ION EXCHANGE CHROMATOGRAPHY IN FOOD ANALYSIS (Báo cáo khoa học)

Báo cáo được tác giả thực hiện tại trường Đại học Bách khoa, môn Ứng dụng sắc ký trong phân tích thực phẩm. Báo cáo nêu đầy đủ chi tiết về những đặc điểm của sắc ký trao đổi ion, nguyên lý của nó và định hướng ứng dụng trong phân tích thực phẩm. Những ưu và nhược điểm của sắc ký trao đổi ion cũng được nêu cụ thể. Ví dụ điển hình cũng được tác giả nêu để cụ thể hóa lý thuyết được nêu. Bài báo cáo là tài liệu tham khảo hay và ý nghĩa cho các bạn đang học sau đại học có học môn “Ứng dụng của sắc ký trong phân tích thực phẩm”. Tài liệu được viết hoàn toàn bằng Tiếng Anh dễ hiểu giúp các bạn có đam mê sử dụng Tiếng Anh trong học tập.

HCM UNIVERSITY OF TECHNOLOGY FACULTY OF CHEMICAL ENGINEERING

&&& ESSAY TO SUBJECT OF APPLICATION OF CHROMATOGRAPHIC METHODS

IN FOOD ANALYSIS

TOPIC ION EXCHANGE CHROMATOGRAPHY IN FOOD ANALYSIS

Supervisor: Dr Nguyễn Thị Lan Phi

Ho Chi Minh City, 2016

TABLE OF CONTENTS

INTRODUCTION 3

CONTENTS 4

I ION EXCHANGE CHROMATOGRAPHY (IEC or IC) 4

1 Ion exchange chromatography 4

2 Classification 5

II PRINCIPLE OF IC 5

1 Net surface charge and pH 5

2 Principle in IC separation 7

2.1 Equilibration 7

2.2 Sample application and wash 7

2.3 Elution 7

2.4 Regeneration 9

3 Resolution 9

4 IC system 10

III PROS AND CONS OF IC 11

IV APPLICATIONS 11

CONCLUSION 15

REFERENCES 16

There are a number of chromatographic methods utilized in food analysis, including Gas Chromatography, High Performance Liquid Chromatography, Size Exclusion Chromatography, etc Ion Exchange Chromatography or Ion Chromatography is a technique rather commonly used in many fields in industry, and

it is of many applications in food analysis in particular

Together with the current developments of continuous detectors, Ion Chromatography is more and more useful in food analysis, especially corporated with modern chromatographic techniques Although there have been only limited applications of Ion Chromatography in food industry, today it appears to be a hopeful technique as a simple, unexpensive method for food industry, also for other ones

I ION EXCHANGE CHROMATOGRAPHY (IEC or IC)

1 Ion exchange chromatography

Ion exchange chromatography is a chromatography technique that

separates ions and polar molecules based on their affinity to the ion exchanger Analytes often used in IC are large proteins, small nucleotides, and amino acids IC is often used in protein purification, water analysis, and quality control The water-soluble and charged molecules such as proteins, amino acids, and peptides bind to oppositely charged stationary phase by forming covalent bonds The equilibrated stationary phase consists of an ionizable functional group where the targeted molecules of a mixture to be separated and quantified can bind while passing through the column [1]

The history of IC primarily began between 1935-1950 through the Manhattan project that applications and IC were significantly extended IC was originally introduced by two English researchers, agricultural Sir Thompson and chemist J T Way It was in the fifties and sixties that theoretical models were developed for further understanding and it was not until the seventies that continuous detectors were utilized, giving the way to the development from low-pressure to high-performance chromatography Not until 1975 was "ion chromatography" established as a name of a technique, and was thereafter used as a name for marketing purposes

Fig 1 Some critical timelines in the history of IC

2 Classification

Acording to the analytes, IC is often categorized into two main types, namely Anion and Cation Chromatography Cation exchange chromatography is used when the desired molecules to separate are cations, and an anion exchange chromatography

is to separate anions meaning that the beads in the column contain positively charged functional groups to attract the anions

Similarly, the stationary phase of IC (often called IC media or IC exchanger) is also divided into Anion and Cation Exchager Anion Exchanger is used for Anion Chromatography whreas Cation Chromatography uses Cation Exchanger for separating the targeting molecules

Fig 2 Commonly used IC media [2]

II PRINCIPLE OF IC

1 Net surface charge and pH

IC separates molecules on the basis of differences in their net surface charge Molecules vary considerably in their charge properties and will exhibit different degrees of interaction with charged chromatography media according to differences in their overall charge, charge density, and surface charge distribution The charged groups within a molecule that contribute to the net surface charge possess different

pKa values (acid ionization constant) depending on their structure and chemical microenvironment

Since all molecules with ionizable groups can be titrated, their net surface charge is highly pH dependent In the case of proteins, which are built up of many different amino acids containing weak acidic and basic groups, net surface charge will change gradually as the pH of the environment changes, that is, proteins are amphoteric Each protein has its own unique net charge versus pH relationship which can be visualized as a titration curve This curve reflects how the overall net charge of the protein changes according to the surrounding pH [2]

Fig 3 Theoretical titration curves, showing how net surface charge varies with pH

IC takes advantage of the fact that the relationship between net surface charge and pH is unique for a specific analyte In an IC separation, reversible interactions between charged molecules and oppositely charged IC media are controlled in order to favor binding or elution of specific molecules and achieve separation A certain molecule that has no net charge at a pH equivalent to its isoelectric point (pI) will not interact with a charged medium However, at a pH above its pI, an analyte will bind to

a positively charged medium or anion exchanger and, at a pH below its pI, an analyte will bind to a negatively charged medium or cation exchanger In addition to the ion exchange interaction, other types of binding can occur, but these effects are very small and mainly due to van der Waals forces and nonpolar interactions

2 Principle in IC separation

An IC medium comprises a matrix of spherical particles substituted with ionic groups that are negatively or positively charged The matrix is usually porous to give a high internal surface area The medium is packed into a column to form a packed bed The bed is then equilibrated with buffer which fills the pores of the matrix and the space among the particles [2]

2.1 Equilibration

The first step is the equilibration of the stationary phase to the desired start conditions When equilibrium is reached, all stationary phase charged groups are bound with exchangeable counterions, such as chloride or sodium The pH and ionic strength of the start buffer are selected to ensure that, when sample is loaded, analytes bind to the medium and as many impurities as possible do not bind

2.2 Sample application and wash

The second step is sample application and wash The goal in this step is to bind the target molecules and wash out all unbound one The sample buffer should have the same pH and ionic strength as the start buffer in order to bind all charged target molecules Oppositely charged items bind to ionic groups of the IC medium, becoming concentrated on the column Uncharged items, or those with the same charge as the ionic group, pass through the column at the same speed as the flow of buffer, eluting during or just after sample application, depending on the total volume of sample loaded

2.3 Elution

When all the sample has been loaded and the column washed with start buffer

so that all nonbinding molecules have gone out of the column, conditions are altered in order to elute the bound analytes Most frequently, analytes are eluted by increasing the ionic strength (salt concentration) of the buffer or, occasionally, by changing the

pH As ionic strength increases the salt ions (typically Na+ or Cl-) compete with the bound components for charges on the surface of the medium and one or more of the bound species begin to elute and move down the column The molecules with the lowest net charge at the selected pH will be the first ones eluted from the column as ionic strength increases Similarly, the components with the highest charge at a certain

pH will be most strongly retained and will be eluted thereafter The higher the net charge of the target molecules, the higher the ionic strength that is needed for elution

By controlling changes in ionic strength using different forms of gradient, components are eluted differently in a purified, concentrated form

Fig 4 Description of steps in IC separation

2.4 Regeneration

A final wash with high ionic strength buffer regenerates the column and removes any molecules still bound This ensures that the full capacity of the stationary phase is available for the next run The column is then re-equilibrated in start buffer before starting the next run

Alternatively, conditions can be chosen to maximize the binding of contaminants to allow the target analytes to first pass through the column to be collected

3 Resolution

The resolution of an IC separation is a representation of the degree of separation between the peaks eluted from the column (the selectivity of the medium), the ability of the column to produce narrow, symmetrical peaks (efficiency) and, of course, the amount (mass) of sample applied These factors depend upon practical issues such as matrix properties, binding and elution conditions, column packing, and flow rates

Resolution (Rs) is defined as the distance between peak maxima compared with the average base width of the two peaks Rs can be determined from a chromatogram,

as shown in Figure 5 [2]

Fig 5 Theoretical determination of Rs in IC separation [2]

Rs gives a measure of the relative separation between two peaks and can be used to determine if further optimization of the chromatographic procedure is necessary

If Rs = 1.0 then 98% purity has been achieved at 98% of peak recovery, provided the peaks are symmetrical and approximately equal in size Baseline resolution requires that Rs ≥1.5 At this value, peak purity is 100% (Fig 6)

Fig 6 Separation result with different Rs, showing if further optimization is needed

4 IC system

Similar to some other chromatography systems, IC has some fundamental components as the following figure:

Fig 7 A typical IC system

The eluent generator is used to generate the proper buffer for analysis, follwed

by the separation collumn with the charged stationary phase The analysis data is obtained by combination with the detector connected to a screen

III PROS AND CONS OF IC

IC is a very powerful separation technique that is used not only for preparative chromatography but also for analytical chromatography However, like all other chromatography modes, IC does have some limitations

One of the main disadvantages of IC is its buffer requirement: because binding

to IC media is dependent on electrostatic interactions between analytes of interest and the stationary phase, IC columns must be loaded in lowsalt buffers For some applications, this restriction may require a buffer exchange step prior to IC analysis

IC, unlike some other chromatography methods, also permits high flow rates, which in some cases can be crucial to the recovery of active protein Finally, a limitation of weak ion exchangers is their pH dependence When working outside of their optimal pH range, these resins rapidly lose capacity, and more importantly, resolution as table below [3]

- Permits high flow rate - Sample must be loaded at low ionic strength

- Concentrates samples - Clusters of positively charged residues cancause a net negatively charged protein to bind a

cation exchanger, and vice versa

- High yield - Small changes in pH can greatly alter bindingprofile of IC resin -Buffers are non-denaturing - Particle size greatly influences resolution

IV APPLICATIONS

IC is a powerful technique in a number of field, such as environment analysis, water treatment, pharmaceutical and drugs analysis and food analysis IC has been utilized in environment and water treatment which the charged resins was used to separate metallic ion from the natural resources Though applications in

pharmaceutical, drugs and food analysis are less than two fields above, IC gradually becomes hopeful technique corporated to other chromatography methods [4-6]

To illustrate, a remarkable investigation conducted in 2005 by C Gu´erin-Dubiard et al separated useful proteins in hen egg white into separate fractions Due to the dependence of IC upon pH, researchers changed the pH to get individual components that have the different pI value This separation procedure was depicted as following figure:

Fig 8 Procedure of hen egg white separation [7]

The analytical results and the confirmation of analytes was determined by HPLC to delight the separation efficiency and the accuracy of this method (Fig 9)

Based upon the HPLC analysis, proteins in hen egg white was separated successfully with the high efficiency This paper also showed the importance of changing pH in IC in which flexibly changing pH of buffer can completely purify the analytes of interest in a mixture

Ion exchange chromatography is a technique that separates charged molecules from the mixture basing upon their net surface charge at specific pH of the surroundings Anion exchange chromatography uses the anion exchagers formed by functionalized the stationary phase with cationic groups for separation of anion analytes By contrast, cation exchage chromatography separates the cationic molecules from the mixture while they pass through the collumn filled by negatively charged matrices

IC is a useful technique used in a wide number of fields, especially in environment and water treatment In food industry, together with the developments of continuous detectors currently, IC has been utilized with other chromatographic methods for separation of charged molecules such as proteins, amino acids and peptides Like other modes of chromatography, IC has advantages and disavantages which can be noted to enhance the efficiency and resolution of this method

1 Wikipedia Ion Chromatography Available from:

https://en.wikipedia.org/wiki/Ion_chromatography.

2 Healthcare, G., Ion Exchange Chromatography: Principles and Methods,

GE Healthcare: GE Healthcare.

3 BioRad, Ion Exchange Chromatography: Applications & Technologies

BioRad: BioRad.

4 Jackson, P.E., Ion Chromatography in Environmental Analysis

Encyclopedia of Analytical Chemistry, 2000: p 2779–2801.

5 Rohrer, L.B.a.J.S., Application of IC for pharma and biological products

A JOHN WILEY & SONS, INC., PUBLICATION, 2012.

6 MICHALSKI, R., Industrial applications of ion chromatography

CHEMIK, 2014 68: p 478-485.

7 al, C.G.e.-D.e., Hen egg white fractionation by ion-exchange

chromatography Journal of Chromatography A, 2005 1090: p 58-67.