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HL Chemistry - Option A :

Modern Analytical
Chemistry

Chromatography

CHROMATOGRAPHY
Chromatography basically involves the
separation of mixtures due to differences in the
distribution coefficient (equilibrium distribution)
of sample components between 2
different
phases.
One of these phases is a mobile phase and
the other is a stationary phase.

Stationary Phase: Alumina

OH

OH

OH

OH

Al

Al

Al

Al

Al

Acidic: -Al-OH
Neutral: -Al-OH + -Al-OBasic: -Al-O-

Stationary Phase: Silica (SiO2)


OH
Si
O

Si
O
O
Si

OH

OH

Si
O
O
Si

O
O
O

Si
O
O
Si

O
O
Si

OH

OH
O
O
Si
O
O
Si

O
O

Si

O
O

O
O

O
O
Si

O
O

Distribution Coefficient (Equilibrium Distribution )


Definition:
Concentration of component A in stationary phase
Concentration of component A in mobile phase
Different affinity of these 2 components to stationary
phase causes the separation.

Some Types of Chromatography


1. Liquid Column Chromatography
(Reverse Phase too)
2. High Pressure (performance) Liquid
Chromatograph (HPLC)
3. Paper Chromatography
4. Thin-layer Chromatography (TLC)
5. Gas Liquid Chromatography

LIQUID COLUMN CHROMATOGRAPHY


A sample mixture is passed through a column
packed with solid particles which may or may
not be coated with another liquid.
With the proper solvents, packing conditions,
some components in the sample will travel the
column more slowly than others resulting in
the desired separation.

Diagram of Simple Liquid Column Chromatography


DIAGRAM OF SIMPLE LIQUID C OLUMN C HROMATOGRAPHY
S olvent(mobile or
moving phas e)
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
A
OOOOO
OOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
Column
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO S olid Particles OOOOOOOOOO
B OOOO
OOOOOOOOOOO
(packing material- OOOOO
OOOOOOOOOO s tationary phas e) OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
C OOOO
OOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
A+B +C

S ample
(A+B+C)

Eluant (eluate)

BASIC LIQUID CHROMATOGRAPHY


The 4 basic liquid chromatography modes are named according to the mechanism
involved:

1. Liquid/Solid Chromatography (adsorption chromatography)


A. Normal Phase LSC
B. Reverse Phase LSC
2. Liquid/Liquid Chromatography (partition chromatography)
A. Normal Phase LLC
B. Reverse Phase LLC
3. Ion Exchange Chromatography
4. Gel Permeation Chromatography (exclusion chromatography)

LIQUID SOLID CHROMATOGRAPHY

Normal phase LS
Reverse phase LS

Si - O - H

Silica Gel

The separation mechanism in LSC is based on the


competition of the components of the mixture sample
for the active sites on an absorbent such as Silica Gel.

LIQUID SOLID CHROMATOGRAPHY

OH

HEXANE

Si - OH

CH3

OH CH
3
C-CH3
CH3

CH3- C
CH3

CH3

WATER-SOLUBLE VITAMINS
1.

Niacinamide

2.

Pyridoxine
H3C

N
HO
CONH 2
Riboflavin
CH 2OH
HOCH
HOCH
HOCH
CH 2
H 3C
N
N

CH 2OH
CH 2OH

3.

H 3C

4. Thiamin

O
NH

N
O

H3C

N
N

NH 2
CH 2

S
N

CH 2CH 2OH
Cl
CH 3

WATER-SOLUBLE VITAMINS
2

3
Inject
1

10

15

20

Column: u Bondapak C18


Solvent: MeOH
Sample: Water-Soluble Vitamins

LIQUID-LIQUID CHROMATOGRAPHY

ODPN(oxydipropionylnitrile)
Normal Phase LLC
Reverse Phase LLC

NCCH3CH2OCH2CH2CN(Normal)
CH3(CH2)16CH3 (Reverse)

The stationary solid surface is coated with a 2nd liquid (the


Stationary Phase) which is immiscible in the solvent (Mobile) phase.
Partitioning of the sample between 2 phases delays or retains some
components more than others to effect separation.

Chromatography Schematic
LIQUID

MOBILE PHASE

FORMAT

Liquid-Liquid
Chromatography
(Partition)

STATIONARY
PHASE

Normal Phase
Mobile Phase Nonpolar
Stationary phase Polar

Liquid

Reverse Phase
Mobile Phase Polar
Stationary phase Nonpolar

Liquid-Solid
Chromatography
(Adsorption)

Solid

Normal Phase

Reverse Phase

ION-EXCHANGE CHROMATOGRAPHY

SO3 Na

Separation in Ion-exchange Chromatography is based on the


competition of different ionic compounds of the sample for the
active sites on the ion-exchange resin (column-packing).

REMEMBER

The stationary phase is POLAR


The more polar component interacts
more strongly with the stationary
phase
The more polar component moves
more slowly.
The non-polar component moves more
rapidly.

MECHANISM OF ION-EXCHANGE
CHROMATOGRAPHY OF AMINO ACIDS

pH2

SO3

Na

H3N

COOH

Ion-exchange Resin

SO3

H3N
Na

+
-

COO

pH4.5

Chromatography of Amino Acids


Stationary Phase

Mobile Phase
H3N

SO3 Na+

COOH
+

Na
SO 3

OH
H3 N

COOH
Exchange Resin
-

SO3 H3N+
COOH
SO 3

pH3.5

OH

H3 N
+

+
-

Na

COO

OH = H 2 O

Na
SO3

H3 N

+
-

COO

OH = H 2 O

SO 3Na+
pH4.5

GEL-PERMEATION CHROMATOGRAPHY

Gel-Permeation Chromatography is a mechanical sorting of molecules


based on the size of the molecules in solution.
Small molecules are able to permeate more pores and are, therefore,
retained longer than large molecules.

SOLVENTS
Polar Solvents
Water > Methanol > Acetonitrile > Ethanol >
Oxydipropionitrile
Non-polar Solvents
N-Decane > N-Hexane > N-Pentane >
Cyclohexane

SELECTING AN OPERATING MODE


Sample Type

LC Mode

Positional isomers

LSC or LLC

Moderate Polarity Molecules

LSC or LLC

Compounds with Similar Functionality

LSC or LLC

Ionizable Species

IEC

Compounds with Differing Solubility

LLC

Mixture of Varying Sized Molecules

GCC

Detectors

1.

Ultraviolet Detector
200-400nm
254 nm

2.

Reflective Index Detector

Liquid Chromatography Set


Up

HPLC Chromatography
1. Pump System. Mobile phase pressures up to 6000 psi are

necessary to achieve reasonable column elution times (~


minutes). Typical flow rates are 0.1 to 10 mL/minute.

2. Injection System. Used to introduce small samples (0.1

to 500 L) into the carrier stream under high pressure.

3. Reservoirs (Solvents). Multiple solvents are necessary

for performing gradient elution (i.e. changing the polarity of


the mobile phase during a run).

4. Chromatographic Column. Typically 10-30 cm in length

containing a packing of 5-10 m diameter. Many types of


columns are available, depending on the type of liquid
chromatography desired.

5. Detector. Many types are available including UV, IR,

refractive index, fluorescence, conductivity, mass


spectrometry, and electrochemical. Diode array detectors
are used when wavelength scans are desired.

Schematic of an HPLC
System

HPLC System

Pump System
Desirable Features:
Must generate pressures
up to 6,000 psi

Flow-rates range from


0.1 to 10 mL/minute
Limited pulsing in the
system

To allow for separation in


reasonable time frames

Many HPLC systems have


a dual pump system to
minimize pulsing

Flow control and


reproducibility < 0.5%
Corrosion resistance

Sample Injection System


Used to
introduce
small samples
(0.001 to 0.5
mL) into the
carrier stream
under high
pressure

HPLC Detectors

No universal or versatile detector


Types

General respond to mobile phase bulk


properties which vary in the presence of
solutes (e.g. refractive index)
Specific respond to some properties of
the solute (not possessed by the mobile
phase (e.g. UV adsorption)
Hyphenated detector LC-MS

Absorbance Detectors

The UV/Vis source usually comes


from a monochromator so the
wavelength can be selected, or
scanned.
Absorbance increases as eluate
passes through the cell.
If wavelength scanning is desired,
the flow is stopped long enough for
the scan to take place.
Its possible to have the same
setup using IR light, although not
as common since many useful
solvents are not IR transparent.

Diode
Array
Detecto
r

HPLC Detectors

HPLC Column

Must operate in high pressure

Typical dimensions

10-30 cm long
1-3 cm ID

Contains packing material


which holds the stationary
phase

Usually constructed of metals

Many types exist


Typical packing materials are 510 m in diameter

Guard column used to extend


life of main column

Type of
HPLC
Depends
on:
1. Molecular

weight of
solute
2. Water solubility
of the solute
3. Polarity of the
solute
4. Ionic/non-ionic
character of
the solute

Separation Principles in
HPLC
General Rule of Thumb:
Polarity of analytes polarity of stationary
phase polarity of mobile phase
To achieve good separation, the analytes
should interact with the stationary phase,
but not too strongly or the retention time
will become very long

Reversed order
of elution

Increasing Mobil
phase Polarity,
Decreases
Elution Time

Typical Applications of
HPLC Chromatography
Field of Application Separation
Pharmaceuticals

Antibiotics, Sedatives, Steroids,


Analgesics

Biochemical

Amino acids, Proteins, Carbohydrates,


Lipids

Food Products

Artificial Sweeteners, Antioxidants,


Preservatives

Industrical Chemicals Condensed Aromatics, Surfactants,


Propellants, Dyes
Forensic Chemistry

Drugs, Poisons, Blood Alcohol,


narcotics

Clinical Medicine

Bile Acids, Drug Metabolites, Urine


Extracts, Estrogens

Pollutants

Pesticides, Herbicides, Phenols, PCBs

HPLC of Orange Juice Compounds

How to Increase HPLC Resolution


1.

2.
3.
4.
5.
6.
7.
8.
9.
10.

Increase column length


Decrease column diameter
Decrease flow-rate
Pack column uniformly
Use uniform stationary phase (packing material)
Decrease sample size
Select proper stationary phase
Select proper mobile phase
Use proper pressure
Use gradient elution

Separating Proteins from Mixtures


In order to understand and study proteins it is
essential to separate them from the biological fluid.

The inside of a cell. White shapes are


proteins (several 10s of thousands per
cell).

Proteins can be separated from each other based on


differences in physical properties
Due to different amino acid sequences proteins differ
in solubility, size, charge, and binding affinity and can
be separated on either of these properties.

Water, Chemical bonds and groups

Amino acids, pH dependence

R
C

H 3 N+

COO-

Protein primary sequence, peptide bonds, secondary structures

Protein studies: Understanding protein


structure and function relationships
All proteins have a distinctive 3D structural
conformation
This unique structure enables its function
Amino acid sequence determines structure
A major goal of biochemistry is to determine how
amino acid sequences specify the 3D conformations
of proteins and to catalogue all proteins in cells.

Characterization
cell

Protein purification:
general experimental
setup
Column
chromatography

Homogenize

Centrifugation

Characterization

Gel permeation chromatography:


separating on basis of size
Mixture of proteins
1.
2.
3.
4.

A mixture of proteins in a small volume


is applied to a column filled with
porous beads
Because large proteins cannot enter the
beads, they emerge sooner than do
small ones
A detector (e.g. UV) is used to detect
protein fragments
Fragments are collected separately

UV
time

Affinity Chromatography: separating on


the basis of affinity

XX X X
X

XX
X

XX
XX

X
X

To separate proteins that recognize


a chemical group X
1. X is covalently attached to beads that
are packed in a column
2. Sample of proteins is added
3. Washed with buffer to remove non
specifically bound protein
4. Eluted with high concentration of
soluble X

Separation on the basis of charge


All proteins are charged. Their charges depend on the
relative number of acid and basic amino acids in their
primary structure.
All proteins have a pH value where they are uncharged:
the isolelectic point (pI)

- Met Ala Asn Cys His Glu Ser Thr Glu Arg-COOH

H2N

Ionic amino acids

Separation on the basis of charge (continued)

- Met Ala Asn Cys His Glu Ser Thr Glu Arg-COOH

H2N

His: 6.0
Glu: 4.1
Arg: 12.5
N-terminal amine: 8.0
C-terminal acid: 3.1

For this peptide:


pI=pKa/N= 6.3

Positively charged at pH < 6.3


Negatively charged at pH > 6.3

Ion Exchange Chromatography:


separation on basis of net charge
---

--

+ -

+-+
+

+-

-+
1.

Positive or negatively charged resin can be


used for separation of positive or
negatively charged proteins

2.

Sample of proteins is added

3.

Washed with buffer to remove non


specifically bound protein

4.

Elute with increasing concentration of salt

5.

Proteins with highest net charge come of


last

-+

--+

-- - --

Why hydrogels are used for protein separations


1.

2.

3.

1. Correct protein folding in aqueous environment


2. Proteins can denature on surfaces
3. Hydrogels are >90% water, good environment for proteins

Compare Reverse Phase to


Normal Phase Column
Chromatography
In Normal Phase Liquid Chromatography:
The column packing in the column is very polar!
Polar compounds are going to be attracted to the polar
column packing by hydrogen bonding or dipole-dipole
attractions. Polar compounds are going to move slowly!
Non-polar compounds are going to come off the column first,
while the polar compounds are going to come off column last.
Usually, one starts will a less polar solvent to remove
the less polar compounds, and then you slowly
increase the polarity of the solvent to remove the more
polar compounds.

Reverse Phase
Column Chromatography

The stationary phase (column packing)


is now NON-POLAR
Non-polar compounds will move more
slowly because they are attracted to the
column packing.
The more polar component moves more
quickly down the column.
Polar solvents, such as water and
methanol are used in reverse phase
chromatography
Used mainly in columns, such as HPLC

Reverse phase
Silica is chromatography
alkylated with long chain hydrocarbon groups, using 18
carbons long. This is usually referred to as C-18 silica.

Summary of Methodology
One of the main aims of biochemistry is to
characterize and catalogue all proteins in the cell
We have discussed some important tools for
separating proteins based on physical properties
such as size, affinity, charge.
Chromatography methods: ion exchange, affinity,
gel permeation chromatography
Electrophoresis: iso electric focusing, SDS PAGE,
2D gels (in the Biochemistry lecture series)

Overview of Paper
Chromatography

Works on principle of
Partition.

Separates dried liquid


samples.

Mobile phase is solvent


used.

Stationary phase is water


bound to surface of paper.

Advantage : its cheap!

Important Concepts in
Paper Chromatography

Capillary Action the movement of liquid within the spaces


of a porous material due to the forces of adhesion, cohesion,
and surface tension. The liquid is able to move up the filter
paper because its attraction to itself is stronger than the force
of gravity.

Solubility the degree to which a material (solute) dissolves

into a solvent. Solutes dissolve into solvents that have similar


properties. (Like dissolves like) This allows different solutes
to be separated by different combinations of solvents.
Separation of components depends on both their solubility in
the mobile phase and their differential affinity to the mobile
phase and the stationary phase.

Paper/TLC
Chromatography
Animation

Simple Example of Paper


Chromatography using Sharpie
Pens

Dye Separation in a Black


Sharpie
1. Dyes separated purple and
black
2. Not soluble in low
concentrations of isopropanol
3. Partially soluble in
concentrations of isopropanol
>20%

0%

20%

50%

70%

Concentration of Isopropanol

100%

Thin Layer Chromatography

Works mainly on
principle of
adsorption.

Mobile phase is the


solvent

Stationary phase is
the solid on the plate.

TLC vs. Column Chromatography

Thin-layer chromatography and column


chromatography and are different types of
liquid chromatography.
The mobile (moving) phase is a liquid.
The stationary phase is usually silica or
alumina. This phase is very polar.
The principle of operation is the same!

Thin Layer Chromatography


The surface of a plate consists of a very thin layer of silica on a plastic or
aluminum backing. The silica is very polar. This is the stationary phase. Material
is spotted at the origin (bottom) of the TLC plate.
The plate is placed into a glass jar with a small amount of a solvent in a glass jar.
This solvent acts as the moving phase.
The plate is removed from the bottle when the solvent is close to the top of the
plate.
The spots are visualized (explanation to follow).
Non-polar compounds will be less strongly attracted to the plate and will spend
more time in the moving phase. This compound will move faster and will appear
closer to the top of the plate.
Polar compounds will be more strongly attracted to the plate and will spend less
time in the moving phase and appear lower on the plate.

Thin-Layer Chromatography: A
Two-Component Mixture
solvent front

component B

Less polar!

component A

More polar!

solvent front

component B

component A
origin mixture
solvent front

origin

Increasing Development Time

origin

Thin-Layer Chromatography:
Determination of Rf Values
solvent front

Rf of component A =
component B

dA
dS
dS

Rf of component B =

dB

dB
dS
The Rf value is a decimal
fraction, generally only
reported to two decimal
places

component A
dA
origin

Thin-Layer
Chromatography
: Qualitative
Analysis

B unknown

Visualization Method

The previous slide shows colored spots.


Most of the time, the spots wont show
unless they are visualized!
Vizualization is a method that is used to
render the TLC spots visible.
A visualization method can be:

Ultraviolet light
Iodine vapors to stain spots
Colored reagents to stain spots
Reagents that selectively stain spots while
leaving others unaffected.

TLC Advantages
Advantages over paper:
Its faster
It gives a better separation.
It is more versatile as the solid on the
plate can be varied.

Uses of TLC

To determine how many components there


are in a mixture (is it really pure?)
To determine the best solvent conditions
for separation on a column
To identify the substances being studied
To monitor the composition of fractions
collected from column chromatography
To monitor the progress of a reaction

Gas-Liquid Chromatography

Works on principle of Partition.


Mobile phase is the carrier gas.
Stationary phase is oil bound to
surface of beads within the column.

Most Common Stationary


Phases
1. Separation of mixture of polar compounds
Carbowax 20M (polyethylene glycol)
2. Separation of mixtures of non-polar compounds
OV101 or SE-30 (polymer of methylsilicone)
3. Methylester of fatty acids
DEGS (diethylene glycol succinate)

Gas-Liquid Chromatography

Gas-Liquid Chromatography

Retention time is used to identify a


component of a mixture. It depends
on:The temperature of the column.
The length of the column.
The material used to pack the column.
The gas pressure.

Gas Liquid
Chromatography

The area under a peak is proportional to


the amount of substance present.

Gas Chromatography
Filters/Traps

Data system
H

RESET

Regulators

Syringe/Sampler
Inlets
Detectors

Gas Carrier

Hydrogen

Air

Column

gas system
inlet
column
detector
data
system

Schematic Diagram of Flame Ionization


Detector
Collector

Detector electronics

- 220 volts
Flame

Chassis ground

Jet
Column

Signal output

Gas-Liquid Chromatography

Gas-Liquid Chromatography is often combined


with mass spectroscopy. The GC separates
the components then the MS analyses them.

One possible Use of GC:


SEMI- QUANTITATIVE ANALYSIS OF FATTY ACIDS
Detector Response

C18

Peak Area (cm2 )


10

C16

8
6

C14

4
2
0.5

1.0

1.5

2.0

2.5

Sample Concentration (mg/ml)

Retention Time
The content % of C fatty acids =
14

C + C + C

= the content % of C14 fatty acids

3.0

Gas Chromatogram of Methyl Esters of Fatty Acids

Another GC Use:
TENTATIVE IDENTIFICATION OF UNKNOWN COMPOUNDS
Response
Mixture of known compounds

Octane
1.6 min = RT
Hexane

GC Retention Time on Carbowax-20 (min)

Response

Unknown compound may be Hexane

1.6 min = RT

Retention Time on Carbowax-20 (min)

Decane

GLC ADVANTAGES
1.

Very good separation

2.

Time (analysis is short)

3.

Small sample is needed - l

4.

Good detection system

5.

Quantitatively analyzed

DISADVANTAGES OF GAS CHROMATOGRAPHY


Material has to be volatilized at 250 C without decomposition!
Methylester

Fatty Acids

R C OH + CH3 OH + H2 SO4

Reflux

R C O CH3
Volatile in Gas
Chromatography

O
CH 2 O C R
O
CH

O C R
O

CH 2

O C R

CH 3 ONa
+

CH 3 OH

O
3 R C O CH3
Volatile in Gas
Chromatography

Summary of Some Chromatographic Techniques


Technique

Stationary Phase

Paper

Trapped water
in the paper

Thin Layer

Oxide Coating

Column

Oxide packing
or resin

Gas-Liquid

Oxide or
volatile liquid
on a solid
support

High Performance
Liquid

Mobile Phase

amino acid
mixtures
food colors or
dyes

Organic Solvent

detect amino
acids
composition of
dyes and food
colors

Organic Solvent

Oxide Packing
or Resin or
Molecular Sieve

Typical Application

preparative
separation of
plant pigments

Organic Solvent

analysis of oil
mixtures
detect drugs &
steroids
fruit esters

Gas

analyze foods,
pesticides, etc
detect iron in
body fluids

Liquid

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