CHROMATOGRAPHIC TECHNIQUES

Program of lectures
1. Introduction to Separation techniques

1.1. Basic types of separation

1.2. Modes of separation techniques
1.3. Introduction to chromatography; basic
classification of chromatographic methods
1.4. Theories of Chromatography
2. Gas Chromatography – principles and applications
3. Liquid Chrmomatography

4. Hyphenated tehcniques : GC-MS and LC-MS

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 Analytical Separations
1.) Introduction

Sample Purity
➢ Many chemical analysis are not specific for one compound
- Actually respond to many potential interferences in the sample

➢ Often it is necessary to first purify the compound of interest

- Remove interfering substances before a selective analysis is possible
- This requires a separation step.

• What is separations science?

– A collection of techniques for separating complex mixtures of analytes.

– Most separations are not an analytical technique in their own right, until combined
with an analytical detector (often a type of spectrometer)

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Concept Map Showing Some Common Separating Technique

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Common Separation Techniques

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Magnetic separation

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Filtration

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Is a technique used for the separation of particles from a solution according to their size, shape,
density, viscosity of the medium and rotor speed.

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Types of centrifugues

Microcentrifuges Small Benchtop Centrifuges

•Research Applications: Cellular

Biology, Microbiology, Genomics / Molecular
Biology, Proteomics, Biochemistry, Pharmaceutical Studies.
•Clinical Applications: Clinical Chemistry, Clinical
Microbiology, Hematology, Immunology, Clinical Studies.

General Purpose Centrifuges Large Capacity Centrifuges

Provide reproducible
separations for
high‐throughput applications
such as blood banking and
bioprocessing.

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Superspeed Centrifuges
Allow you to collect
micro-organism, cellular
debris, larger cellular
organelles and
precipitated proteins.
Ultracentrifuges
Separation of
macromolecules/ligand
binding kinetic studies,
separation of various
lipoprotein fractions from
plasma and deprotonisation
of physiological fluids for
amino acid ananlysis.
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Simple distillation

Fractional distillation

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The bases of
separation for the
different types of
separation techniques

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Solid-Phase Microextraction (SPME)

SOLVENTLESS EXTRACTION
• High sensivity
• Reproducibility
• Solventless nature
• Sampling/isolation/enrichment
into one step

• Fast, easily automated

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 Solid-Phase Microextraction (SPME)

SOLVENTLESS EXTRACTION
• Little sample manipulation
Drawbacks
• Shorter analysis time • Lot-to-lot variation

• Wide range of sampling techniques • Sensibility against organic

▪ Field solvents
▪ On site
• Limited range of stationary
▪ Air sampling phases

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Sampling VOMs Extraction GC-qMS

SPME Optimization
parameters

Fibre
Extraction time
Extraction temperature
Ionic strength
pH
Sample volume
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Microextraction in Packed Syringe (MEPS)

MEPS Analytical Procedure UHPLC

to barrel
PTFE
sealing
ring

frits
MEPS
packed
eVol - MEPS
bed
end
plug needle

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 QuEChERS

Tomato, garlic, onium, red Transfer 10g sample + Centrifugation

pepper, green pepper, carrot, 10mL solvent to a for 5min
beetroot and brocolli 50mL tube

Transfer 2mL to 2mL

tube containing150mg
Filter (0.22m) Resuspend in 100L MgSO4 + 25 mg PSA +
and inject mobile phase 25 mg C18

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Extractions

1.) Definition
➢ The transfer of a compound from one chemical phase to another
- The two phases used can be liquid-liquid, liquid-solid, gas-solid, etc
- Liquid-liquid is the most common type of extraction

Immiscible
[ S ]2
K=
liquids

[ S ]1

- The partitioning of solute s between two chemical phases (1 and 2) is

described by the equilibrium constant K

K is called the partition coefficient

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Extractions

2.) Extraction Efficiency

➢ The fraction of moles of S remaining in phase 1 after one extraction can be
determined
- The value of K and the volumes of phases 1 and 2 need to be known

V1
q=
(V1 + KV2 )
where: q = fraction of moles of S remaining in phase 1
V1 = volume of phase 1
V2 = volume of phase 2
K = partition coefficient

➢ The fraction of S remaining in phase 1 after n extractions is

n
 V1 
qn =  
( )
Assumes V2 is constant

 1V + KV2 

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Extractions

4.) Example #1:

➢ Solute A has a K = 3 for an extraction between water (phase 1) and benzene
(phase 2).

If 100 mL of a 0.01M solution of A in water is extracted one time with 500 mL

benzene, what fraction will be extracted?

Solution:
First determine fraction not extracted (fraction still in phase 1, q):

n 1
 V1   100 mL 
qn =   =   = 0.062 = 6.2%
 (V1 + KV2 )  100 mL + ( 3 )  ( 500 mL ) 

The fraction of S extracted (p) is simply:

p = 1 − q = 1 − 0.062 = 0.938 = 93.8%

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 ANALYTICAL SEPARATIONS

Extractions

4.) Example #2:

➢ For the same example, what fraction will be extracted if 5 extractions with 100
mL benzene each are used (instead of one 500 mL extraction)?

Solution:
Determine fraction not extracted (fraction still in phase 1, q):
n 5
 V1   100 mL 
qn =   =   = 0.00098 = 0.98%
 (V1 + KV2 )  100 mL + ( 3 )  ( 100 mL ) 

The fraction of S extracted (p) is:

p = 1 − q = 1 − 0.00098 = 0.99902 = 99.902%

Note: For the same total volume of benzene (500 mL), more A is extracted
if several small portions of benzene are used rather than one large portion
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