In the name of God

Size Exclusion Chromatography

Presented by

Esmael MoradiKambiz AhmadiReza ShabankarePeyman AsefiFarshid 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

IntroductionSize-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

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

Theory background

DON’T 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

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

Principle

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 7.5–8mm for analytical columns and 22–25mm 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 SW-type columns are silica-based)

125Å pore size for analysis of small proteins and peptides 250Å pore size for most protein samples 450Å pore size for very large proteins and nucleic acids

Product pH stability Particle sizeSuperdex Peptide Long term: 1–14

Short term: 1–1413–15 μm

Superdex 75 Long term: 3–12Short term: 1–14

13–15 μm

Superdex 200 Long term: 3–12Short term: 1–14

13–15 μm

Superdex 30 prep grade Long term: 3–12Short term: 1–14

22–44 μm

Superdex 75 prep grade Long term: 3–12Short term: 1–14

22–44 μm

Superdex 200 prep grade Long term: 3–12Short term: 1–14

22–44 μm

Commercially available columns and properties:

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 (20–90 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

(35mgL−1 DOC) and Harris Dam (3.6mgL−1 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 (1mgL−1 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 wascalculated by adding the mean of the area of the noise to threetimes the standard deviation of this noise and converting thispeak area to concentration using the line of best fit determinedby calibration [8]. Using this method, the LOD was calculated as6.5 ng, equivalent to 3.6gL−1 at the instrument’s maximum injectionvolume of 1800L. The LOQ, calculated similarly, but usingten times the standard deviation of the noise [8], was 22.8 ng,equivalent to 12.7gL−1 at the instrument’s maximum injectionvolume of 1800L. These values were substantially lower thanthose calculated for the same instrument when a lightpipe FTIRspectrophotometer was used as the DOC (as CO2) detector [3]. Inthis case the values calculated using the same method [8] were31 ng for the LOD and 68 ng for the LOQ. The lower LOD and LOQvalues for the MS demonstrate its increased sensitivity over thelightepipe FTIR as a post-SEC organic carbon detector in this instrumentalsetup

Fig. 2. AMWprofiles ofwater fromHarris Dam,Western Australia (DOC 3.6mgL−1),utilising MS (thick line) and FTIR (thin line) to detect CO2 produced upon oxidationof the organic matter in the water sample.

Fig. 1. AMW profiles of water from Quickup Dam, Western Australia (DOC35mg L−1), utilising MS (thick line) and FTIR (thin line) to detect CO2 produced uponoxidation of the organic matter in the water sample.

ConclusionsThe instrument using the mass spectrometer as the DOC (as

CO2) detector was calibrated by analysing a potassium hydrogen

phthalate solution (1mgL−1 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