Chromatography

Pharmaceutical Chemistry Department

Faculty of Pharmacy / Umm- Alqura University
 What Is Chromatography?
ØIs a technique used to separate and identify the
components of a mixture.

ØWorks by allowing the molecules present in the

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

ØThe mixture separates as it interacts with the

two phases.
 Basic Principle
ØIn the animation below the green molecules are
more soluble in the liquid (LC) or less volatile
(GC) than are the red molecules.

ØOver time, the molecules will become physically

separated from each other
 Basic Principle
ØWhen the molecules Sample M obile phase

reach the far end of the

surface, they are detected
or measured one at a time Column

as they emerge.

ØChromatography is non Detector

destructive
does not alter the molecular
structure of the compounds
Time
 Terminology
Ø Mobile Phase: Carrier of the sample, moving
it through the stationary chromatographic
packing material.
Ø Stationary Phase: the chromatographic
packing material which is held in a fixed
position within column hardware, and
performs the chemical separation.
Ø The stationary phase and the mobile phase
will have OPPOSITE properties to set up a
competition for sample analytes.
Classification according to the packing of the stationary phase:

1- Thin layer chromatography (TLC): the stationary phase is a thin layer supported
on glass, plastic or aluminium plates.

2- Paper chromatography (PC): the stationary phase is a filter paper strips as

carrier or inert support.

3- Column chromatography (CC): stationary phase is packed in a glass column.

Classification according to the force of separation:

1- Adsorption chromatography.
2- Partition chromatography.
3- Ion exchange chromatography.
4- Gel filtration chromatography.
5- Affinity chromatography.
You’ve Got a Problem to Solve

I need a quantitative
separation of I’ll get
carbohydrates in some on it!
of our products
as soon as possible.

I’ll need a separation

technique.

8
Separation Techniques

I have two separation techniques in my lab,

High Performance Liquid Chromatography
and Gas Chromatography. Which should I use?
9
Comparison of HPLC and GC

Sample Volatility Sample Polarity

HPLC HPLC
• No volatility requirement • Separates both polar and
non polar compounds
• Sample must be soluble
in mobile phase • PAH - inorganic ions

GC GC
• Samples are nonpolar
• Sample must be volatile and polar

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Comparison of HPLC and GC

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Comparison of HPLC and GC

Sample Thermal Lability Sample Molecular Weight

HPLC HPLC
• Analysis can take place
at or below room • No theoretical upper limit
temperature
• In practicality, solubility is
limit.

GC GC
• Sample must be able
to survive high • Typically < 500 amu
temperature injection
port and column

12
Comparison of HPLC and GC

Sample Preparation Sample Size

HPLC HPLC
• Sample must be filtered
• Sample size based upon
column i.d.
• Sample should be in
same solvent as mobile
phase

GC GC

• Solvent must be volatile • Typically 1 - 5 µL

and generally lower
boiling than analytes

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Comparison of HPLC and GC

Separation Mechanism Detectors

HPLC HPLC
• Both stationary phase • Most common UV-Vis
and mobile phase take • Wide range of non-
part destructive detectors
• 3-dimensional detectors
• Sensitivity to fg (detector
dependent)

GC GC
• Most common FID,
•Mobile phase is a universal to organic
sample carrier only compounds

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How can We Analyze the Sample?

Carbohydrates
1. fructose
2. Glucose
3. Saccharose
4. Palatinose
5. Trehalulose
6. isomaltose 5

2
3
Zorbax NH2 (4.6 x 250 mm) mAU
4
70/30 Acetonitrile/Water 1
6
1 mL/min Detect=Refractive Index

time

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Separations
Separation in based upon differential
Injector
migration between the stationary and
mobile phases.

Mixer Stationary Phase - the phase which

remains fixed in the column, e.g. C18,
Silica

Pumps Mobile Phase - carries the sample

through the stationary phase as it
moves through the column.
Column

Detector

Waste
Solvents

High Performance Liquid Chromatograph

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Separations
Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

High Performance Liquid Chromatograph

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Separations
Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

30
Separations
Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector
Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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The Chromatogram
Typical response obtained by chromatography (i.e., a chromatogram):

Chromatogram - Response versus elution time

to - elution time of unretained peak

tR- retention time - determines sample identity
tR

tR
mAU Area or height is proportional
to the quantity of analyte.

to

Injection time

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 Chromatographic Parameters
Ø V= Retention volume.
Ø V0= Void volume.
Ø tr= retention time.
Ø W= Peak width.
Ø k= retention factor or capacity factor.
Ø α= Selectivity factor.
Ø N= Plate count.
Ø R= resolution.
Ø These parameters are used to develop and optimize
separations.
 Elution Volume & Retention time
tr

V0
V1
V2
V0= Elution volume of unretained compound , equal roughly one
column volume (column size)
Vn= Elution volume of compound n

tr = retention time ( it is the time from the injection to the emergence

of the peak maximum)
 k= Retention Factor, a measure of retention

V0=2
V1=4 V2=8

kx= Vx-V0 or kx= tx-t0 k1= 1 and k2= 3

V0 t0
Ø Describes how far the peak from V0.
ØDirect measure of how strongly the sample interacts with the packing
Material.
ØIt is dependent of flow rate, time and column size.
ØIn HPLC could be increase by changing mobile phase polarity, in GC by
changing temperature
 R = Resolution
Degree of Magnitude of Separation
Ø Resolution is how far apart the peaks are relative to how
board they are!!

2% overlap Complete separation

 R = Resolution
Degree of Magnitude of Separation

With computerize instrument Rs could be calculated

 Column Efficiency
Ø Number of theoretical plates (N): used to determine the
performance and effectiveness of columns. It could be
calculated by instrument’s software.
 Column Efficiency as a Function of
Flow Rate

Ø This curve shows that every column has an optimum

operation flow rate where N is largest.
Ø The relationship between N and flow rate is expressed by
the van Deemter equation.
 GENERAL FACTORS INCREASING
RESOLUTION
1. Increase column length.
2. Decrease column diameter.
3. Decrease flow-rate.
4. Pack column uniformly.
5. Use uniform stationary phase (packing material).
6. Decrease sample size.
7. Select proper stationary phase.
8. Select proper mobile phase.
9. Use proper pressure.
10.Use gradient elution
 Van Deemter Equation
H = A + B/u + uC
ØH is Plate Height; Also called height equivalent to a
theoretical plate (HETP).
“Need H to be as small as possible so you can fit more plates into a column”.

Øu is flow rate, or linear velocity.

Ø A inter-particle channels, velocity independent.
Ø B molecular axial diffusion, inversely proportional to
velocity.
Ø C mass transfer kinetics, directly proportional to
velocity.
 Analyte Bands and the Van Deemter A term,
interstitial volume or channel
Ø A is the multipath term.

Ø The smaller the packing, the smaller the differences in paths.

Ø The more even the packing, the smaller the differences in
paths.

Particle size A H Efficiency

Analyte Bands and the Van Deemter B
term (Diffusion of Band)

u B/u H Efficiency
 Analyte Bands and the Van Deemter C
term (mass transfer)
Ø The most important aspect of the C term is that efficiency
will increase as particles size decrease, and decrease as flow
rate increase. This why flow rate cannot just be increased to
reduce the analysis time without suffering decreased
resolution.

u C H Efficiency

d C H Efficiency
 Ultra Performance Liquid Chromatography
(UPLC)
Ø With particles less than 2 µm, there is a significant gain in efficiency,

FYI
which is not lost as the flow rate is increased.
Ø Thus can do good separations in very short times.
Ø Special particles were developed for this application.