency : two factors contribute to how well components are separated :

� the widths of the peaks :

� the wider the peak, the poorer

separation.

� b) Selectivity:

� the spacing in time :

� the further apart, the better separation.

Resolution of Column

• Tells us how far apart 2 bands are relative to their widths.

• Provides a quantitative measure of the ability of the column to separate 2 analytes.

Resolution

Factors Governing Resolution

• Low flow rates favour increased resolution

• Long column and smaller dia particles packing increase resolution

• Temperature

• Mobile phase composition

Solution

• Change column conditions during elution

-change in liquid mobile phase composition - gradient elution

or solvent programming

-change in temperature for gas chromatography- temperature programming

Effect of Mobile Phase Composition

Effect of Temperature on Resolution

Elution Method

Gradient Elution

Advantage of Gradient Elution