Chromatography

Chromatography

• Chromatography is used routinely in almost every (bio)chemical laboratory for a

large number of tasks. These range from the separation of mixtures on an
analytical as well as preparative scale, from purification and pre-concentration of
an analyte, to controlling the progress of a chemical reaction. There are many
types of chromatography based on the separation principle, e.g. thin layer
chromatography (TLC), reverse phase (RP), Gas chromatography (GC), ion-
exchange, affinity column, and etc.

(B) Thin layer chromatography (TLC)

(A) Typical set up for

manual column
chromatography.
 The Principle of Chromatography

• Chromatography is a separation method where the analyte is contained within a

liquid or gaseous mobile phase, which is pumped through a stationary phase.
Usually, one phase is hydrophilic and the other lipophilic. The components of the
analyte interact differently with these two phases. Depending on their polarity,
they spend more or less time interacting with the stationary phase and are thus
retarded to a greater or lesser extend. This leads to the separation of the different
components present in the sample.

The sample components interact

differently with the stationary and
mobile phase and elute at their
specific retention time
 Gas Chromatography
• Gas chromatography can be applied only to gaseous or volatile substances that are
heat stable. The mobile phase, an inert carrier gas such as nitrogen, hydrogen or
helium, is pumped through a heated column. This column can be packed with a
silicon oxide based material or is coated with a polymeric wax. The sample is
vaporised, pumped through the column and the analytes are detected in the gas
stream as they exit the column. Analyte detection can be achieved by either flame
ionisation or thermal conductivity. GC is not commonly used for the analysis of
biomolecules since large molecular weight compounds such as peptides and
proteins are thermally destroyed before evaporation. Some cell cultures produce
volatile metabolites such as aldehydes, alcohols or ketones. These can be analysed
readily via GC.

Diagram of a gas chromatograph.

 Thin Layer Chromatography

• Thin-layer chromatography (TLC) is a chromatography technique used to separate

non-volatile mixtures. Thin-layer chromatography is performed on a sheet of glass,
plastic, or aluminium foil, which is coated with a thin layer of adsorbent material,
usually silica gel, aluminium oxide, or cellulose. This layer of adsorbent is known as
the stationary phase.

• After the sample has been applied on the plate, a solvent or solvent mixture
(known as the mobile phase) is drawn up the plate via capillary action. Because
different analytes ascend the TLC plate at different rates, separation is achieved.

Development of a TLC plate, a purple spot separates into a red and blue spot.
 Thin Layer Chromatography Staining

As the chemicals being separated may be colorless, several methods exist to visualize
the spots:

• Fluorescent analytes like quinine may be detected under UV light (254 or 366 nm)
• Manganese-activated zinc silicate, is added to the adsorbent that allows the
visualization of spots under UV-C light (254 nm). The adsorbent layer will thus
fluoresce light-green by itself, but spots of analyte quench this fluorescence.
• Iodine
• Potassium permanganate
• Cerium Molybdate Stain
• Ninhydrin

Once visible, the Rf value, or retardation factor, of each spot can be determined by
dividing the distance the product traveled by the distance the solvent front traveled
using the initial spotting site as reference. These values depend on the solvent used
and the type of TLC plate and are not physical constants.

http://www.chemistry.mcmaster.ca/adronov/resources/Stains_for
_Developing_TLC_Plates.pdf
 Liquid Chromatography
• In liquid chromatography, the sample is dissolved and pumped through a column
containing the stationary phase. LC is more versatile than GC as it is not restricted
to volatile and heat-stable samples; the sample only has to dissolve completely in
the mobile phase. Common detection methods are UV spectroscopy,
measurement of refractive index, fluorescence, electrical conductivity and mass
spectrometry.

Instrumentation setup of an liquid chromatography system

 Application of Liquid Chromatography for Bioanalysis

• Chromatography is an essential part of almost any biomolecules purification. They

can be classified according to the physical principle involved in the separation
process as shown in the table.
 Normal Phase Liquid Chromatography

• In normal phase chromatography, the stationary phase consists of a hydrophilic

material such as silica particles and the mobile phase is a hydrophobic organic
solvent such as hexane and ethyl acetate.

• NP-HPLC fell out of favor in the 1970s because of poor reproducibility of retention
times due to the presence of a water or protic organic solvent layer on the surface
of the silica or alumina chromatographic media. This layer changes with any
changes in the composition of the mobile phase (e.g. moisture level) causing
drifting retention times.

In normal phase chromatography,

non polar material elutes first.
Silica gel column
 Reversed Phase Liquid Chromatography

• Reversed phase HPLC (RP-HPLC) has a non-polar stationary phase and an aqueous,
moderately polar mobile phase. One common stationary phase is a silica which has
been surface-modified with RMe2SiCl, where R is a straight chain alkyl group such
as C18H37 or C8H17. With such stationary phases, retention time is longer for
molecules which are less polar, while polar molecules elute more readily (early in
the analysis).

(A) Surface group used for

stationary phases in reversed phase
Chromatography

(B) Solvent ordered according to

polarity and elution speed of the
analytes.
 Adsorbent Phase Type

• The best organic phase to use in separating polypeptides depends on the size and
hydrophobicity of the polypeptide. Large macromolecules, e.g. proteins (>5000
Da), are generally best separated on column with short, less hydrophobic C4
column.

Hydrophobic phase selection

 Pore Diameter, Particle Size and Column Length

• Pore diameter: The RP-HPLC for polypeptides separation is normally performed on

column with having pores size of 300Å.

• Particle size: Smaller diameter particles produce sharper peaks and better
resolution with higher back pressure. 5 µm materials are common for analytical
and small-scale preparative separations.

• Column length: The resolution of column increases with longer column. Column
back pressure is a function of column length, and longer columns do result in
higher back pressure.
 Column Diameter

• The column diameter affects sample loading, solvent usage and detection
sensitivity. The diameter of standard analytical columns is 4.6 mm. Shorter and
smaller columns with smaller particles offer faster analysis times, decreased
solvent consumption and require less sample.
 Column Diameter and Eluent Peak Concentration

• The eluent peak volume from a chromatographic column is inversely proportional

to the square of the column diameter and directly proportional to the column
length. If the column diameter is reduced from 4.6 mm to 1.5 mm, the flow rate
must be reduced by (4.6/1.5)2, to achieve the same linear flow velocity . For
concentration dependent detectors, this will result in an equivalent increase in
detector signal.

Principles of microbore column chromatography

 Mobile Phase

• Desorption and elution of analytes from RP-HPLC columns are accomplished with
aqueous solvents containing an organic modifier and an ion-pair reagent or buffer.
The most commonly used organic modifier is acetonitrile. It is volatile and has a
low viscosity minimizing column back pressure. Temperature affects solvents
viscosity and column back pressure.
 Gradient Elution

• The organic solvent in the mobile phase helps to solubilize the polypeptide and
desorbs it from the hydrophobic adsorbent surface. Polypeptide is very sensitive to
subtle change in the organic modifier concentration. As a consequence, isocratic
elution is difficult for polypeptide analysis in RP-HPLC.
• A typical mobile phase for the separation of proteins is a gradient from 20% to 80
% acetonitrile in water with 0.1% TFA. For polypeptide separation, a typical
gradient is from 10% to 60% acetonitrile in water with 0.1% TFA.

Standard chromatography conditions for RP-HPLC of protein standards

 Adding TFA in the Mobile Phase

• The separation of analytes by reversed phase (RP) chromatography may be

modified by changes in the mobile phase composition, including: pH, and by the
addition of ion-pairing agents, typically at the low mM level. Trifluoroacetic acid
(TFA), a commonly used ion pairing reagent, is also strongly acidic (pKa = 0.3) and
helps to maintain the mobile phase at low pH even when used at low nM (<0.1%
v/v) concentrations. TFA has several other desirable characteristics:
• It can sharpen peak shape and improve analyte resolution by suppressing the
interaction of basic compounds with active silanol sites on silica-based HPLC
columns.
• It is volatile and can be used with HPLC detectors that use nebulization techniques
such as MS. Formic acid is more commonly used than TFA in MS.
• It has low molar absorptivity and can be used with optical detectors.
• For polypeptide separation, it can polarize the amide bond to give better
resolution.
 Ion-pairing Reagents

• Ion Pair Chromatography is a method for improving the separation of charged

analytes. In the resolution of organic ions with conventional HPLC methods, use of
ion pair reagents can enhance peak shape and retention time when common
remedies such as modifying eluent ratios or changing stationary phase fail.
• The addition of the ion-paring reagents to the mobile phase should be very
careful, as the reagent might not be washed completely from the column.

(A) Ion-pairing mechanism (B) How fast various ion pairing reagents
are washed out of the column.
 RP-HPLC Instrumentation

Schematic representation of an HPLC unit. (1) Solvent reservoirs, (2) Solvent degasser, (3)
Gradient valve, (4) Mixing vessel for delivery of the mobile phase, (5) High-pressure
pump, (6) Switching valve in "inject position", (6') Switching valve in "load position", (7)
Sample injection loop, (8) Pre-column (guard column), (9) Analytical column, (10)
Detector (i.e. IR, UV), (11) Data acquisition, (12) Waste or fraction collector.
 Ion Exchange Chromatography (IEC)
• Ion exchange chromatography separates and purifies analytes according to their
overall charge. It can be used for almost any kind of charged molecule including
large proteins, small nucleotides and amino acids. It is often used as a first step in
protein purification. The principle of ion exchange chromatography is based on the
competitive interaction between charged sample molecules and salt ions for the
charged functional groups on the stationary phase.

IEC has the following features:

• High capacity
• High resolving power
• Mild separation conditions
• Relatively low cost

http://www.youtube.com/watch?v=q3fMqgT1do8
 Stationary Phase in Ion Exchange Chromatography
• The stationary phase used in ion exchange chromatography is often referred to as
a gel. It consists of agarose or cellulose beads with covalently attached charged
groups. Anion exchangers feature positively charged functional surface groups
whereas cation exchangers feature negatively charged surface groups. Commonly
used ion exchangers are diethyl aminoethyl (DEAE) and carboxymethyl (CM). The
charge and thus the capacity of these ion exchangers depends on the pH of the
mobile phase that is used. For example, an anion exchanger like DEAE will be
deprotonated and thus neutralised at high pH and lose its activity. Both CM and
DEAE work sufficiently well at pH values between 4 and 8, the range of greatest
relevance for biomolecular applications.

Desorption of negatively charged analytes from

an anion exchanger by increasing the salt
concentration. Analytes with a low net charge are
eluted at medium salt concentration, whereas
analytes with a high net charge require a mobile
phase with a high ionic strength before they are
eluted.
 Choosing An IEC Buffer

• Avoid choosing a buffer salt that may interact with the medium. For example, a phosphate
buffer together with an anion exchange. In this case, the phosphate group will bind to the
column and the equilibrium will be distorted, leading to a change in pH sufficient to desorb
the protein.
• Characteristics of a good buffer are high buffering capacity at the working pH and high
solubility. The concentration of buffer salts usually ranges from 10 to 50 mM.

(A)

(B)
 Buffer pH
1. As a rule, the pH of the mobile phase buffer must be between the pI (isoelectric point)
or pKa (acid dissociation constant) of the charged molecule and the pKa of the charged
group on the solid support. For example, in cation exchange chromatography, using a
functional group on the solid support with a pKa of 1.2, a sample molecule with a pI of
8.2 may be run in a mobile phase buffer of pH 6.0. In anion exchange chromatography a
molecule with a pI of 6.8 may be run in a mobile phase buffer at pH 8.0 when the pKa
of the solid support is 10.3.

• Many chromatographers also use changes in pH to affect a separation. In cation

exchange chromatography, raising the pH of the mobile phase buffer will cause the
molecule to become less protonated and hence less positively charged. The result is
that the protein no longer can form a ionic interaction with the negatively charged solid
support, which ultimately results in the molecule to elute from the column. In anion
exchange chromatography, lowering the pH of the mobile phase buffer will cause the
molecule to become more protonated and hence more positively (and less negatively)
charged. The result is that the protein no longer can form a ionic interaction with the
positively charged solid support which causes the molecule to elute from the column.

http://www.separations.us.tosohbioscience.co
m/ServiceSupport/TechSupport/ResourceCent
er/PrinciplesofChromatography/IonExchange
 What Salt to Use for Elution?

• After binding, salt concentrations for elution are chosen so the target molecule
does not co-elute with contaminants that have also bound to the ion exchange
matrix. Ions of the eluting salt must displace other molecules from the charged
groups on the stationary phase with either a gradient or step in the 0 to 1.0 M
range. The effectiveness of displacement for commonly used cations is:
Ca2+ > Mg2+ > Na+ > K+ >NH4+
The order of displacement effectiveness for commonly used anions:
PO43- > SO42- > COO- > Cl-

• The strongest eluting salt is not always the best. Ideally, several salts should be
tested, and finding optimum elution conditions often involves trial and error. Most
users will start with either NaCl or KCl simply because they are readily available in
the lab. However, CaCl2 or MgCl2 may be used. For some proteins, those salts may
actually end up being a better choice. Regardless of eluting salt selection, the
effect on the purity, stability, and activity of the target molecule will have to be
assessed.

http://www.pall.com/main/laboratory/literatu
re-library-details.page?id=38542
 Ionic Strength of Salts

• The ionic strength of a solution is a measure of the concentration of ions in that

solution. Ionic compounds, when dissolved in water, dissociate into ions. The total
electrolyte concentration in solution will affect important properties such as the
dissociation or the solubility of different salts. One of the main characteristics of a
solution with dissolved ions is the ionic strength.

• The ionic strength, I, of a solution is a function of the concentration of all ions

present in that solution.

• where ci is the molar concentration of ion i (M, mol/L), zi is the charge number of
that ion, and the sum is taken over all ions in the solution. For a 1:1 electrolyte
such as sodium chloride, the ionic strength is equal to the concentration, but for
MgSO4 the ionic strength is four times higher. Generally multivalent ions
contribute strongly to the ionic strength.

http://en.wikipedia.org/wiki/Ionic_strength
Functional Groups for Ion Exchange Stationary Phases

• The functional groups substituted onto a chromatographic matrix (Table 2) determine the
charge of an IEX medium i.e. a positively-charged anion exchanger or a negatively-charged
cation exchanger.

• The active end of the charged group is the same for DEAE and ANX. The difference between
them is in the length of the carbon chain of the charged group. DEAE has a
diethylaminoethyl-group bound to the agarose. ANX has a diethylaminopropyl-group
attached which prevents the formation of quaternary groups, giving a different selectivity
compared to DEAE.

Ion Exchange Chromatography and

Chromatofocusing Handbook from GE Healthcare
Functional Groups for Ion Exchange Stationary Phases

• The terms strong and weak refer to the extent that the ionization state of the functional
groups varies with pH. The terms strong and weak do not refer to the strength with which
the functional groups bind to proteins. Strong ion exchangers show no variation in ion
exchange capacity with change in pH. These exchangers do not take up or lose protons with
changing pH and so have no buffering capacity, remaining fully charged over a broad pH
range. Strong ion exchangers include Q (anionic), S and SP (cationic).
 Functional Groups for Ion Exchange Stationary Phases
• There are several advantages to working with strong ion exchangers:
– development and optimization of separations is fast and easy since the charge characteristics
of the medium do not change with pH.
– the mechanism of interaction is simple since there are no intermediate forms of charge
interaction.
– sample loading (binding) capacity is maintained at high or low pH since there is no loss of
charge from the ion exchanger.
• The majority of proteins have isoelectric points within the range 5.5 to 7.5 and can be separated on
either strong or weak ion exchangers. An advantage of a weak ion exchanger, such as DEAE
(anionic), ANX (anionic) and CM (cationic), is that they can offer a different selectivity compared to
strong ion exchangers. A disadvantage is that, because weak ion exchangers can take up or lose
protons with changing pH, their ion exchange capacity varies with pH.

Try a weak ion exchanger such as DEAE, CM or ANX

Sepharose Fast Flow, if a strong ion exchanger
(substituted with Q, S or SP) does not give the
required selectivity.
 Affinity Chromatography

• Affinity chromatography makes use of the highly specific molecular recognition of certain
biomolecules. By attaching a specific ligand such as an antigen to the stationary phase
material, the matching antibody can be specifically and reversibly adsorbed. To elute the
target molecule from the affinity medium the interaction can be reversed, either specifically
using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity.
• Affinity chromatography has the highest specificity and selectivity of all chromatographic
methods and is a powerful method for the purification and isolation of biomolecules even at
low concentration.

(A) Steps of affinity chromatography: (B) Adsorption and specific desorption

sample addition, adsorption, washing and elution for affinity chromatography
 Binding, Washing and Elution

• Simple procedure used to perform affinity purification on pre-packed columns. columns may
be used with a syringe, a peristaltic pump or a liquid chromatography system.

Gradient and step elution

 Types of Affinity Chromatography

• Ligands used for affinity chromatography can be divided into mono-specific and
group-specific ligands.
 Application of His-Tag in Protein Purification

10µM imidazole
Binding Mechanism

20-50µM imidazole

Lane 1: Molecular weight

500µM imidazole standards
Lane 2: E.coli lysate
Lane 3: E.coli lysate
flowthrough
Lanes 4, 5: Protein purified
Purification Procedures on Ni-NTA resin
 Size Exclusion Chromatography

• In size exclusion chromatography (or gel filtration for biomolecules separation),

dissolved molecules are separated according to their size. The chromatographic
column is filled with a porous material such as polymeric gel or agarose beads with
diameters of typically 10 to 40 µm.

The principle of size exclusion chromatography: large molecules are un-retained

and eluted first, smaller molecules are retarded by the pores of the stationary phase.
 The Shape of a Molecule Affects its Elution Time

• Solutes of similar molecular weights but different shapes elute with different
retention volumes. Rod-shaped molecules elute more quickly than flexible-coil
molecules, which in turn elute more quickly than spherically shaped molecules
(Figure A).
• Analysis of the levels of dimers and oligomers in a protein sample is possible with
size exclusion chromatography (Figure B).

(A) (B)
 Factors Affect the Resolution of Eluting Proteins

• Bead Size: The smaller the beads, the narrower the peak width and so the better the
resolution.

• Flow Velocity: Excessive high flow velocities lead to incomplete partitioning

equilibrium, resulting in peak broadening, especially for large molecules.

• Column Length: Increasing the column length increases the resolution. However,
doubling the column length does not result in doubling the resolution. Rather, the
increase in resolution will be 2 = 1.414.

• Sample Volume: In SEC, the solute does not become concentrated during sample
application as occurs in ion-exchange chromatography and other adsorptive techniques.
Practically, 0.5% of the column volume is used for nominal 10 µm gels and 2-5% of the
column volume for 100 µm gels. Sample volume can be up to 30% in groups
separations.

• Viscosity: Samples that are much more viscous than the eluent form unstable sample
zones, which quickly become very broad. For optimum results, protein sample
concentrations below 70mg/mL are advised.
 Basic Components of FPLC

• Gel filtration columns are often used together with FPLC. A typical laboratory FPLC
consist of one or two high-precision pumps, a control unit, a column, a detection
system and a fraction collector.
 Pumps: Most SEC columns are used at low pressure (<0.5 MPa) or moderate
pressure (< 2.0 MPa). As comparison, HPLC has about 40 MPa.
 Injection loop: Loop volume can range from a few microliters to 50ml or more.
 Injection valve: A motorized valve which links the mixer and sample loop to the
column.
 Column: normally with size exclusion column or ion exchange column.
 Flow Cells: The effluent from the column passes through one or more flow cells to
measure the concentration of protein in the effluent (by UV light absorption at
280nm). The conductivity cell measures the buffer conductivity, usually in
millisiemens/cm, which indicates the concentration of salt in the buffer.
 Fraction collector: It allows samples to be collected in fixed volumes, or can be
controlled to direct specific fractions detected as peaks of protein concentration,
into separate containers.

So! What is the difference between HPLC and FPLC

 Superdex Size Exclusion Columns

• Superdex (sold by GE healthcare) are gel filtration media with a unique composite
medium of dextran and agarose. Superdex is a composite medium based on highly
cross linked porous agarose particles to which dextran has been covalently
bonded.

Separation examples
 Sephacryl Size Exclusion Columns

• Sephacryl High Resolution (HR) media provide a useful alternative to Superdex

prep grade for applications that require a slightly broader fractionation range.