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):

1. Membrane Dialysis – passive diffusion through a semi-permeable membrane – 100 to 1,000,000 Daltons

2. Membrane Filtration – applied pressure across a selective membrane. 3 types based on size range:

a. Macrofiltration – 5 μm and up (Woven Mesh Screens)

1. Microfiltration – 0.05 – 5 μm(Direct Flow and Tangential Flow)

a. Ultrafiltration (Molecular filtration) – 5 kD to 0.05 μm(Tangential Flow only)

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

Understanding DialysisPassive 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 GRADIENTThe higher the concentration gradient the faster the rate of dialysis.

Opposing Force:EQUILIBRIUMAs you approach equilibrium, the rate of dialysis slows down.

Mode of Operation: Gentle diffusion across a

membrane

(high conc. in sample)

(low conc. in buffer)(low conc. in buffer)

(equilibrium)

(equilibrium) (equilibrium)

(high conc. in sample)

4 Fundamental Dialysis Applications

1. Purification – elimination of low MW solutesProtein purificationHPLC sample prepRemoval of free amino acids or nucleotides

2. Modification – changing ionic / chemical environment to affect activityDesaltingpH ChangeBuffer Exchange

3. Isolation – controlled exposure for reactivitySeparation of small target speciesElectro-elution

4. Equilibrium Dialysis – Binding StudiesAntibody & LigandEnzyme Activity

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

Molecular Weight Cut-Off (MWCO)

Indirect measure of MW (solute size) retained by approximately 90-95%.

Below the curve retainedAbove the curve passes

Selecting MWCO for Max Yield and Purity

1.Max Yield: MWCO smaller than product size.

2. And Max Purity: MWCO 100-1000X bigger than low MW contaminants.

Ex: 100 kD to retain 98% IgG AND remove 99% Vit B12

Select Lower MWCO: Yield vs. Purity

If only 10 X size difference between product (Cyt C) & contaminant (Vit B12):

1.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)

Select Higher MWCO: Yield vs. Purity

If only 10 X size difference between product (Cyt C) & contaminant (Vit B12):

1.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)

2.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)

Spectrum Dialysis Membranes: 2 Grades

1. Standard Grade - natural cellulosic polymer: 1 Typea. Regenerated Cellulose (Standard-RC)

MWCO - DISADVANTAGE: Limited range with loose selectivityPURITY - DISADVANTAGE: Trace levels of heavy metals & sulfidesTOLERANCE - ADVANTAGE: Good temp (121 C), pH (2-12) & chemical resistanceCOST - ADVANTAGE: Most economicalAPPLICATIONS: General dialysis, eliminating small ions (desalt, pH change, Buffer exchange, etc.)

2. Biotech Grade – synthetic cellulosic polymer: 2 Typesa. Cellulose Ester (Biotech-CE)

MWCO - ADVANTAGE : Broad range (100 – 1,000,000 D) with best selectivityPURITY - ADVANTAGE: Ultra-pure – no trace heavy metals & sulfidesTOLERANCE - DISADVANTAGE: Limited temp (37 C), pH (2-9) & chemical resistanceCOST - DISADVANTAGE: More costlyAPPLICATIONS: Critical separations & molecular purifications (protein, oligos, macromolecules, etc.)

b. Regenerated Cellulose (Biotech-RC)MWCO ADVANTAGE: Small range of MWCO’s with good selectivityPURITY ADVANTAGE: Ultra-pure – no trace heavy metals & sulfidesTOLERANCE ADVANTAGE: Good temp (121 C), pH (2-12) & chemical toleranceCOST DISADVANTAGE: Most costlyAPPLICATIONS: Requiring purity and tolerance (sample prep with organics, etc.)

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

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

DisposableDialysis 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

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)

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

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)

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

–

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:

1. Standard Closure – Aids in buoyancy2. Weighted Closure – Aids in vertical orientation3. Magnetic Closure – Aids in buffer stirring

• Paired Standard & Weighted – buoyancy & orientation• 4 colors available for sample identification

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

Disposable Dialysis DevicesSpectrum offers 3 kinds of pre-assembled dialysis devices:

1. Float-A-Lyzer G2 (FAL-G2)For ultimate convenience of small volumes

2. Micro Float-A-Lyzer (μFAL)For ultimate convenience of micro volumes

3. Tube-A-Lyzer (TAL)For increased efficiency combined with convenience

Float-A-Lyzer G2 Video Clip

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

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

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

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

Disposable Dynamic Dialysis Device: Tube-A-Lyzer

Purpose: Dialysis in 4-12 hoursProvides 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

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

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

Disposable Dynamic Dialysis Device: Tube-A-Lyzer

Flat-sheet Dialysis MembraneStandard RC Discs for Spectrum Dialysis Systems:• 3 diameters: 33 mm, 47 mm & 100 mm (50/pk)• 4 membranes:

1. Spectra/Por 1 (6-8 kD)2. Spectra/Por 2 (12-14 kD high perm)3. Spectra/Por 3 (3.5 kD)4. Spectra/Por 5 (12-14 kD)

Standard RC Sheets for other dialysis systems:• Square sheets for cutting (25/pk)• 5 membranes:

1. Spectra/Por 1 (6-8 kD)2. Spectra/Por 2 (12-14 kD high perm)3. Spectra/Por 3 (3.5 kD)4. Spectra/Por 5 (12-14 kD)5. Spectra/Por 5 (12-14 kD large volume)

Flat-sheet Dialysis SystemsDesign 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:

1. MacroDialyzerFor larger sample volumes

2. MicroDialyzerFor multiple micro volume samples

3. Equilibrium Dialyzer For drug and ligand binding studies

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.

Flat-Sheet Dialysis System: MicroDialyzerPurpose: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

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

Hollow Fiber Micro-dialysisPurpose:• 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 um20 fibers, 15 cm length

• Available as loose fibers or potted bundle for easier loading

Membrane Dialysis AccessoriesDialysis 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

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).

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

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

4 Molecular Separation Technologies in the LabTechnology

(force)Material

Substrate Sep Yield Cost Advantage Disadvantage

Membrane Dialysis

(diffusion)

macromolecules, micromolecules, low MW solutes,

salts & ions

good bestvery low

very gentle best recovery inexpensive

easiest

very slow need 100X size diff

Membrane Filtration(pressure)

cells (parts), virus, micro/nanoparticles & macromolecules

good goodlow 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 volumepoor recovery

expensive

Aff/Ion Chrom(attraction &

pressure)

macromolecules, micromolecules & low MW solutes

best poor highspecificity resolution

similar sizes

alter activity & stability poor recovery

expensive

Centrifugation(centripetal)

cells, virus, micro/nano particles &

macromolecules

good poor highfast

easy

pellet forming damage poor recovery

expensive

(reference table for technology comparison)

Thank You

谢谢