1 / 40

Laboratory Dialysis

Laboratory Dialysis. Cassidy Markee Director of Laboratory Division. Spectrum’s Core Technology: Membrane Separation. Spectrum offers products for two kinds of Membrane Separation (membrane, systems, components and accessories):

galvin-lester
Download Presentation

Laboratory Dialysis

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Laboratory Dialysis Cassidy Markee Director of Laboratory Division

  2. Spectrum’s Core Technology: Membrane Separation • Spectrum offers products for two kinds of Membrane Separation (membrane, systems, components and accessories): • Membrane Dialysis – passive diffusion through a semi-permeable membrane – 100 to 1,000,000 Daltons • Membrane Filtration – applied pressure across a selective membrane. 3 types based on size range: • Macrofiltration – 5 μm and up • (Woven Mesh Screens) • Microfiltration – 0.05 – 5 μm • (Direct Flow and Tangential Flow) • Ultrafiltration (Molecular filtration) – 5 kD to 0.05 μm • (Tangential Flow only)

  3. Why use laboratory dialysis? Many Advantages: • Simple, easy & quick set-up • Inexpensive & disposable • Very gentle separation (no pressure or shear stress) • Doesn’t require monitoring • Dissolved solutes remain in solution • Solutes are unaltered or damaged • Maintains macromolecule concentration • Purify low MW solutes • Total product recovery Only 2 Major disadvantages: • Very slow (1 – 2 days) • Molecules should be 100 X size difference

  4. Understanding Dialysis Passive diffusion of solutes across a semi-permeable membrane from an area of high concentration (sample) to an area of low concentration (dialysate/buffer) until equilibrium is reached. Driving Force: CONCENTRATION GRADIENT The higher the concentration gradient the faster the rate of dialysis. Opposing Force: EQUILIBRIUM As you approach equilibrium, the rate of dialysis slows down.

  5. Mode of Operation: Gentle diffusion across a membrane (high conc. in sample) (low conc. in buffer) (low conc. in buffer) (equilibrium) (equilibrium) (high conc. in sample) (equilibrium)

  6. 4 Fundamental Dialysis Applications • Purification – elimination of low MW solutes Protein purification HPLC sample prep Removal of free amino acids or nucleotides • Modification – changing ionic / chemical environment to affect activity Desalting pH Change Buffer Exchange • Isolation – controlled exposure for reactivity Separation of small target species Electro-elution • Equilibrium Dialysis – Binding Studies Antibody & Ligand Enzyme Activity

  7. Typical Dialysis Parameters • Sample & Dialysate (buffer) Concentrations • equal except for retained and passing molecular species • Sample to Dialysate (buffer) Ratio • at least 1 : 100 - 500 by volume • Membrane MWCO (pore rating) • 50-90% of the retained species molecular size • Volume to membrane surface area (SA) ratio • Length (10-15 cm) x FW (24-30 mm) = 2-5 ml/cm • Temperature • Room Temperature (20-25 Celsius) • “Static” Dialysis with stirring • Buffer Changing • Three times at 3-4 hrs, 6-8 hrs & 16-24 hours • Duration • 1 – 2 Days

  8. Molecular Weight Cut-Off (MWCO) Indirect measure of MW (solute size) retained by approximately 90-95%. Below the curve retained Above the curve passes

  9. Selecting MWCO for Max Yield and Purity • Max Yield: MWCO smaller than product size. • And Max Purity: MWCO 100-1000X bigger than low MW contaminants. Ex: 100 kD to retain 98% IgG AND remove 99% Vit B12

  10. Select Lower MWCO: Yield vs. Purity If only 10 X size difference between product (Cyt C) & contaminant (Vit B12): • Lower MWCO with 80-100% product retention for max yield BUT sacrifice purity. (Ex: 20 kD MWCO- 88% Cyt C yield & 12% Vit B12 contaminant)

  11. Select Higher MWCO: Yield vs. Purity If only 10 X size difference between product (Cyt C) & contaminant (Vit B12): Lower MWCO with 80-100% product retention for max yield BUT sacrifice purity. (Ex: 20 kD MWCO- 88% Cyt C yield & 12% Vit B12 contaminant) Higher MWCO with 50-80% product retention for max purity BUT sacrifice yield.(Ex: 50 kD MWCO- 76% Cyt C yield & 2% Vit B12 contaminant)

  12. Spectrum Dialysis Membranes: 2 Grades • Standard Grade - natural cellulosic polymer: 1 Type • Regenerated Cellulose (Standard-RC) MWCO - DISADVANTAGE: Limited range with loose selectivity PURITY - DISADVANTAGE: Trace levels of heavy metals & sulfides TOLERANCE - ADVANTAGE: Good temp (121 C), pH (2-12) & chemical resistance COST - ADVANTAGE: Most economical APPLICATIONS: General dialysis, eliminating small ions (desalt, pH change, Buffer exchange, etc.) • Biotech Grade – synthetic cellulosic polymer: 2 Types • Cellulose Ester (Biotech-CE) MWCO - ADVANTAGE : Broad range (100 – 1,000,000 D) with best selectivity PURITY - ADVANTAGE: Ultra-pure – no trace heavy metals & sulfides TOLERANCE - DISADVANTAGE: Limited temp (37 C), pH (2-9) & chemical resistance COST - DISADVANTAGE: More costly APPLICATIONS: Critical separations & molecular purifications (protein, oligos, macromolecules, etc.) • Regenerated Cellulose (Biotech-RC) MWCO ADVANTAGE: Small range of MWCO’s with good selectivity PURITY ADVANTAGE: Ultra-pure – no trace heavy metals & sulfides TOLERANCE ADVANTAGE: Good temp (121 C), pH (2-12) & chemical tolerance COST DISADVANTAGE: Most costly APPLICATIONS: Requiring purity and tolerance (sample prep with organics, etc.)

  13. Laboratory Dialysis: 4 Membrane Configurations • Dialysis Tubing • Most Economical • Many Flat Widths (FW) • Large Qty – bulk rolls • Evaluation – Trial kits • Requires closures • 3 Types: Standard-RC, • Biotech-RC & Biotech-CE • Disposable • Dialysis Devices • Built-in convenience • Easy-to-use • Saves time • Disposable • Ultra-pure membrane • 1 Type: Biotech-CE • Flat Sheet & • Dialysis Systems • Chamber separations • Large volume samples • Small volume samples • Equilibrium studies • 1 Type: Standard-RC • Hollow Fiber • MicroDialysis • Microvolume dialysis • Encapsulation w/ epoxy • Implant for in vivo studies • Potted bundle or loose HF • 1 Type: Standard-RC

  14. Dialysis Tubing: Standard Grade RC • Dry Standard Grade RC • Spectra/Por 1: 6-8 kD MWCO, general application • Spectra/Por 2: 12-14 kD MWCO, high perm / special FW • Spectra/Por 3: 3.5 kD MWCO, general application • Spectra/Por 4: 12-14 kD MWCO, general application • Spectra/Por 5: 12-14 kD MWCO, paper for wet strength • Flat Width (diameters): Many for different sample volumes • Quantity: Bulk 15 or 30m; and Trial Kits 5m (S/P 1, 2 & 3) • Preparation: Soak 30 min in water to remove glycerin • Storage: Dry room temp (avoid humidity) • Pre-treated Standard Grade RC • Spectra/Por 6 – Pre-wetted for convenience; 10 meters • Spectra/Por 7 – Pre-cleaned to remove trace heavy metals & sulfides; 5 meters • 8 MWCO’s: 1, 2, 3.5, 8, 10, 15, 25 & 50 kD; Flat Width (diameter): Many for different sample volumes • Quantity: S/P 6- 10m/roll & S/P7- 5m/roll • Preparation: Flush with water to remove preservative (0.05% sodium azide & 1% NaCl) • Storage: 4°C in azide, benzoate or formaldehyde preservative (avoid drying out or freezing)

  15. NEW Spectra/Por 1, 2 & 3 Dialysis Trial Kits Six Standard RC Dialysis Tubings in 5 meter Lengths (dry) • Spectra/Por 1: 32 mm & 50 mm FW • Spectra/Por 2: 25 mm & 45 mm FW • Spectra/Por 3: 18 mm & 54 mm FW • “T” at end of standard PN designates a trial size length, Ex: 132720T • Small Quantity Benefits: • Costs less than 15 and 30 m/roll • Ideal for Small Quantity Experiments • Qualify membrane for application • Compare performance of multiple MWCO’s

  16. Dialysis Tubing: Biotech Grade • Biotech CE – Best selectivity & ultra-pure • 9 concise MWCO’s • 0.1-0.5, 0.5-1, 3.5-5, 8-10, 20, 50, 100, 300 & 1000 kD • 4 tubing flat-widths (10, 16, 24 & 31 mm) • Bulk (10 meter) and Trial (1 meter) sizes • Pkg’d wet in 0.05% sodium azide (rinse w/ water) • Store at 4 C in azide, benzoate or formaldehyde • Biotech RC – Best compatibility & ultra-pure • 5 MWCO’s • 3.5, 8, 10, 15 & 25 kD • 2 tubing flat-widths (10 & 16 mm) • Bulk (10 meter) • Pkg’d dry with glycerin (soak in water to remove) • Store dry at room temp (avoid humidity)

  17. NEW Biotech CE Dialysis Trial Kits 9 Biotech CE Dialysis Tubings in 1 meter Length (wet) • Trial size available for all 9 MWCO’s • 0.1-0.5 kD, 0.5-1 kD, 3.5-5 kD, 8-10 kD, • 20 kD, 50 kD, 100 kD, 300 kD & 1000 kD • Biotech CE: 16 mm FW only • “T” at end of standard PN designates a trial size length, Ex: 131270T • Small Quantity Benefits: • Costs less than 10m/roll • Ideal for Small Quantity Experiments • Qualify membrane for application • Compare performance of multiple MWCO’s

  18. Closures for Dialysis Tubing: 2 Classes Universal Closures: All tubing grades & types • Use with Standard RC, Biotech CE & Biotech RC • 5 sealing width sizes (50, 70, 90, 110 & 150 mm) • Nylon – sinks in water • 6 colors available for sample identification Spectra/Por Closures: Standard RC tubing only • 5 sealing width sizes (12, 23, 35, 55 & 75 mm) • Polypropylene – floats in buffer • 3 functional types available: • Standard Closure – Aids in buoyancy • Weighted Closure – Aids in vertical orientation • Magnetic Closure – Aids in buffer stirring • Paired Standard & Weighted – buoyancy & orientation • 4 colors available for sample identification

  19. Why use pre-assembled dialysis device? Disadvantages of Dialysis Tubing: • Requires closures • More likely to leak • Requires headspace for buoyancy and vertical orientation • More difficult to load & retrieve sample Advantages of Dialysis Devices: • Very easy handling & convenient • Self-contained & complete • Self-buoyant and self-orienting • Allows in-process testing • Total sample recovery • Disposable

  20. Disposable Dialysis Devices • Float-A-Lyzer G2 (FAL-G2) • For ultimate convenience of small volumes • Micro Float-A-Lyzer (μFAL) • For ultimate convenience of micro volumes • Tube-A-Lyzer (TAL) • For increased efficiency combined with convenience Spectrum offers 3 kinds of pre-assembled dialysis devices:

  21. Float-A-Lyzer G2 Video Clip

  22. Disposable Dialysis Device: Float-A-Lyzer G2 Purpose: Provide ultimate convenience for dialysis of common volumes Design Features: • Biotech CE preformed tubing (16 mm FW, dry) • Pot-sealed on top & bottom in open configuration • Leak-proof screw cap lid with sealing O-ring • 9 MWCO’s (color-coded cap): 0.1-0.5, 0.5-1, 3.5-5, 8-10, 20, 50, 100, 300 & 1000 kD • 3 volume sizes: 1 ml, 5 ml & 10 ml • Floatation ring for buoyancy (included) • Pipette loading & retrieval for 5 & 10 ml (included) and micropipettor loading & retrieval for 1 ml • 12 units/pkg

  23. Disposable Dialysis Device: Float-A-Lyzer G2 Benefits: • Superior handling and ease-of-use • Leak-proof with sample protection • Best MWCO selectivity and range (100 – 1,000,000 Dalton) • Ultrapure Biotech CE membrane • Volume specific dilution control • Easy sample loading and retrieval • Total sample recovery • Complete & ready-to-use

  24. Disposable Dialysis Device: Micro Float-A-Lyzer Purpose: Provide ultimate convenience for dialysis of micro volumes Design Features: • Biotech CE preformed tubing (10 mm FW, dry) • Pot-sealed on top and clamp sealed on bottom • Leak-proof Luer-lok™ cap • 7 MWCO’s (color-coded cap): 0.1-0.5, 0.5-1, 3.5-5, 8-10, 20, 50 & 100 kD • 2 volume sizes: 100-200 μl & 400-500 μl • Self-floating (“air bladder”) & self-standing • Syringe (no needle) loading & retrieval (included) • Interlocking housing for multi-sample dialysis • 12 units/pkg New - January 2012

  25. Disposable Dialysis Device: Micro Float-A-Lyzer Benefits: • Superior handling and ease-of-use • Leak-proof with sample protection • Best MWCO selectivity and range (100 - 100,000 Dalton) • Ultrapure Biotech CE membrane • Open to clamped configuration to maximize SA and minimize volume hold-up • Easy sample loading and retrieval • 100% sample recovery • Complete & ready-to-use New - January 2012

  26. Disposable Dynamic Dialysis Device: Tube-A-Lyzer Purpose:Dialysis in 4-12 hours Provides the convenience of a disposable device and the efficiency of dynamic dialysis for faster dialysis Mechanism of Efficiency: • Two membrane defined chambers: sample & buffer • Solutes diffuse through membrane • Circulating buffer sweeps away solutes • Maintains max concentration gradient • Prevents equilibrium • Achieves higher rate of dialysis

  27. Disposable Dynamic Dialysis Device: Tube-A-Lyzer Design Feature: Membrane Sample Chamber • Biotech CE preformed tubing (16 mm FW, dry) • Pot-sealed on top & bottom in open configuration • 2 sample volume sizes: 8-10 ml & 25-30 ml • 6 MWCO’s: 0.1-0.5, 3.5-5, 8-10, 20, 50 & 100 kD • Leak-proof Luer-lok™ sample port & cap • Syringe loading & recovery Design Feature: Flow-through Buffer Chamber • Tubular housing encases the membrane chamber • Upper & lower hose-barb ports (3/8” or 6 mm) • Operates with standard peristaltic pump & flexible tubing

  28. Disposable Dynamic Dialysis Device: Tube-A-Lyzer Benefits: • Reduces dialysis time from days to hours! • Ready-to-Use: no preparation required • Easy sample loading and in-process testing • Larger buffer volumes • Maintains maximum concentration gradient • Prevents polarization and membrane fouling • Achieves higher sample purities

  29. Flat-sheet Dialysis Membrane Standard RC Discs for Spectrum Dialysis Systems: • 3 diameters: 33 mm, 47 mm & 100 mm (50/pk) • 4 membranes: • Spectra/Por 1 (6-8 kD) • Spectra/Por 2 (12-14 kD high perm) • Spectra/Por 3 (3.5 kD) • Spectra/Por 5 (12-14 kD) Standard RC Sheets for other dialysis systems: • Square sheets for cutting (25/pk) • 5 membranes: • Spectra/Por 1 (6-8 kD) • Spectra/Por 2 (12-14 kD high perm) • Spectra/Por 3 (3.5 kD) • Spectra/Por 5 (12-14 kD) • Spectra/Por 5 (12-14 kD large volume)

  30. Flat-sheet Dialysis Systems Design concept: Sample chamber and buffer chamber separated by a flat membrane disc. Assembly clamped together by pressure. (a membrane sandwich) • 3 system types depending on application: • MacroDialyzer • For larger sample volumes • MicroDialyzer • For multiple micro volume samples • Equilibrium Dialyzer • For drug and ligand binding studies

  31. Flat-Sheet Dialysis System: MacroDialyzer Purpose: Dialysis of larger volumes (200 ml +) Features & Operation: • Equal size sample & buffer chamber (acrylic) • 4 volume sizes: 1 ml, 5 ml, 10 ml & 50 ml • Spectra/Por Standard RC membrane discs • Sealing chamber O-rings • Clamps assure leak-proof assembly • Flow through ports on sample & buffer chambers for circulation of larger volumes • Should be equal pressure or closed system to avoid filtration mode.

  32. Flat-Sheet Dialysis System: MicroDialyzer Purpose: Dialysis of multiple micro volumes Features & Operation: • 10-well dialyzer, 500 μl sample volumes • 96-well dialyzer, 150 μl sample volumes • Common buffer chamber with flow through ports • Spectra/Por Standard RC Disc Membranes • Sealing chamber O-ring or gasket • Clamps or screws for leak-proof assembly • Cover plate to avoid sample contamination • Spectra/Por Standard RC membrane sheets

  33. Flat-Sheet Dialysis System: Equilibrium Dialyzer Purpose: Studying pharmacokinetics and ligand binding. Compare up to 20 independent reactions to determine drug or binding efficacy. Features & Operation: • 20 reaction cells loaded with equal sample & buffer volumes (1.4 ml half-cell chambers) • 5 cells stacked & spaced per carrier • 4 carriers mounted to drive unit and submerged in temperature controlled water bath. • Continuous rotation of 20 reaction cells • Spectra/Por Standard RC Membrane Discs

  34. Hollow Fiber Micro-dialysis Purpose: • Dialysis of micro-volume samples (5 ul/fiber) • Encapsulation & implantation for in vivo studies Features & Benefits: • Standard RC hollow fiber membrane with glycerin • 2 MWCO’s: 13 kD & 18 kD • Fiber dimensions: ID x OD - 200 x 216 um 20 fibers, 15 cm length • Available as loose fibers or potted bundle for easier loading

  35. Membrane Dialysis Accessories Dialysis Reservoirs • Polystyrene cylindrical containers ideal for stirring dialysis tubing or devices in buffer • Magnetic stir bar included • Nylon sealing lid prevents evaporation • Two volume sizes – 600 ml & 1800 ml • Spectra/Gel Absorbent: • Concentration of dialysis samples in tubing or device without product loss or contamination • Inert polyacrylate-polyalcohol complex draws and binds water from the sample • Pack with dry powder & then brush away gel • 10 fold sample concentration in 1 hour

  36. How do we overcome complicationswith atypical applications and increase efficiency? • Physically modify aspects or components of dialysis. • Membrane • Volumes • Concentrations • Buffer changes • Manipulating operating parameters to perpetuate the Driving Force (Concentration Gradient) and eliminate the Opposing Force (Equilibrium).

  37. Parameters to Increase Rate of Dialysis • Sample & Buffer Concentrations • equal except for retained and transferred species • Sample to Dialysate Ratio • 1 : 1000+ by volume • Membrane MWCO • 90-130% of the retained species molecular size • Volume to membrane surface area (SA) ratio • Length (30 cm) x FW (10-16 mm) = 0.1 – 1ml / cm • Temperature • Elevated (36-60 Celsius) • Dynamic Dialysis • Buffer Changing • 4-5 times at 2, 4, 6-8, 8-10, 14-16 & 20 hours • Duration (Resulting) • 0.5 – 1.5 days

  38. Common Complications and Issues Associated with Dialysis and Atypical Applications • Membrane impurities • Product purity • Product yield • Membrane Handling • Sample volume dilution • Concentrating the sample • Gel electro-elution • High salt samples • Large volume samples • Small volume dialysis • in vivo implantation research • Dialysis takes too long • Biotech Grade or S/P 7 Membrane • Membrane with higher MWCO • Membrane with lower MWCO • Ready-to-Use Dialysis Device (RDD) • RDD or fully loaded tubing • Spectra/Gel Absorbent • Float-A-Lyzer G2 • Step-down salt concentration of buffer • S/P 5, MacroDialyzer or consider TFF • Micro Float-A-Lyzer • Microdialysis hollow fiber or PVDF implant • Tube-A-Lyzer, Dynamic Dialysis or modify operating parameters

  39. 4 Molecular Separation Technologies in the Lab (reference table for technology comparison)

  40. Thank You谢谢

More Related