Theory of Ion Chromatography from Metrohm

There are many different ways of determining ions qualitatively and

quantitatively. One such technique that is widely used is ion
chromatography.

Ion chromatography is one member of the large family of

chromatographic methods. It is used to determine all ions which carry one or two
charges. In the past ion chromatography (IC) was a very expensive method but today
it is much more favorably priced, thanks to the quality Compact IC's available from
Metrohm.

Which Ion Chromatography Column?

There are many important fields of application today for ion chromatography such
as:

1. the routine investigation of aqueous systems such as drinking water, rivers,

effluents and rain water.
2. for the analysis of ions in chemical products, foods, cosmetics, pharmaceuticals
etc
3. ultratrace analysis such as in the semi-conductor and power industry.

Ion Chromatography can be used for the analysis of anions, cations, organic acids and
amines plus analytes such as carbohyrates

Schematic of an Ion Chromatography System

The above schematic represents a non-suppressed ion chromatography system. The

sample is introduced onto the system via a sample loop on the injector. When in the
inject position the sample is pumped onto the column by the eluent and the sample
ions are then attracted to the charged stationary phase of the column. The charged
eluent elutes the retained ions which then go through the detector (which is most
commonly conductivity) and are depicted as peaks on a chromatogram.

Three main modes of Ion Chromatography Columns.

The different modes of chromatography (anion exchange, cation exchange and ion
exclusion) simply relate to the different types of columns used to achieve the
separation of the ions. The eluent used depends on the column type and also the
mode of detection - however unless stated the following is all based on conductivity
detection.

Ion exchange

Ion exchange chromatography (IC) is based on a stoichiometric chemical reaction

between ions in a solution and the oppositely charges groups functional groups on the
column resin. In the simplest case in cation chromatography these are sulfonic acid
groups or carboxylic acid groups (such as maleic acid) and in anion chromatography
quaternary ammonium groups.

Anion exchange

Anion exchange chromatography forms the largest group of IC methods mainly

because there are few alternatives with such simplicity, sensitivity or selectivity -
particularly for sulphate. The two forms are anion exchange with or without
suppression and of these two suppressed methods are the most widely used. Eluents
for suppressed chemistries tend to be either carbonate based or hydroxide.

chromatogram showing an anion exchange separation followed by direct

conductivity detection (non-suppressed)

Suppression in Ion Chromatograph

Often, a device called a suppressor is used and is placed between the column and
detector as shown above. When suppression is used the detector is almost certainly
conductivity.The chromatogram below shows a sample with a suppressor unit placed
between the column and detector. The greatest achievment of suppression is to
increase the sensitivity of the anion, however at the same time the background
conductivity of the eluent is greatly reduced. The same suppressor units can also be
used to increase the sensitivity of organic acids.
Chromatogram showing an anion exchange separation followed by suppression and
then conductivity detection

The suppressor used in anion chromatography is simply a cation exchanger and its job
is to remove cations and replace them with an H+. So a sodium carbonate eluent
(~800uS) would be converted to carbonic acid (~18uS) by the suppressor and the
analyte, for example NaCl (~126uS without suppression) would become HCl (~426uS
with suppression). The ways in which this can be done are varied but the two common
ways are as follows:

1: The Metrohm MSM (Metrohm Suppressor Module)

contains 3 separate suppressor units. At any one time, one
will be in-line with the eluent and conductivity detector,
one will be in-line with dilute sulphuric acid (replacing the
removed cations with H+) and the third is washed with
water. The benefits of this technique are lack of baseline
noise and a ruggedness that is reflected in the fact that the MSM comes with a ten
year warranty and is not, for example, adversely affected by the transition metals
which can cause precipitation problems for other types of suppressor technology.

This suppressor module is present in all the Metrohm Compact ICs (792 Basic, 790
Personal, and 861 Compact) and is available in the modular system as the 833
Advanced IC Liquid Handling Unit.

The anion chemical suppression can be taken a stage further with the new 853 MCS
instrument (fitted inline after the MSM) which removes carbon dioxide from the
suppressor reaction and carbonate from the sample, which means that the sodium
carbonate/sodium bicarbonate mobile phase is converted to water instead of carbonic
acid so a background conductivity approaching 1uS is achieved. The 853 MCS can be
fitted to all Modular and 761/861 Compact IC’s and the benefits include no injection
peak, no system peak, superb linearity and an enhancement in the peak areas
allowing lower limits of detection to be achieved.
2. The Metrohm Dual Suppressor is a continuous suppression device which removes the
cations and replaces them with H+ (which is provided by electrolysis of water) so with
a carbonate eluent it forms carbonic acid. The Dual Suppressor then reduces the
conductivity of the carbonic acid (~18uS) by removing it to leave water (~1uS). As the
concentration of the eluent increases throughout the gradient, the baseline rise is a
result of the increasing conductivity of the eluents suppression product. The
importance of this is that carbonate eluents can now be used for gradient elution of
anions which means more versatility, no system peak, less corrosive eluents and no
gases required.

Schematic of the Metrohm Gradient System with two 818 Advanced IC Pumps
forming the high pressure gradient pump (P1 and P2) and the 828 IC Dual
Suppressor.

Chromatogram showing gradient elution with carbonate eluents.

Cation Exchange

There is a variety of cation columns available, however the modern ones contain
carboxylic acid functional groups. A large number of applications for silica-gel-based
ion exchangers exist. These columns allow simultaneous separation of alkali metals
and alkaline earths plus the separation of transitional metal and heavy metal ions is
also possible. Small amines can also be analysed using cation exchange columns.
 Chromatograms showing cation exchange
with two different tartaric acid/dipicolinic acid eluents

The eluents used for non-suppressed cation exchange are weak acids with a
complexing agent such as dipicolinic acid, the concentration of which can effect the
elution of calcium and heavy metals such as iron, zinc and cobalt.

Cations become less sensitive when suppressed and so are analysed with direct
conductivity detection which also allows heavy metals to be analysed as shown above.

Ion exclusion

Ion exclusion chromatography (IEC) is mainly used for the separation of weak acids or
bases. The greatest importance of IEC is for the analysis of weak acids such as
carboxylic acids, carbohydrates, phenols or amino acids.

Chromatogram showing ion exclusion

with an acid eluent

For a more detailed explanation of the theory of ion chromatography and detection
see the Metrohm Monograph 'Practical Ion Chromatography' available free of charge
from Metrohm UK. HYPERLINK enquiry@metrohm.co.uk

For compact ion chromatography units containing all components see 792 Basic IC,
790 Personal IC and 861 Compact IC. For modular IC see 819 Advanced IC
Detector, 818 Advanced IC Pump, 833 Advanced IC Liquid Handling Unit and 820
Advanced IC Separation Centre. For on-line IC see the 811 Online IC and the 821
Compact Online IC.

Other modes of Detection

Amperometric detection (see 791 IC-VA Detector and 817 Bioscan)

(Amperometric detection (see 791 IC-VA Detector and 817 Bioscan)

In principle voltammetric detectors can be used for all compounds which have
functional groups which are easily reduced or oxidized. The amperometric detector is
the most important version. Amperometry is very sensitive. Apart from a few cations
(Fe3+, Co2+) it is chiefly anions such as nitrite, nitrate, thiosulfate as well as halogens
and pseudo-halogens which can be determined in the ion analysis sector. The most
important applications lie, however, in the analysis of sugars by anion
chromatography and in clinical analysis.

Photometric detection (see UV-Vis Spectrophotometer, 844 Compact UV-

Vis and Post Column Reactor)

Chromatogram showing analysis of 1ppb chromate using the 844 Compact UV-Vis

Because of its extremely wide range of application photometric or UV/VIS detection is

the most important detection method used in HPLC, as many organic molecules
contain chromophore groups, or can have one introduced or added, which are able to
absorb in the UV or VIS spectrum. In the field of inorganic ion analysis UV/VIS
detection plays a smaller role. While of the simple anions only analytes such as
nitrate, bromide or iodide absorb, important analytes such as fluoride, sulfate or
phosphate can only be measured indirectly. Many cations do not absorb at all, but
multivalent and transitional metals in particular can be converted in a post-column
derivatization with chelate formers such as 4-(2-pyridylazo)-resorcinol (PAR) or Tiron
to form colored complexes. Redox-active analytes such as bromate and other
oxohalide ions can be analyzed by UV/VIS detection after undergoing a post-column
reaction with an electrochemically active indicator.
Sample Preparation Techniques for Ion Chromatography

Introduction to Sample Preparation Techniques

Quite often with problematic ion chromatography applications, the matrix of the
sample makes it difficult to accurately quantify the species of interest with the
standard ion chromatography set-up and some form of sample preparation then
becomes necessary.

Schematic Diagram of Standard Ion Chromatography Set-up

The sample preparation may be as straightforward as simply diluting the sample with
deionised water or can involve injection of the sample through a solid phase
extraction cartridge to remove the interference. In the case of more difficult forms of
sample matrices it may be necessary to add additional dedicated sample preparation
modules to the standard ion chromatography configuration.

What is Ion Chromatography?

Chromatography is a method for separating mixtures of substances using two phases,

one of which is stationary and the other mobile moving in a particular direction.
Chromatography techniques are divided up according to the physical states of the two
participating phases. The term Ion Exchange Chromatography or Ion Chromatography
(I.C) is a subdivision of High Performance Liquid Chromatography (H.P.L.C).
A general definition of ion chromatography can be applied as follows;” ion
chromatography includes all rapid liquid chromatography separations of ions in
columns coupled online with detection and quantification in a flow-through
detector”.

A stoichiometric chemical reaction occurs between ions in a solution and a solid

substance carrying functional groups that can fix ions as a result of electrostatic
forces. For anion chromatography these are quaternary ammonium groups and for
cation chromatography sulphonic acid groups. In theory ions with the same charge can
be exchanged completely reversibly between the two phases. The process of ion
exchange leads to a condition of equilibrium, the side to which the equilibrium lies
depends on the affinity of the participating ions to the functional groups of the
stationary phases.

Different Types of Sample Preparation Techniques Employed

Dilution of the Sample

Dilution of the sample is performed when the concentration of the analytes of

interest either exceed the working capacity of the separation column chosen, or there
are sample matrix effects that can often be minimised by a dilution usually with
water but eluent can also be used.

Filtration of the Sample

It is recommended to filter all samples prior to injection with 0.45mm filters to

ensure that any particulate material from the samples don’t make their way onto the
injection valve or the analytical column where they can cause blockages and
considerably reduce the lifetime of the column(s).

Solid Phase Extraction Cartridges

Passage of the sample through one or more solid phase extraction cartridges prior to
injection will often retain selectively certain species within the homogeneous sample.
Quite often the retained species are substances that would interfere with the
chromatography had they not been previously removed. There are a number of
different cartridges whose suitability depends upon the type of chemistry undertaken.

For anion analysis, the sample can be treated with a cation exchanger in the H+ form
that removes divalent cations that can mask any fast eluting anions. This type of
exchange cartridge removes carbonate/bicarbonate and is also useful for the removal
of cations from samples being determined by ion exclusion chromatography. Another
option is the use of a cation exchanger in the Ag+ form for the removal of any halides
present in the sample.

Similarly for cation analysis, one can employ an anion exchanger in the OH- form to
remove any interfering anions present in the sample.
Another common type is the non-polar exchange cartridge (reversed phase) that often
utilises C18 groups to remove organic substances that would otherwise interfere with
the chromatography.

Digestion of the Sample

If digestion techniques are to be employed then analyte content should be changed as

little as possible and any organic matter present should ideally be destroyed
completely. One can obtain analytical inaccuracies due to an exaggerated blank value
as a result of the chemicals used during the digestion. Different types of digestion
include wet, microwave and UV, the suitability of each depends on both the sample
matrix and the analytes of interest being determined.

Instrumental Sample Preparation Modules

Often with more complex sample matrices, one has to add additional dedicated
sample preparation modules to the standard ion chromatography configuration. There
are a number of different instrument options available within the Metrohm range
depending on the type of sample treatment required prior to analysis. Metrohm has
actually been a pioneer of inline sample preparation modules with the release of the
754 IC Dialysis Unit in 1997, since then the technology has been optimised and
considerably improved so that today the Metrohm IC portfolio contains many different
sample preparation instruments.

833 Advanced IC Liquid Handling Dialysis Unit (2.833.0040)

The Metrohm 833 Dialysis Unit is a module for

online sample preparation in ion chromatography
permitting the use of automatic sample dialysis
directly before sample injection. It consists of a
dual channel peristaltic pump for conveying the
sample and acceptor solutions and the actual
dialysis cell in which the ions from the flowing
sample solution are enriched in the resting
acceptor solution. The 833 Dialysis Unit is strongly
recommended for demanding applications that
contain strongly loaded samples. Dialysis is the
diffusion of ions from a sample solution into an acceptor solution to achieve a
concentration equilibrium. The sample solution is constantly being renewed resulting
in no depletion of the sample. The ions pass through the membrane without hindrance
but larger sample particles – the sample matrix - are transported past the membrane
to waste reducing matrix effects to an absolute minimum. Calibration of the
standards is easily done requiring no extra outlay. The reported dialysis recovery rates
have been found to be in excess of 98% using the patented stopped flow method.
838 Advanced IC Ultra-Filtration Sample Processor (2.838.0210)

The aim of sample filtration is to protect the

separation columns from contamination and
blockage from particulates that may be present in
the sample. The 838 IC Ultra-Filtration sample
processor combines inline filtration with automatic
sample injection through the use of an ultra-
filtration cell. It is eminently suitable for those
samples with a light to medium load such as surface
waters and digestion solutions.

The samples are placed onto the sample carousel

before being processed automatically. Sample
filtration and introduction to the injection valve is
achieved by means of an integrated double channel
peristaltic pump meaning that it is possible to
aspirate slightly viscous samples.

The sample is conveyed by one channel of the pump

through the ultra-filtration cell passing the
membrane. At the same time the filtrate is aspirated off from the rear of this
membrane and transferred to the sample loop by the second channel of the pump.
Only a small fraction of the sample is removed as filtrate so the contaminants remain
mainly in the sample stream preventing the regenerated cellulose membrane from
becoming blocked too quickly.

833 Advanced IC Liquid Handling Sample Preparation Unit (2.833.0030)

Picture of Metrohm 833 Advanced IC Liquid Handling Sample Preparation Unit

Inline sample preparation is rapidly becoming the method of choice for eliminating
difficult sample matrices in ion chromatography and Metrohm has developed the 833
Advanced IC Liquid Handling Sample Preparation Unit based upon its Metrohm
Suppressor Module (M.S.M) for difficult anion analyses such as those found in
concentrated alkaline solutions. The modules consists of a reactor block that houses
the cation exchangers with a control unit that contains a two channel peristaltic
pump that conveys the regenerant and rinse solutions.
Schematic Diagram of Inline Sample Preparation

The matrix elimination occurs inline whilst the regeneration and rinsing of the packed
bed suppressor occur simultaneously offline. A fresh suppressor channel is used for
each new analysis and because the rinse and regeneration occurs after each
determination, the capacity of the 833 is unlimited.

The sample solution is transferred to the 833 module from an autosampler via a loop
injection and rinsed with deionised water. The sample cations are exchanged against
protons (H+). If sodium hydroxide constitutes the sample matrix, water is formed by
neutralisation. The sample solution then passes onto the preconcentration column
where the trace anions to be determined are retained and then eluted by the eluent
flowing in a counter flow direction. The analyte anions are then separated on the
analytical column before quantification using chemical suppression with conductivity
detection.
Example Chromatogram Showing Matrix Elimination upon a Sample of Caustic Soda
for the Determination of Chloride, Chlorate and Sulphate

Metrohm Inline Sample Preparation (MISP)

With Metrohm it is possible to perform the time consuming sample

preparation inline using the 838 Advanced IC Sample Processor
equipped with MISP technology. The 838 comes in a number of
different variants that mean it is possible to dialyse, ultra-filtrate
or even dilute the sample automatically inline.

Located on the side of the tower is the relevant sample

preparation technology for example an ultra-filtration cell or an
injection valve which is utilised for dilution along with the proven
Dosino™ liquid handling technology.

The new Range of Anion Columns from Metrohm include:-

The disinfection by-products generated in water

processing plants are suspected to be not only a health
hazard but could even be carcinogenic. For this reason
the oxo halides, above all bromate that is generated
from bromide during the ozone-treatment of drinking
water, have become the object of many investigations
and standard methods (e.g., EPA 300.1 Part B, EPA
317.0). Metrohm now presents a high-performance
separation column for the simultaneous determination
of the standard anions, the oxo halides and
dichloroacetic acid.

With the Metrosep A Supp 7 these ions can be determined reliably and precisely
down to lower ppb range. The outstanding detection sensitivity is obtained by
applying the 5-µm polyalcohol polymer, which yields extremely high plate numbers
and accordingly excellent separation and detection characteristics. The separations
require a temperature of 45 °C.

The high-capacity Metrosep A Supp 8 – 150 allows the

determination of nitrite, bromide and nitrate in
concentrated salt solutions. UV detection at 215 nm opens up the determination of
concentrations in the one-digit ppb range. A special sodium chloride eluent is used for
these applications.

The Metrosep Dual 4 columns contain an entirely

novel carrier material, namely a functionalized
monolith based on silica. This monolith allows flow
rates of up to 5 mL/min. Even at these high flow
rates, the column's counter-pressure remains small.
Compared to conventional materials, the monolith
with its structure made up of macro- and mesopores
has a much larger surface area. This contributes to the
high capacity of the Metrosep Dual 4 column, whose
dead volume is very low.
The high-capacity Metrosep Dual 4 – 100 column is the separation column of choice for
the detection of very small amounts of toxic perchlorate. It allows to determine 0.5
ppb perchlorate in the presence of a total of 3 g/L of chloride, carbonate and sulfate.
The Dual 4 – 100 also easily achieves the base-line separation of chloride and nitrite
present at a concentration ratio of 1000 to 1.

The Metrosep A Supp 10 – 100 separation column is

based on a high-capacity polystyrene/divinylbenzene
copolymer having a particle size of only 4.6 µm.
Metrohm has optimized this time-tested column
concept, which is characterized by its robustness, high
selectivity and excellent separation performance. By
variation of temperature, flow rate and eluent
composition, the column characteristics can be
adapted to the application at hand.
The A Supp 10 – 100 is the column of choice for routine
applications. Thanks to its robustness, excellent price-performance ratio and very
good separation performance, combined with moderate run times, the A Supp 10 –
100 is a universally applicable anion separation column.

Dialysis – An Overview

Dialysis is a successful method for the separation of low molecular substances from
high molecular ones by means of a semi-permeable membrane and is used on patients
with kidney deficiencies.

Low molecular substances in the blood refer to ions that disturb the electrolyte
balance. As the kidneys can not functioning correctly, then the concentrations of
these ions increases impairing the metabolic functions. The concentrations of these
ions must be reduced at frequent intervals and this is achieved by continuous flow
dialysis.

An acceptor solution of a low ionic strength (usually deionised water) is pumped along
the semi-permeable membrane with the blood flowing past on the other side. As the
ions pass through the membrane virtually unhindered they diffuse from the high
molecular strength blood into the low ionic strength acceptor solution. The acceptor
solution is permanently renewed in its continuous flow ensuring a steep concentration
gradient with relatively large efficiency. Deliberate care is taken to ensure that no
concentration equilibrium can be established between the two solutions.

Metrohm has developed the patented stopped flow method for dialysis where the
sample solution is continuously pumped past a semi-permeable membrane but the
acceptor solution lies at rest and here lies the inherent difference from continuous
flow dialysis. This ensures that equilibrium is attained between the sample solution
and the acceptor solution usually in less than 10 minutes. Once the equilibrium has
been set up the dialysed acceptor solution is transferred to the sample loop and
injected onto the separation column.

Diagram Illustrating Stopped Flow Dialysis Technique

Applications of Dialysis

The benefit of dialysis is that there are no complicated, time consuming sample
preparation steps such as digestion that can potentially destroy the analytes of
interest. This is particularly applicable to foodstuffs and other complex matrices that
carry high organic loads such as waste waters or soil eluates.

In the food industry the ionic contents of milk and other diary products can easily be
determined using an ion chromatography system incorporating a dialysis module. It is
no longer necessary to separate the proteins from the milk using Carrez precipitation
ensuring that the sample preparation is reduced to a simple dilution step. Other
difficult matrices include the analysis of fruit juices that contain fruit pulp, cutting oil
emulsions and inks, with dialysis these types of samples no longer represent a
problem for the ion chromatography user.

Method for Analysis of Milk Samples

The modular system used for the determination of anions present in samples of milk
comprised the Metrohm modules 818 Advanced IC Pump, 819 Advanced IC Detector,
820 Advanced IC Separation Center, 833 IC Suppressor Module, 830 IC interface and
838 Advanced IC Dialysis Sample Processor.

Picture Showing Modular System Configuration for Dialysis of Milk Samples

The milk sample was diluted 1:5 with deionised water and placed in the sample vials
upon the rack of the 838 Advanced IC Sample Processor. The subsequent dialysis of
the sample and injection of the dialysed sample onto the separation column was fully
automated and the response for the peaks recorded using a mobile phase eluent of
sodium carbonate/sodium bicarbonate. The calculation was carried out automatically
using integration software IC Net 2.3 against a previously prepared calibration plot.
Chromatograph of Anions Found in Milk Samples after Dialysis

Ion chromatography as an analytical technique has seen an enormous surge in

popularity due partly to the simplicity of the method as well as other factors such as
market forces driving down the expenditure costs of the initial instrumentation and
improved reliability and power. For a sample in a homogeneous, ionic form then very
little sample preparation is required and quantified results can be obtained within a
matter of minutes.

As in any industry, the consumer places ever more stringent demands and
requirements upon the manufacturer and the world of ion chromatography is no
different. Because of the ease of use at which ion chromatography as a method can
be manipulated, the end user today wishes to analyse ions within increasingly
complicated sample matrices which until recently would not have been possible.

Metrohm has developed a range of instrumental sample preparation modules that can
be added to standard ion chromatography configurations to allow quantification even
with the most difficult sample matrices. The analysis is fully automated so once the
sample is loaded, the analyst is then free to carry out other functions within the
laboratory affording an increase in efficiency of the employed manpower.
Even for those samples requiring sample preparation by dialysis or ultra-filtration, still
only a relatively small volume of sample is required. This coupled with the low
running costs of ion chromatography using Metrohm instruments really does mean that
ion chromatography is the method of choice for the analyst even with the most
difficult and problematic sample matrices.

References

1. Fundamentals of Analytical Chemistry, (1992) 6th edition, D.A Skoog, D.M West and
F.J Holler, Saunders College Publishing, ISBN 0-03-075397-X.

2. Principles and Practice of Analytical Chemistry, (1992) 3rd edition, F.W Fifield and
D. Kealey, Blackie Academic & Professional, ISBN 0-216-92920-2.

3. The Essential Guide to Analytical Chemistry, (1999) 2nd edition, G. Schwedt, John
Wiley & Sons, ISBN 0-471-97412-9.

The following internet site was also used extensively as a reference and can be used
to obtain further information:-

www.metrohm.com

Article written by Jonathan Bruce, Product Application Manager (IC/VA Division) for
Metrohm UK Ltd.
Coupling techniques

So-called coupling techniques represent the link-up of a chromatography system with

an independent analytical method, such as mass spectroscopy

Ion chromatography with Metrohm means

High sensitivity
Accurate and reproducible results
Low acquisition cost
All instruments backed by the COOL guarantee (Cost of Ownership is Lower)
Very low service expenditure
And don't forget:
Swiss quality from over 60 years as a pioneer of Ion Analysis
Metrohm – First Class Ion Chromatography

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age&ved=0CCsQ9QEwBQ

Gel Permeation Chromatography

Conventional versus multiple detection

Mark A. Jordi, Ph.D. and Maricel De Mesa, Ph.D.

Size exclusion chromatography (SEC), also known as gel permeation

chromatography (GPC) or gel filtration chromatography (GFC), is a widely
accepted analytical method used in the separation, purification and
characterization of biopolymers and synthetic polymers. One of the primary
uses of SEC is for the determination of polymer molecular weight. Currently,
most GPC analyses are performed by comparing the molecular weight of a
sample against standards of known molecular weight. This method is often
described as classical GPC. A newer method is becoming increasingly
common, which uses multiple detectors to provide absolute molecular weight
information. In our experience, both methods have their own distinct
advantages, and a thorough understanding of their strengths can aid in
selecting the most beneficial method for a particular analysis.

Background

Unlike other chromatographic methods, SEC utilizes a non-interactive mode

of separation. It employs a stationary phase composed of a macromolecular
gel containing a porous network. As the polymer traverses the column
containing the gel, the components of the sample are sieved based on
differential pore permeation. Molecules with a hydrodynamic volume larger
than the largest pores of the stationary phase cannot penetrate the pores of
the gel, and then pass through the spaces between the gel particles
unretarded. On the other hand, molecules with smaller hydrodynamic volume
enter the pores and the open network of the gel, and are retained in the
stationary phase to varying degrees, depending on their size and shape. This
results in an elution order based on decreasing molecular size.

Figure 1: Classical GPC chromatogram (left) for

 Articles

 Light Scattering Completes GPC and SEC

 Application of a GPC-LC-MS-MS Method
for the Determination of Various Mycotoxins in
Edible Oils
 Xylene Solubles in Polypropylenes by FIPA
 ViscoGEL PolyCAT GPC Columns

Products

 GP-SEC Characterizes Complex Food

Ingredients
 GPC-SEC Multi-Angle, Multi-Detector
Software
 4-Column GPC with Parallel Processing
Increases Sample Clean-Up Throughput
 GPC-SEC Detector Allows Polymer and
Biopolymer Measurement

News

 GPC Identifies Polysaccharide Health

Effects
 New GPC System for Polymers
 Dionex Introduces the New SPE System
 Polymer ApNotes Released for GPC

a partially agglomerated sample and

multidetector signal (right) for the same sample.

Through the years, SEC has gained popularity, not only because it can
separate biomolecules such as proteins, enzymes, nucleic acids,
polysaccharides and hormones, but also because it can be used to determine
the molecular weight averages and molecular weight distribution of synthetic
and naturally occurring polymers. The most widely used method in SEC is
conventional GPC, which makes use of a single concentration detector, most
often a differential refractive index (RI) or a UV spectrophotometric detector.
RI detection is more universal than UV and has become the detector of choice
for most GPC separations. Molecular weight determinations by conventional
GPC, using either RI or UV detection, rely on comparison of the sample with
standards of known molecular weight. In this method, a calibration curve (log
Mw versus retention volume) is created, which allows determination of sample
molecular weight based on the sample retention volume. The primary
limitation of conventional GPC is that the molecular weights obtained are
relative values. Thus, the accuracy of the method depends upon the
standards and sample having the same relationship between their
hydrodynamic volume and molecular weight.

In recent years, the use of multidetector GPC systems has become

increasingly more common. These systems typically consist of multiangle light
scattering coupled with refractive index and viscometry detection. These
systems provide a significant increase in the amount of information that can
be obtained during the GPC experiment. Parameters such as the radius of
gyration, radius of hydration, intrinsic viscosity and the various molecular
weight averages can all be obtained from a single experiment. Light scattering
detectors measure the light scattered inelastically (i.e., Rayleigh scattering)
and, with the use of the Zimm relationship Mw, this can be obtained directly.
The viscosity detector measures the pressure drop and, in combination with a
concentration detector, allows calculation of the intrinsic viscosity (inverse
molecular density). This structural information can be used to probe such
important features of the polymer system as its shape and branching
characteristics.

Classical versus multidetector

Multidetector GPC provides significant advantages in terms of the information

obtained and, based on this fact, one might assume that this technique is
superior for all purposes. In our experience, both techniques have
advantages, and classical GPC remains a viable alternative for many
analyses. The primary factor that determines the best method is the purpose
for the analysis. Most GPC analyses are performed for the purpose of
determining the molecular weight, but the reasons for determining molecular
weight are also varied. Some of the most common reasons to determine
molecular weight include:

• theoretical studies of polymer systems,

• routine quality control,
• troubleshooting polymer failures, and
• regulatory concerns for polymer exemptions.

One of the clearest cases that can be made for multi-detection is for
theoretical studies of polymers. Polymer research is a very exciting field that is
exploring such fascinating topics as supramolecular chemistry,
nanotechnology, controlled polymer architecture, biodegradable polymers and
the mimicking of biological systems. Polymer systems of ever-increasing
complexity are being produced. This array of new molecules often requires
additional information beyond molecular weight. Furthermore, the choice of
the standards used for a conventional GPC analysis has become increasingly
important if accurate molecular weights are to be obtained. This is especially
true for polymers with non-linear molecular architecture and for charged
polymer systems. Polymer systems are now commonly being produced with
non-linear geometries such as stars, combs, dendritic and hyperbranched
materials. Even more mature polymer systems also have the possibility of
containing short- or long-chain branching. In such cases, multidetector GPC
offers clear advantages. Classical GPC provides molecular weight information
solely based on hydrodynamic volume (molecular size). Two molecules with
differing polymer architecture can have the same hydrodynamic volume but
have very different molecular weights. Classical GPC cannot distinguish these
cases. Light scattering detection coupled with viscometry provides the
absolute molecular weight and the intrinsic viscosity. The Mark-Houwink plot
can be accessed showing trends in chain branching as a function of molecular
weight and the general shape of the molecule (rod, sphere, random coil) can
be determined. The polymer size in solution can also be determined.
Theoretical work clearly benefits from the increased information obtained
using multidetector systems.

GPC is often used as a quality control measure for polymer production. This
includes both product release testing as well as product failure analysis. Many
of the most crucial properties of a polymer are dependent upon molecular
weight, including strength, elongation and crucial processing parameters such
as melt temperature and viscosity. Molecular weight determination is a good
way to predict a polymer’s behavior, or to determine why a material is not
performing. Multidetection and classical GPC analysis both play an important
role in serving this function. Some of the important considerations for any
routine analysis include reproducibility, cost, instrument reliability, analysis
time and the ability of the operator to competently interpret the data. The
balance of these factors determines which technique is best for a particular
application. Multidetector GPC is often the best choice for high-end
applications such as drug delivery polymers. These systems tend to be more
complex and include a more unique polymer architecture. In our experience,
absolute molecular weight determination also has improved reproducibility for
analyses that are ongoing over long periods of time.

Classical GPC offers the advantage of reduced cost both in terms of initial
instrument setup and during ongoing maintenance. This latter cost should be
considered carefully as the complexity of multidetector systems may require
more routine maintenance than a classical GPC system. Without a well-
implemented maintenance program, these same reliability issues can lead to
increased downtime for the more complicated multidetector systems.

The final factor that should be considered is the complexity of the data
interpretation. For more routine applications, it is sometimes the case that
operators with limited experience in GPC may be performing the analysis.
Interpretation of multidetector GPC results does require a reasonably high
level of proficiency in order to accurately determine the meaning of the various
signals. This is especially true in cases where the meaning is only clear when
considering the relationship between the signals.

Analyses that are performed to satisfy regulatory requirements are an

example of an analysis in which classical GPC is often preferred. A number of
regulatory bodies currently offer exemptions for materials that meet the
requirements.

This includes the EPA and the European REACH legislation. The
classification as an exempt polymer is generally based on an analysis of the
monomeric and oligomeric content of the material, among other things. In the
case of REACH, the legislation exempts polymeric materials so long as the
monomer does not make up more than 2 percent by weight of the final
material. Multidetector systems are not suitable for these analyses, as
molecules of under 2,000 molecular weight do not scatter sufficient light to be
detected. This provides an artificially low estimate of the low molecular weight
content. Multidetector systems will provide an indication that the oligomeric
and monomeric materials are present due to the RI signal, but they will have
to be operated in classical GPC mode to perform the weight percent
determination. This concept is a general one and the analysis of oligomeric or
other low-molecular-weight materials by GPC is often best done using the
classical method.

Closing

Multidetector GPC analysis is an exciting tool providing the researcher with

increased information over classical GPC methods. In spite of the increased
information this technique offers, we believe that it is not superior in all
applications. The best method for a particular application requires an
understanding of the strengths and weaknesses of each technique.

Mark A. Jordi, Ph.D., is the Vice President, and Maricel De Mesa, Ph.D., is a
Senior Scientist, both at Jordi FLP. They may be contacted at
ChromatographyTechniques@advantagemedia.com. Company’s Other Products

Jordi FLP
4 Mill St.
Bellingham MA 02019
Phone: 508-966-1301
Fax: 508-966-4063
http://www.jordiFLP.com

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Gel permeation chromatography

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Gel permeation chromatography is a term used for when the separation technique
size exclusion chromatography (SEC), that separates analytes on the basis of size, is
applied to polymers in particular. As a technique, SEC was first developed in 1955 by
Lathe and Ruthven.[1] The term gel permeation chromatography can be traced back to
J.C. Moore of the Dow Chemical Company who investigated the technique in 1964.[2]
While polymers can be synthesized in a variety of ways, it is often necessary to
separate polymers, both to analyze them as well as to purify the desired product.

When characterizing polymers, it is important to consider the polydispersity index

(PDI) as well the molecular weight. Polymers can be characterized by a variety of
definitions for molecular weight including the number average molecular weight (Mn),
the weight average molecular weight (Mw) (see molar mass distribution), the size
average molecular weight (Mz), or the viscosity molecular weight (Mv). GPC allows
for the determination of PDI as well as Mv and based on other data, the Mn, Mw, and
Mz can be determined.

Contents
[hide]

• 1 How GPC Works

• 2 Application
• 3 Material and Methods
o 3.1 Instrumentation
o 3.2 Gel
o 3.3 Eluent
o 3.4 Pump
o 3.5 Detector
• 4 Data Analysis
• 5 Advantages of GPC
• 6 Disadvantages of GPC

• 7 References

[edit] How GPC Works

GPC separates based on the size or hydrodynamic volume (radius of gyration) of the
analytes. This differs from other separation techniques which depend upon chemical
or physical interactions to separate analytes. [3] Separation occurs via the use of porous
beads packed in a column (see stationary phase (chemistry)).

Schematic of pore vs. analyte size

The smaller analytes can enter the pores more easily and therefore spend more time in
these pores, increasing their retention time. Conversly, larger analytes spend little if
any time in the pores and are eluted quickly. All columns have a range of molecular
weights that can be separated.

Range of molecular weights that can be separated for each packing

material

If an analyte is either too large or too small it will be either not retained or completely
retained respectively. Analytes that are not retained are eluted with the free volume
outside of the particles (Vo), while analytes that are completely retained are eluted
with volume of solvent held in the pores (Vi). The total volume can be considered by
the following equation, where Vg is the volume of the polymer gel and Vt is the total
volume:[3] Vt = Vg + Vi + Vo

As can be inferred, there is a limited range of molecular weights that can be separated
by each column and therefore the size of the pores for the packing should be chosen
according to the range of molecular weight of analytes to be separated. For polymer
separations the pore sizes should be on the order of the polymers being analyzed. If a
sample has a broad molecular weight range it may be necessary to use several GPC
columns in tangent with one another to fully resolve the sample.
[edit] Application

GPC is often used to determined the relative molecular weight of polymer samples as
well as the distribution of molecular weights. What GPC truly measures is the
molecular volume and shape function as defined by the intrinsic viscosity. If
comparable standards are used, this relative data can be used to determine molecular
weights within ± 5% accuracy. Polystyrene standards with PDI of less than 1.2 are
typically used to calibrate the GPC. [4] Unfortunately, polystyrene tends to be a very
linear polymer and therefore as a standard it is only useful to compare it to other
polymers that are known to be linear and of relatively the same size.

[edit] Material and Methods

[edit] Instrumentation

A typical Waters GPC instrument including A. sample holder,

B.Column C.Pump D. Refractive Index Detector E. UV-vis Detector
The inside of sample holder of Waters GPC instrument

Gel permeation chromatography is conducted almost exclusively in chromatography

columns. The experimental design is not much different from other techniques of
liquid chromatography. Samples are dissolved in an appropriate solvent, in the case of
GPC these tend to be organic solvents and after filtering the solution it is injected onto
a column. A Waters GPC instrument is shown above. The separation of multi-
component mixture takes place in the column. The constant supply of fresh eluent to
the column is accomplished by the use of a pump. Since most analytes are not visible
to the naked eye a detector is needed. Often multiple detectors are used to gain
additional information about the polymer sample. The availability of a detector makes
the fractionation convenient and accurate.

[edit] Gel

Gels are used as stationary phase for GPC. The pore size of a gel must be carefully
controlled in order to be able to apply the gel to a given separation. Other desirable
properties of the gel forming agent are the absence of ionizing groups and, in a given
solvent, low affinity for the substances to be separated. Commercial gels like
Sephadex, Bio-Gel (cross-linked polyacrylamide), agarose gel and Styragel are often
used based on different separation requirements. [5]

[edit] Eluent

The eluent (mobile phase) should be a good solvent for the polymer, should permit
high detector response from the polymer and should wet the packing surface. The
most common eluents in for polymers that dissolve at room temperature GPC are
tetrahydrofuran (THF), o-dichlorobenzene and trichlorobenzene at 130–150 °C for
crystalline polyalkines and m-cresol and o-chlorophenol at 90 °C for crystalline
condensation polymers such as polyamides and polyesters.

[edit] Pump

There are two types of pumps available for uniform delivery of relatively small liquid
volumes for GPC: piston or peristaltic pumps.

[edit] Detector

In GPC, the concentration by weight of polymer in the eluting solvent may be

monitored continuously with a detector. There are many detector types available and
they can be divided into two main categories. The first is concentration sensitive
detectors which includes UV absorption , differential refractometer (DRI) or
refractive index (RI) detectors, infrared (IR) absorption and density detectors.
Molecular weight sensitive detectors include low angle light scattering detectors
(LALLS), multi angle light scattering (MALLS).[6] The resulting chromatogram is
therefore a weight distribution of the polymer as a function of retention volume.

GPC Chromatogram; Vo= no retention, Vi= complete retention, A

and B = partial retention

The most sensitive detector is the differential UV photometer and the most common
detector is the differential refractometer (DRI). When characterizing copolymer, it is
necessary to have two detectors in series.[4] For accurate determinations of copolymer
composition at least two of those detectors should be concentration detectors.[6] The
determination of most copolymer compositions is done using UV and RI detectors,
although other combinations can be used. [7]
[edit] Data Analysis

Gel permeation chromatography (GPC) has become the most widely used technique
for analyzing polymer samples in order to determine their molecular weights and
weight distributions. Examples of GPC chromatograms of polystyrene samples with
their molecular weights and PDIs are shown on the left.

GPC Separation of Anionically Synthesized Polystyrene; Mn=3,000

g/mol, PDI=1.32

GPC Separation of Free-Radical Synthesized Polystyrene; Mn=24,000

g/mol, PDI=4.96
Standardization of a size exclusion column.

Benoit and co-workers proposed that the hydrodynamic volume, Vη, which is
proportional to the product of [η] and M, where [η] is the intrinsic viscosity of the
polymer in the SEC eluent, may be used as the universal calibration parameter. If the
Mark-Houwink-Sakurada constants K and α are known (see Mark-Houwink
equation), a plot of log [η]M versus elution volume (or elution time) for a particular
solvent, column and instrument provides a universal calibration curve which can be
used for any polymer in that solvent. By determining the retention volumes (or times)
of monodisperse polymer standards (e.g. solutions of monodispersed polystyrene in
THF), a calibration curve can be obtained by plotting the logarithm of the molecular
weight versus the retention time or volume. Once the calibration curve is obtained, the
gel permeation chromatogram of any other polymer can be obtained in the same
solvent and the molecular weights (usually Mn and Mw) and the complete molecular
weight distribution for the polymer can be determined. A typical calibration curve is
shown to the right and the molecular weight from an unknown sample can be obtained
from the calibration curve.

[edit] Advantages of GPC

As a separation technique GPC has many advantages. First of all, it has a well-defined
separation time due to the fact that there is a final elution volume for all unretained
analytes. Additionally, GPC can provide narrow bands, although this aspect of GPC is
more difficult for polymer samples that have broad ranges of molecular weights
present. Finally, since the analytes do not interact chemically or physically with the
column, there is a lower chance for analyte loss to occur. [3] For investigating the
properties of polymer samples in particular, GPC can be very advantageous. GPC
provides a more convenient method of determining the molecular weights of
polymers. In fact most samples can be thoroughly analyzed in an hour or less.[8]. Other
methods used in the past were fractional extraction and fractional precipitation. As
these processes were quite labor intensive molecular weights and mass distributions
typically were not analyzed. [9] Therefore, GPC has allowed for the quick and
relatively easy estimation of molecular weights and distribution for polymer samples

[edit] Disadvantages of GPC

There are disadvantages to GPC, however. First, there is a limited number of peaks
that can be resolved within the short time scale of the GPC run. Also, as a technique
GPC requires around at least a 10% difference in molecular weight for a reasonable
resolution of peaks to occur.[3] In regards to polymers, the molecular masses of most
of the chains will be too close for the GPC separation to show anything more than
broad peaks. Another disadvantage of GPC for polymers is that filtrations must be
performed before using the instrument to prevent dust and other particulates from
ruining the columns and interfering with the detectors. Although useful for protecting
for instrument, the pre-filtration of the sample has the possibility of removing higher
molecular weight sample before it can be loaded on the column.

[edit] References

1. ^ Lathe, G.H.; Ruthven, C.R.J. The Separation of Substance and

Estimation of their Relative Molecular Sizes by the use of
Columns of Starch in Water. Biochem J. 1956, 62, 665–674.
PMID:13249976
2. ^ Moore, J.C. Gel permeation chromatography. I. A new
method for molecular weight distribution of high polymers. J.
Polym. Sci., 1964, 2, 835-
843.DOI:10.1002/pol.1964.100020220
3. ^ a b c d Skoog, D.A. Principles of Instrumental Analysis, 6th ed.;
Thompson Brooks/Cole: Belmont, CA, 2006, Chapter 28.
4. ^ a b Sandler, S.R.; Karo, W.; Bonesteel, J.; Pearce, E.M.
Polymer Synthesis and Characterization: A Laboratory Manual;
Academic Press: San Diego, 1998.
5. ^ Helmut, D. Gel Chromatography, Gel Filtration, Gel
Permeation, Molecular Sieves: A Laboratory Handbook;
Springer-Verlag, 1969.
6. ^ a b Trathnigg,B. Determination of MWD and Chemical
Composition of Polymers by Chromatographic Techniques.
 Prog. Polym. Sci. 1995, 20, 615-650.DOI:10.1016/0079-
6700(95)00005-Z
7. ^ Pasch, H. Hyphenated Techniques in Liquid Chromatography
of Polymers. Adv. Polym. Sci. 2000, 150, 1-66.DOI:10.1007/3-
540-48764-6
8. ^ Cowie, J.M.G.; Arrighi, V. Polymers: Chemistry and Physics of
Modern Materials, 3rd ed. CRC Press, 2008.
9. ^ Odian G. Principles of Polymerization, 3rd ed.; Wiley
Interscience Publication, 1991.

Retrieved from
"http://en.wikipedia.org/wiki/Gel_permeation_chromatography"

(GPC continued)

The sample is loaded onto the column with the

Introduction
In order to determine contaminant levels
in the blubber samples under
examination, we used Gas
Chromatagraphy--an analytical technique
which separates the compounds within a
sample, over time. In GC, different
chemicals and pollutants are separated-
out or eluted at different, known times.
This allows the identification and
quantification of various contaminants in
our samples.
Before we could test our blubber samples
by gas chromatagraphy, however, we
had to go through several purification
stages.
Below is the general methodology we
used.
The various stages in our high-resolution
eluent, which run through the column together.
Relatively smaller molecules like PCBs and OCs
are able to pass through the pores in the beads,
however, relatively larger molecules like lipids
cannot. Larger molecules are said to be
excluded (from passing through the pores in the
beads) and as a result they move more quickly
through the column (and leave the column
sooner) than do smaller molecules.
By collecting the liquid that leaves the column -
the eluant - the researcher achieves a
successful fractionation of the sample--
separating in our study, lipids and contaminants.
c) Sub-fractionation by silica-gel clean-
up
The final stage of sample clean-up in our
procedure, was sub-fractionation using
silica-gel columns. As discussed above,
the first fraction from our GPC
separation was used for determining
lipid weight. The second fraction,
however, is used in this stage -- sub-
gas chromatographic analysis, included: fractionation -- for contaminant analysis.

a) Extraction Here, the contaminant fraction from GPC

separation was rotovapped to ~1mL and
b) Lipid removal run on a silica column.As the name
suggests, the stationary phase is silica -
a finely divided white solid; the mobile
c) Silica gel clean-up /
phase, like in GPC, is a liquid that runs
subfractionation
through the column.
d) Gas chromatography

e) Analysis of chromatograms

Methods

a) Extraction

In order to remove the contaminants from

the blubber samples, we used an
extraction protocol which put them, and
the lipid material, into solution. First we
weighed each sample for later reference,
and cut the blubber samples into small
pieces. These pieces were then
homogenized with a mortar and pestle in
sodium sulphate, to remove any water.

The homogenate was transferred to a

cellulose extraction thimble, and covered
with glass wool. We ran these samples in
a Soxhlet extractor for 4 hours; reflux
events occurred every 7-10 minutes. Diagrammatic representation of a
Silica column.

Molecular separation in silica gel

chromatography is based upon the
polarity of a particular molecule. Polar
molecules, like amino acids, will adsorb
or stick to the silica and these are left
behind in the column as the non-polar
molecules are collected in the eluant. By
changing the polarity of the mobile
phase a researcher can remove
molecules adsorbed to the silica.

We recovered two fractions for analysis.

The first (A) fraction contained:

PCBs, aldrin, HCB, heptachlor,

mirex, and most of the p,p' DDE
 A soxhlet extractor. (ie. primarily PCBs)

What is a soxhlet extractor? The sample,
which has been homogenized in sodium
sulphate, is covered with glass wool and
contained in a porous cellulose thimble.
The thimble is placed in the extraction
tube, which itself sits on a flask
containing an organic solvent (like
hexane).
while the second (B) fraction contained:
heptachlor epoxide, HCHs,
chlordanes, dieldrin, p,p' DDT,
p,p' DDD, methoxychlor, and a
small proportion of p,p' DDE (ie.
primarily OC pesticides).
Each of these two fractions were later
analysed by high resolution gas
chromatography on a Varian model 3500
HRGC with splitless injection, using
electron capture detection (ECD). The
results of which are explained
elsewhere.

Gas Chromatography
Gas chromatography is one of the most widely
used methods to determine the chemical make-
up of a complex, volatile mixture. In order to
perform this technique a researcher uses a gas
chromatograph or GC.

Diagrammatic representation of a
soxhlet extractor.

The solvent is boiled, and its vapor Diagrammatic representation of a

travels upward through the extraction
tube into the condenser tube. The cool
water flowing around the outside of the
condenser tube condenses the vapor,
which then drips into the thimble,
containing the sample.
Because the contaminants and lipid are
soluble in organic solvents, they move
into the condensed solvent as it
accumulated in the thimble. The solution,
generalized gas chromatograph.
Before gas chromatography can be
performed, the sample must be disolved
in a volatile solvent like iso-octane. Gas
chromatography begins by injecting a
very small amount of the sample (e.g. 1
uL) into the GC. This small amount of
sample is then volatilized by the heat of
the oven and carried into the column by
an inert gas like helium (He).
now containing the contaminants and
dissolved lipid, build up in the thimble.
Once the liquid reaches the level of the
bypass arm, it is siphoned back into the
flask. This continuous condensation,
buildup, and siphoning is known as the
reflux event.
The advantage of the soxhlet is
that once the contaminants and
lipid material are brought into
solution, and siphoned back into
the flask, they stay in the flask--
so that the sample in the
extraction thimble is continuously
re-exposed to fresh, heated
solvent--thus greatly increasing
the extraction rate.
After extraction, we reduced the volume
of solvent in our samples by rapid rotary
evaporation down to about 2mL, using a
Rotovap.
b) Lipid removal
Next we separated the contaminants
from the lipid material, by fractioning
them using GPC (gel-permeation
chromatography). GPC is a size-
exclusion technique, as it works by
slowing the flow of smaller molecules by
trapping them in pores which are too
small for large molecules--thus the larger
compounds are excluded (see below).
It is inside the column where the separation of
the individual chemical components takes place.
There are two "phases" inside the column which
control actual separation: the mobile phase and
the stationary phase.
The mobile phase is simply the carrier
gas, helium, and is so-called because it
moves through the column.
The stationary phase is some non-
volatile liquid like silicone rubber, wax, or
oil, and is so-called because it does not
move through the column.
Some chemicals have a relatively high affinity for
the mobile phase, and some have a relatively
high affinity for the stationary phase, but each
chemical is unique.
These affinities are based on the molecular
weight of the chemical and its polarity. If a
chemical has a high affinity for the mobile phase,
it will tend to move quickly through the column. If
a chemical has a high affinity for the stationary
phase it will tend to move slowly through the
column. Since each chemical has unique
degrees of affinity, each will eventually leave the
column at a different time.
Inside the GC
The column
The column itself is a long thin tube
which is coiled so that it may be
contained easily within the oven. In
many of the pioneering studies which
used gas chromatography, columns
were 2 to 3 metres long, with a diameter
of 2 to 4 mm, and made of glass or
metal. These columns were packed, that
is they contained many small spherical
particles known generally as "solid
support." The solid support was coated
with a liquid stationary phase and it
provided a great deal of surface area for
exposure to the mobile phase.
More modern studies have turned away
from this method and use the higher-
resolution capillary column. Capillary

The GPC apparatus.
Separation into two fractions was
acheived in our study--the first used for
lipid weight determination, and the
second for PCB and OC pesticide
determination and quantification. Upon
obtaining the first fraction (containing the
much larger lipid molecules), we reduced
the solvent volume to approximately 2mL,
and allowed it to completely evaporate
over a few days in pre-weighed vessels--
leaving a lipid-only residue which could
be weighed.This is important later,
because as mentioned previously,
contaminants such as PCBs and OCs are
associated with lipid; because, however,
different organisms have different
proportions of lipid in their tissues
(including blubber), we divide the
concentrations by the amount of lipid to
Analysing the chromatogram
get a standardized measure -- allowing
comparison with other data..
What is GPC? Gel permeation
chromatography (GPC) is a technique
used to separate molecules based on
size differences. It is also referred to as
gel filtration chromatography, or size
exclusion chromatography.
Molecular separation occurs in
the GPC column. Inside the
column there are two phases:
a) The stationary phase
columns are 10 to 60 metres long, have
diameters ranging from 100 to 320 um,
and are made of fused silica. Capillary
columns have the stationary phase
coated on the inside of the tube.
The detector
In order to determine what and when
chemicals leave the column, (and
therefore how much of each chemical is
present), the GC is equipped with a
detector. Many types of detectors exist,
but for our purposes an Electron-
Capture Detector (ECD) was used. This
detector was chosen because it is
especially useful for detecting
halogenated compounds like PCBs and
pesticides.
The ECD works by passing the
chemicals and a different gas (e.g.
nitrogen) over a radioactive source as
the chemicals leave the column. The
radiation produces a series of events at
the sub-atomic level which result in a
change in electrical potential between
two electrodes when chemicals of
interest pass between them.
The detector reports to a recorder, and a
"gas chromatogram" is produced for the
researcher to inspect.
A representation of a
 consists of an inert gel of
porous beads-- so-called
because it does not
move in the column
b) A mobile phase, which
is the eluent or liquid
which is run through the
column.
Diagrammatic representation of
Gel Permeation Chromatography.
(continued above - click here)
Click here to see the results of our study.
Click here to learn abo
chromatogram.
The chromatogram shown above is
representative of the PCB fraction from
sample #2539. The location of the peaks
along the x-axis of the graph correspond
to the time at which certain chemicals
left the column and were detected by the
ECD. These elution times are
considered constant for specific
chemicals and contaminants. This
constancy facilitates the individual
identification of specific contaminants
For example PCB congener 138
left the column 73.05 minutes
after the method began;
congener 194 left later, at 88.24
minutes.
While the height of a particular peak
directly corresponds to the electrical
charge induced by a chemical at the
electrodes of the ECD, the area under
the peak (that is its size) can be used to
calculate how much of a particular
chemical there was in the sample: the
larger the peak, the more of the
chemical was present.
The actual quantity of each chemical is
determined with the help of computer
software. Essentially, the computer
takes the information given on the
chromatogram and compares it to values
for known concentrations--thus
calibrating concentration information
against standard reference data.
Ultimately such software provides the
concentration of each chemical in the
sample.