Chapter 3- CHROMATOGRAPHY CLASSIFICATION BASED ON THE MOBILE b) Gas Chromatography
PHASE o Gas mobile phase
CHEM o Applied only to gaseous or volatile
a) Liquid chromatography
Mikhail Tsweet, Russian, 1872-1919- used substances that are heat stable.
o Mobile phase is a liquid
chromatography to separate plant pigments o Not commonly used for the analysis
o More versatile than gas
of biomolecules since large
Chromatography chromatography molecular weight compounds such as
- MODES OF OPERATION: peptides and proteins are thermally
- a process for separating components of a o Normal phase chromatography- destroyed evaporation.
mixture the stationary phase consists of a
- Chroma means color and graphein means write hydrophilic material such as silica
- To get the process started, the mixture is particles and the mobile phase is a
dissolved in a substance called the mobile hydrophobic organic solvent such as
phase, which carries it through a second hexane.
substance called the stationary phase. o Reversed phase chromatography-
the stationary phase is hydrophobic,
and the mobile phase is a mixture of
polar solvents, for example water and
acetonitrile
CLASSIFICATION ACCORDING TO THE c.) Column chromatography (CC) - the
PACKING OF THE STATIONARY PHASE stationary phase is packed in a glass column
a.) Thin layer chromatography (TLC)-
stationary phase is a thin layer supported on
glass, plastic or aluminum plates
b.) Paper chromatography (PC) - the stationary
phase is a thin film of liquid supported on an
inert support
CLASSIFICATION ACCORDING TO THE FORCE
OF SEPARATION
a. Adsorption chromatography
b. Partition chromatography
c. Ion exchange chromatography
d. Gel filtration chromatography
e. Affinity chromatography
APPLICATION OF LIQUID
CHROMATOGRAPHY FOR BIOANLYSIS
- In bioanalytical chemistry, chromatography is
mainly used for the separation, isolation and
purification of proteins from complex samples
matrices.
REVERSE PHASE LIQUID CHROMATOGRAPHY
Normal phase chromatography- was developed many
years before reversed phase chromatography was
investigated
- Stationary phase: Paper, cellulose or silica gel
- Mobile phase: Non-polar solvents such as
hexane or chloroform
Gradient elution- is a chromatography technique that
separates mixtures of solutes by changing the
composition of the mobile phase
Reversed phase chromatography- is the method of
choice for the separation of smaller biomolecules such as Buffer systems in liquid chromatography
peptides, amino acids, carbohydrates and steroids which
are soluble in water/acetonitrile mixtures. - Common buffers: Ammonium Acetate,
Phosphate, Hydrogen Carbonate
- Stationary phase: Porous silica particles with - Typical concentration: 20mM
non-polar surface groups - Purpose: adjust pH of the mobile phase
- Mobile phase: polar solvent, aqueous buffer, - pH Range: 2 to 8
acetonitrile or methanol
Why adjust the pH?
- Enhances analyte stability
- Improves peak shape and resolution
- Optimizes analyte interaction with the
stationary phase
Types of ion pairing reagents - Gradient Elution
o in gradient elution, the composition
- Anionic Reagents- (e.g., Trifluoroacetic Acid- of solvents is gradually altered during
TFA): bind to positively charged analytes separation
- Cationic Reagents- (e.g., Tetraalkyl - Column and Temperature control
Ammonium Salts): Bind to negatively charged o Columns are tightly packed with
analytes
small particles (1-5 um in diameter)
Mechanism of Ion Pairing o Column often situated inside a
thermostatically regulated oven to
- Ion-pair complexes are more hydrophobic maintain constant temperature
- Retained longer by the stationary phase - Detection Methods
- Easier separation compared to unretained o UV Detection
charged analytes Fixed wavelength
detection: A=210nm for
HIGH PERFORMANCE LIQUID peptides. A=254 nm or
CHROMATOGRAPHY A=280nm for proteins
- To achieve ambient flow rates in these o Diode Array Detectors (DAD)
columns, high pressure of up to 300-400 bar More expensive
must be generated. A typical instrumental setup instruments feature DAD
for this high pressure or high-performance Capture several spectra per
liquid chromatography (HPLC) second
Allows for more
unambiguous identification
o Fluorescence Detection
High sensitivity detection
of derivatized amino acids
and peptides.
- Advanced Detection- ESI-MS
o Coupling liquid chromatography to
electrospray ionization mass
spectrometry (ESI-MS).
o Provides universal detection at high
sensitivity
- Mobile phase control o Offers structural information about
o Computer-controlled pumps move the analyte
the mobile phase through the system
o Aqueous solvent A and organic
Solvent B are mixed to the desired
composition
o To adsorb onto a cation exchange
(CM), the protein must be positively
ION EXCHANGE CHROMATOGRAPHY charged (pH<pl)
What is ion exchange chromatography? o To adsorb onto an anion exchanger
(DEAE), the protein must be
- A technique to separate and purify analytes negatively charged (pH >pl)
based on their overall charge.
- Applicable to large proteins, small nucleotides,
and amino acids
Step in Ion Exchange Chromatography
1. Column preparation
- Column composition
2. Sample Application
o Contains Porous particles with
3. Adsorption of charged molecules
4. Elution of Bound Molecules positively charged functional groups
- Binding and elution mechanism on the surface
o Negatively charged molecules are o Negatively charged molecules bind to
adsorbed onto the stationary phase these groups
o Neutral and positively charged - The stationary phase
o often referred to as gel, made of
molecules elute without binding
- Principle - How are molecules eluted? agarose or cellulose beads with
o Based on competitive interaction o Gradual increase in salt covalently attached charged groups
between charged samples molecules o anion exchangers have positively
concentration displaces bound
and salt ions. molecules. charged functional groups, cation
o Charged functional groups on the o Changing pH can decrease protein exchangers have negatively charged
station phase attract opposite charges groups
net charge or neutralize ion
from the sample. o Common ion exchangers: diethyl
exchanger groups.
- Protein adsorption aminoethyl (DEAE) and
o Adsorption is minimal at pH close to carboxymethyl (CM). The charge
capacity depends on the pH of the
the pl.
mobile phase
Steps in Affinity Chromatography
1. Sample introduction
2. Adsorption: Target molecules bind to the
ligand
3. Washing: Removes non-specifically bound
components
4. Desorption: Elution of the bound molecules
Types of Ligands
- Monospecific ligands: Bind only one analyte
(e.g., peptide hormone to its receptor).
AFFINITY CHROMATOGRAPHY - Group-Specific Ligands: Bind similar
proteins (e.g., immobilized lectins binding
Introduction to affinity chromatography
glycoproteins).
- Highly specific molecular recognition
techniques are used for purification and
isolation of biomolecules.
- Utilizes interactions like antigen-antibody,
enzyme-coenzyme, and receptor-hormone.
- High Specificity & Selectivity: Can isolate Adsorption and Desorption
biomolecules even at low concentrations.
- Binding Partners: - Adsorption: Target biomolecules bind
o Antigen and antibody specifically to ligands
o Enzyme and Co-enzyme - Desorption Methods:
o pH decrease
o Receptor Protein and hormones
o Increase in ionic strength
o Single strands of Oligonucleotides
o Addition of denaturing agents (.eg.,
and their matching counterparts
urea)
o Organic solvents
SIZE EXCLUSION CHROMATOGRAPHY
What is Size Exclusion Chromatography?
- Size exclusion chromatography separates
dissolved molecules based on size, related to
molecular weight.
- Used for:
o Polymers in on-aqueous solutions
(Gel Permeation Chromatography,
GPC)
o Biomolecules in aqueous solutions
(Gel Filtration Chromatography) - Mobile Phase
- Principle o Acts as a solvent, not influencing
o Column filled with porous material separation.
(polymeric gel, agarose beads) o Aqueous buffers with 50-100mM
o Separation occurs based on molecule ionic strength
size relative to pore size o Typical flow rates: 0.1-1 mL/min.
- Advantages
o Gentle method: No harsh pH or ionic
strength conditions
o No sample loss on the stationary
phase
o Solvent gradients do not alter
retention volume
- Applications
o Separation of proteins from peptides
and amino acids
o Separation of biomolecules (proteins,
- Pore Size and Molecular weight Range
o Typical SEC range: 2 kDa to 200 fatty acids, nucleotides) by size.
o Molecular weight determination
kDa (can extend up to 1,000 kDa
with specialized gels) using calibration standards
o Retention time correlates with - Compared with other MW Determination
Methods
molecular size within a critical range.
o MALDI-TOF/MS: More accurate
- Retention and Elution
o Large molecules: unretained, elute and faster but semi-quantitative
o SEC with UV detection: Quantitative
first
o Small molecules: retained longest, MW determination and sample
quantification.
elute later
