In the name

of
God

Size Exclusion
Chromatography
Presented by

Esmael Moradi
Kambiz Ahmadi
Reza Shabankare
Peyman Asefi
Farshid Fasihi
Dr. A. Golshan

Discovery
The technique was invented by Grant Henry and Colin C
Ruthven in London. They used starch gel as matrix.
In 1964 J.C.Moore published his
work on the preparation of
Gel permeation chromatography
columns based polystyrene
with controlled pore size

Background
Size exclusion chromatography is used primarily
for analytical assays and semi-preparative
purifications

It is not commonly used for process scale work

due to the low capacity of the size exclusion mode

SEC chromatography

Stationary phase

Mobile phase

Introduction
Size-exclusion chromatography (SEC) is a
chromatographic method in which molecules in solution
are separated by their size, and in some cases molecular
weight
It is usually applied to large molecules or macromolecular
complexes such as proteins and industrial polymers

Fig 1. A size exclusion column

Theory background
The medium is a porous matrix in the form of spherical
particles that have been chosen for their chemical and
physical stability, and inertness
When an aqueous solution is used as mobile phase the
technique is known as gel filtration
When an organic phase is used as mobile phase the
technique is known as permeation chromatography

DONT CONFUSE! Gel filtration by Gel electrophoresis

where an electric field is used to pull or push molecules
through the gel depending on their electrical charge .

Mechanism of action
samples that contain few components or partially
purified by other chromatography techniques will
give the best result
Single buffer system, packed bed(chemically and
physically stable and inert), pore size in stationary
phase separates proteins according to their
molecular weight

Basic of size exclusion

chromatography

Fig 4. Schematic of a size-exclusion chromatography column

Fig.2 Typical chromatogram of a group separation

REMEMBER THAT

To have a good resolution there has to be

10% difference in molecular mass

Less than 10% of molecular weight the peaks will overlap

Principle

One requirement for SEC is that the analyte does not interact with
the surface of the stationary phases
Differences in elution time are based solely on the volume the
analyte
The underlying principle of SEC is that particles of different sizes will
elute (filter) through a stationary phase at different rates. Particles of
the same size should elute together

Molecules larger than the pore size can not enter the
pores and elute together as the first peak in the
chromatogram
Molecules that can enter the pores will have an average
residence time in the particles that depends on the
molecules size and shape
Different molecules therefore have different total transit
times through the column
Molecules that are smaller than the pore size can enter all
pores, and have the longest residence time on the
column and elute together as the last peak in the
chromatogram

A believable example!

Analysis
The collected fractions are often examined by
spectroscopic techniques to determine the concentration
of the particles eluted
Common spectroscopy detection techniques are
refractive index (RI) and ultraviolet (UV)
Columns are ohten calibrated using 4-5 standard
samples ( protein of known molecular mass)

Fig. 5. Theoretical chromatogram of a high resolution

fractionation (UV absorbance)

Commercially avaiable columns

The typical column diameters are 7.58mm for analytical
columns and 2225mm for (semi)preparative columns; usual
column lengths are 25, 30, 50, and 60 cm
The packings are based on either porous silica or semirigid
(highly crosslinked) organic gels, in most cases copolymers
of styrene and divinylbenzene
For example: TSKgel GFC columns for protein analysis (TSKgel SWtype columns are silica-based)
125 pore size for analysis of small proteins and peptides
250 pore size for most protein samples
450 pore size for very large proteins and nucleic acids

Commercially available columns and properties:

Product
Superdex Peptide
Superdex 75
Superdex 200
Superdex 30 prep grade
Superdex 75 prep grade
Superdex 200 prep grade

pH stability
Long term: 114
Short term: 114
Long term: 312
Short term: 114
Long term: 312
Short term: 114
Long term: 312
Short term: 114
Long term: 312
Short term: 114
Long term: 312
Short term: 114

Particle size
1315 m
1315 m
1315 m
2244 m
2244 m
2244 m

Superdex 200 - the molecular weight of the protein of interest is unknown

Superdex 200 or Superdex 200 prep grade - especially suitable for the
separation of monoclonal antibodies from dimers and from contaminants
of lower molecular weight

Advantages

Unlike ion exchange or affinity chrom. molecules do

not bind to the medium so buffer composition does
not directly affect resolution
is well suited for biomolecules that may be sensitive to
changes in pH, conc. of metal ions or co-factors and
harsh environmental conditions
conditions can be varied to suit the type of sample or
the requirements for further purification, analysis or
storage without altering the separation
Can be used after any chrom. tech. bcz components of
any elution buffer will not affect the final separation

Disadvantages

Scale of chromatogram is short and a

limited number of bands can be
accommodated.

to have a good resolution there has to be

10% difference in molecular weight

Size-exclusion chromatography with

organic carbon detection
using a mass spectrometer

BenWarton, Anna Heitz, Bradley Allpike, Robert Kagi

Curtin Water
Quality Research Centre, Centre for AppliedOrganic Geochemistry and CRC for Water
Quality and Treatment,Department of Applied Chemistry, Curtin University of
Technology, GPO Box U1987, Perth, WA 6845, Australia

Introduction
Size-exclusion chromatography (SEC) is an important and
widely used technique for studying dissolved organic carbon
(DOC) present in aquatic environments. The molecular size profile
(expressed as apparent molecular weight (AMW)) is useful for
comparing DOC in a variety of situations, including different water
sources containing different organic matter inputs, and different
drinking water treatment processes (e.g. [1,2]). The technique has
many practical advantages, in that minimal sample preparation
is needed, the sample volumes required are small (<2 mL), and
analysis times are relatively short (2090 min). A major limitation
of conventional SEC analysis is that generally only ultraviolet
(UV) absorbance detection has been used: these detectors are not
quantitative for organic carbon in natural waters because different
chemical functionalities within the organic carbon structure give
different signal responses.

Water Samples
Two raw surface water samples were
collected for SEC analysis
with quantitative organic carbon detection:
Quickup Dam
(35mgL1 DOC) and Harris Dam
(3.6mgL1 DOC) are both in the
south-west of Western Australia.

Results and discussion

The sensitivity of the instrument using the mass spectrometer
as the detector for organic carbon (as CO2) was compared
with the sensitivity of the instrument when the lightepipe FTIR was
used to detect the evolved CO2.
spectrometer was calibrated by analysing a potassium hydrogen
phthalate solution (1mgL1 as C) with five injection volumes from
20L to 500L. This equated to masses of organic carbon injected
onto the column of 20 ng to 500 ng, respectively. The calibration
curve of peak area versus mass of C injected (ng) showed a high
degree of linearity (R2 = 0.9995), and the trendline equation was
y = 0.0164x. The limit of detection (LOD) and limit of quantification
(LOQ) (also known as limit of determination) were also calculated
using the results of 10 blank (ultra-pure laboratory water) injections
and the calibration results described above.

The LOD was

calculated by adding the mean of the area of the noise to three
times the standard deviation of this noise and converting this
peak area to concentration using the line of best fit determined
by calibration [8]. Using this method, the LOD was calculated as
6.5 ng, equivalent to 3.6gL1 at the instruments maximum injection
volume of 1800L. The LOQ, calculated similarly, but using
ten times the standard deviation of the noise [8], was 22.8 ng,
equivalent to 12.7gL1 at the instruments maximum injection
volume of 1800L. These values were substantially lower than
those calculated for the same instrument when a lightpipe FTIR
spectrophotometer was used as the DOC (as CO2) detector [3]. In
this case the values calculated using the same method [8] were
31 ng for the LOD and 68 ng for the LOQ. The lower LOD and LOQ
values for the MS demonstrate its increased sensitivity over the
lightepipe FTIR as a post-SEC organic carbon detector in this instrumental
setup

Fig. 2. AMWprofiles ofwater fromHarris Dam,Western Australia (DOC 3.6mgL1),

utilising MS (thick line) and FTIR (thin line) to detect CO2 produced upon oxidation
of the organic matter in the water sample.

Fig. 1. AMW profiles of water from Quickup Dam, Western Australia (DOC
35mg L1), utilising MS (thick line) and FTIR (thin line) to detect CO2 produced upon
oxidation of the organic matter in the water sample.

Conclusions
The instrument using the mass spectrometer as the DOC (as
CO2) detector was calibrated by analysing a potassium hydrogen
phthalate solution (1mgL1 as C) and values for LOD and LOQ
were calculated. These values were substantially lower than those
calculated for the same instrument with a lightpipe FTIR spectrophotometer
and demonstrate its improved sensitivity over the
lightpipe FTIR as an organic carbon detector for SEC. The SEC AMW
profiles of raw water samples derived from using the mass spectrometer
were similar to those produced by the same instrument
using the lightpipe FTIR detector. The S/N ratios for both of the
AMW profiles were calculated and the MS response had a greater
S/N ratio, providing further evidence that this is a more sensitive
detector than the FTIR in this application. In addition, this study
shows that MS can be readily coupled with the SEC-organic carbon
detection system to analyse the evolved CO2

Thanks
For Your
Attention