Selection Guide: Separation Products for Centrifugal and Tangential Flow Filtration

The biomolecule of interest in your sample is called a product. Separation can occur by choosing a membrane that retains the product while passing any low molecular weight contaminants. Alternatively, a membrane can be chosen that passes the product while retaining higher molecular weight components in the sample. It is also possible to combine both separations in a two-stage process that will fractionate out the product from both higher and lower molecular weight components. In the first stage, a membrane is chosen that passes the product and retains the higher molecular weight components. The filtrate from the first stage then becomes the sample for the second stage. For the second stage, the membrane is chosen to concentrate the product and remove lower molecular weight substances. You will need to define your separation goals – concentration, diafiltration, or fractionation. You must also consider the process volumes that you have to work with and any scale up requirements. It is important to know the concentration factor or the level of salt reduction required in order to choose the most appropriate membrane and system for the process.

Pall offers membrane types to meet the challenges of molecular separations with superior performance and stability. Our separation products feature these versatile membranes:

Membrane	Key Characteristics	Available Platforms
Ultrafiltration
Omega™ Membrane (modified PES)	Low non-specific protein binding Low DNA binding Yields extremely high recoveries Superior chemical and physical properties	Centrifugal TFF
Alpha™ Membrane (modified PES)	Antifoam resistant High flow rates and extracellular passage	TFF
Microfiltration
Bio-Inert® Membrane (modified Nylon 6,6)	Low protein binding	Centrifugal
GH Polypro (GHP) Membrane (hydrophilic polypropylene)	Low protein binding Low extractables	Centrifugal
Supor® Membrane (hydrophilic polyethersulfone)	Low protein binding HIgh flow rates and throughputs	TFF

Once sample volume is determined, the next step is to select the appropriate MWCO (for ultrafiltration) or pore size (for microfiltration). MWCOs are nominal ratings based on the ability to retain > 90% of a solute of a known molecular weight (in Kilodaltons). The tables below provide retention characteristics of different MWCO membranes for some solutes. For proteins, it is recommended that a MWCO be selected that is three to six times smaller than the molecular weight of the solute being retained. If flow rate is a consideration, choose a membrane with a MWCO at the lower end of this range (3X); if the main concern is retention, choose a tighter membrane (6X).

It is important to recognize that retention of a molecule by a UF membrane is determined by a variety of factors, among which its molecular weight serves only as a general indicator. Therefore, choosing the appropriate MWCO for a specific application requires the consideration of a number of factors including molecular shape, electrical charge, sample concentration, sample composition, and operating conditions. Because different manufacturers use different molecules to define the MWCO of their membranes, it is important to perform pilot experiments to verify membrane performance in a particular application. 

Common Variables that Increase Molecule Passage

• Sample concentration less than 1 mg/mL.
• Linear versus globular molecules.
• High transmembrane pressure created by g-force in centrifugal concentrators. (This is especially important in the case of linear molecules, for example DNA fragments. Decreasing the g-force can increase retention of molecules by a membrane.)
• Buffer composition that favors breakup of molecules.
• pH and ionic conditions that change the molecule (for example, cause conformational changes or aggregation).

Common Variables that Decrease Molecule Passage

• Sample concentration higher than 1 mg/mL.
• Buffer conditions that permit molecules to aggregate.
• Presence of other molecules that increase sample concentration.
• Lower transmembrane pressure (in the case of centrifugal concentrators, lower g-force).
• Adsorption to the membrane or device.
• Low temperature (4 °C versus 24 °C).

Centrifugal devices from Pall Life Sciences are available in a range of MWCOs color coded for easy identification.

MWCO Selection for Nucleic Acid Applications

MWCO	Base Pairs Double Strands (DS)	Bases Single Strands (SS)
1K	5 - 16 (Base Pairs) Bp	9 - 32 (Base Strands) Bs
3K	16 - 32 Bp	32 - 65 Bs
5K	25 - 50 Bp	50 - 95 Bs
10K	50 - 145 Bp	95 - 285 Bs
30K	145 - 285 Bp	285 - 570 Bs
50K	240 - 475 Bp	475 - 950 Bs
100K	475 - 1,450 Bp	950 - 2,900 Bs
300K	1,450 - 2,900 Bp	2,900 - 5,700 Bs
1000K	4,800 - 9,500 Bp	> 9,500 Bs

MWCO Selection for Protein Applications

MWCO	Membrane Nominal Pore Size*	Biomolecule Size	Biomolecule Molecular Weight
1K			3K - 10K
3K			10K - 20K
5K			15K - 30K
10K			30K - 90K
30K			90K - 180K
50K	5 nm	15 - 30 nm	150K - 300K
100K	10 nm	30 - 90 nm	300K - 900K
300K	35 nm	90 - 200 nm	900K - 1,800K
1000K	100 nm	300 - 600 nm	> 3,000K

MWCO Selection for Virus Applications

MWCO	Membrane Nominal Pore Size*	Virus or Particle Diameter
50K	5 nm	15 - 30 nm
100K	10 nm	30 - 90 nm
300K	35 nm	90 - 200 nm
1000K	100 nm	300 - 600 nm

A 100K device should be used to concentrate PCR** products (regardless of size) if primer removal is required or if adapters are to be recovered from restriction digests.

*Nominal pore size as measured by electron microscopy (50K is an estimate).
**PCR technique is a proprietary technology of Roche.

Starting Volumes from < 100 µL to < 150 mL

Device	Sample Volume
AcroPrep™ 384 filter plates	< 100 µL
AcroPrep 96 filter plates	< 350 µL
Nanosep® centrifugal devices	< 0.5 mL
Microsep™ centrifugal devices	0.5 - 3.5 mL
Macrosep® centrifugal devices	3 - 15 mL
Jumbosep™ centrifugal devices	15 - 60 mL

Starting Volumes from 25 mL to 25 Liters

TFF Capsule or Cassette*	Membrane Area/Capsule or Cassette	Typical Filtrate Flow Rate** at 50 LMH and 20 °C	Recommended Retentate Flow Rate/ Capsule or Cassette for Screen Channel	Starting Sample Volume Range	Minimum Concentrated Volume***
LAB SCALE / SCALE UP
Minimate™ capsules	50 cm2 (0.05 ft.2)	4 mL/min	30 - 40 mL/min	25 - 1000 mL	< 10 mL
LV Centramate™ system	0.01 m2 (0.1 ft.2)	8 mL/min	60 - 80 mL/min	40 - 2000 mL	10 mL
LV Centramate system	0.02 m2 (0.2 ft.2)	15 mL/min	120 - 160 mL/min	60 - 4000 mL	15 mL
PROCESS DEVELOPMENT/ SMALL-SCALE PRODUCTION
Ultrasette™ devices	0.084 m2 (0.9 ft.2)	4 L/hr	1200 - 1500 mL/min	0.2 - 5 L	100 mL
Centramate system	0.093 m2 (1.0 ft.2)	4.6 L/hr	600 - 800 mL/min	0.2 - 25 L	100 mL

* Data is per unit or cassette. Centramate holder can hold five cassettes. Other column data can be calculated by multiplying table values by the number of cassettes installed in the holder.
** Typical filtrate flow rate is based on an average filtrate flow rate of 50 LMH and a process time of about four hours. Actual value may be higher or lower depending on the MWCO of membrane, sample composition and viscosity, operating conditions, i.e., transmembrane pressure, cross flow rate, temperature, etc.
*** Minimum concentrated volume depends on system hold-up volume, reservoir design and pump type and speed. Smaller volumes can be achieved by minimizing tubing lengths and use of properly sized components, tubing, fittings, etc.

The sample concentration and solution characteristics (viscosity, particulates, etc.) determine the type of channel configuration required for the application. TFF devices and cassettes are available with two types of flow channel separators to provide maximum control of flow rates and membrane fouling. While all of these configurations have a woven support in the filtrate channel, they differ in the type of spacers and supports found in the retentate channel, as illustrated (see below). Not all configurations are available in the various TFF system formats.

Tangential Flow Channel Configurations

Screen Channel

Screen channel available in:
• Minimate™ capsule
• Ultrasette™ devices
• LV Centramate™ cassettes
• Centramate cassettes
In the screen channel configuration, a woven separator creates gentle turbulence in the retentate flow, minimizing membrane fouling. Screen channel configuration is used with a clean filtered 0.2 µm solution (free of particles or aggregates that can get trapped in the screen).

Suspended Screen

Suspended screen available in:
• Ultrasette devices
• LV Centramate cassettes
• Centramate cassettes
The suspended screen channel configuration has a more open structure in the retentate channel that provides better performance when highly viscous or particle-laden solutions are being processed.