Cellulose Phosphate

For the best separations . . .

Whatman cellulose phosphates P1 and P11 are bi-functional cation exchangers containing both fully ionized and weak acid groups based on an ester-linked orthophosphate functional group.

These media are used for the separation of biopolymers and, in particular, enzymes catalyzing ATP-dependent phosphate group interactions such as protein kinases, restriction enzymes, deaminases, nucleases and polymerases.

The exceptionally high binding strength and selectivity provided by these highly substituted bi-functional exchangers may also be utilized to the best advantage for the separation of relatively small biopolymers bearing positive charges over the pH 3 to 7 range, such as peptides, small polypeptides and proteins (i.e. MW less than 15,000).

They possess a particularly high capacity and an affinity for large multivalent inorganic cations.

MEDIA RANGE

PHYSICAL AND CHEMICAL PROPERTIES

where (a) = strong ionized group and (b) = total of strong and weak groups.

(1) dg = dry gram (2) O.O5M pH 5.0 acetate buffer

EXCHANGER PREPARATION

PRE-CYCLING

Stir the weighted cellulose phosphate into 25 volumes of O.5N NaOH and leave for 5 minutes.

Filter or decant off the supernatant and wash the medium in a funnel until the filtrate pH is 11.0 or below.

Stir the cellulose phosphate into 25 volumes of O.5N HCI and leave for 5 minutes.

Filter or decant off the supernatant and wash in a funnel until the filtrate pH is above 3.0. In order to avoid excessive hydrolysis and swelling changes, it is important not to exceed the recommended 5-minute contact times.

NOTES ON GENERAL HANDLING

• DO Store the cellulose phosphate in a cool place.
• 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 the generation of fines. Since cellulose phosphate is an ester derivative of cellulose, in order to minimize the degree of hydrolysis of the product, special care with regard to the conditions of handling this product in the wet state are required.

• DO NOT leave cellulose phosphate in contact with aqueous solutions for periods of time beyond those necessary to complete the separation, unless the medium is stored in the presence of O.5M phosphate buffer at pH 7.0 at 5 xifC. In the latter case and for bulk use the medium may be stored for up to one week. For small laboratory scale use, in order to ensure reproducible results, fresh media should be employed on each occasion.

EQUILIBRATION

For batch separations

1. Transfer the pre-cycled cellulose phosphate immediately into 20 volumes of a more concentrated (say 3 to 10 times) buffer solution of the same pH as that to be used in the subsequent separation.

2. Titrate the stirred slurry with the base component of the high concentration buffer to the correct pH.

3. Decant or filter off the supernatant.

4. Stir the cellulose phosphate into 20 volumes of the starting elution buffer.

5. Leave for 3 or 4 minutes.

6. Repeat steps 3 and 4 until the filtrate of the supernatant has exactly the same pH and conductivity as the starting buffer. This may require as many as 5 or 6 changes of buffer.

For column separations

Equilibration may be completed in situ by pumping the starting buffer until the pH and conductivity of the eluent are constant and equal to those of the buffer.

COLUMN PACKING (For P11 only)

1. Column packing

1. Disperse the equilibrated cellulose phosphate into starting buffer and allow the slurry to settle in a suitable measuring cylinder. Note the "settled volume" occupied by the cellulose phosphate after settling. Adjust the total volume of liquid in the measuring cylinder to the "settled volume" plus 20 %.

2. Set up the column vertically, and fit an extension tube if the slurry volume is greater than the column volume.

3. Pour the stirred slurry into the column. Allow the eluent from the column to run to waste.

4. When all the slurry is added, attach or insert the top column end. Pump or run the buffer solution through the column at a flow rate of at least 45 mL/hour/cm2 of the internal cross-sectional area of the column until the column bed height is constant. Stop the flow of buffer into and out of the column.
5. Remove the extension tube (if fitted) and replace the top column end.

1. Sample Loading

1. Dissolve or equilibrate the sample mixture in the starting buffer.
2. Check the pH and adjust if necessary.
3. Load the sample.

CHROMATOGRAPHIC ELUTION CONDITIONS

Elution conditions will depend upon the type of separation and the mode selected:

1. Separation mode - as a normal high binding strength cation exchanger (i.e. for peptides, small proteins etc.) Elute adsorbed components by increasing ionic concentration gradients (i.e. buffer or starter buffer plus neutral salt). Alternatively, for well-defined systems, stepwise elution may be applied.

2. Separation mode - As a phosphate group interaction specific affinity medium (e.g. for protein kinases, restriction enzymes, polymerases, deaminases, etc.).

When possible it may be advantageous to select pH conditions whereby the components of interest are not bound by ionic interactions, i.e. at a pH level above their iso-electric points.

Subsequently elute the components of interest either by substrate elution (e.g. with ATP), or alternatively with increasing concentrations of phosphate buffer.

BATCH TECHNIQUE

1. Add a known quantity of the pre-cycled equilibrated cellulose phosphate to the biopolymer 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 cellulose phosphate from the slurry by filtration or centrifugation.

4. Wash the cellulose phosphate with the equilibration buffer.

5. Redisperse the cellulose phosphate in the buffer solution required to elute the components of interest and stir for up to 60 minutes. The nature of this buffer solution, which should be predetermined, will depend upon the particular system and the separation mode selected.

6. Separate the cellulose from the slurry by filtration or centrifugation. Collect the filtrate or supernatant.

7. Wash the cellulose phosphate cake with at least 2 bed volumes of elution buffer and combine this with the filtrate or centrate from stage 6 above.

Alternatively, after the batch adsorption stage the P11 medium may be packed into a column and eluted according to the particular separation requirements. This technique is especially useful when the initial volume of the biopolymer solution is large and combines the advantages of batch adsorption and column elution for the controlled release of bound components in the minimum volumes.

The long fibered P1 product is unsuitable for column packing and may only be used in batch adsorption/desorption techniques.