1. Director of Laboratory Division
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 : 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 & 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 X 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 % 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 % 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
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: kD MWCO, high perm / special FW
Spectra/Por 3: 3.5 kD MWCO, general application
Spectra/Por 4: kD MWCO, general application
Spectra/Por 5: 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: T
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
, 0.5-1, 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
kD, kD, 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: T
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
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

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):
, 0.5-1, 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):
, 0.5-1, 3.5-5, 8-10, 20, 50 & 100 kD
2 volume sizes: μl & 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
( ,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 & ml
6 MWCO’s: , 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 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 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 complications with 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 : 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, & 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
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
low to med
fast easy
large volume
shear stress damage membrane plugging
Size Chrom
(gravity or pressure)
macromolecules, micromolecules & low MW solutes
poor
high
poor recovery expensive
Aff/Ion Chrom
(attraction & pressure)
specificity resolution similar sizes
alter activity & stability poor recovery expensive
Centrifugation
(centripetal)
cells, virus, micro/nano particles & macromolecules
pellet forming damage poor recovery expensive
(reference table for technology comparison)

40. Thank You 谢谢