Laboratory Dialysis

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Laboratory Dialysis
Cassidy Markee Director of Laboratory Division
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Spectrums 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)

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Why use laboratory dialysis?

• Many Advantages
• Simple, easy quick set-up
• Inexpensive disposable
• Very gentle separation
• (no pressure or shear stress)
• Doesnt 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

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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.
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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)
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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

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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

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

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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)

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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)

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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 MWCOs 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

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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

• Hollow Fiber
• MicroDialysis
• Microvolume dialysis
• Encapsulation w/ epoxy
• Implant for in vivo studies
• Potted bundle or loose HF
• 1 Type Standard-RC

• Flat Sheet
• Dialysis Systems
• Chamber separations
• Large volume samples
• Small volume samples
• Equilibrium studies
• 1 Type Standard-RC

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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 MWCOs 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 4C in azide, benzoate or formaldehyde
  preservative (avoid drying out or freezing)

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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 MWCOs

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Dialysis Tubing Biotech Grade

• Biotech CE Best selectivity ultra-pure
• 9 concise MWCOs
• 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
• Pkgd 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 MWCOs
• 3.5, 8, 10, 15 25 kD
• 2 tubing flat-widths (10 16 mm)
• Bulk (10 meter)
• Pkgd dry with glycerin (soak in water to remove)
• Store dry at room temp (avoid humidity)

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NEW Biotech CE Dialysis Trial Kits

• 9 Biotech CE Dialysis Tubings in 1 meter Length
  (wet)
• Trial size available for all 9 MWCOs
• 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 MWCOs

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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

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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

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Disposable Dialysis Devices

• Spectrum offers 3 kinds of pre-assembled 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

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Float-A-Lyzer G2 Video Clip
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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 MWCOs (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

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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

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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 MWCOs (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
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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
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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

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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 MWCOs 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

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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

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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)

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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

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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.

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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

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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

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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 MWCOs 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

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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

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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).

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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

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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

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4 Molecular Separation Technologies in the Lab
Technology (force) Material Substrate Sep Yield Cost Advantage Disadvantage
Membrane Dialysis (diffusion) macromolecules, micromolecules, low MW solutes, salts ions good best very low very gentle best recovery inexpensive easiest very slow need 100X size diff
Membrane Filtration (pressure) cells (parts), virus, micro/nanoparticles macromolecules good good low to med fast easy large volume shear stress damage membrane plugging
Size Chrom (gravity or pressure) macromolecules, micromolecules low MW solutes good poor high large volume poor recovery expensive
Aff/Ion Chrom (attraction pressure) macromolecules, micromolecules low MW solutes best poor high specificity resolution similar sizes alter activity stability poor recovery expensive
Centrifugation (centripetal) cells, virus, micro/nano particles macromolecules good poor high fast easy pellet forming damage poor recovery expensive
(reference table for technology comparison)
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Thank You??