Gel Filtration columns and media

Transcription

1 GE Healthcae Gel Filtation columns and media Selection guide and poduct pofile

2 Technical infomation Choice of eluent Eluent composition does not diectly influence esolution To avoid ionic inteactions, the ionic stength of the eluent should be at least 0.5 M Choose an eluent poviding good solubility and stability fo the sample potein/s The eluent can often be chosen to simplify a late sepaation stage e.g. the column can be equilibated and eluted with the stat buffe fo a subsequent ion exchange sepaation Eluent pepaation Use distilled wate Use HPLC o analytical gade solvents, salts, and buffes Make and stoe the eluents in clean glasswae Filte the eluents though a 0. µm steile filte and de-gas befoe use Stoe the eluents at 4 C when not in use; to pevent bacteial gowth an antimicobial agent can be added Equilibate the eluents to ambient tempeatue befoe use to pevent fomation of ai bubbles (it is ecommended to de-gas befoe use if the eluent has been in stoage) Sample pepaation Choose a sample volume of <0.5% of the column volume fo media with aveage paticle sizes in the ange of 0 5 µm and <5% of the volume fo media with aveage paticle sizes in the ange 0 00 µm In goup sepaations (desalting) the sample volumes can be up to 0% of the column volume When epeating uns, pepae the sample using the same method and keep the sample concentation and volumes constant Choose chomatogaphic conditions unde which the sample is stable and soluble Centifuge (e.g., g fo 0 min) o filte sample to emove micopaticles (be sue to select a solvent-esistant filte if samples ae dissolved in oganic solvents) If the sample has high viscosity, dilute it with the eluent (avoid >0 mg potein/ml sample) Neve apply a tubid solution to the column; tubidity indicates sample insolubility which may be due to incoect ionic stength o ph Sample application Make sue the sample is ecently filteed o centifuged befoe applying it to the column A pefilte between injecto and column is not ecommended unless automated injections ae pefomed (a pefilte educes the esolution) ptimization Paametes to change ae:. Sample volume a smalle sample volume gives bette esolution. Flow ate a lowe flow ate gives bette esolution fo high molecula weight components (poteins), but the opposite may be tue fo small components (small peptides) since they diffuse moe quickly, and the longe the sepaation time, the wide the sample zones become. Column length the esolution of two sepaated zones inceases by the squae oot of the column length; the effective bed height can be inceased by coupling two columns in seies Buffe choice and ph ae nomally of mino impotance. Howeve, low ph may enhance hydophobic inteactions and can be used to impove sepaation in some cases (e.g., peptides; mixed-mode sepaation) Cleaning potocols Thee cleaning potocols ae ecommended: A. Simple: fo egula cleaning B. Rigoous: when the column is contaminated C. Hash: to be used as a last esot A. Simple cleaning The packing efficiency is cucial to avoid losing any pefomance, only clean the column when the contamination causes an incease in backpessue. Wash with /5 column volume (CV) 0. M NaH. Wash with /5 CV M acetic acid. Equilibate with eluent until the baseline is stable B. Rigoous cleaning Clean a column if you obseve: An incease in backpessue befoe cleaning, make sue that the high backpessue in the system is in fact caused by the column: disconnect one piece of equipment at a time (stating at the faction collecto) with the pumps woking, check the pessue eading afte each piece is disconnected to detemine the souce of the backpessue (you will often find that a dity pefilte causes the incease in backpessue) check the backpessue at the same stage duing each un since the backpessue can vay within one un (e.g., injecting a sample and mixing diffeent eluents may cause an incease in backpessue) A space visible between the adapto and filte A colo change at the top of the column A loss of esolution The following steps should be pefomed in sequence (NEVER exceed the column pessue limits):. Change the filte at the top of the column (Ticon and HiLoad columns; see instuctions fo individual columns o filte kits) since the contaminants ae intoduced with the liquid flow, many of them ae tapped in the filte. Set the pessue limit contol to the pessue limit given in the column instuctions. Wash with column volume (CV) M acetic acid 4. Wash with CV wate 5. Wash with CV 0% ethanol (un at a low flow ate) 6. Wash with CV 0. M NaH 7. Rinse with CV wate and a few injections of M acetic acid 8. Equilibate with buffe until the baseline is stable Cleaning volume of CV is only a guideline the pactical equiements ae best detemined by monitoing the baseline, which should be stable at the end of each step C. Hash cleaning. Chemical opeations when planning a ecovey opeation, always take into account what caused the poblem in the fist place; vaious altenatives ae given fo each type of contaminant choose the most convenient accoding to the eagents you have available: if this does not wok, ty anothe Hydophilic poteins and peptides: Wash the column (ovenight, at low flow ate 5 0 cm/h) with the solution which peviously dissolved the mateial duing sample pepaation fo example, an extaction solution o detegent Wash the column ovenight in M acetic acid at 5 0 cm/h Fill the column with mg/ml pepsin in 0. M acetic acid and 0.5 M NaCl and incubate ovenight at oom tempeatue, o h at 7 C. Afte enzymatic digestion, thooughly inse the column with equilibation buffe Hydophobic poteins and peptides: These ae usually soluble in pola oganic solvents such as 90% ethanol, 0% acetonitile o 0% isopopanol If the pecentage of oganic solvent that best dissolves the contaminant is known, un this ovenight at a low flow ate Nucleic acids: Geneal: RNA and DNA ae vey soluble in solutions of low ionic stength: wash with 0 mm Tis-HCl, mm EDTA, ph 8.0 at low flow ate fo 4 h at oom tempeatue to dissolve pecipitated nucleic acids RNA: Inject 0. M NaH (see Instuction fo NaH stability) and leave fo h, inse with wate, inject 0. column volume of ibonuclease I solution ( mg/ml in 0. M NaCl, 50 mm Tis-HCl, ph 7.5), incubate the column fo h at 7 C, inse with at least column volumes of 0 mm Tis-HCl, mm EDTA, ph 8.0 DNA: Add 0. column volume Deoxyibonuclease I solution ( mg/ml in 0. M NaCl, 0 mm MgCl, 50 mm Tis-HCl, ph 7.5) to the column, incubate the column fo h at 7 C, inse with at least column volumes of 0 mm Tis-HCl, mm EDTA, ph 8.0. Afte enzymatic digestion, thooughly inse the column with equilibation buffe to emove tace amounts of enzyme emaining in the system; special caution is ecommended if subsequent sepaations of RNA o DNA ae planned. Lipids: Wash the column ovenight at a flow ate of 5 0 cm/h with detegent such as 0.% % Beol 85 in a basic o acidic solution. Remove detegent by washing with methanol, ethanol, o isopopanol. Cabohydates: Wash with column volume of 0. M sodium tetaboate (boax) titated to ph 8 with HCl. Note that boax pecipitates with some metal ions such as Cu +.. Mechanical opeations Change the bottom filte note that this may educe the column efficiency Stoage of unused medium +4 C to +0 C Make sue the unused medium is potected against bacteial gowth (e.g., stoe in 0% ethanol o buffe containing an antimicobial agent) Stoage of column +4 C to +0 C Shot tem stoage (e.g., ovenight) Stoe the column connected to the system; a low flow ate though the column will pevent bacteial gowth Stoe in the eluent used in sepaation Long-tem stoage The following steps should be pefomed in sequence (NEVER exceed the column pessue limits):. Clean the column accoding to Simple cleaning. Rinse the column and the system thooughly with wate to emove salt. Equilibate the column with column volumes of 0% ethanol: stat the equilibation at a low flow ate and check the backpessue while equilibating the column (mixing wate and ethanol inceases the backpessue) 4. Disconnect the column fom the system 5. Seal the column inlet and outlet Antimicobial teatment Pepacked columns: Sanitize Bulk media: Sanitize/autoclave Sanitization is the inactivation of micobial populations. When a packed column is washed with a sanitizing agent, the isk of contaminating the puified poduct with viable micooganisms is educed. The most commonly used sanitization method in chomatogaphy today is to wash the column with NaH. NaH has a vey good sanitizing effect and also has the additional advantage of cleaning the column; See poduct specific instuctions Bulk medium may be autoclaved in a wet fomat at ph 7 fo up to 0 minutes at +0 C Stoe samples in the cold unless this leads to pecipitation; avoid long stoage unless you can stoe at 70 C do not exceed pessue limits given in the column instuctions Use distilled wate Remove the top mm of medium and discad Use HPLC o analytical gade solvents, salts, and buffes Always check the efficiency of the column afte mechanical opeations

3 Poduct deing infomation Pepacked column/bulk media Code No. Column dim. id Pack size bed height (mm) Supedex Peptide PC./ Supedex Peptide 0/00 GL (Ticon ) Supedex 75 PC./ Supedex 75 0/00 GL (Ticon) Supedex 75 5/50 GL (Ticon) Supedex 00 PC./ Supedex 00 0/00 GL (Ticon) Supedex 00 5/50 GL (Ticon) HiLoad 6/60 Supedex 0 pg HiLoad 6/60 Supedex 0 pg HiLoad 6/60 Supedex 75 pg HiLoad 6/60 Supedex 75 pg HiLoad 6/60 Supedex 00 pg HiLoad 6/60 Supedex 00 pg Supedex 0 pep gade * ml ml Supedex 75 pep gade * ml ml Supedex 00 pep gade * ml ml Supeose PC./ Supeose 0/00 GL (Ticon) Supeose 6 PC./ Supeose 6 0/00 GL (Ticon) Supeose pep gade ml Supeose 6 pep gade ml HiPep 6/60 Sephacyl S-00 HR HiPep 6/60 Sephacyl S-00 HR HiPep 6/60 Sephacyl S-00 HR HiPep 6/60 Sephacyl S-00 HR HiPep 6/60 Sephacyl S-00 HR HiPep 6/60 Sephacyl S-00 HR Sephacyl S-00 HR * ml ml Sephacyl S-00 HR * ml ml Sephacyl S-00 HR * ml ml Sephacyl S-400 HR * ml ml Sephacyl S-500 HR * ml ml Sephacyl S-000 SF ml HiTap Desalting HiTap Desalting HiPep 6/0 Desalting HiPep 6/0 Desalting PD-0 Desalting Columns Sephadex G-0 * g g Sephadex G-5 Supefine * g g Sephadex G-5 Fine * g g Sephadex G-5 Medium * g g g Sephadex G-50 Fine * g g Sephadex LH-0 * g g g BioPocess Media Made fo biopocessing. * Pocess scale quantities ae avaible. Please contact GE Healthcae fo futhe infomation. Pack size available by special ode. Ticon columns ae designed fo analytical and semi-pepaative use with ÄKTAdesign systems and othe high pefomance chomatogaphy systems. Factionation ange Factionation ange Exclusion globula poteins dextans limit DNA M (elative molecula M (elative molecula (base pais) weight, Daltons) weight, Daltons) No data No data No data No data < No data No data < No data No data No data No data < No data No data No data No data No data No data No data No data No data No data No data No data No data No data No data No data > < 700 < No data < 5000 No data PC./0 is designed fo analytical and micopepaative use with SMART System. Pecision Column Holde (Code No ) also enables the columns to be used with ÄKTAdesign systems and othe high pefomance chomatogaphy systems. Paticle size Numbe of ph ange (µm) theoetical stability plates/mete (woking and long tem) 5 > > > > > > > > > > > > > > > > > > > > > > > > Not specified 0 80 (dy) Not specified Not specified 40 0 (dy) 0 50 (dy) 0 80 (dy) (dy) 0 80 (dy) (dy) HiLoad and HiPep columns ae designed fo pepaative applications with high pefomace systems such as ÄKTAdesign systems as well as low pessue systems. LabMate buffe esevoi (Code No ) can be used with PD-0 Desalting Columns fo easie and moe convenient equilibation. Maximum Maximum Recommended Bed volume opeating opeating sample volume appox. (ml) back pessue flow ate (MPa/psi) appox..0/ ml/min 5 5 µl.4.8/60. ml/min 5 50 µl 4.4/50 0. ml/min 5 µl.4.8/60.5 ml/min 5 50 µl 4.8/ ml/min 4 50 µl.5/0 0. ml/min 5 µl.4.5/0.0 ml/min 5 50 µl 4.5/0 0.8 ml/min 4 50 µl 0./44.7 ml/min 5 ml 0 0./ ml/min ml 0 0./44.7 ml/min 5 ml 0 0./ ml/min ml 0 0./44.7 ml/min 5 ml 0 0./ ml/min ml 0 0./44 90 cm/h 0./44 90 cm/h 0./44 90 cm/h.4/50 0. ml/min 5 µl.4 /45.5 ml/min 5 50 µl 4./75 0. ml/min 5 µl.4.5/0.0 ml/min 5 50 µl 4 0.7/00 40 cm/h 0.4/58 40 cm/h 0.5/.0 ml/min 5 ml 0 0.5/.7 ml/min ml 0 0.5/.0 ml/min 5 ml 0 0.5/.7 ml/min ml 0 0.5/.0 ml/min 5 ml 0 0.5/.7 ml/min ml 0 0./9 60 cm/h 0./9 60 cm/h 0./9 60 cm/h 0./9 60 cm/h 0./9 50 cm/h Not detemined 40 cm/h 0./44 5 ml/min ml 5 0.5/ 40 ml/min.5 5 ml ml cm/h 0 cm/h Can be 60 cm/h calculated using Dacy s Law 50 cm/h 60 cm/h 0.5/ 0 cm/h Pepacked HiTap columns ae designed fo convenient, fast and easy pepaative sepaations. The columns can be used eithe alone o connected in seies. Use the columns with a syinge, peistaltic pump, o chomatogaphy system. Applications Micopepaative analytical high esolving sepaation of peptides and othe small biomolecules Semi-pepaative and analytical high pefomance sepaation of peptides and othe small biomolecules Micopepaative analytical high esolving sepaation of poteins, peptides, polynucleotides and othe biomolecules Semi-pepaative and analytical high pefomance sepaation of poteins, peptides, polynucleotides and othe biomolecules Rapid size analysis of potein homogeneity in sceening expeiments Micopepaative analytical high esolving sepaation of poteins, DNA fagments and othe biomolecules Semi-pepaative and analytical high pefomance sepaation of poteins, DNA fagments and othe biomolecules Rapid size analysis of potein homogeneity in sceening expeiments Pepaative sepaation of peptides and othe small biomolecule Rapid, pepaative sepaation of poteins, peptides, polynucleotides and othe biomolecules Rapid, pepaative sepaation of poteins, DNA fagments and othe biomolecules Pepaative sepaation of peptides and othe small biomolecules Rapid, pepaative sepaation of poteins, peptides, polynucleotides and othe biomolecules Rapid, pepaative sepaation of poteins, DNA fagments and othe biomolecules Micopepaative analytical high esolving sepaation of poteins, peptides, oligonucleotides and polysacchaides Semi-pepaative and analytical high pefomance sepaation of poteins, peptides, oligonucleotides and polysacchaides Micopepaative analytical high esolving sepaation of poteins, peptides, oligonucleotides, polysacchaides and nucleic acids Semi-pepaative and analytical high pefomance sepaation of poteins, peptides, oligonucleotides, polysacchaides and nucleic acids Pepaative high pefomance sepaation of poteins, peptides, oligonucleotides and polysacchaides Pepaative high pefomance sepaation of poteins, peptides, oligonucleotides, polysacchaides and nucleic acids Pepaative sepaation of poteins and peptides Pepaative sepaation of poteins e.g. small seum poteins such as albumin Pepaative sepaation of poteins e.g. membane poteins and seum potein such as antibodies Pepaative sepaation of poteins and peptides Pepaative sepaation of poteins e.g. small seum poteins such as albumin Pepaative sepaation of poteins e.g. membane poteins and seum potein such as antibodies Pepaative sepaation of polysacchaides and othe macomolecules with extended stuctues e.g. poteoglycans and liposomes Pepaative sepaation of lage macomolecules e.g. goup sepaation of DNA estiction fagments Pepaation of DNA and sepaation of vey lage polysacchaides, poteoglycans and small paticles e.g. membane-bound vesicles and viuses Fast and convenient goup sepaation between high and low molecula weight substances Fast and convenient goup sepaation between high and low molecula weight substances Disposable column fo goup sepaation and buffe exchange Fast and convenient goup sepaation between peptides and low molecula weight substances Fast and convenient goup sepaation between high and low molecula weight substances Sepaation of natual poducts, such as steoids, tepenoids and lipids, in oganic solvents. ph stability long tem efes to the ph inteval whee the medium is stable ove a long peiod of time without advese effects on its subsequent chomatogaphic pefomance. All anges given ae estimates based on ou knowledge and expeience.. At oom tempeatue in aqueous buffe. The flow ate giving optimal esolution depends on the sample. Refe to instuctions fo each column and media.. Flow ate is calculated fom measuement in packed columns with an i.d. of.6 cm. A column heigth of 60 cm is used fo Supeose, Supedex and Sephacyl. Fo Sephadex the column i.d. is.6 cm and the height 0 cm.

4 Selection Guide Gel Filtation Media T his selection guide povides some geneal guidelines, based on the biomolecule of inteest and the pefeed factionation ange. Additional infomation can be found in Gel Filtation, Pinciples and Methods, available fom GE Healthcae. Stat fom the bottom left. Analytical (M ) Pepaative (M ) High selectivity (M ) Wide ange (M ) High selectivity (M ) Supedex Uppe - medium pessue systems High ecovey High stability High selectivity Supeose Medium pessue systems High ecovey Wide M factionation ange M (appox) Peptide M (appox) 00 Supeose 6 Supeose. Thyoglobulin (M ). Feitin (M ). Aldolase (M ) 4. Albumin (M ) 5. valbumin (M 4 000) 6. Chymotypsinogen A (M 5 000) 7. Ribonuclease A (M 700) 8. Cytochome C (M 500) 9. Apotinin (M 6500) 0. Gastin I (M 6). Substance P (M 48). (Gly) (M 6 60). (Gly) (M 89) 4. Gly (M 75). Thyoglobulin (M ). Feitin (M ). Aldolase (M ) 4. Albumin (M ) 5. valbumin (M 4 000) 6. Chymotypsinogen A (M 5 000) 7. Ribonuclease A (M 700) Semi-pepaative Analytical sepaation Pepaative sepaation Semi-pepaative Analytical sepaation Pepaative sepaation Peptides Small poteins Polynucleotides Poteins DNA-fagment Peptides Small poteins Polynucleotides Poteins DNA-fagment Wide factionation ange Intemediate factionation ange Wide factionation ange Intemediate factionation ange Supedex Peptide Supedex 75 Supedex 00 Supedex 0 pep gade Supedex 75 pep gade Supedex 00 pep gade Supeose 6 Supeose Supeose 6 pep gade Supeose pep gade Factionation ange (globula poteins) High esolution Factionation Pepaative & analytical (M ) Pepaative/ Maco factionation (M ) Wide ange (M ) Sephacyl Lowe - medium pessue systems Macomolecule sepaation Poduct line coveing wide factionation ange M (appox) 6 S - 00 S Thyoglobulin (M ). Feitin (M ). Aldolase (M ) 4. Albumin (M ) 5. valbumin (M 4 000) 6. Chymotypsinogen A (M 5 000) 7. Ribonuclease A (M 700) S - 00 Poteins Maco molecules Small poteins Poteins Lage poteins Factionation of macomolecules Puification of macomolecules Small paticles Vius Sephacyl S-00 HR Sephacyl S-00 HR Sephacyl S-00 HR Sephacyl S-400 HR Sephacyl S-500 HR Sephacyl S-000 SF NaCl <70% oganic solvents Goup sepaation Desalting Sephadex Desalting Goup sepaation 0 BSA Time (seconds) Small peptides Peptides/small poteins Poteins Sephadex G-0 Sephadex G-5 SF Sephadex G-5 F Sephadex G-5 M Sephadex G-50 F Exclusion limit Exclusion limit Exclusion limit >70% oganic solvents. C H Stat hee Sephadex LH Sepaation in (nonpola) oganic solvents 50 mg 54 mg. NH C CH CH C HN C H Low molecula steoids Tepenoids, lipids and peptides Sephadex LH-0 68 Time (h)

5 deing Infomation Columns Code No. Media Pack size Code No. Supedex Peptide PC./ Supedex Peptide 0/00 GL (Ticon) Supedex 75 PC./ Supedex 75 0/00 GL (Ticon) Supedex 75 5/50 GL Supedex 00 PC./ Supedex 00 0/00 GL (Ticon) Supedex 00 5/50 GL HiLoad 6/60 Supedex 0 pg HiLoad 6/60 Supedex 0 pg HiLoad 6/60 Supedex 75 pg HiLoad 6/60 Supedex 75 pg HiLoad 6/60 Supedex 00 pg HiLoad 6/60 Supedex 00 pg Supeose PC./ Supeose 0/00 GL (Ticon) Supeose 6 PC./ Supeose 6 0/00 GL (Ticon) HiPep 6/60 Sephacyl S-00 HR HiPep 6/60 Sephacyl S-00 HR HiPep 6/60 Sephacyl S-00 HR HiPep 6/60 Sephacyl S-00 HR HiPep 6/60 Sephacyl S-00 HR HiPep 6/60 Sephacyl S-00 HR HiTap Desalting (5 5 ml) HiTap Desalting (00 5 ml)* HiPep 6/0 Desalting ( 5 ml) HiPep 6/0 Desalting (4 5 ml) PD-0 Desalting Columns (0 pcs) *Pack size available by special ode Supedex 0 pep gade 5 ml ml Supedex 75 pep gade 5 ml ml Supedex 00 pep gade 5 ml ml Supeose pep gade 5ml Supeose 6 pep gade 5ml Sephacyl S-00 HR 50 ml ml Sephacyl S-00 HR 50 ml ml Sephacyl S-00 HR 50 ml ml Sephacyl S-400 HR 50 ml ml Sephacyl S-500 HR 50 m ml Sephacyl S-000 SF 750 ml Sephadex G-0 00g g Sephadex G-5 Supefine 00g g Sephadex G-5 Fine 00g g Sephadex G-5 Medium 5g g g Sephadex G-50 Fine 00g g Sephadex LH-0 5g g g Related poducts Gel Filtation Calibation Kit, LMW Gel Filtation Calibation Kit, HMW Handbook: Gel Filtation Pinciples and Methods BioPocess Media Made fo biopocessing Secue Supply Validated Manufactue Regulatoy suppot Captue to Polishing High Poductivity Sanitization & CIP/Scalability Custom Designed Media Lage capacity poduction integated with clea odeing and delivey outines-means availability in the ight quantity, at the ight place, at the ight time. Chomatogaphy is ou business; making BioPocess Media a safe investment fo long-tem poduction. Validated methods fo manufactuing & quality contol within IS900 cetified quality system. A cetificate of analysis is available fo evey lot and an MSDS fo evey poduct. Regulatoy Suppot Files detail pefomance, stability, extactable compounds, and analytical methods. The essential infomation in these files is an invaluable stating point fo pocess validation, as well as suppot fo clinical and maketing applications submitted to egulatoy authoities. BioPocess Media ae designed fo each chomatogaphic stage in a pocess fom Captue to Polishing. Take a systematic appoach to method development by using BioPocess Media fo evey stage. High flow ates, capacities, and ecoveies available with BioPocess Media contibute to the oveall economy of industial pocesses. All BioPocess Media can be cleaned and sanitized in place. Packing methods ae established fo a wide ange of scales. Use the same BioPocess Media fo development wok, pilot studies and outine poduction fo a diect scale up. Povide lage-scale uses with media designed fo specific applications though vaiations in ligand, coupling chemisty. and base matix. Custom Designed Media (CDM) ae fully tested and quality contolled. Some CDM s ae made on an exclusive basis fo specific customes; othes ae available on eceipt of ode.

6 GE Healthcae Bio-Sciences AB Bjökgatan Uppsala Sweden GE, imagination at wok, and GE monogam ae tademaks of Geneal Electic Company. ÄKTAdesign, BioPocess, HiLoad, HiPep, HiTap, Sephacyl, Sephadex, Supedex, Supeose and Ticon tademaks of GE Healthcae companies. The Ticon column and components ae potected by US design patents USD500856, USD5066, USD500555, USD and thei equivalents in othe counties. All thid paty tademaks ae the popety of thei espective ownes Geneal Electic Company All ights eseved Fist published 000. All goods and sevices ae sold subject to the tems and conditions of sale of the company within GE Healthcae which supplies them. A copy of these tems and conditions is available on equest. Contact you local GE Healthcae epesentative fo the most cuent infomation. GE Healthcae Euope GmbH Munzinge Stasse 5, D-79 Feibug, Gemany GE Healthcae UK Limited Amesham Place, Little Chalfont, Buckinghamshie HP7 9NA, UK GE Healthcae Bio-Sciences Cop. 800 Centennial Avenue, P.. Box 7 Piscataway, NJ , USA GE Healthcae Bio-Sciences KK Sanken Bldg. -5-, Hyakunincho Shinjuku-ku Tokyo , Japan Asia Pacific Tel: Fax: Austalasia Tel: Fax: Austia Tel: 0 / Fax: 0 / Belgium Tel: Fax: Canada Tel: Fax: Cental, East, & South East Euope Tel: Fax: Denmak Tel: Fax: Eie Tel: Fax Finland & Baltics Tel: Fax: Fance Tel: Fax: Gemany Tel: Fax: Geate China Tel: Fax: Italy Tel: Fax: Japan Tel: Fax: Koea Tel: Fax: Latin Ameica Tel: Fax: Middle East & Afica Tel: Fax: Nethelands Tel: Fax: Noway Tel: Fax: Potugal Tel: Fax: Russia & othe C.I.S. & N.I.S Tel: Fax: Spain Tel: Fax: Sweden Tel: Fax: Switzeland Tel: Fax: UK Tel: Fax: USA Tel: Fax: imagination at wok AE 05/007