Ion-Exchange Chromatography

Indah Solihah

• Merupakan pemisahan senyawa senyawa polar dan ion berdasarkan muatan

• Dapat digunakan untk hampir semua molekul bermuatan termasuk protein, nukleotida, dan asam-asam amino

• Sering digunakan untuk pemurnian protein, analisis air dan kontrol kualitas

Penggolongan dan Sifat dasar penukar ion:

- Penukar kation : membebaskan dan menukar kation (+)

- Penukar anion : membebaskan dan menukar anion (-)

- Penukar ion amfoter : mengandung gugus fungsi kation dan anion dlm satu matriks

Prinsip Kromatografi Pertukaran Ion

• Fase diam mampu menukar ion, pada permukaannya mempunyai muatan listrik. Pada resin atau gel itu terikat gugus ionik : SO3

=, COO-, NR3+

• Muatan dinetralkan oleh ion balik (counter ion) dari fasa gerak

• Fasa gerak mengandung ion balik dan molekul cuplikan ionik bersaing dengan ion2 itu untuk mendapat tempat pada permukaan fasa diam

• Separation in ion exchange chromatography depends upon the reversible adsorption of charged solute molecules to immobilized ion exchange groups of opposite charge.

• Separation is obtained since different substances have different degrees of interaction with the ion exchanger due to differences in their charges, charge densities and distribution of charge on their surfaces.

• These interactions can be controlled by varying conditions such as ionic strength and pH

• In ion exchange chromatography one can choose whether to bind the substances of interest and allow the contaminants to pass through the column, or to bind the contaminants and allow the substance of interest to pass through.

The Matrix • The matrix may be based on inorganic compounds,

synthetic resins or polysaccharides

• An ion exchanger consists of an insoluble matrix to which charged groups have been covalently bound

• The charged groups are associated with mobile counter-ions

• These counter-ions can be reversibly exchanged with other ions of the same charge without altering the matrix

The Matrix

• Positively charged exchangers have negatively charged counter-ions (anions) available for exchange and are called anion exchangers.

• Negatively charged exchangers have positively charged counter-ions (cations) and are termed cation exchangers

Resin atau gel

SO3- SO3- SO3- SO3-

Na+ Na+ Na+ Na+

SO3- SO3- SO3- SO3-

Na+ Na+ Na+ P+

Ion balik

Gugus penukar ion

Satu ion cuplikan pengganti ion Na+

P+ ion cuplikan dlm FG

Charged Groups

• The presence of charged groups is a fundamental property of an ion exchanger

• The type of group determines the type and strength of the ion exchanger; their total number and availability determines the capacity

There are several advantages to working with strong ion exchangers: • Development and optimization of separations is fast and easy since the charge characteristics of the medium do not change with pH. • The mechanism of interaction is simple since there are no intermediate forms of charge interaction. • Sample loading (binding) capacity is maintained at high or low pH since there is no loss of charge from the ion exchanger.

The majority of proteins have pI within the range 5.5 to 7.5 and can be separated on either strong or weak ion exchangers. An advantage of a weak ion exchanger is that they can offer a different selectivity compared to strong ion exchangers. A disadvantage is that because weak ion exchangers can take up or lose protons with changing pH, their ion exchange capacity varies with pH

Charged Groups

• Sulphonic and quaternary amino groups are used to form strong ion exchangers; the other groups form weak ion exchangers

• The terms strong and weak refer to the extent of variation of ionization with pH and not the strength of binding

• Strong ion exchangers are completely ionized over a wide pH range whereas with weak ion exchangers, the degree of dissociation and thus exchange capacity varies much more markedly with pH

• Some properties of strong ion exchangers are:

1. Sample loading capacity does not decrease at high or low pH values due to loss of charge from the ion exchanger

2. A very simple mechanism of interaction exists between the ion exchanger and the solute.

3. Ion exchange experiments are more controllable since the charge characteristics of the media do not change with changes in pH. This makes strong exchangers ideal for working with data derived from electrophoretic titration curves.

• No single ion exchanger is best for every separation. The choice of matrix and ionic substituent depends on:

1. The specific requirements of the application

2. The molecular size of the sample components

3. The isoelectric points of the sample components

Step in IEC

• Equilbration

• Sample application and wash

• Elution

• Regeneration

The resolution of an IEX separation is a combination 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 are influenced by 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.

RESOLUTION

If Rs = 1.0 (Fig 1.4) 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%.

A single, well-resolved peak is not necessarily a pure substance, but might represent a series of components that could not be separated under the chosen elution conditions.

VR = volume eluted from the start of sample application to the peak maximum wh = peak width measured as the width of the recorded peak at half of the peak height

H (height equivalent to a theoretical plate, HETP) is calculated from the expression:

L = height of packed bed

Column packing and efficiency

As a general rule, a good H value is about two to three times the average particle diameter of the medium being packed. For a 90 µm particle, this means an H value of 180 to 270 µm

Column Efficiency

Although resolution in terms of efficiency can be improved by decreasing the particle size of the matrix, using a smaller particle size often creates an increase in back pressure so that flow rates need to be decreased, lengthening the run time. Hence the need to match the medium with the requirements for the purification (speed, resolution, recovery, and capacity).

The viscosity of highly concentrated samples might reduce resolution if large sample volumes are loaded onto columns packed with small particles. Samples may be diluted or, alternatively, a larger particle size should be used.

Selectivity

Good selectivity (the degree of separation between peaks) is a more important factor than high efficiency in determining resolution and depends not only on the nature and number of the functional groups on the matrix, but also on the experimental conditions, such as pH (influencing the protein charge), ionic strength, and elution conditions.

Selectivity and pH

A B

C D

Selectivity and elution

A

B

C D