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Dialysis
Technical tips: Principle of dialysis | Advantages and main applications
Choosing the right membrane:
Cut-Off MWCO | Membrane type | Porosity | Permeation rate | Membrane compatibility chart
Applications: Desalting / Buffer exchange
Sterile dialysis
Dialyse à l'équilibre - Fraction liée/libre : information | Equilibrium Dialysis products

FAQ – choosing the right membrane

What dialysis membrane should I choose?(type, MWCO, size/format)
Which membranes minimize protein binding the best?
What is the shelf life for dialysis membranes?
Are dialysis membranes endotoxin free?
What size of tubing size to choose / or dialysis device?
What MWCO of tubing size to choose?
How accurate are the membrane pore sizes and what is MWCO?
What are Daltons (MW) –why not microns unit?- and how do you convert Da/µm?
What is the difference between Spectra/Por 2 and 4 dialysis membranes?
Membrane Porosity – convertion Dalton / Microns?
and see Tech tip: choosing the right membrane(Cut-Off, Porosity, Membrane type)

FAQ – using dialysis membranes

How do you prepare dialysis membrane tubing?
How long should I cut the dialysis tubing?
Should I close the dialysis tube using closures or tyieng knots?
How to select the right closure and closure size for membrane tubing?
Is there a "rule of thumb" regarding membrane surface area to sample volume?
How much volume of dialysate is needed to dialyze a sample and how often does the dialysate need to be changed?
How how long does dialysis take to complete -does dialysis work-?
How how long does dialysis take to complete -does dialysis work-?
Can dialysis membranes be re-used for the same protein samples?
Can dialysis membranes be chemically or heat sealed?
Which dialysates (buffers) are commonly used in dialysis?
How to sterilize dialysis membranes / devices
How much pressure can a dialysis membrane withstand if used for ultrafiltration?
Why is the volume of my sample increasing during dialysis / my dialysis has leaked or has burst!
and see Tech tip: using dialysis membranes | Sterile dialysis

FAQ – deeper insight in dialysis

What is dialysis versus osmosis?
How good is the mass transfer across the membrane if the osmolarity is equal on both sides but concentration gradients
still exist?
and see Tech tips: Principle of dialysis | Permeation rate | Membrane compatibility chart

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Dialysis principle and operating
Dialysis principle
The dialysis process of a solute occurs when its concentration
differs between each side of an hemipermeable membrane oncotic
surface. This process is affected by the variables of pressur
temperature, viscosity and mixing rate of a solution. e
The movement of a solute across a semipermeable membrane is the result of
random molecular motion. As the solute molecules in a solution move, they
will collide from time to time with the membrane until they diffuse.
Diagram: The permeation of a given small solute (•) from a solution on the
right of the membrane to the left, and back again, will depend upon the
frequency of collisions between the molecules on either side of the membrane.
Larger molecules (●) will not cross the membrane.

For example, if the concentration of solute •, in a s&ample solution (right) is

100 mM, and solution left is 10 mM, the probability of Y-solute • colliding and
crossing the membrane is much higher than in opposite sens. Therefore, the net rate of transfer of a given solute (at a certain temperature, viscosity
and mixing rate) will increase with greater concentration differences between the two solutions.

FAQ: What is dialysis versus osmosis?

Dialysis is the passage of molecules with molecular/size across an hemipermable membrane, from a compartment with
a higher solute content to the lower concentration compartment (see above).
Osmosis is similarly, the passage of water (or other solvent) across an hemipermeable membrane from a compartment
with a higher water content to the lower content compartment. In fact, one should consider the difference of "oncotic
pressure", that is driven by solutes dissolved in the buffer in each compartment.
other said, the osmosis process is linked to dialysis one, being a diffusion of molecules through an hemipermeable
membrane, but applies to the solvent (water) and not to the small samples compound to be removed by dialysis.
Dialysis usually restrains osmosis, because it participates to equilibrate small hydrophilic compounds in the 2
compartments. It can be more rapid or, most often, longer to take place.
+
Theses consideration are generally with few importance when performing dialysis, provided one know that the sample
volume may inflate 1-20% and consequently have forseen the dialysis chamber volume to avoid burst and leaking of
sample. See FAQ

FAQ: How good is the mass transfer across the membrane if the osmolarity is equal on both sides but
concentration gradients still exist?
Most dialysis is done with no osmotic pressure across the membrane. The dialysis process is driven by the concentration
gradient from the inside and outside of the dialysis tubing. If there is a large difference in osmotic pressure, water will
move across the membrane. If too much water migrates across the membrane, the dialysis tubing can potentially burst
or collapse, depending upon the direction of water movement.

Advantages and applications

ADVANTAGES OF DIALYSIS DIALYSIS APPLICATIONS
 Very Gentle Conditions  Macromolecular Purification
 Easy Operation  Protein Concentration
 Wide Range of Sample Volumes  Solute Fractionation
 Many Membrane Types & MWCO's  Contaminant Removal
 Inexpensive Materials  pH Change
 Disposable Membranes & Devices  Desalting
 Buffer Exchange
 Binding Studies
 Electro-elution
 Sample preparation analysis (MS, electrophoresis,
Immuno- or Cell- assays)
 Cell culture

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Selecting "the Right Membrane"

FAQ: What type of dialysis membrane should I choose?
The key to a successful dialysis separation is using the RIGHT MEMBRANE under the right conditions.
In most applications, the choice amongst available membrane types is not difficult. It is driven essentially by the MW of molecules to keep and MW
of molecules to remove to define membrane MWCO, typically 10KDa with RC type membraens. Additionally, special membranes may be preferred
for demanding application, when considering undesired adsorption of biomolecules of interest (i.e. proteins), the compatibility (resistance) toward
solvents, requirement of strongest dialysis rate or mechanic strength, ...

On can follow below steps to select a membrane:

Select membrane type/grade (CE, RC or PVDF) based on application: Choose typically

CE membranes for standard applications
RC membranes for more accurate molecular separation and compatibility with organic solvents
Standard RC membranes (CelluSep T1) are ideal for routine dialysis (economic), or S/P 1 to 7
Biotech CE,RC (CelluSep H1, S/P Biotech) when no cleaning required (purer synthetic membranes without metals traces).
PVDF or PE or PES membranes for large MW molecules, and harsh conditions
(compatibility with acids, bases, high temperature/autoclave...) – available now only in HC or RTU formats
 See more insight below at 'choice of membrane type'.
 See also if you need sterile dialysis: sterile membranes are available now only in

Select membrane MWCO based on solutes sizes (Molecular Weight),

the one(s) to be retained iin dialysis compartement, and (often less critical) the one that should be removed.
Choose typically a MWCO at mid-range between the MW of solutes to remove and analytes to keep.
10KDa suits mots applications, provide MS of samples >15KDa and compounds to remove are <8Da
 See below Choice of Cut Off, Membrane Porosity/MWCO chart.

Select membrane configuration/format/size based on sample volume and number:

Tubings for routine and economic dialysis such as CelluSep –
Disposable ready-to use devices such as FloatALyser & GebaFlex for small samples and limited series, and convenient use
Sacks for large volumes, Flat sheets for dialysis systems
Microfibers for process dialysis and filtration systems (easy scaling, higher through flow).
Tubing for medium to large volumes, RTU devices for (very) small volumes, fiber for Liters
See suitable sample volumes in each product line presentation.

Please see at Dialysis selection guide.

Please ask [email protected] if you don’t find enough help in below technical notice.

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Choice of Membrane type – Nature, Compatibility

● Quel type de membrane choisir ?Les membranes sont proposée en 2 matériaux principaux, l’acétate de cellulose (CE)
et la cellulose régénérée (RC). Elles se distinguent par leur résistance physique et chimique. Les matériaux plus
résistants (PVDF, Nylon) ne sont désormais disponibles qu'en microfibres.
Résistance à la température:
Biotech Cellulose Ester wide range of selectivity & purity RC (autoclavable) > Biotech RC > CE
(CE) Résistance aux solvents acides, alcalins :
Biotech Regenerated Cellulose combined selectivity, purity & organic PVDF > RC ≥ CE.
(RC) resistance. Fixations non spécifiques: (protéines globulaires):
Available in RTU devices (FAL, TAL) CE < RC < PVDF.
Standard Regenerated Cellulose " same. + broader option: MWCO, flat sheet..
(RC) Dialysis sacks. lower cost lab applications. See also the detailled chemical compatibility chart
CelluSep Regenerated Cellulose " same. + More economic. But less MW below.
(RC) options.

Chemical nature and structure of membranes

Cellulose Ester (CE) is the first intention choice for biochemistry works (proteins), but may have limitation for diluted
proteins because of low resistance to some organic solvents, and even alcohols or other compounds*.
CE is a generic term, including Cellulose Acetate (CA) –its main component-, and Mixed Cellulose (ME), depending
on the CA content.

Regenerated Cellulose (RC) provides more accurate molecular separation and better compatibility with organic
solvents: RC is more resistant to chemicals*. It is also purer and has narrower pore size.
RC is thus preferred for nucleic acids and for proteins, when they should be precipitated with organic solvents, or which
structure or state can be affected by traces of metals. They absorption of protein is low or higher than CE (depends on
protein nature. Binding of globular protein is slightly higher).
Limitations may come from the fact they are a little more expensive, and may not be available with low or large
MWCO (<3000Da, >50 000Da), and they need to be soaked and rinsed before use (except the products offered already
cleaned and wetted (S/P6; FAL.) or specially pure grades (CelluSep H1; S/P7, Biotech RC).

The polymers in the cellulose ester (CE) membrane cross-link to form a more rigid molecular lattice.. The opaqueness comes from the pores in a
more rigid frame. The larger the pore size, the more opaque the membrane. In comparison, regenerated cellulose (RC) polymers form a more flexible
lattice structure, hence translucid.

PVDF, PE, PES, Nylon and PC are for dialysis of large MW compounds, and harsh conditions (compatibility with
acids, bases, high temperature/autoclave...), and are available now only in HC or RU formats. They have higher protein
adsorption level than CE and RC. PVDF material can be sealed by heating.

see compatibility chart for compatibility level of thedifferent membranes toward different substances.

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Grades of membranes
Standard membranes (CE, RC: CelluSep T1, S/P 1 to 7) for routine applications
Biotech CE,RC and PVDF (CelluSep H1, S/P Biotech) for more pure membranes (synthetic membranes without metals
traces: no cleaning
Please ask [email protected] for more information.

FAQ: Are dialysis membranes endotoxin free?

Most membranes are intended for laboratory use, not checked for endotoxin. Please inquire +

FAQ: Which membranes minimize protein binding the best?

Each type of membrane displays a different affinity for various molecules. For globular proteins, the relative binding
affinity is CE < RC < PVDF.

FAQ: What is the shelf life for dialysis membranes?

The dry packaged dialysis membranes have a shelf-life of 5 years. The wet packaged (0.05% sodium azide solution)
membranes have a shelf-life of 3 years. The irradiated membranes have a shelf life of 1.5 years.

Choice of dialysis tubing size /format

The size of the tubing or dialysis device depends directly on your sample size, and eventually of general consumption in
your lab. Dialysis devices are a more flexible choice in labs with lower et more various applications, and also more
convenient: ready to use!

FAQ: What size fof tubing size to choose?

● Quel dimension de tube de dialyse choisir ?

Il s’agit de choisir un diamètre adapté à la taille de
l’échantillon (tenir compte du volume contenu par une
longueur de 1cm de tube de dialyse indiquées dans les
tableaux de référence) pour avoir une hauteur (et surface
d’échange) suffisante, plus une longueur en bas et en haut
pour fermer le tube (clamp ou nœud)

Les tableaux de référence indiquent le volume contenu par une longueur de 1cm de tube de dialyse. Choisir le diamètre
du tube pour avoir 1-3cm de hauteur d’échantillon pour les microvolumes et diamètres, et jusqu’à 50cm maximum (ou
la hauteur de vos récipients pour le bain de dialyse). Prévoir que l’échantillon occupe en fait plus que cette longueur
théorique, et notamment après dialyse (osmose : jusqu’à 10-30% de volume), et qu’il faut une longueur en bas et en
haut pour fermer le tube (2-3cm pour un clamp, 3-7cm pour un nœud)

Le choix dépend aussi éventuellement d'options liées aux besoins du labo,

-opter pour du tubing, en rouleau de 10m ou plus, est interessant pour une consommation régulière. Prendre plus un
diamètre correspondant aux volumes supérieurs moyens traités.
-opter pour des dispositifs de dialyse permet plus de flexibilité selon les besoins du labo, sur les tailles d'échantillons
traités (et les MWCO + coté prêt à l'emploi).

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Choice of MW Cut Off

FAQ: What MWCO of tubing size to choose?
As a general advice, choose a MWCO intermediate between the MW of the compound(s) to eliminate, and the
compound(s) to retain.
I.e. choose a 10KDa membrane to separate salts (<1000Da, and even peptides (2-6kDa) from large proteins (>20KDa). But a 10KDa membrane will
give poor dialysis rate if too close from the MW of compound to eliminate, and mediocrous yield if too close ftom the MW of the compound to retain.

Ideally, the MWCO should be 10-fold that of the solute to eliminate, and at least 2-fold less that the molecule to retain.
For large compounds or particules, choose a MWCO depending on rather the MW of the substance to be removed.
I.e. one typicall y choose a 10 000Da MCO to remove salts, whatever the size of large compound is. But a MW of 100 KDa(ca0.01µm) up to 1 000
KDa(ca 0.1µm) may be preferable if you should remove efficiently bulky salts (500-2000Da) or peptides (1-10KDa), or even proteins (10-50KDa).

More: fundamentals:
Since the dialysis membrane consists of a spongy matrix of crosslinked
polymers, the pore rating referred to as Molecular Weight Cut Off
(MWCO), is an indirect measure of the retention performance. More
precisely, the membrane MWCO is determined as the solute size that is
retained by at least 90%. However, since a solute's permeability is also
dependent upon molecular shape, degree of hydration, ionic charge and
polarity, it is recommended to select a MWCO that is half the size of the
MW of the species to be retained and/or twice the size of the MW of the
species intended to pass through.

The MWCO should be chosen as high as possible in order to maximize the dialysis rate. However, in order to achieve a
higher sample recovery you can select the MWCO that is about half of the molecular weight of the macromolecules that
need to be retained. Alternatively, choice a MWCO intermediate between large molecules to be retained , and smaller
molecules to be removed.
For Applications in which separation of molecules is required, there must be at least a 5x difference between the
molecular weight of both species for membrane dialysis to be effective. Otherwise, you may require other separation
techniques such as chromatography or TFF filtration.

FAQ: How accurate are the membrane pore sizes and what is MWCO?
Since dialysis membrane consists of a spongy matrix, it is more appropriate and practical to measure the "pore size"
indirectly by rating its retention performance characterized by its "Molecular Weight Cut Off" (MWCO). The MWCO
is defined by the molecular weight solute that is 90% retained by the membrane during a 17 hour period. For this
reason, you should select a MWCO that is just smaller than the size of the solutes you want to retain.

FAQ: What are Daltons (MW) –why not microns unit?- and how do you convert Da/µm?
While the size of dissolved molecules is defined typically by molecular weight (MW) units in Daltons (but alos
molecular shape, that depends on its structure and on environment), th size of particles and cells is defined by metric
diameter because the MW units become impractical and do not account for shape in the microscopic range. Since
microns are a measure of a 2-dimensional distance and Daltons are a measure of 3-dimensional size based on atomic
weight units, there is no direct conversion from one to the other.
For this reason, many common biological materials were characterized for dialysis, ultrafiltration and microfiltration
purposes and plotted on a conversion chart to correlate the approximate scales as a reference for estimating conversions.
Above Pore Size Chart may helps to converting between Daltons and metric units.
See Membrane Porosity – convertion Dalton to Microns

FAQ: What is the difference between Spectra/Por 2 and 4 dialysis membranes?

Both Spectra/Por 2 and 4 have a MWCO of 12-14 kD. While Spectra/Por is more suited for general dialysis,
Spectra/Por 2 offers special higher and lower FW's and/or higher permeability; i.e. water permeability of Spectra/Por 2
is superior to that of Spectra/Por 4.

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Porosity and Permeation rate
The permeation/exclusion of a molecule across/from a semipermeable membrane depends mainly on the shape, charge, and size of the solute, driving
the dialysis performence i.e. speed and size exlusion. Typically with globular biomolecules, i.e. most proteins, the size of a solute correlates highly
with the molecular weight. The flux, or rate of transport across a semipermeable membrane of solutes in solution, is then inversely related to the
molecular weight. As the molecular size approaches and exceeds the size of the membrane pores (MWCO), passage of solutes will completely or
partially be prevented.

Small molecules collide more often with the membrane, thus, their rate of molecular migration through the membrane will be high. Large molecules,
moving at low velocities collide infrequently with the membrane. Therefore, their rate of migration through the semi-permeable membrane will be
low (even those that fit through the membrane pores).

The permeation rate is increased with :

-larger difference of solute concentrations between dialysis compartments
-smaller and more spheric molecules
-higher temperatures
-thiner membrane thickness
-larger membrane surface area over sample volume.

Membrane Porosity – convertion Dalton to Microns

See FAQ: What are Daltons (MW) –why not microns unit?
This table gives indicative correspondence of MWCO and membrane porosity or molecular size for globular molecules.
Take correspondence with care because this may vary on many factors (shape, charge, viscosity...).

Note: RC membranes have more uniform and pores, and are thus recommended for more precise separations. See above.

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Membrane compatibility chart
This chemical resistance chart is intended for use as a guide, not as a guarantee of chemical compatibility. Variations in
temperature, concentrations, durations of exposure and other factors may affect the performance of the product. It is
recommended to test under your own conditions.

The following codes are used to rate chemical resistance:

R = Recommended L = Limited Exposure NR = Not Recommended U = Unknown

Cellulose
Polysulfone
Ester (CE) PolyVinyli
Regenerated (PS) / Polypropyl dene Stainless Polyester Fluorocarb
Substance / Cellulose PolyetherS Nylon (N)
Mixed ene (PP) DiFluoride Steel (SS) (P) on (F)
(RC) ulfone
(PVDF)
Cellulose
(PES)
(ME)
Acetic acid (diluted-5%) L R R R R NR L L R
Acetic acid (med conc- NR R R R R NR L NR R
25%)
Acetic acid (glacial) NR R R R R L L NR R
Acetone NR R NR R L R R R R
Acetonitrile NR R NR R L U U U U
Ammonium hydroxide NR R R R R R R U R
(diluted)
Ammonium hydroxide NR L R R R R R U R
(med conc)
Amyl acetate NR R NR R R L R L R
Amyl alcohol L R L R R R R R R
Aniline NR R NR R R R R U R
Benzene NR R L R R R L R R
Benzyl alcohol NR R NR R L U L NR R
Boric acid R R R R R L L R R
Brine R R R R R R R R R
Bromoform NR R NR R R U U U U
Butyl acetate NR R NR R R R L R R
Butyl alcohol L R R R R L R R U
Butyl cellosolve NR L NR U R U U U U
Butylaldehyde NR R NR R R U U U R
Carbon tetrachloride NR R NR R R NR L R U
Cellosolve NR L R R R U U U R
Chloroacetic acid NR R NR R R NR L U R
Chloroform L R L R R R R R R
Chromic acid NR NR NR L R NR L U U
Cresol NR R NR R NR NR R U R
Cyclohexane L R L R R R R U R
Cyclohexanone NR R NR R L R R R R
Diacetone alcohol NR R NR R R R L U R
Dichloromethane L R L R R L L NR R
Dimethyl formamide NR L NR R NR R R NR U
Dimethylsulfoxide NR R NR R L U U U U
1,4 Dioxane NR L L R R U U R R
Ethyl acetate NR R NR R R R L U R
Ethyl Alcohol L R R R R R R R R
Ethyl alcohol (15%) R R R R R R R R R
Cellulose Regenerate Polysulfone Polypropyl PolyVinyli Stainless Polyester Fluorocarb
Ester (CE) d (PS) / ene (PP) dene Nylon (N) Steel (SS) (P) on (F)

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/ Cellulose PolyetherS DiFluoride
Mixed (RC) ulfone (PVDF)
Cellulose (PES)
(ME)
Ethyl alcohol (95%) L R L R R R R R R
Ethylene dichloride NR R NR L R R L U R
Ethylene glycol L R R R R R L R R
Ethylene oxide NR L R R R R L U R
Formaldehyde (2%) L R R R R R R R R
Formaldehyde (30%) L R R R R R R R R
Formic acid (25%) NR R R R R NR L NR R
Formic Acid (50%) NR R R R R NR L NR R
Freon® R R R R R NR R R R
Gasoline R R L R R R R R R
Glycerine R R R R R R R R R
Glycerol R R R R R R R R R
Hexane R R R R R L R R R
Hexanol L R R R R R R R R
Hydrochloric acid R R R R R L NR R R
(diluted-5%)
Hydrochloric a. (25%) NR NR R R R NR NR R R
Hydrochloric a. (37%) NR NR R L R NR NR R R
Hydrofluoric a. (25%) NR L L NR R L NR NR R
Hydrogen peroxide R R R R R NR L R R
(30%)
Iodine solutions NR NR NR R R L NR U R
Isobutyl alcohol R R R R R NR R U R
Isopropanol L R R R R NR L R R
Isopropyl acetate NR R NR R R L L R R
Isopropyl alcohol L R R R R NR L R R
Isopropyl ether L R R L R R R U R
Jet Fuel 640A R R R R R L R U R
Kerosene R R R R R R R L R
Lactic acid R R R R R L L R R
Methyl acetate NR R NR R R R R L R
Methyl alcohol L R L R R L R U R
Methyl alcohol (98%) L R R R R L R U R
Methyl cellosolve L L R R R L L U R
Methyl chloride NR R NR R L L R U R
Methyl ethyl ketone NR R NR R L R R U R
Methyl formate NR L NR R R U U U U
Methyl isobutyl
NR R NR R L L L R R
ketone
Methylene chloride L R L R R L L NR R
N-methyl-2-
NR R NR R R U L U U
pyrrolidone
Mineral spirits R R R R R R R U R
Monochlorobenzene L R NR L R U U U U
Nitric acid (diluted-5%) L R R R NR NR R R R
Nitric acid(mi-conc.25%) NR NR R R NR NR R L R
Nitric acid (6N) NR N R L R NR R R R
Cellulose Regenerate Polysulfone Polypropyl PolyVinyli Stainless Polyester Fluorocarb
Ester (CE) d (PS) / ene (PP) dene Nylon (N) Steel (SS) (P) on (F)

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/ Cellulose PolyetherS DiFluoride
Mixed (RC) ulfone (PVDF)
Cellulose (PES)
(ME)
Nitric acid (conc-70%) NR NR NR NR NR NR R NR R
Nitric acid
NR NR R NR L NR R NR R
(concentrated)
Nitrobenzene NR L NR NR R L L NR R
Nitropropane NR L NR L R U U U U
Oils, mineral R R R R R R R U R
Pentane R R R R R R L R R
Perchloric acid (25%) NR L NR NR R NR L U R
Perchloroethylene NR R NR L R L L U R
Petroleum based oils R R R R R R R R R
Petroleum ether R R R R R U U R U
Phenol (0.5%) R R R R R NR L L R
Phenol (10%) NR R L R R NR L NR R
Phosphoric acid (25%) NR L R R R L NR U R
Potassium hydroxide L L NR R R L L R R
(1N)
Potassium hydrox(25%) NR R R R R L L R R
Potassium hydrox(50%) NR NR R R R L L L R
Propanol R R R R R NR R R R
Pyridine NR R NR R L L R NR R
Silicone oil R R R R R R R U R
Sodium hydroxide L R R R R R L R R
(0.1N)
Sodium hydroxide NR L R R R R L L R
(diluted-5%)
Sodium hydroxide NR L R R R R L NR R
(25%)
Sodium hydrox (50%) NR NR R R R R L NR R
Sodium Hydrox.(conc) NR NR R R R L L NR R
Sodium hypochlorite R R R L R NR NR U R
Sulfuric acid (dil.-5%) L R R R R L NR NR R
Sulfuric acid (mico.25%) NR L R R R NR NR NR R
Sulfuric acid (6N) NR L R R R NR NR NR R
Sulfuric Acid (conc) NR NR R NR L NR NR NR R
Tetrahydrofuran NR R NR R R R R R R
Toluene R R L R R R R U R
Trichloroacetic acid NR NR R R R L NR NR R
(25%)
Trichlorobenzene NR R NR R R U U U U
Trichloroethane L R L R R L L L R
Trichloroethylene R R R R NR L L R R
Triethylamine NR R NR L R R R U R
Turpentine NR R NR R R L R U R
Urea R R R R R R L R R
Urea (6N) NR R NR R R R L R R
Water R R R R R R R R R
Xylene NR R NR R R R L NR R
Cellulose Regenerate Polysulfone PolyVinyli
Polypropyl Stainless Polyester Fluorocarb
Ester (CE) d (PS) / dene Nylon (N)
Cellulose ene (PP) DiFluoride Steel (SS) (P) on (F)
/ PolyetherS
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Mixed (RC) ulfone (PVDF)
Cellulose (PES)
(ME)

Dialysis operating – using dialysis membranes

FAQ: How do you prepare dialysis membrane tubing?
Preparation instructions will vary based on the membrane type as follows. Note that boiling membrane is not
recommended as it can damage the membrane and alter the pore rating.

A) CelluSep, Biotech RC, CE, and PVDF membranes should be rinsed in DI water for 15 to 30 minutes to remove
sodium azide preservative.

B) Spectra/Por® 7 Standard RC has been pretreated to remove the trace levels of heavy metals and sulfides and only
requires a 15 to 30 minute soak in DI water to remove the sodium azide preservative.

C) Spectra/Por 1 through 6 Standard RC membranes may require some extra preparation. While rinsing Spectra/Por 1
through 6 in water is typically sufficient to remove glycerin or preservative, Spectrum offers two membrane pre-
treatment solution kits for the removal of the trace levels of heavy metals and sulfides introduced during manufacturing.
Heavy Metal Cleaning Solution and Sulfide Removal Solution Kits are recommended for ultra-sensitive dialysis
applications like binding studies or when low level presence of these contaminants may interfere with downstream
analysis of the dialysis sample. Refer to the Membrane Dialysis Accessories webpage for more product information.

FAQ: How long should I cut the dialysis tubing?

Total length = (sample volume) / (vol/length) + (additional 10-20%) + 4 cm (for the knot or clamp)

Along with each tubing flat width, the correlating volume/length ratio are generally indicated, and so can be used to
calculate how much length is required to contain your sample volume. For example if the Flat Width is 16 mm, the
volume/length ratio is 0.79 ml/cm. To contain a sample of 5 ml, you will need a length of approximately 6.5 cm.
However, you also need to add about 10 to 20% more length ato prevent osmosis, and as head space (an air buble willto
keep your sample buoyant). Lastly you need to add enough about 2 cm at each end to allow for applying two closures.
The total tubing length would be at least 11.5 cm. The simple equation to calculate total required tubing length is as
follows:
Dont hesitate to ask at [email protected] if you need help!

FAQ: Should I close the dialysis tube using closures or tyieng knots?
Tying knots with the tubing is for many an easy and economic way to close the dialysis chamber. Knots however may
lead to sample leakage if not properly done. Additionally, one might feel it is not convenient to make knots, there is a
risk to leave the tubing fall and loose precious sample. After dialysis, opening tied tubing need scissors that may
contaminate the opening, hence the sample when filling it out. Sample recovery may also be lowered because folded
tubing keep more sample on its surface.
To these points, tubing clamps are recommended for more convenient operating.
Yet, people familiar with dialysis can see no real advantages using clamps, and prefer doing knots.

FAQ: How to select the right closure and closure size for membrane tubing?
Standard RC membrane (CelluSep, Spectra/Por 1-7) and Biotech RC is constructed of flexible regenerated cellulose
polymers and can be sealed using any of the dialysis tubing closures. You just have to choose the right clamp width
depending on the flat width of you tubing. Large clamps can be used for smaller tube width, but with caution because
the tubing may be no so good maintained properly.
Biotech CE and PVDF are constructed of a more rigid polymer requiring gentler Universal closures.
Since these work well for all dialysis tubing; when in doubt, use Universal Closures. Standard Closures should ONLY
be used with Standard RC tubing.

It is recommended to use a closure with a sealing width of 4-10 mm longer than the flat width of the dialysis tubing.
The smallest Universal Closure has a sealing width of 50 mm. This will seal all flat widths .

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FAQ: Is there a "rule of thumb" regarding membrane surface area to sample volume?
The surface area to volume ratio is a function of the tubing flat width. If you have a two equal length pieces of tubing
with two different flat widths, the smaller flat width piece possesses a higher surface area to volume ratio and dialyzes
quicker while the larger flat width piece possesses a lower surface area to volume ratio and dialyzes slower. The smaller
flat width has a shorter distance for diffusion and less solute "competition" through the membrane pores. Larger flat
widths have a longer distance to the membrane and more solute competition through the pores. In general, the greater
the surface-area-to-volume ratio, the quicker the dialysis.
The membrane volumes, the dialysis bath volume to sample volume should be considered.

FAQ: How much volume of dialysate is needed to dialyze a sample and how often does the dialysate need to be
changed?
Dialysis buffer volume depends on the number of dialysis steps you are doing, and their duration! The larger the dialysate volume, the greater the
driving force for diffusion of small molecules.
We generally recommend a +100:1 buffer to sample volume ratio. By replacing the buffer just as the rate of diffusion slows down and the solutions
are approaching equilibrium, you can maintain the driving force and the rate of dialysis.

We generally recommend two or three buffer changes over the period of 12 - 24 hrs as follows:

First buffer change: volume 100:1 during 0.5-3 hours

Second buffer change: volume 100-300:1 during 4-5 hours
Last buffer change: volume 500:1 overnight.

You may consider a final/total volume of bath of at least 100 to 1000 and even 10 000 over the volume sample, in similar proportion of the desired
dilution you expect for undesired substance(s) removed by dialyzis.
Typically, it is more economic in buffer to perform a shorter dialysis steps and and at least a second dialysis step for a longer period to reach an
equilibrium in a larger bath (overnight). I.e. a fist dialysis with a volume ratio 250 : 1 of buffer/sample and a secund one of 1000:1 would ideally
dilute removed salts by 1:250 000 if both dialysis were complete. Even with a 50% efficiency during the first dialysis step, this is generally sufficient
in most applications. But by precaution one may dialyze even more, because it is so easy to increase volume, duration and even add a 3rd step!

FAQ: How how long does dialysis take to complete -does dialysis work-?
Dialysis is the diffusion of dissolved solutes across a selectively permeable membrane against a concentration gradient
in an effort to achieve equilibrium. While small solutes pass through the membrane larger solutes are trapped on one
side.
By exchanging the dialysate buffer on the outside side of the membrane, you can continually pull away the smaller
solutes to purify the trapped larger molecules. In general, dialysis will be most effective when the buffer is replaced a
few times over the course of a day and then left overnight at room temperature on a stir plate. A standard protocol for
dialysis is 16 to 24 hours. Many factors affect the rate dialysis, including: diffusion coefficients, pH, temperature, time,
concentration of species, sample volume, dialysate (buffer) volume, number of dialysate changes, membrane surface
area, membrane thickness, molecular charges and dialysate agitation (stirring).

FAQ: Can I still use the membrane if it dries out or freezes?

Should I be concerned about the creases, roller marks, or fold lines on membranes?
The roller marks or fold lines along the delivered membranes will not affect the diffusion properties as long as the
membrane is integral. However, attention should be payed when unproper storage:

If wetted membrane dries out, the pore size is adversely affected and the membrane becomes brittle and will likely leak.
The membrane should be discarded.

If the membrane freezes, the ice crystals may rupture the membrane and also cause leaking. It is recommended not to
use the membrane. However, you can try slowly increasing the temperature until the storage solution completely melts.
The possibility remains that the membrane is not longer integral.

FAQ: Can dialysis membranes be re-used for the same protein samples?
We do not recommend re-using dialysis membranes since they can be contaminated through handling and dialysis
conditions (pH, temperature, chemical exposure, etc.) can alter the membrane integrity and/or cause leaking, especially
when removing and reapplying closures. Dialysis membranes are designed for single use.

FAQ: Can dialysis membranes be chemically or heat sealed?

The CE and RC dialysis membranes can only be mechanically sealed.

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However, the PVDF dialysis membranes can be mechanically sealed or heat sealed and is often used in this manner for
the purposes of sample "encapsulation".

FAQ: Which dialysates (buffers) are commonly used in dialysis?

Biomolecules must be maintained under strict pH control to stabilize their molecular properties. The typical pH range
for dialysis buffers is 6 to 8. The following are some of the common solutions/buffers found in biochemical solutions:

Water
PBS: Phosphate buffer saline
TBS: Tris buffered saline
HEPES
Amino Acid Buffers

FAQ: How to sterilize dialysis membranes / devices

You can order sterilized dialysis membranes or devices – available in certain formats-. Please inquire.

You can sterilize dialysis membranes or devices

By irradiation (the mostly recommanded method)
Chemically. See 3 protocols in FT-CelluSep. *
By autoclaving (not possible for CE membranes; not recommended with RC membranes) *
On custom: please inquire
* Chemical sanitization and autoclaving may affects slightly the cut off.

FAQ: How much pressure can a dialysis membrane withstand if used for ultrafiltration?
Dialysis membranes are not designed for pressure filtration. The maximum recommended pressure is 1.5 psi without
affecting the MWCO.
Alternatively to supported (reinforced) dialyiss membranes, one may overlay it with a paper filter.

More / trouble shhoting

FAQ: Why is the volume of my sample increasing during dialysis / my dialysis has leaked or has burst!
A sample often contains more solutes than the dialysis buffer. This drives the solvent (water) to cross the membrane in
order to equilibrate the concentration in the 2 compartment, and so the sample volume to increase. This phenomenon is
called Osmosis (see FAQ What is dialysis versus osmosis?), and normal. It is generally limited, causing a few% to 20%
increase in sample volume, up 20% with very concentrated samples like 3/M glycine, urea or glycerol, and
SulfateAmmonium precipitated globulins). That's why it is recommended to always leave unfilled tubing (see FAQ
How long to cut the dialysis tubing?)

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Dialysis Applications in R&D

Desalting / Buffer exchange
Desalting / Buffer exchange is the main application of dialysis, to keep purified protein free of undesired small
compounds that are present in biological samples, or added during upstream purifications steps.

Sterile dialysis
Same samples require dialysis to be performed in sterile conditions, notably with cells to be cultured free of some
componends form medium of that are released.
There are very few commercial sterile dialysis membranes and devices (please inquire), but
lab-made sterilization is possible. See FAQ: How to sterilize dialysis membranes / devices

Dialyse à l'équilibre - Fraction liée/libre

Une application importante de la dialyse correspond à l'étude de la répartition de petits composés entre compartiments,
et notamment entre la fraction qui reste libre en solution et la fraction qui se lié aux molécules ou particules du milieu.
La méthode présentée ci après correspond à l'application typique de quantification de la fraction d'un médicament liée
aux protéines plasmatiques.

1) Méthode approximative par ultrafiltration/dessalage

Ultrafiltrer un échantillon (plasma+médicament) permet de séparer rapidement le médicament en solution. C'est une
méthode rapide pour apprécier la quantité de médicament lié, approximativement (Clin Chem. 1985 Jan;31(1):60-4): car cette
méthode ne mesure pas forcément toute la fraction de médicament liée, pour différentes raisons: l'équilibre (et taux de
médicament lié) est modifié par la concentration réalisée lors de l'UF; tout le médicament libre n'est pas éliminé en une
étape d'UF; le biais n'est pas gommé si vous faites une 2eme séparation UF après ajout de plasma sans médicament (et
pire si ajout de PBS): au contraire le médicament peut se décrocher en partie durant le process d'UF; ...
Regardez nos dispositifs d'UF centrifugeables VivaSpin[]:

2) Méthode par dialyse à l'équilibre.

La dialyse à l'équilibre permet de mesurer précisément la fraction lié, au moyen d'une durée de manip plus longue et de
quelques calculs (équation avec lles concentrations [liée] et [dialysée] -méthode préférée en pharma-:

Equilibrium Dialysis products

Dispositifs de dialyse pour ces études (& voir 'Dialysis Products Guide)[]:
RED (rapid Equilibrium Device)[]:
DispoDialyser devices (equilibrium version)[]:
HTD Equilibrium Dialysis System(96 samples)[]:
96-Well DispoDialyser plate(equilibrium version)[[]:

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Equilibrium Dialysis information

(r)

Equilibrium dialysis is a specific application of the general phenomenon of dialysis that is important for the study of the
binding of small molecules and ions by proteins. It is one of several methods currently available but its attractive
feature continues to be its physical simplicity.
The objective of an equilibrium dialysis experiment is usually to measure the amount of a ligand bound to a macro-
molecule. This is typically done through an indirect method because in any mixture of the ligand and macro-molecule,
it is difficult to distinguish between bound and free ligand.

If, however, the free ligand can be dialyzed through a Data obtained from several experiments at a range of
membrane, until its concentration across the membrane is temperatures and with varying initial concentration of
at equilibrium, free ligand concentration CL(f) and the ligand can provide other binding parameters:
following data can be measured: Association constant K
Temperature (absolute) T Number of binding sites n
Concentration of binding component, e.g. protein CP(o) Binding capacity N
Starting concentration of ligand CL(o)
Final concentration of free ligand CL(f) Further, the thermodynamics of the binding reaction can
be derived:
From which the following parameters can be derived Change of free energy ∆G
directly: Enthalpy change ∆H
Concentration of bound ligand CL(b) Entropy change ∆S
Free fraction (of ligand) ƒ
Bound fraction (of ligand) b
Degree of binding or saturation fraction r

Since equilibrium exists, the value CL(f) is the same on both sides of the membrane.
(Note: where charged species are involved the Gibbs-Donnan effect can upset the equilibrium unless moderately
concentrated salts are in solution; say 0.6% NaCl).
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Hence: CL(o) = C L (f) + CL(f) + CL(b) *

*It is essential to correct this equation to take account of any ligand which might be bound to the membrane.
CL(b) = CL(o) – 2 x CL(f)
The free fraction ƒ is given by: ƒ = CL(f) / [ CL(o) – CL(f) ]
The bound fraction b is: b = 1 – ƒ

The degree of binding or saturation fraction r is: . r = CL(b) / CP(o .

If the protein concentration is known, the Scatched plot can be used to determine binding constants and the number of
binding sites. If the protein concentration is unknown, the absolute number of binding sites is replaced by binding
capacity N .
In the former case, values of r would be plotted on the abscissa against r/CL(f) on the ordinate. If only one class of
binding sites is present, the Scatched plot results in a straight line with slope equal to –K see Fig. 1.
The intercept on the abscissa give the value n . If two classes of binding sites are involved, the plot takes the form of
an hyberbola. In this case, the asymptotes have slopes equal to –K for each class of site, and their intercepts on the
abscissa give the two values for n . The intercept between the curve and the abscissa is equal to the sum of the two
values for n , see Fig. 2.
The free energy change is obtained simply by substituting the appropriate values in the following equation:
∆G = - RT In K where R is the gas constant.
∆H can be obtained from a graph based upon an integrated form of the van/t Hoff equation.
Ln K = - ∆H/RT + C
In this case a plot In K versus 1 /T has a slope of - ∆H/R . Once a value for ∆H has been found it can be substituted
into: ∆G = s H - T ∆S to obtain a result for the entropy change ∆S.
Fig. 1. Fig. 2.

References:
Scatchard, G., Am. N.Y. Acad. Sci. 51, 660-672 (1949)
Rosenthal, H.E., Anal. Biochem. 20, 525-532 (1967)
Weder, H.G., Schildknecht, J., Lutz, R.A. and Kesselring, P., Eur. L. Biochem. 42, 475-481 (1974)

(a) Clin Chem. 1985 Jan;31(1):60-4. Equilibrium dialysis, ultrafiltration, and ultracentrifugation compared for determining
the plasma-protein-binding characteristics of valproic acid. Barré J, Chamouard JM, Houin G, Tillement JP.
Abstract: Equilibrium dialysis, ultrafiltration, and ultracentrifugation were compared to determine their reliability and applicability in the study of
binding of an anticonvulsant drug, valproic acid, by plasma proteins. We studied drug binding with pooled serum and with solutions of human serum
albumin at physiological concentrations. We compared binding characteristics such as number of binding sites, affinity constants, and percent of
binding as measured by each method in the therapeutic range for valproic acid. Results by ultracentrifugation differed from those by equilibrium
dialysis and ultrafiltration, which agreed reasonably well with each other.

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Process Dialysis – Applications & Tips

Large-volume Process Dialysis is an efficient and economic
technology driven by the increasing demand for gentle and
consistent multi-batch purification at the production scale. While
Laboratory Dialysis for research and analytical testing typically
involves static stirring of small volumes for sample prep or solute
release studies, process dialysis facilitates dynamic buffer flow
around the membrane-encased sample to increase purification
efficiency and improve large buffer handling for the production of
purified or synthetic compounds or particucles. Dialysis is notably a
nice choice for fragile proteins, viscous fluids and polymer gels,
such as hyaluronic acid.

Spectra/Por® Products Designed for Process Dialysis

Large Volume Dynamic Buffer Flow
Largest Membrane Selection Smart Dialysis
Customized Dialysis Tubing & Membrane
Applications, tips
=> see Process Dialysis catalog [BB111p]

Contact Interchim for any question Rev. S02E

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