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 exchangewith 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 exclusionwith 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 meansHigh sensitivityAccurate and reproducible resultsLow acquisition costAll instruments backed by the COOL guarantee (Cost of Ownership is Lower)Very low service expenditureAnd don't forget:Swiss quality from over 60 years as a pioneer of Ion AnalysisMetrohm – First Class Ion Chromatography

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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

Click to enlarge.

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"

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 gas chromatographic analysis, included:

(GPC continued)

The sample is loaded onto the column with the 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-fractionation -- for contaminant analysis.

Here, the contaminant fraction from

a) Extraction

b) Lipid removal

c) Silica gel clean-up / subfractionation

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.

GPC separation was rotovapped to ~1mL and run on a silica column.As the name suggests, the stationary phase is silica - a finely divided white solid; the mobile phase, like in GPC, is a liquid that runs through the column.

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 (ie. primarily PCBs)

A soxhlet extractor.

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).

Diagrammatic representation of a soxhlet extractor.

The solvent is boiled, and its vapor 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,

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 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

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).

an inert gas like helium (He).

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

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 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

from this method and use the higher-resolution capillary column. Capillary 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.

Analysing the chromatogram

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)

A representation of a 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.

Click here to see the results of our study.

Click here to learn abo