Laboratory Dialysis
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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

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:


Float a lyzer g2 video clip

Float-A-Lyzer G2 Video Clip


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


Laboratory dialysis

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)


Thank you

Thank You谢谢


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