Ion Exchange Cellulose

For the best separations . . .

The range of Whatman ion exchange celluloses have been developed specifically for the efficient separation for biopolymers, e.g. proteins, enzymes and nucleic acid fragments. Optimum results can only be achieved if certain precautions are taken in their use.

Physical and Chemical Properties

EXCHANGER PREPARATION

Pre-swollen (51, 52 and 53 types) can be used without the initial pre-cycling but must be equilibrated completely. Pre-swollen ion exchangers should never be dried, by any method, at any stage of pretreatment, or in subsequent use.

For dry ion exchange celluloses (23 and 32 types) all stages of pretreatment must be carried out as described below in order to obtain the best possible performance.

DE 51, QA 52, SE 52 contains a preservative. This is automatically removed during equilibration and column packing. If the ion exchanger is used without equilibration, the preservative may be removed by washing with distilled or de-ionized water.

PART I - FOR ION EXCHANGE CELLULOSES IN DRY FORM

PRE-CYCLING

1. Stir the weighed ion exchanger into 15 vols. (i.e. w/v) of `first treatment' acid or alkali (see below).
2. Leave for at least 30 minutes for Microgranular or 60 minutes for fibrous products.
3. Filter or decant off supernatant and wash until the filtrate is at the ‘intermediate pH' stated below.
4. Stir ion exchanger into 15 vols. of `second treatment' acid or alkali and leave for a further 30 minutes.
5. Repeat second treatment (4) and follow by washing until the filtrate is near neutral.

Order of Treatment

Type First Treatment Intermediate pH Second Treatment

NB This stage is not required for pre-swollen microgranular grades (51,52, 53) and therefore

these materials are to be preferred in large scale uses.

NOTES ON GENERAL HANDLING

DO Store the ion exchanger at room temperature. DO Always keep pack closed when not in use. DO Always use distilled or preferably, de-ionized water. DO NOT Macerate or stir slurries too vigorously in order to avoid

DO NOT Subject ion exchangers to concentrated acids, alkalis or strong oxidizing agents. Limits O.SN acid or alkali for 2 hours.

DO NOT Leave ion exchangers in contact with buffers or polyelectrolytes for longer than one week without the addition of a preservative. For cation exchangers (CM and SE) use 0.1% w/v sodium azide or 0.1% w/v 2,2' dithiobis (pyridine-N-oxide) or 0.02% w/v benzalkonium chloride.

PART II - FOR ION EXCHANGE CELLULOSES IN PRE-SWOLLEN FORM AND FOR DRY FORMS AFTER PRE-CYCLING.

SLURRY PREPARATION

In general, fully ionized exchangers, e.g. QA 52 and SE 52 require less processing time to attain equilibrium with buffers of low ionic concentrations than the weaker DE 52 and CM 52 alternatives. Further, equilibration volumes required for all media are less when buffers are used in which the buffering ion is the co-ion, i.e. carries a charge of the same sign as the ion exchanger (e.g. when TRIS hydrochloride buffers are used for DE or QA the equilibration time and volume will be less than for with acetate-based buffers). In all cases, it is advantageous to pre-equilibrate the exchanger initially with a buffer of substantially higher concentration (at least tenfold) than the required initial starter buffer concentration. This is the basis for the preferred equilibration method described below. After column packing the ion exchanger must, of course, be equilibrated with buffer of the required lower ionic concentration before sample application. This is normally readily achieved by passing 2-4 bed volumes of the low concentration buffer through the packed column.

(a) Preferred Method

1. Stir the ion exchanger with a concentrated solution (0.2.0M) of the buffer to be used in the initial separation stage for 2 to 3 minutes. About 15-30mL of buffer are used for every dry gram of cellulose initially taken, or about 6mL/g of wet ion exchanger.
2. Adjust the pH of the buffer/ion exchanger slurry, while stirring, to the desired pH value with the acidic or basic component of the buffer.
3. Allow the slurry to settle and decant the supernatant containing any fines.
4. Redisperse the ion exchanger in the buffer. The total volume of the slurry should be 30mL/g of dry ion exchanger used or about 6mL/g of wet ion exchanger.
5. Allow the slurry to settle in a suitable measuring cylinder in an area free of draughts, direct sunlight, heaters, etc. The time allowed for settling is calculated from

t=nh

where t = time (min.)

where h = the total height of slurry in the measuring cylinder (cm)

where n = a factor between 1.3 and 2.4

6. Note the ‘wet settled volume'. This is the volume occupied by the ion exchanger after settling under prescribed conditions. Immediately remove the supernatant containing fines so that the final volume remaining in the measuring cylinder is the `wet settled volume' plus 50%.

(b) Alternative method - aliquot buffer changes

1. Stir the ion exchanger into a volume of the buffer as in (a) 1.

2. Leave for ten minutes and decant off or filter the supernatant.

3. Repeat the treatment until the filtrate or the supernatant has exactly the same pH and conductivity as the buffer. This must be checked after a further buffer change. This method may require many changes and be time consuming when buffers of low concentrations are used.

4. Fines removal and preparation of column packing slurry is carried out according to instruction (a) 4, 5, and 6 above.

COLUMN PACKING

Convection currents in the slurry must be avoided during the actual column packing.

It is essential that from the moment of pouring the slurry into the column to the stage of having a settled column bed of ion exchanger, the operations are carried out as quickly as possible; otherwise, convection currents in the slurry have sufficient time to be set in motion.

1. Set up the column vertically in an area free of drafts, direct sunlight, heaters, etc.
2. Fit an extension tube if the slurry volume is greater than the column volume.
3. Pour the stirred slurry into the column.
4. Allow the eluent from the column to run to waste.
5. When all the slurry has been added, attach or insert the top column end.
6. Pump or run the buffer through the column at a flow-rate of at least 45 mL/hour/cm of the internal cross-sectional area of the column until the column bed height is constant.
7. Stop the flow of buffer into and out of the column.
8. Remove the extension tube (if fitted) and replace the top column end.

EQUILIBRATION

It must be emphasized that readings of pH and conductivity must be exact. With true equilibrium the equilibrated solution will be identical to the starting buffer solution. It is essential that readings of two consecutive equilibration solutions are not only the same, but are identical to the starting buffer. Incorrect equilibration is the most frequent cause of irreproducible results. Pass the starting buffer through the column until the conductivity and pH of the eluent are exactly the same as the starting buffer. This method is suitable for column separations starting with low concentration of buffer.

SAMPLE PREPARATION AND LOADING

Dissolve the sample in the starting buffer and adjust the pH of the solution. Cell extracts and ammonium sulphate precipitates should be dialyzed against the chromatographic starting buffer. Lack of attention to detail at this stage may give non-reproducible results. The mixture is normally loaded onto the column at a controlled flow rate.

ELUTION

Start the elution immediately or at a standard time after sample loading. Generally, there are three methods by which chromatographic separations are achieved.

1. Isocratic elution

The buffer used for the equilibration of the ion exchanger and the sample mixture may also be used for elution, which may be accomplished in two ways:

1. Target molecule is unbound. The amount of ion exchanger required would depend on its capacity for the unwanted, bound, components in the mixture. A short, fat, column may be used.

2. Target molecule is bound. When the mixture consists of chromatographically similar components a relatively long column is required in order to obtain the optimum resolution. It is advisable to use only a small part ( ~ 5 % ) of the total capacity of the column.
   
   Alternatively, different buffers are used for the equilibration of ion exchanger and for the chromatographic separations on the column. This method should be avoided unless the system has been well defined already as misleading results may be obtained.

2. Gradient elution

A buffer of a continuously or step-wise changing composition is used to effect the separation. The variation in the composition of the buffer may be one to higher ionic concentrations or to the appropriate pH or both. Since the buffer itself is the main factor in the achievement of the separation, the amount of ion exchanger required would depend on the capacity of the ion exchanger for the target compounds. Should the mixture contain any chromatographically similar components, however, some additional length to the column would be required in order to obtain resolution.

SPECIAL TECHNIQUES

STERILIZATION

All Whatman ion exchange media, with the exception of P11, may be autoclaved for sterilization purposes. This is best carried out on a slurry of the ion exchanger buffered with a non-volatile buffer of pH 6.5 to 7.5. The recommended conditions are 10 psi for 15 minutes, or 15 psi for 10 minutes.

Alternatively, also with the exception of P11, all products may be chemically sterilized by dispersion in 0.5 NaOH followed by washing with sterile water. All products may also be treated with ethanol-water mixtures containing up to 20-25 % by volume of ethanol.

DEGASSING (FOR ANIONIC EXCHANGERS)

For the most delicate work, removal of carbon dioxide absorbed by DE and QA cellulose exchangers, may he required in obtaining reproducible results. Generally this is not necessary if the ‘Preferred Method’ for slurry preparation is followed.

1. Place cellulose in the acid compartment of the buffer.

2. Check that the pH is below 4.5. If not, then a higher concentration of the acid component must be used.

3. Apply a good vacuum (down to below IO cm Hg pressure) with stirring until no more bubbles are noticed, but stop before boiling occurs. This may conveniently be carried out by stirring the slurry with a magnetic stirrer in a stoppered Buchner flask connected to an aspirator.

4. Add the basic component of the buffer to give the desired pH. For more critical work buffer solutions must be made up from CO₂-free water and subsequently kept free of CO_2.

USE OF ALCOHOLIC BUFFE RS

When alcoholic buffers are used with SE 53, the equilibration procedure is to use the aqueous buffer component first and then complete by equilibration with the alcoholic buffer system.

BATCH USE

1. Add a known quantity of the pre-cycled, equilibrated (and fines free) ion exchanger to the crude solution.

2. Stir for a standard time depending on the rate at which the component(s) to be separated is adsorbed (this should be pre-determined).

3. Separate the ion exchanger from the buffer by filtering or centrifuging.

4. Wash the ion exchanger with the buffer used for equilibration.

5. Desorb in stirred fluidized bed or in centrifuge; alternatively, harvest and pack the cellulose into a column and desorb using column elution techniques previously described..