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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):

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Laboratory Dialysis

Cassidy Markee

Director of Laboratory Division

spectrum s core technology membrane separation
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)
why use laboratory dialysis
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 dialysis
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.

mode of operation gentle diffusion across a membrane
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)

4 fundamental dialysis applications
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

typical dialysis parameters
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
Molecular Weight Cut-Off (MWCO)

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

Below the curve retained

Above the curve passes

selecting mwco for max yield and purity
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

select lower mwco yield vs purity
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)

select higher mwco yield vs purity
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)

spectrum dialysis membranes 2 grades
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.)

laboratory dialysis 4 membrane configurations
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
dialysis tubing standard grade 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
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
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
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
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
why use pre assembled dialysis device
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 devices
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:

disposable dialysis device float a lyzer g2
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 g21
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
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 lyzer1
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
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
disposable dynamic dialysis device tube a lyzer1
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
slide28

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
flat sheet dialysis membrane
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)
flat sheet dialysis systems
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
flat sheet dialysis system macrodialyzer
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 microdialyzer
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
flat sheet dialysis system equilibrium dialyzer
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 dialysis
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
membrane dialysis accessories
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
how do we overcome complications with atypical applications and increase efficiency
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
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
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 lab
4 Molecular Separation Technologies in the Lab

(reference table for technology comparison)

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