INSTRUMENTAL METHODS

OF ANALYSIS
Presentation by:
R.Pushpalatha
Dept. of Chemical Engineering
SSN College of Engineering
 Classification
1. Physical configuration of stationary
phase & contacting type of mobile
phase: Planar & Column
2. Nature of the mobile phase: Liquid, Gas
& Supercritical fluid
3. Mechanism of separation
 Types of Chromatography

LIQUID
MOBILE PHASE

Liquid-Liquid Liquid-Solid
FORMAT Chromatography (Partition) Chromatography (Adsorption)

Liquid Solid

Normal Phase Reverse Phase Normal Phase Reverse Phase

Mobile Phase - Nonpolar Mobile Phase - Polar

Stationary phase - Polar

Stationary phase - Nonpolar
 Mobile Phase
• gas (GC)
• water (LC)
• organic solvent (LC)
• supercritical fluid (SCFC)
 Classification based on Mobile
Phase

Gas Chromatography
Pyrolysis GC -
heat solid materials
to 500 - 10000C
Gas - solid Gas - liquid so they decompose
into gaseous products
Stationary Phase

Sample MUST be volatile at temperatures BELOW 3500C

Classification based on Mobile
Phase

Liquid chromatography (LC)

Column High performance Thin layer

(gravity flow) (pressure flow) (adsorption)
Classification Based on Mechanism

1. Adsorption
2. Partition
3. Ion exchange
4. Size exclusion
5. Affinity
HPLC is amenable to introduce all the mechanisms
by suitable selection of the operating mode
GC : only partition or adsorption
for GC & LC for GC
resin-SO3- resin- gel filtration
N(CH3)3+ by size
 Affinity Chromatography

pH, and ionic strength

(A) uses charge, (B) uses
pores, and (C) uses covalent
bonds to create the
differential affinities among
the mixture components for
the stationary phase.
 Classification based on
Attractive Forces
• Adsorption - for polar non-ionic compounds

• Ion Exchange - for ionic compounds

– Anion - analyte is anion; bonded phase has positive
charge
– Cation – analyte is cation; bonded phase has
negative charge

• Partition - based on the relative solubility of

analyte in mobile and stationary phases
– Normal – analyte is nonpolar organic; stationary
phase MORE polar than the mobile phase
– Reverse – analyte is polar organic; stationary phase
LESS polar than the mobile phase

• Size Exclusion - stationary phase is a porous

matrix; sieving
Planar Chromatography
 Planar Chromatography
• A separation technique in which the stationary
phase is present as or on a plane.
• The plane can be a paper, serving as such or
impregnated by a substrate as the stationary bed
(paper chromatography, PC) or a layer of solid
particles spread on a support e.g. a glass plate
(thin layer chromatography, TLC).
• Sometimes planar chromatography is also termed
open-bed chromatography.
• Both paper and thin-layer chromatography
provide remarkably simple and inexpensive
means for separating and identifying the
components of small samples of complex
inorganic, organic, and biochemical substances.
Examples of Chromatography

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

Paper Chromatography
Can be used to separate the
components of inks, dyes, plant
compounds (chlorophyll), make-up,
and many other substances
 Paper Chromatography
• Paper chromatography is an analytical chemistry technique
for separating and identifying mixtures that are or can be
colored, especially pigments.

• This method has been largely replaced by thin layer

chromatography, however it is still a powerful teaching tool.

• Operation:
– Filter paper – stationary phase, mobile phase – solvent,
solute
– Solvent moves up by capillary action
– Different compounds in the sample mixture travel at
different rates due to competition between the paper fibers
and solvent for the solutes. Since paper is composed of
cellulose, a polar substance, polar substances have a
high affinity for the paper.
Paper Chromatography

Tape – Label with marker

Pencil

Filter
Paper

Ink
Mark
Thin Layer Chromatography (TLC)
• Thin-layer chromatography (TLC) is one of the
most popular and widely used separation
techniques because of its ease of use, cost
effectiveness, high sensitivity, speed of
separation, as well as its capacity to analyze
multiple samples simultaneously

• TLC is related to paper chromatography (PC) as

both use a stationary phase and a liquid phase to
move the sample
 Sorbents for TLC
• Silica gel, cellulose, alumina,
polyamides, ion exchangers, chemically
modified silica gel, and mixed layers of
two or more materials, coated on a
suitable support.

• Silica gel is by far the most commonly

used sorbent supported on a glass plate
 Operation of TLC
• In TLC, the sample is applied as a small spot or streak to the
marked origin of stationary phase supported on a glass, plastic, or
metal plate.

• It is then placed in a closed chamber containing a shallow pool of

mobile phase at the bottom.

• The mobile phase moves through the stationary phase by capillary

forces. The components of the sample mixture migrate at
different rates during movement of the mobile phase through the
stationary phase.

• The migration of each component in a mixture during TLC is a

result of two opposing forces: capillary action of the mobile phase
and retardation action of the stationary phase. Both forces
contribute to achieve differential migration of each component.
 Operation of TLC (contd.)
• When the mobile phase has moved an
appropriate distance, the plate is removed from
the chamber and the solvent front is marked.

• Developed TLC plates can be detected by various

means, based on the nature of the sample. They
could be nondestructive (UV densitometer),
destructive (derivatizing agents), or the
combination of both.

• The results can be documented by photography

and saved electronically for archiving and future
reference.
 Important Features of TLC over
HPLC
• While HPLC is widely used for separation and quantification,
TLC remains a valuable and commonly used separation
technique because of its features that are complementary
to HPLC. Some of the most important features of TLC
compared to HPLC are:

– Open format of stationary phase

– Simple sample preparation: Samples for TLC separation
often involve fewer cleanup steps because every sample
is separated on fresh stationary phase, without cross-
contamination or carryover
– High sample throughput: The simultaneous but
independent application and separation of multiple samples
in TLC results in higher sample throughput and less time
consumption, as well as lower costs
– Flexible and versatile dissolving solvent and mobile
phase
 Qualitative Analysis of Amino
Acids by TLC
• Eluent: [Mix n- butanol, acetic acid (purity 98 – 100 %) and
distilled water in volume ratio 5:1:5. Stir for 10 minutes, then let
the layers separate. Use upper layer as eluent.]

• Solution on ninhydrin. [Dissolve 0.3 g of ninhydrin in 100 ml n-

butanol. Add 3 ml of glacial acetic acid.] The separated amino
acids are visualized using solution of ninhydrin. Purple color
develops upon reaction of amino acid with ninhydrin.

• 0.02 M solutions of amino acids (leucine, methionine, alanine and

serine). [Dissolve: 0.026 g leucine, 0.030 g methionine, 0.018 g
alanine and 0.021 g serine in distilled water and bring the volume
to 10 ml.]

• Chromatographic paper with dimensions 85 x 50 mm

• Elution chamber with approximate internal dimensions: 7 cm high

and with 5.5 cm diameter.
• The spots of individual amino acids and sample solutions are applied to
the chromatographic paper. Use separate clean and dry glass capillary for
each solution. The spot on the paper should not be bigger than 2-3 mm.

• After application of samples let the spots dry. Meanwhile measure with a
graduated test-tube 5 ml of eluent into the elution chamber. Cover the
chamber with lids and let the chamber atmosphere saturate with eluent
vapours for 10 min.

• To start the analysis, insert the chromatographic paper. Check if the paper
reaches the eluent surface. Elution is stopped when the solvent front has
traveled up the plate until 7-10 mm from the lid.

• Remove the paper from elution chamber and place it on a sheet of filter
paper. After 2-3 minutes mark the eluent front with pencil and dry the
paper in oven. When the paper is dry, take it into the fume hood and
spray it with solution of ninhydrin until the paper is slightly wet. The
chromatographic paper is again put in the drying oven for 15 min to speed
up the reactions.

• Remove chromatographic paper from drying in the oven, draw the

contours and centers of the chromatographic bands. Compare retention of
standard substances and components in sample and determine which
amino acids were present in the sample.
Paper Chromatography

Tape – Label with marker

Pencil

Filter
Paper

Ink
Mark
 Detectors
• UV-vis
• Refractive Index (RI)
• Mass spectrometry (MS)
• Electrochemical (EC)
– amperometric
• NMR - novel