Ion Exchange Chromatography - Amersham

8/12/2019 Ion Exchange Chromatography - Amersham

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• Based on well documented and w ell proven

Sephadex base matrix

• Simple and economical to use

• Very high capa cities even at elevated ionicstrengths

• Very low non-specific adsorption

• Separa te molecules over broa d molecularw eight a nd pH ranges

• Wide range of applications at laboratoryand process scales

• Suitable for column or batch techniques

IntroductionIon exchange is probably the most frequently usedchromat ographic technique for separa ting andpurifying prot eins, po lypeptides, nucleic acids a ndother charged biomo lecules. In ad dition to itswidespread usefulness, ion exchange is also easy toperform. Results are reliable and reproducible.Furthermore, ion exchange media generally

combine high capacity with high resolving power.The popularity and general applicability of thetechnique has resulted in a wide range of ionexchange media being available toda y. O ne groupof media, Sephadex ion exchangers, has for ma nyyears held a key position in several importantapplication areas, especially at process scale.Typical process scale applications include thefractionation of plasma components such asprothrombin complex using the ba tch ion exchangetechnique, and the purifi cation of insulin by

traditional column chromatography.

Sephadex ion exchangers

General descrip tion

Sephadex ion exchangers are produced byintroducing functiona l groups onto Sephadex,a cross-linked d extran mat rix. These groups areat tached to glucose units in the matrix b y stab leether linkages

A m e r s h a m B i o s c i e n c e

D ata FileI on exchange chromatography

Sephadex® ionexchange media

Sephadex ion exchange media are w idely used for process scaleapplications. Their separa tion pow er, ease of use and overalleconomy make them suitable for both column and batchtechniques.

Table 1 show s the full range of eight Sephadex ionexchangers. Anion a nd ca tion exchangers aredesignated A-25 or A-50 and C -25 or C -50

respectively.

18-1117-58

Edition AA



The terms strong and weak refer to the extent ofvariation of ionization w ith pH and not to t hestrength of binding. Strong exchangers (QAE and

SP Sephadex) are completely ionized over a widepH range w hereas the charge of w eak exchangers ispH dependent. Weak anion exchangers loosecharge when pH increases w hereas w eak cationexchangers gain charge.

Table 2 summarizes the main cha racteristics ofSephadex ion exchangers.

Bead form

Sephadex is a dry, bead-formed gel prepared bycross-linking dext ran w ith epichloro hydrin. The gelswells in aqueous solutions. Different types ofSephad ex belonging to the G -series differ in theirdegree of cross-linking and hence in their degree ofswelling.

Sephadex ion exchangers are derived from eitherSephad ex G -25 or Sephadex G -50. The G -25matrix is more highly cross-linked tha n the G -50.Ion exchangers based o n Sephadex G -25 havegreater rigidity and thus swell less than those basedon G -50, w hich are less rigid (see Table 6).

These properties mean t ha t A-25 and C -25 type ionexchangers are a good choice for small moleculesup to about 30 000 MW (e.g. peptides,oligosaccharides and oligonucleotides) whereasA-50 and C -50 types are better suited to larger

biomo lecules, such as pro teins in the molecularw eight ra nge 30 000 to 100 000. H igh molecularweight molecules, which are excluded from thebead, ma y be ad sorbed to some extent on t he outersurface.

As described later, Sephadex A-50 and C-50 ionexchangers are ideal for b at ch separa tions.

DEAE Sephadex QAE Sephadex CM Sephadex SP Sephadex

Type o f ex cha nger M ix ed w ea k a nd St ro ng a nio n Wea k ca tio n St rong ca tionstrong* anion exchanger exchanger exchanger exchanger

M ain functional group D iethylaminoethyl D iethyl-(2-hydroxy- Carboxymethyl Sulphopropylpropyl)aminoethyl

Counter ion Chloride Chloride Sodium Sodium

Rec. pH w orking range 2–9 2–10 6–10 2–10

D ynamic capacity See Tables 3 and 4.

M ol w t range A-25 and C -25 types: up to 30 000.A-50 and C-50 types: 30 000–100 000.

Bead form Spherical, 40-125 µm in dry form

Bead structure Cross-linked dextranC hemica l sta bility Sta ble in w a ter, sa lt solutions, orga nic solvents, a lka line a nd

weakly acidic solutions and denaturing solvents

Physica l sta bility A-25 a nd C -25 types: robust – ha ve good flow properties a ndnegligible volume changes due to cha nges in pH and ionic strength.A-50 and C-50 types: volume may va ry due to changes in pH a nd ionic strength.

Autoclavable Sw ollen, in sa lt form for 30 min a t 120 ° C .

Availability Supplied dry in a range of laboratory and process scale pack sizes.See ordering informat ion

Table 2. G eneral cha racteristics of Sephadex ion exchange media.

* See Table 1.

Type Description

DEAE Sephadex A-25 Mixed weak and st rongA-50 an ion exchanger*

QAE Sephadex A-25 Strong anion exchangerA-50

C M Sepha dex C -25 Wea k ca tio n ex cha ngerC-50

SP Sepha dex C -25 St ro ng ca tio n ex cha ngerC-50

Table 1. The range of Sephadex ion exchangers. See Table 2for more detailed information.

* DEAE Sephadex contains functional groups that display both

strong and w eak ion excha nge groups (1/3 strong, 2/3 w eak).



Capacity

Due to the differences in swelling, ion exchangersba sed on Sephadex G -25 have a higher ioniccapacity per ml sw ollen gel than tho se based o nG -50. Dy namic capacity a lso depends on the pH ,ionic strength and the nature of the sample (seeTables 3 and 4).

Stability

Sephadex ion exchangers are insoluble in allsolvents commonly used in liquid chromatography.They are stable in water, salt solutions, organicsolvents, and a lkaline and w eakly acidic solutionsin the pH range of 2–10. The media can beauto claved a s a slurry in the salt fo rm for 30minutes at 120 ° C and pH 7. Exposure to strongoxidising agents or dextranases should be avoided.When media are stored in the swollen state, a non-ionic antimicrobial agent should be included in thebuffer.

The high mechanica l stability o f Sephadex A-25and C -25 media gives outstanding flo w properties,w hich makes them ideal for packing and operatingin large scale chromat ography columns.

The pressure/flow ra te curve show n in Figure 1illustra tes the relationship betw een the pressuredrop over a packed bed of DEAE Sephadex A-25and the linear flow rate in a 5 cm i.d. column with

a bed height of approximately 10 cm. The curve isessentially linear in the typical operating range upto 0.15 ba r w here the fluid velocity is abo ut380 cm/h for this ba tch. The maximum pressuredrop i.e. that pressure drop where the flo w rat elevels off was not determined in this case.

Thyroglobulin HSA α-lactalbuminProtein (MW) (669 000) (68 000) (14 300)

D EAE Sephadex A-25 1.0 30.0 140.0A-50 2.0 110.0 50.0

Q AE Sephadex A-25 1.5 10.0 110.0A-50 1.2 80.0 30.0

Table 3. D ynamic capa city (mg/ml swo llen gel) da ta for D EAE and Q AE Sephadexion exchangers.

Ca pacities were determined at a fl ow rat e of 75 cm/h. The buffer w as 0.05 M Tris, pH 8. 3.

IgG Bovine COHbProtein (MW) (160 000) (69 000)

CM Sephadex C-25 1.6 70.0C-50 7.0 140.0

SP Sephadex C-25 1.1 70.0C-50 8.0 110.0

Table 4. D ynamic capa city (mg/ml swollen gel) data for C M and SP Sephadex ionexchangers.

Ca pacities w ere determined a t a flo w rate of 75 cm/h. The buffer wa s 0.1 M acetate buffer,pH 5.0.

Flow rate(cm/h)

450

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50

0

0 0.05 0.1 0.15 0.2

bar

Fig. 1. The pressure/flo w rat e curve for a 10 cm bed of D EAESephadex A-25 in a 5 cm i.d. column is essentially linear,w hich demonstrates the suita bility of Sephadex A-25 and C-25ion exchangers for column use.



Sephadex A-50 and C-50 media, which are lessrigid and therefore more prone to volume changes,are better suited t o t he batch t echnique. Ba tchseparation is simple to perform and no technicaldifficulties are caused by the swelling or shrinkageof the ion excha nger.

Batch to batch reproducibil it y C onsistent qua lity and performance from bat ch-to-ba tch is important f or a ll separa tion media, but itassumes extra significance w hen the media arerout inely used in industrial pro cesses, a s is the casew ith Sephadex ion exchangers. Some randomlyselected qua lity control da ta gat hered fro m over30 years prove beyond do ubt tha t these media ha vebeen produced w ith outstanding bat ch-to-batchconsistency (see Table 5).

Swell ing vari es w it h ionic str engthand pH

Ion exchangers based on Sephadex swell inaq ueous solutions. As an example, Table 6 show sdry a nd hydra ted bead sizes of Sephadex cat ionand anion exchangers.

As dry media are swollen before use, it is the wetbead diameter that is of practical importa nce w henchoosing the correct type of equipment to use, e.g.mesh size of t he column nets.

Initial swelling of dry Sephadex ion exchangersshould be performed in 0.2 M salt. In distilledw at er the beads w ill swell too much, to o q uickly,w hich may cause breakage.

Swelling varies with ionic strength and pH. A-25and C -25 exchangers have a fa irly rigid beadstructure and size variations due to changes in pH

or ionic strength are therefore small. A-50 andC -50 exchangers, on t he other hand, swell morethan their A-25 and C-25 equivalents due to theirless rigid structure. In addition, swelling at a givenpH is highest at low ionic strengths as repulsionbetw een similarly charged groups on the mat rix isthen ma xima l (see Fig 2). Fluctuat ions in swellingwill be minimized in buffers with ionic strengthsab ove 0.1.

A similar pa ttern can be seen in Figure 3, w hichshow s how pH aff ects the degree of swelling. Not e,

how ever, t hat the swelling of the strong ionexchangers QAE and SP Sephadex is virtuallyindependent of pH since, as strong exchangers,they remain charged over a w ide pH range.

Separation principleThe separation effect of ion exchange chromato-graphy depends on the reversible adsorption ofcharged solute molecules to immobilized groups ofopposite charge. Most separations can be describedin five stages.

Flow rate at Function; salt conc. in elution peak; M NaCl Total capacityLot. No. ∆p/L=10 [cm/h] CO Hb1 CO Hb2 HSA mmolCl–/g

(Limits) – (min. 60) (0.04–0.07) (0.06–0.10) (0.16–0.22) (3.0–4.0)5042 1972 – 0.06 0.09 0.20 3.40

18896 1981 97 0.06 0.09 0.19 3.302783 1986 83 0.05 0.07 0.16 3.40

13799 1991 103 0.05 0.07 0.17 3.42

226550 1994 90 0.05 0.07 0.17 3.29

Table 5. Batch to batch reproducibility is demonstrated by some typical data from the quality control of DEAE Sephadex A-50.

Hydrated median Diameter ratioProduct Medium diameter µm* hydrated/dry*

D EAE Sephadex A-25 0.15 M NaCl 127 1.83D EAE Sephadex A-25 0.50 M NaCl 121 1.75D EAE Sephadex A-50 0.15 M NaCl 214 3.17D EAE Sephadex A-50 0.50 M NaCl 182 2.69CM Sephadex C-25 0.15 M NaCl 131 1.94CM Sephadex C-25 0.50 M NaCl 125 1.86

CM Sephadex C-50 0.15 M NaCl 250 3.41CM Sephadex C-50 0.50 M NaCl 221 3.02

Table 6. H ydra ted bead sizes of Sephadex ion exchangers. No te tha t low er ionic strengths w ill give higher hydra ted/drydiameter ratios.

* Average values from three product batches of each ion exchanger.



Stage one is equilibration. The pH and ionicstrength o f the equilibra tion buffer a re adjusted tofavour binding of the molecules of interest in thesolute. Weak ion exchangers have their ownbuffering capacities and need longer equilibrationtimes. At this stage the immobilized groups areassociated w ith exchangeable counter ions.

Stage tw o is sample application and ad sorption.

Solute molecules of interest d isplace the counterions and bind reversibly to the gel. Unboundsubstances can be washed out using starting buffer.

(An a lternat ive stra tegy sometimes used is to b indthe contaminant s at this sta ge, in w hich case, it isthe the substance of interest tha t is w ashed out o fthe gel.)

Not e that contaminants may compete w ith thesubstance of interest and a lso bind a t stage tw o,thus reducing the capacity for binding the intended

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Ionic strength

Effect of ionic strength on swelling

Fig. 2. Bed volumes obta ined from 1 gram d ry gel as a f unction of ionic strength. DEAE and QAE Sephadex w ere measured in

Tris HCl buffer pH 7.6 with varying NaCl concentration. CM and SP Sephadex were measured in acetate buffer pH 4.3 withvarying Na Cl concentration.

product. If this occurs, the experimental conditionsand/or the chosen ion excha nger should be cha ngedto favour binding the substance of interest.

Stage three involves increasing the ionic strength ofthe eluting buffer or changing its pH to conditionstha t fa vour the elution of the bound substance ofinterest.

The fourth and fi fth sta ges are the remova l fromthe column of substances not eluted under theprevious experimental conditions followed by re-equilibration t o t he sta rting conditions for the nextseparation cycle.

Ion exchange separa tions can b e carried o ut in acolumn or by a batch procedure. The basicprinciple outlined in the five stages described ab oveapplies to bot h methods.



Working with Sephadex ion exchangersSephadex A-25 and C-25 ion exchangers arehighly suitable for column chromatography a ndSephadex A-50 and C-50 for batch techniques.This versatility combined with low non-specificinteraction, consistent qua lity, batch to ba tchreliability and excellent price performance, makesSephadex ion exchangers an obvious choice for

large scale applications.

Column chromatography

G ood column design and good packing techniquesare important in realising the full separationpotential of ion exchange chromato graphy.

Amersham Biosciences has columns available for ionexchange chromatography at laboratory, pilot andfull production scales.

Our range of process columns covers all stages

from process development to full scale production

and includes INdEX ™ columns, BPG ™ columns,Process Stack co lumns (PS370), B ioPro cess™

Stainless Steel (BPSS) columns andCHROMAFLOW™ columns. BPG , BPSS andCHROMAFLOW are designed for applicationstha t d emand t he highest standa rds of hygiene, suchas in pharma ceutical production.

As in most adsorptive techniques, ion exchange is

carried out in short columns. Bed heights aretypically 5–15 cm. Once the separation has beenopt imized, scale up is achieved by increasing thecolumn diameter.

Column packi ng

As with any other column chromatographictechnique, packing is a critical step in theexperiment. A w ell-packed co lumn gives even flo w,minimizes band bro ad ening and promotes highresolution.

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Effect of pH on swelling

Fig. 3. Bed volumes obtained from 1 gram dry gel as a function of pH. DEAE and QAE Sephadex were measured in imidazole

and ethylenediamine buffers of varying pH at a constant ionic strength of 0.05. CM and SP Sephadex were measured inphosphate buffers of varying pH at a constant ionic strength of 0.05.



Fig. 4. CHROMAFLOW columns are designed andconstructed t o meet the stringent demands o f process scalechromatography.

7

elution a nd a ba tch procedure. As described earlier,either the substance of interest or contaminantsmay be att ached to the ion exchanger.

Although batch procedures are less efficient thancolumn techniques they ma y o ffer adva ntages inpart icular ca ses. When very large sample volumeswith low protein concentration have to be

processed, the sample application time on a columncan be very long and filtration of such a largesample can also be ra ther difficult to perform.Binding the sample in batch mode will be muchquicker and there will be no need to removeparticulate matter.

A batch procedure can a lso be an a ttra ctiveapproach if high sample viscosity generates highba ck-pressure in a column pro cedure or if highback-pressure is generated by contaminants such aslipids, which may cause severe fouling and clogging

of the column.When w orking with ba tch ion exchange, thestarting conditions are selected in the same w ay asin column chromatography, i.e. choose buffer pHand ionic strength to bind the substance of interestbut to prevent a s many cont aminants as possiblefrom b inding.

To maximize recovery, the start ing condit ionsshould be selected so tha t t he protein of interestbinds much stronger than is necessary in columnchromat ography. U nless the proteins adsorb to

100%, losses during subsequent washing will beinevitable, especially if t he volume of liquid is largecompa red to the volume of a dsorbent. To keeprecovery high, the pH in a ba tch experiment ma yhave to be several units away from the isoelectricpoint of the protein.

Bat ch separa tion is carried o ut by stirring the ionexchanger previously equilibrated in theappropriate buffer w ith the solution to b e treat eduntil the mixt ure has reached equilibrium. Thisusually ta kes abo ut o ne hour. The slurry is then

separated by fi ltration and w ashed w ith the buffersolution. In cases of incomplete adsorption, thisprocedure should be repeated on the fi ltrate w ithfresh ion excha nger. Then elution b uffer is added(1–2 times the volume of the sedimented gel) andstirred until desorption is complete, which can take30 minutes or more. Finally, suction is used to fi lterthe buffer containing the desorbed product ofinterest from the ad sorbent.

Figure 5 show s a ba tch ta nk suitable for use w ithSephad ex A-50 and C -50 ion excha ngers.

Prior to packing, Sephadex ion exchangers shouldbe swollen at the pH to be used in the experiment.C omplete swelling takes 1–2 da ys at roomtemperat ure or 2 hours in pH 7 buffer atapprox imately 100 ° C . The required amount of ionexchanger should be stirred into an excess ofstarting buffer. The supernata nt should be removed

and replaced with fresh buffer several times duringthe swelling period. To a void generat ing fi nes, theionic strength during swelling should be 0.2 M salt.

Appli cati ons of process scale column

techniques

The excellent chemical a nd physical stability plusthe ease of packing and cleaning have madeSephad ex A-25 and C -25 types popular cho ices fora wide range of applications. They are usedespecially fo r separa ting sma ll proteins, peptides

and carbo hydrat es because of the low non-specificadsorption of these types of biomolecules.

D EAE and QAE Sephadex A-25 have been usedextensively for the large scale production of peptidehormones as, to a lesser extent, has SP SephadexC-25. CM Sephadex C-25 is e.g. used for the largescale purificat ion of oligosaccharides.

Batch separation

There is essentially no difference in separation

principle between a column desorbed by stepwise



Product Quantity/Pack size Code No.

D EAE Sephadex A-25 100 g 17-0170-01D EAE Sephadex A-25 500 g 17-0170-02D EAE Sephadex A-25 5 kg 17-0170-03D EAE Sephadex A-25 40 kg 17-0170-07

D EAE Sephadex A-50 100 g 17-0180-01D EAE Sephadex A-50 500 g 17-0180-02D EAE Sephadex A-50 5 kg 17-0180-03D EAE Sephadex A-50 40 kg 17-0180-07

Q AE Sephadex A-25 100 g 17-0190-01Q AE Sephadex A-25 500 g 17-0190-02Q AE Sephadex A-25 5 kg 17-0190-03

Q AE Sephadex A-50 100 g 17-0200-01Q AE Sephadex A-50 500 g 17-0200-02Q AE Sephadex A-50 5 kg 17-0200-03

CM Sephadex C-25 100 g 17-0210-01CM Sephadex C-25 500 g 17-0210-02CM Sephadex C-25 5 kg 17-0210-03

CM Sephadex C-50 100 g 17-0220-01CM Sephadex C-50 500 g 17-0220-02

CM Sephadex C-50 5 kg 17-0220-03SP Sephadex C-25 100 g 17-0230-01SP Sephadex C-25 500 g 17-0230-02SP Sephadex C-25 5 kg 17-0230-03SP Sephadex C-25 40 kg on request

SP Sephadex C-50 100 g 17-0240-01SP Sephadex C-50 500 g 17-0240-02SP Sephadex C-50 5 kg 17-0240-03

Ordering information

Fig. 5. A stainless steel batch t ank eq uipped w ith a stirringsystem.

The gel can also be packed in a column after thewashing step and be eluted stepwise in the sameway as during normal column chromatography.Resolution w ill how ever be low er for such acombined b at ch and column procedure comparedwith a normal column procedure, since the sampleis bound uniformly throughout the gel slurry andthe subsequent chromat ographic bed.

Under these conditions stepwise elution isrecommended since gradient elution w ill give

broa der bands a nd of ten poorer resolution. Alsothe amo unt of eluent w ill be low er in stepw iseelution.

Bat ch chromato graphy is useful for concentra tingdilute solutions and separating the substances ofinterest from gross contaminants during the initialcapture stage of a purifi cation scheme.

Ion exchangers used in batch processes arediscarded a fter use, so the operat or do es not haveto be concerned with regeneration or cleaning-in-place procedures. The overa ll economy ofSephadex ion exchangers also favours their onetime use.

Appl ications of the batch technique

Batch ion exchange with Sephadex A-50 and C-50is ideal for the simple and economical Capture ofproteins and complexes from large volumes offeedstocks. They a re part icularly useful forprocessing crude feedstocks that are not suitablefor loading onto a column.

IgG and prothrombin complex are representat iveof a number of plasma components purifi ed onDEAE Sephadex A-50. Removal of detergents and

the purifica tion of w hey proteins are commonapplicat ions of C M Sephadex C -50.

Further information is available in the AmershamBiosciences handb ook “ Ion Exchange Chroma to-graphy – Principles and M ethods” (article no.18-1114-21), which is a practical guide to thetechnique, its applications and experimentalprocedures. Ask your loca l representa tive ofAmersham Biosciences for d etails.

Columns

For more information a bout t he range of processscale columns, please contact you local sales officeof Amersham Biosciences.

Ion Exchange Chromatography - Amersham

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