Chromatography- Theory, FPLC and Beyond.

Biochromatography, the separation of biological molecules, traces back to the

turn of the 20th century, when M. Tswett coined the term "chromatography" to
describe his process of separating colorful leaf pigments in a column made of
chalk (M. Tswett, Proceedings of the Warsaw Society for Natural Sciences,
Section 14, Minute 6, 1903). Liquid chromatography techniques-in which
substances in a moving liquid (the mobile phase) are separated or "partitioned"
between the mobile phase and a stationary phase (usually linked to an inert
matrix in a column)-were under development by the early 1940s and during the
decades that followed (A.J.P. Martin, R.L.M. Synge, Journal of Biochemistry,
35:1358-68, 1941; L.R. Snyder, J.J. Kirkland, Introduction to Modern Liquid
Chromatography, 1st and 2nd eds. New York, Wiley-Interscience, 1974 and
1979).

The early liquid chromatography techniques were performed in large columns

with "soft" (gels of silica or cellulose) supports under atmospheric conditions. As a
result, separations were achieved slowly and were not always well resolved.
Currently, commercially available media for liquid chromatography applications
provide faster, higher-resolution separations for a wide range of applications,
including several for chromatography of such biological molecules as proteins,
amino acids, and DNA.

For purifying proteins, life scientists can use a number of different

chromatography techniques. These include size-exclusion chromatography (in
which molecules are separated according to their size), ion-exchange
chromatography (for charged molecules), and reverse-phase and hydrophobic-
interaction chromatography (which distinguish between molecules based on
hydrophobic interactions between the sample and the stationary phase).

In choosing which type of chromatography method to use, a researcher must

consider the volume and complexity of the starting material, the substance or
substances to be separated, and the level of purification that he or she hopes to
achieve.

Starting with a crude cell extract, for example, may require several
chromatography steps before the desired substance is purified. Devising
a purification scheme for a particular protein may involve more than one
chromatography step. For each step, we need to consider the chemistry of the
molecule being separated and the nature of the chromatography medium
employed in the separation. The type of chromatography also you may wish to
use also depends on the separation. Chromatography columns which have a
high number of theoretical plates can yield a great resolution between protein
bands. However this does come at a price. The more separations per area
requires very small particles for the increased surface area necessary for the
separation. This means higher pressures are required for the column and more
expensive columns and systems (columns of about $800 to $1000 and systems
starting at $25,000).
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General Principles
The basis of all forms of chromatography is the partition or distribution
coefficient. (Kd), which describes the way in which a compound distributes itself
between two immiscible phases. For the most part, all chromatography systems
consist of the stationary phase which for most of biochemical purposes are solids
or gels. The mobile phase is liquid or gaseous. The mobile phase flows over and
around the stationary phase. In practice separating molecules requires that the
various molecules will have a different partition between the two phases.
Examples include:
 Adsorption Equilibrium - hydrophobic chromatography is an example.
Proteins with a high concentration of hydrophobic amino acids or
modified with lipids will often distribute themselves more with the mobile
phase
 Ion Exchange Chromatography – A flexible technique used mainly for the
separation of ionic or easily ionizable species. The stationary phase is
characterized by the presence of charged centers bearing exchangable
counterions. Ion Exchange chromatography is one of the most used and
well defined chromatographic methods.
 Gel Filtration - For this type of chromatography the stationary phase is the
liquid trapped inside of the matrix.
 Affinity Chromatography - is an equilibrium between a stationary
immobilized ligand and a mobile liquid phase.

Factors which influence chromatography

 Theoretical Plates – the

N=1000 number of equilibrations
that a compound makes
with the stationary phase
will increase the separation
of similar but unique
compounds.
Chromatography columns
N=100 are considered to consist
of a number of adjacent
plates or zones where there
is enough space for a
compound to achieve
complete equilibrium
between the mobile and
N=10 stationary phase. Each
zone is called a theoretical
plate and the length of the
column the plate height.
Relative distribution on the column The more theoretical plates
the better the resolution of

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protein. Consider three columns each with a different number of

theoretical plates

 It is important to remember that the plates do not really exist; they are a
figment of the imagination that helps us understand the processes at work
in the column.They also serve as a way of measuring column efficiency,
either by stating the number of theoretical plates in a column, N (the
more plates the better), or by stating the plate height; the Height
Equivalent to a Theoretical Plate (the smaller the better).

 Peak resolution – there are several factors which alter the ability of a
column to separate or resolve proteins. Separation of compounds on a
liquid chromatography column are dependent on the nature of the
solute, the type of chromatography and the method of elution. Take for
example two closely related proteins whose isoelectric points are close.
Using a very steep gradient these proteins will not resolve. However a
shallower gradient which will effect the equilibrium between the solid and
mobile phases to be discriminated between the two proteins can often
increase the separation.
Another factor that will effect resolution is peak broadening. This will
result in overlap of closely eluted samples. Column matrix with large void
volumes leads to preak broadening. The cost of using very small matrix is
increasing back pressure. That is the same kind of thing that happens
when you put your finger over the garden hose.

 Column velocity –
Since the solute
molecules are
carried through the
column by the
mobile phase, it is
natural that its
speed has an
influence on the
process in the
column. A slow
flow rate will
increase the time a
protein is in the
mobile phase and diffusion occurs resulting in peak broadening. On
the other hand a very high flow rate will decrease the interaction of
the protein with the stationary phase and decrease the number of
possible equilibration, resulting in a reduction of theoretical plates
possible. A Van Deemter plot is a plot of plate height vs. average
linear velocity of mobile phase. Such plots are of considerable use in
determining the optimum mobile phase flow rate.

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Additional note: One major problem with pressurising chromatography systems

using liquid solvents is that pressure reductions can cause dissolved gases to
come out
of solution. The two locations where this occurs are the suction side of the pump
( which is not self-priming, consequently a gas bubble can sit in the pump and
flow is reduced ), and at the column outlet ( where the bubbles then pass
through the detector causing spurious signals). Note that the problem is usually
restricted to solvents that have relatively high gas solubilities - usually involving
an aqueous component, especially if a gradient is involved where the
water/organic solvent ratio is changing. As water usually has a higher dissolved
gas content, then a gradient programme may cause the gases to come out of
solution as the mobile phase components mix.

There are three traditional strategies used to remove problem dissolved gases
from chromatographic eluants. Often they are used in combination to lower the
dissolved gases.
a. Subject the solvent to vacuum for 5-10 mins. to remove the gases.
b. Subject the solvent to ultrasonics for 10-15 mins. to remove the gases.
c. Sparge the solvent with a gas that has a very low solubility compared
to the oxygen and nitrogen from the atmosphere. Helium is the preferred
choice - 5 minutes of gentle bubbling from a 7um sinter is usually
sufficient, although maintaining a positive He pressure is even better.
Note that most aqueous-based solvents usually have to be degassed every 24
hours. Also remember that solubility of gases increases as temperature
decreases, so ensure eluants are at instrument temperature prior to degassing.
Helium is preferred as the degassing solvent because it has
relatively low solubility in water, and the solubility is less affected by temperature.

Types of matrixes and chromatographies

Perfusion Chromatography - A chromatography technique that utilizes

convective flow of the liquid phase through the particles of the
chromatographic support is patented by PerSeptive Biosystems Inc. of
Framingham, Mass. It’s use is licensed with the purchase of products in
PerSeptive's POROS chromatography line.

During conventional liquid chromatography, molecules move through the

column by convection of the molecules around and between the particles or
beads that make up the solid support matrix. Transport through the particles'
pores (to the inner surfaces) occurs relatively slowly by diffusion. POROS media
consists of particles with considerable mechanical strength that are engineered
with two classes of pores, which enhances the transport of molecules by
providing convective flow through the beads as well as around them. This allows
chromatography to occur at flow rates 10 to 100 times faster than that of
conventional chromatography media with high capacity and high resolution.

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High Performance- Before the 1970s, samples were passed through a solid phase
that was generally packed into a large-diameter glass column, to maximize the
surface area of the stationary phase. Because the flow of material through the
column was driven by gravity, separations were generally slow and poorly
resolved. By shrinking the column size and applying pressure,
biochromatographers achieved faster separations with better resolution. "Adding
pressure prevents the sample from diffusing throughout the column as it passes
through, and this improves the resolution of the separation.

From the cumbersome liquid chromatography techniques of the 1960s evolved

low-pressure (LP) liquid chromatography in the latter part of the decade,
followed by high-performance liquid chromatography (HPLC) in the late 1970s.
With these methods, pressure is applied to the column from a peristaltic pump
(LP) or a more powerful pressure-delivery system such as a dual-piston pump
(HPLC), which forces the mobile phase through the column at a much greater
flow rate than simple diffusion alone. Because faster flow rates lead to quicker
separations with better resolution, liquid chromatography performed under
pressure is a powerful analytical and preparative tool for chemists and life
scientists.

In both LPC and HPLC, molecules are separated through chromatographic

columns of uniformly sized microparticles packed into small columns or
cartridges. "For HPLC, the limiting factor for many years was the chromatography
media, because the support matrix must be rigid enough to withstand the
applied pressure and still maintain the desired chromatographic qualities.

Many of the media currently available for chromatography are suitable for both
LPC and HPLC applications. Firms such as Pierce Chemical Co. of Rockford, Ill.,
and Bio-Rad Laboratories in Hercules, Calif., sell chromatography media for HPLC
prepacked in cartridges that are easy to install in a chromatography instrument.
Pierce's Cartridge Column System allows researchers to select the size of the
cartridge to optimize separation speed and resolution. Shorter cartridge columns
reduce analysis time, while longer columns provide higher resolution
(along with a longer separation time). Pierce's Aquapore Columns for reverse
phase and ion-exchange chromatography were designed specifically for the
HPLC separation of proteins, peptides, and other polyfunctional
macromolecules.

Bio-Rad's prepacked Econo-Pac cartridge columns are available in 1 ml or 5 ml

volumes and come filled with a variety of supports. Up to three cartridges can be
connected in tandem to triple the system's sample capacity. For researchers in
the first stages of a purification who need to optimize their separation schemes,
Bio-Rad sells Protein Purification Sampler Packs. These contain five different
media cartridges (included are three ion-exchange cartridges, a hydrophobic
interaction cartridge, and a cartridge containing HTP hydroxyapatite). With the
Sampler Pack, an investigator can perform five different test runs on a sample in
a single day.

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Automated Separations - The separating power of LPC and HPLC are enhanced
with the addition of instruments that automate steps and control the
chromatographic runs. Integrated systems consisting of a pressure-generating
pump, the chromatography column (or columns), a friction collector and
monitor to collect and analyze substances as they elute from the column, and a
computerized control system can be purchased for separations by LPC and
HPLC. The Econo System from Bio-Rad is an integrated system designed for the
purification of milligram quantities of proteins and nucleic acids by low-pressure
chromatography. The fully automated version of the Econo System is a totally
hands-off system, with everything, including sample loading and fraction
collection, under the control of the computer.

Fast Performance Liquid Chromatography- Liquid chromatography is a term

which refers to all chromatographic methods in which the mobile phase is liquid.
The stationary phase may be a liquid or a solid. Fast performance liquid
chromatography (FPLC) is a type of liquid chromatography where the solvent
velocity is controlled by pumps. The pumps control the constant flow rate of the
solvents. The solvents are accessed through tubing from an outside resevoir. The
flow rate of the solvent is set through computer input and controlled by pumps.
There are various columns used in liquid chromatography depending on the
type of separation preferred. Each column contains a small diameter packing
material. The column is a large (mm id) tube containing small (u) particles (gel
beads) known as stationary phase. The chromatographic bed is composed by
the gel beads alone when they are inside the column. The sample is introduced
into the injector and then carried into the column by the flowing solvent. Once in
the column, the sample mixture separates as a result of different components
adhering to or diffusing into the gel. As the solvents is forced into the
chromatographic bed by the flow rate, the sample separates into various zones
of sample components. These zones are referred to as bands.

HIGH RESOLUTION: Bio-Rad's

BioLogic System, a high-pressure
chromatography device. Bio-
Rad's BioLogic System is a high-
pressure chromatography
instrument with Micosoft Corp.'s
Windows-based software that
features pull-down menus and
intuitive icons that simplify
programming and system
operation. The BioLogic System
supports a wide range of
chemistries for chromatography,
including ion-exchange, hydro- phobic-interaction, size-exclusion, reverse-phase,
and affinity chromatography. Bio-Rad's HPLC Series 5000 System is a complete
HPLC unit. Pharmacia Biotech Inc. of Piscataway, N.J., manufactures the FPLC
(Fast Protein Liquid Chromatography) System for both manual and automatic
biochromatography applications. With this system, researchers can program

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multi-step purification schemes into the instrument to control parameters such as

flow rate, gradient formation, and fraction collection. The automated FPLC
System handles both small and large sample volumes and enables fast, high-
resolution separations.

FROM PHARMACIA: FPLC (Fast Protein

Chromatography System

Pharmacia's SMART System is a

micropurification chromatography system
that recovers and purifies biologically active
material present in
a sample at very low concentrations (from
micrograms to picograms). The system
consists of specially designed instruments,
software, and
prepackaged columns to purify biomolecules for applications such as peptide
sequencing and protein structure/function studies.

Beckman Instruments Inc. has introduced the BioSys 500 and 510 protein-
purification systems, which are the first in a series of workstations being
developed by Beckman for bioseparation applications. Unlike the FPLC, the
BioSys 500 series instruments can be operated with both low-pressure and high-
pressure chromatography columns.

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