19/02/2024

ANALYTICAL CHEMISTRY AND

INSTRUMENTATION
3(2-1)

Course Contents:
Principals, instrumentation, applications
Chromatography including paper, thin layer, gel filtration, ion-
exchange, affinity, HPLC, gas chromatography, GC-MS and LC–MS;

Spectroscopy types including NMR, visible, ultraviolet, luminescence,

flame, atomic absorption, fluorescence

Flow cytometry
X-ray diffraction
Dialysis
Ultra-filtration
Lyophilization
Ultracentrifugation
Amino acid analyzer

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Chromatography

History
The Russian botanist Mikhail
Tswett coined the term
chromatography in 1906 to
describe his experiments in
separating different colored
constituents of leaves by passing
an extract of the leaves through a
column

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HISTORY
● Chromatography
(from Greek :chromatos -- color ,
"graphein" -- to write)
● 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- PC
● 1959 Gel permeation
● 1965 instrumental LC (Waters)

Chromatography

Is technique/s used to separate and identify the

components of a mixture.

Works by allowing the molecules present in the

mixture to distribute themselves between a
stationary and a mobile medium.

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Purpose of Chromatography
● Analytical - determine
chemical composition of a
sample

● Preparative - purify and

collect one or more
components of a sample

Uses of 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

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Examples of Chromatography
Liquid
Chromatography
Used to identify unknown plant
pigments & other compounds.

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

Gas Chromatography Paper

Used to determine the chemical composition of
unknown substances, such as the different Chromatography
compounds in gasoline shown by each separate
Can be used to separate the
peak in the graph below.
components of inks, dyes, plant
compounds (chlorophyll), make-up,
and many other substances

Classification of Methods
There are two classification schemes:
– mobile phase
– attractive forces

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Mobile Phase
● Gas chromatography (GC)
● Liquid Chromatography (LC)
Water (LC)
Organic solvent (LC)
Supercritical fluid (SCFC)

A supercritical fluid (SCF) is any substance at

a temperature and pressure above its critical
point, where distinct liquid and gas phases do
not exist. It can effuse through solids like a gas,
and dissolve materials like a liquid.

Classification based on Mobile Phase

Gas Chromatography

Gas - solid Gas - liquid

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Classification based on Mobile Phase

Liquid chromatography (LC)

Column Thin layer

High performance
(gravity flow) (adsorption)
(pressure flow)

Classification based on Attractive

Forces
● Adsorption
● Ion Exchange
● Partition
● Size Exclusion

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Adsorption Chromatography

➢ Separation based on their

adsorption onto the surface of
solid (stationary phase).

➢ Normal phase-like separation

– Nonpolar mobile phase

➢ for polar non-ionic compounds

➢ Ex; Column chromatography (CC)

TLC, HPLC

Ion Exchange Chromatography

➢ Uses ionic stationary phase

– ions separated on the basis of their tendency to
displace counter ions adsorbed on stationary phase
(Depends on charge, hydration, “solubility”…)

➢ Anionic stationary phases: used for cation separation

➢ Cationic stationary phases : for anion separation
➢ for ionic compounds
➢ - Ex : CC, HPLC

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Partition Chromatography
➢ solutes are separated based on their partition
between a liquid mobile phase and a liquid
stationary phase coated on a solid support.

– Paper Chromatography

Size Exclusion Chromatography

➢ Separation is a result of “trapping”
of molecules in the pores of the
packing material

● Very large molecules can’t get into

the pores – unretained

● Very small molecules get hung up in

to pores for a long time - most
retained – longest retention time

● stationary phase is a porous matrix

● Ex: CC, HPLC

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STATIONARY PHASE

Type of Material
chromatography
Paper chromatography Filter paper, cellulose,
water
Thin Layer Chromatography Silica gel, alumina,
polyamide

Gas chromatography Squalene, apezion,

(GC) carbowax M
High Performance Liquid C-8, C-18, Licosorb,
Chromatography Silicone

MOBILE PHASE
Type of chromatography Solvent

Paper chromatography Combination of different

solvents
Thin Layer Hexane, ether petroleum,
Chromatography alcohol.

Gas chromatography He, Ar, N2

(GC)
High Performance Liquid Cyclohexane, n-hexane, carbon
Chromatography tetrachloride, ethanol,
methanol, air

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PAPER
CHROMATOGRAPHY

DEFINITION

technique for separating dissolved

chemical substances by taking
advantage of their different rates
of migration across sheets of paper

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PRINCIPLE

• Certain solvents are used to separate a mixture

i.e. water, alcohol, acetone etc

• Due to capillary action the solvent will move up

to filter paper.

• Movement of a solvent will carry components of

mixture along.

• Each component will move up at characteristic

velocity

● The retention factor, or Rf, is defined as the distance

traveled by the compound divided by the distance
traveled by the solvent

For example, if a compound travels 2.1 cm and the

solvent front travels 2.8 cm, the Rf is 0.75:

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Materials List
• Beakers or jars
• Covers or lids
• Solvent (Distilled H2O,
Isopropanol)
• Graduated cylinder
• Filter paper
• Sample (Different colors
of pins, plant extract)
• Pencil
• Ruler
• Scissors
• Tape

Preparing the solvent solution

• Prepare the solvent solution in various concentration:

5%, 10%, 20%, 50%, and 100%

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Preparing the Chromatography Strips

1. Cut filter paper

2. Draw a line 1 cm above

the bottom edge of the
strip with the pencil

3. Label each strip with its

corresponding solution

4. Place a spot from each

pen on your starting line

Developing the Chromatograms

● Place the strips in the beakers
● Make sure the solution does not
come above your start line
● Keep the beakers covered
● Let strips develop until the
ascending solution front is about
2 cm from the top of the strip
● Remove the strips and let them
dry

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Spot Detection

- Color spot ➔ observed by naked eye

- Non – color spot ➔ color reagent will give
specific colors for different compound.
Example :
➢ Ninhydrin for Amino Acids
➢ Iodine for most Organic Compounds
➢ UV light for fluorescent compounds

THIN LAYER
CHROMATOGRAPHY

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Thin Layer Chromatography

a chromatographic technique used to

separate the components of a mixture
using a thin stationary phase supported by
an inert backing
● This method is simple, rapid and cheap
● Widely used in pharmaceutical & food
stuff industry.

TLC Plate

-A plate of TLC can be made from aluminium or glass

which is coated by a solid matter as a stationary phase.

- The coated material is 0.1-0.3mm in thickness

-some of them has been added with fluorescent

indicator that will make it florescence during the UV
light exposure.

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STATIONARY PHASE

● Silica is commonly used as stationary

phase

● The separation of sample mixture will be

dependent on the polarity of sample.

● Some modified silica is also used in

certain purposes.

STATIONARY PHASE
Stationery phase Description Application

Silica gel G Silica gel with average Used in wide range

particle size 15µm pharmacopoeial test
containing ca 13% calcium
sulfate binding agent

Silica gel G254 Silica gel G with Same application with

fluorescence added Silica gel G where
visualization is to be
carried out under UV
light.

Cellulose Cellulose powder of less Identification of

than 30µm particle size. tetracyclines

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MOBILE PHASE
● The ability of mobile phase to
move up is dependent on the
polarity itself
● Volatile organic solvents is
preferably used as mobile phase.

MOBILE PHASE

SOLVENT POLARITY INDEX

Hexane 0
Butanol 3.9
Chloroform 4.1
Methanol 5.1
Ethanol 5.1
Acetonitrile 5.8

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MATERIALS
• TLC plate
• ‘Developing container’
- chamber/ jar/ glass beaker
• Pencil
• Ruler
• Capillary pipe
• Solvents / mobile phase
- organic solvents
• UV lamp

METHOD

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1.Developing Container Preparation

Solvent is transferred
into the container with
0.5-1cm in dept from the
bottom

2. TLC Plate Preparation

➢ Commercially obtained with 5cm

x 20cm in size
➢ Prepare your size when
necessary
➢ Line 1 cm from the bottom with
a pencil as a part should be
spotted.

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3.Spotting’ TLC plates

➢ Make sure that your sample is

liquified already.
➢ stick it using capillary pipe &
spot onto the line you have made
➢

4.‘Develop the plate’

➢ after spotting, put the plate inside
the chamber in the standing position
➢ Make sure that the solvent doesn't
touch the spots
➢ Let it develop up to the 1-2cm from
the top of plate
➢ After that, pull out the plate from
the chamber and let the solvent be
vaporized

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5. Detection of spots
- The color samples are easy to
be seen and no need to use UV
lamp to detect them

6. DETECTION OF SPOT

1) Iodination-put the plate in which the spots face to

the iodine crystal and see what is the spot color
changing
2) Ninhydrin:
-specific identification of amino acid compounds.
- Ninhydrin solution will show a purple spot when it is
sprayed to the amino acid spot.
3) KMnO4
used to identify a reducing agent such as glucose,
fructose, vitamin C and others.
4) Alkaline tetrazolium blue
specifically used for corticosteroid identification

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The use of Rf as separation parameter

- The distance taken through by the solvent to move up

will be assigned as solvent front
- The distance taken through by the sample to move up
will be assigned as sample front
- Rf value is obtained by dividing the sample front
toward solvent front

Rf = sample front
solvent front

Thin-Layer Chromatography: Determination of Rf

Values

solvent front
Rf of component A =
dA component B

dS Less polar!
dS
Rf of component B = dB

dB
component A
dS More polar!
dA
origin
The Rf value is a
decimal fraction,
generally only
reported to two
decimal places

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7. Quantitative determination of known sample

- Done by scratching the spot using spatula, and
extract the compound using the suitable solvent
- The liquid extract can be determined its content
using other method such as spectroscopy.

Problems commonly occur in TLC and how to solve

a. The spot shape is too broad

- Diameter is supposed to be < 1-2mm
b. The movement of solvent
- should be straight up
- unproportionality in stationary phase surface
will inhibit the movement of solvent
c. streaking formation
- caused by too concentrated sample

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TLC Compared to Paper Chromatography

1. Precise and effective

2. More stable toward various organic
solvents

Advantages

● Cheap
● Simple
● The developing can be monitored
visually
● Able to use various chemical as a
detector

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Identification unknown drugs using standard Reference

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Rf = distance moved by substance

distance moved by solvent front

For substances that are very soluble in the liquid

Rf will be close to ....
1

For substances that are rather insoluble in the liquid

Rf will be close to ....
0

Gas Liquid Chromatography

Technique used for separating and analyzing compounds

that can be vaporized without decomposition

Here the mobile phase is an non-reactive gas ( e.g.

Nitrogen) flowing through a tube.

And the stationary phase is a non-volatile liquid

held on particles of a solid support.

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In the animation below the red molecules are more soluble

in the liquid than are the green molecules.

In practice the Column is contained in a thermostatic oven.

About 1μL of liquid is injected into one end of the column.

As each component reaches the other end it is detected

and registered on a chart recorder.

The Retention Time is characteristic of a particular

substance (for the same column, temperature, gas flow etc.)

The area under each peak indicates the relative quantities.

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Injection
port Recorder

Oven

Detector

Column
Nitrogen
cylinder

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Chromatogram of petrol

Gas Chromatography
Introduction

1.) Gas Chromatography

➢ Mobile phase (carrier gas) is a gas
- Usually N2, He, Ar and maybe H2

➢ Requires analyte to be either naturally volatile or can be converted to a volatile

derivative
- GC is useful in the separation of small organic and inorganic compounds

➢ Stationary phase:
- Gas-liquid partition chromatography – nonvolatile liquid bonded to solid support
- Gas-solid chromatography – underivatized solid particles
- Bonded phase gas chromatography – chemical layer chemically bonded to solid
support

Bonded phase Magnified Pores in activated carbon

Zeolite molecular sieve

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Gas Chromatography
Introduction

2.) Instrumentation
➢ Process:
- Volatile liquid or gas injected through septum into heated port
- Sample rapidly evaporates and is pulled through the column with carrier gas
- Column is heated to provide sufficient vapor pressure to elute analytes
- Separated analytes flow through a heated detector for observation

Gas Chromatography
Instrumentation

1.) Open Tubular Columns

➢ Commonly used in GC
➢ Higher resolution, shorter analysis time, and greater sensitivity
➢ Low sample capacity
➢ Increasing Resolution
- Narrow columns → Increase resolution

- Resolution is proportional to N , where N increases directly with column length

Easy to generate long (10s of meters)

lengths of narrow columns to maximize
resolution

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Gas Chromatography
Instrumentation

1.) Open Tubular Columns

➢ Increasing Resolution

Decrease tube diameter

Increase resolution
Increase Column Length
Increase resolution

Gas Chromatography
Instrumentation

1.) Open Tubular Columns

➢ Increasing Resolution

Increase Stationary Phase Thickness

Increase resolution of early eluting compounds

Also, increase in
capacity factor and
reduce peak tailing

But also decreases

stability of stationary
phase

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Gas Chromatography
Instrumentation

2.) Choice of liquid

stationary phase:
➢ Based on “like dissolves like”

➢ Nonpolar columns for

nonpolar solutes

➢ Strongly polar columns for

strongly polar compounds

➢ To reduce “bleeding” of
stationary phase:
- bond (covalently attached)
to silica

- Covalently cross-link to
itself

Gas Chromatography
Instrumentation

3.) Packed Columns

➢ Greater sample capacity
➢ Broader peaks, longer retention times and less resolution
- Improve resolution by using small, uniform particle sizes

Open tubular column

Packed column

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Gas Chromatography
Instrumentation
500 L chromatography
column
3.) Packed Columns
➢ The major advantage and use is for large-scale or
preparative purification

➢ Industrial scale purification maybe in the kilogram or

greater range
Oil refinery – separates
fractions of oil for
petroleum products

Gas Chromatography
Retention Index

1.) Retention Time

➢ Order of elution is mainly determined by volatility
- Least volatile = most retained
- Polar compounds (ex: alcohols) are the least volatile and will be the most
retained on the GC system

➢ Second factor is similarity in polarity between compound and stationary

phase

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Gas Chromatography
Retention Index

2.) Describing Column Performance

➢ Can manipulate or adjust retention time by changing polarity of stationary
phase

➢ Can use these retention time differences to classify or rate column

performance
➢ Can be used to identify unknown compounds

Gas Chromatography
Temperature and Pressure Programming

1.) Improving Column

Efficiency
➢ Temperature programming:
- Temperature is raised
during the separation
(gradient)

- increases solute vapor

pressure and decrease
retention time

Temperature gradient
improves resolution
while also decreasing
retention time

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Gas Chromatography
Temperature and Pressure Programming

1.) Improving Column Efficiency

➢ Pressure Programming:
- Increase pressure → increases flow of mobile phase (carrier gas)
- Increase flow → decrease retention time

➢ Pressure is rapidly reduced at the end of the run

- Time is not wasted waiting for the column to cool
- Useful for analytes that decompose at high temperatures

Gas Chromatography
Carrier Gas

1.) N2, He and H2 are typical carrier gases

➢ He:
- Most common and compatible with most
detectors
- Better resolution
- Solutes diffuse rapidly → smaller mass
transfer term
➢ N2:
- Lower detection limit for a flame ionization
detector
- Lower resolution and solute diffusion rates
➢ H2:
- Fastest separations
- Can catalytically react with unsaturated
compounds on metal surfaces
- Can not be used with mass spectrometers Flow rate increases N2 < He < H2
Forms explosive mixtures with air
- Better resolution
Diffusion coefficients follow: H2 > He > N2

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Gas Chromatography
Sample Injection

1.) “Sandwich” Injection

➢ Separate sample with air bubbles and solvent

- Solvent is used to push out sample, but bubble prevents mixing

- Final air bubble pushes out solvent

- Gas-tight syringe is required for gas samples

- Injection volume is typically 0.1-2 mL

Gas Chromatography
Sample Injection

2.) “Split” Injection

➢ Injection port

- Inject rapidly ( < 1s) through septum into evaporation zone

- Injector temperature is kept high (350 oC) for fast evaporation

- Rapid gas flows carries sample to mixing chamber for complete vaporization
and complete mixing before entering column

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Gas Chromatography
Sample Injection

3.) On-column Injection

➢ Delivers ~100% of sample to the column
➢ For samples that decompose above their boiling points
➢ Solution injected directly on column
- Warming column initiates chromatography

Lower initial column temperature

to prevent sample decomposition

Raise temperature to volatize

sample and start separation

Gas Chromatography
Detectors

1.) Qualitative and Quantitative Analysis

➢ Mass Spectrometer and Fourier Transform Infrared Spectrometers can identify
compounds as part of a GC system
- Compare spectrum with library of spectra using a computer

➢ Compare retention times between reference sample and unknown

- Use multiple columns with different stationary phases
- Co-elute the known and unknown and measure changes in peak area

➢ The area of a peak is proportional to the quantity of that compound

Are a of Gaus s ian pe ak = 1 .064  peak height  w 1
2

Peak area increases proportional

Peak Area

to concentration of standard if
unknown/standard have the
identical retention time → same
compound

Concentration of Standard

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Gas Chromatography
Detectors

2.) Thermal Conductivity Detector

➢ Measures amount of compound leaving column by its ability to remove heat
- He has high thermal conductivity, so the presence of any compound will lower
the thermal conductivity increasing temperature of filament
➢ As heat is removed from filament, the resistance (R) of filament changes
- Causes a change in an electrical signal that can be measured
➢ Responds to all compounds (universal)
- Signal changes in response to flow rate of mobile phase and any impurities
present
- Not very sensitive

Ohm’s Law: V =IR

Based on Ohm’s law, monitored
potential (V) or current (I) Changes
as resistance (R) of filament changes
due to presence of compound

Gas Chromatography
Detectors

3.) Flame Ionization Detector

➢ Mobile phase leaving the column is mixed with H 2 and air and burnt in a flame
- Carbon present in eluting solutes produces CH radicals which produce CHO + ions
- Electrons produced are collected at an electrode and measured
➢ Responds to almost all organic compounds and has good limits of detection
- 100 times better than thermal conductivity detector
- Stable to changes in flow rate and common mobile phase impurities (O 2, CO2,H2O,NH3)

Burn sample and measure

amount of produced electrons

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Gas Chromatography
Detectors

4.) Mass Spectrometry

➢ Detector of Choice → But Expensive!
➢ Sensitive and provides an approach to identify analytes
➢ Selected ion monitoring – monitor a specific mass/charge (mz) compared to
scanning over the complete spectra
- Simplifies complex chromatogram
- Increases sensitivity by 102-103

Gas Chromatography
Method Development in GC

1.) How to Choose a Procedure for a Particular Problem

➢ Many Satisfactory Solutions
➢ The order in which the decision should be made should consider:
1. Goal of the analysis
2. Sample preparation
3. Detector
4. Column
5. Injection

➢ Goal of the analysis

- Qualitative vs. quantitative
- Resolution vs. sensitivity
- Precision vs. time
- Interest in a specific analyte

➢ Sample preparation
- Cleaning-up a complex sample is essential
- Garbage in → garbage out

➢ Choosing the Detector

- Detect a specific analyte(s) or everything in the sample
- sensitivity
- Identify an unknown (MS, FTIR)

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Gas Chromatography
Method Development in GC

1.) How to Choose a Procedure for a Particular Problem

➢ Selecting the Column
- Consider stationary phase, column diameter and length, stationary phase
thickness
- Match column polarity to sample polarity
- To improve resolution, use a:
a. Longer column
b. Narrower column
c. Different stationary phase

➢ Selecting Injection method

- sandwich
- split
- on column

Biotron Gas Chromatography instrument

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