Ultrafree-CL Centrifugal Filter Units User Guideaix1.uottawa.ca/~sgee/protocols/Millipore Ultrafilters Manual.pdfanalysis (HPLC, MS, IC, GC) ... Xylene KEY: ... Viscous solutions,

Ultrafree®-CL CentrifugalFilter Units

Single-Use Only

NoticeThe information in this document is subject to change without notice and should not beconstrued as a commitment by Millipore Corporation. Millipore Corporation assumes noresponsibility for any errors that may appear in this document. This manual is believed to becomplete and accurate at the time of publication. In no event shall Millipore Corporation beliable for incidental or consequential damages in connection with or arising from the use ofthis manual.

Copyright 1999, Millipore Corporation or an affiliated company.

Millipore, Durapore, and Ultrafree are registered trademarks of Millipore Corporation or anaffiliated company.Biomax is a trademark of Millipore Corporation or an affiliated company.Cello-Solve is a registered trademark of Union Carbide Chemicals & Plastics TechnologyCorporation.Freon is a registered trademark of E. I. du Pont de Nemours and Company.Tyvek is a registered trademark of E.I. du Pont de Nemours and Company.

P17956, Rev C, 9/99

ContentsIntroduction .......................................................................................................................... 1Membrane Types and Applications ........................................................................................ 2Table: % Protein Recovered from Retentate @ 25°C ................................................................ 4Guidelines for Use ................................................................................................................ 7How to Use the Ultrafree-CL Centrifugal Filter Unit ............................................................... 14Specifications ..................................................................................................................... 15Ordering Information .......................................................................................................... 16Technical Assistance ............................................................................................................ 18Standard Warranty ............................................................................................................. 19

IntroductionMillipore Ultrafree-CL centrifugal filter units are disposable, single-use devices used toprocess aqueous biological solutions in the 0.5 to 2 milliliter (mL) range. Ultrafree-CL unitscan be used in standard, variable-speed centrifuges with fixed-angle rotors capable ofholding 15 mL tubes. A complete device consists of the following components:

■ Filter cup

■ Filtrate collection tube with cap

Ultrafree-CL units are available in a range of microporous and ultrafiltration membranes.

2 Ultrafree-CL Centrifugal Filter Units

Membrane Types and ApplicationsUltrafree-CL units are available with ultrafilters and microporous filters.

Ultrafilters Description

BiomaxTM high-flux polysulfone Highest flow rate characteristics for fast sampleultrafiltration series preparation of complex protein samples. Available in

5,000, 10,000, 30,000, 50,000, and 100,000 nominalmolecular weight limit (NMWL).

Low-binding, regenerated Exhibits the lowest binding for amino acids, mostcellulose, Ultracel PL ultrafiltration drugs, peptides, proteins, and nucleic acids. Available inseries 5,000, 10,000, and 30,000 NMWL.

Polysulfone PT ultrafiltration Millipore's original polysulfone UF membrane. Availableseries in 10,000; 30,000; 100,000; and 300,000 NMWL.

Membrane Types and Applications, continued

Microporous Filters DescriptionLow-binding Durapore® Available in 0.1, 0.22, 0.45, 0.65, and 5.0 micron (µm)microporous series sizes.PTFE microporous series Available in 0.2 and 0.5 µm sizes.

Application MembraneProtein concentration or dialysis 5,000; 10,000; 30,000; 50,000; 100,000 NMWLClarification, particulate removal before instrument 0.1, 0.2, 0.45 µm Duraporeanalysis (HPLC, MS, IC, GC)Protein removal before instrument analysis 10,000 NMWLFermentation broth screening (to remove 10,000 or 30,000 NMWLinterfering high molecular weight solutes)Serum or plasma concentrations 10,000 or 30,000 NMWL

http://www.millipore.com/ultrafree 3

% Protein Recovered from Retentate @ 25°Cuuuuu

Biomax High-flux PolysulfoneRetentate MW 5,000 10,000 30,000 50,000 100,000

NMWL NMWL NMWL NMWL NMWLVitamin B-12(0.2 mg/mL) 1.3 kD 5–10% 5–10% 0–5% 0–5% 0–5%Bovine Insulin(0.25 mg/mL) 6 kD 85–90% 60–70% 45–55% 10–20% 5–10%Lysozyme(1 mg/mL) 14 kD 90–100% 80–90% 60–70% 40–50% 5–10%Bovine Serum Albumin(1 mg/mL) 67 kD 95–100% 95–100% 95–100% 90–100% 20–30%Goat IgG(1 mg/mL) 156 kD 95-100% 95–100% 95–100% 95–100% 90–100%


u Passage of proteins with a molecular weight lower than the membrane rating may vary with g-force. Ingeneral, a lower g-force will favor increased passage.


Regenerated Cellulose, PL SeriesRetentate MW PLCC 5,000 PLGC 10,000 PLTK 30,000

NMWL NMWL NMWLVitamin B-12(0.2 mg/mL) 1.3 kD 15–20% 10–15% 5–10%Bovine Insulin(0.25 mg/mL) 6 kD 90–95% 80–90% 55–65%Lysozyme(1 mg/mL) 14 kD 95–100% 90–95% 60–70%Bovine Serum Albumin(1 mg/mL) 67 kD 95–100% 95–100% 95–100%Goat IgG(1 mg/mL) 156 kD 95–100% 95–100% 95–100%




Polysulfone, PT SeriesRetentate MW PTGC PTTK PTHK PTMK

10,000 30,000 100,000 300,000NMWL NMWL NMWL NMWL

Vitamin B-12(0.2 mg/mL) 1.3 kD 5–10% 0–5% 0–5% 0–5%Bovine Insulin(0.25 mg/mL) 6 kD 80–90% 55–65% 5–10% 0–5%Lysozyme(1 mg/mL) 14 kD 90–95% 60–70% 5–10% 0–5%Bovine Serum Albumin(1 mg/mL) 67 kD 95–100% 90–100% 10–20% 0–5%Goat IgG(1 mg/mL) 156 kD 95–100% 95–100% 90–100% 35–45%




Guidelines for UseFollow these guidelines to optimize the performance of Ultrafree-CL units.

Protein Binding

While different membrane materials can nonspecifically bind proteins to varying degrees,many factors can influence protein binding, including pH, buffer strength, contact time,surface tension of the solution, solute concentration, and individual protein character. Ingeneral, when ultrafiltration membranes are soaked in protein solutions, regeneratedcellulose membranes are the lowest binding, Biomax high-flux polysulfone membranes aresomewhat higher binding, and PT series original polysulfone membranes are the highestbinding. However, because Biomax membranes have such high flow rates when used in acentrifugal mode and therefore protein contact with the membrane is so brief, proteinrecoveries with Ultrafree-CL centrifugal filters containing Biomax membranes are often justas high (>95%) as those with regenerated cellulose. For protein concentration or desalinationapplications, we recommend comparing the Biomax membrane to PL and PT type filters.

Extractables

All ultrafiltration membranes contain a humectant. A humectant ensures that the membraneretains enough moisture to maintain its structure. All of the ultrafiltration membranes used inUltrafree-CL devices contain glycerin as their humectant. The exception is the 10,000 NMWLregenerated cellulose membrane, which contains triethylene glycol. If these humectantsinterfere with instrument-based analysis of a sample, flush the Ultrafree-CL device with Milli-Q-system water immediately before use (contact Technical Service for details).

Chemical Compatibility with Ultrafree-CL MembranesDurapore Polysulfone/Biomax Regenerated cellulose

Acetic Acid, glacial ● ● ❍Acetic Acid, 5% ● ● ❍Boric Acid ● ● ●Hydrochloric Acid (conc.) ● ▲ ▲Hydrofluoric Acid ● ● ●Nitric Acid (conc.) ❍ ❍ ❍Sulphuric Acid ❍ ❍ ❍


KEY: ● Recommended, ❍ Not recommended, ▲ Limited use: testing is recommended

Chemical Compatibility with Ultrafree-CL Membranes, continued

Durapore Polysulfone/Biomax Regenerated celluloseAmmonium Hydroxide (6N) ❍ ● ▲Sodium Hydroxide (conc.) ❍ ● ❍Acetone ❍ ❍ ●Acetonitrile ● ● ●Amyl Acetate ● ● ●Amyl Alcohol ● ● ●Benzene ● ▲ ●Benzyl Alcohol (1%) ● ● ●Butyl Alcohol ● ● ●Carbon Tetrachloride ● ▲ ●Cello-Solve® (ethyl) ● ❍ ●Chloroform ● ❍ ●Cyclohexanone ● ❍ ●Dimethylacetamide ❍ ❍ ●Dimethylformamide ❍ ❍ ●Dioxane ● ❍ ●




Durapore Polysulfone/Biomax Regenerated celluloseDMSO ▲ ❍ ●Ethyl Alcohol ● ● ●Ethers ● ❍ ●Ethyl Acetate ● ❍ ●Ethylene Glycol ● ● ●Formaldehyde ● ● ●Freon® TF or PCA ● ● ●Gasoline ● ▲ ●Glycerine (glycerol) ● ● ●Hexane ● ● ●Hydrogen Peroxide (3%) ● ● ●Hypo (photo) ● ❍ ●Isobutyl Alcohol ● ● ●Isopropyl Acetate ● ● ●Isopropyl Alcohol ● ● ●Kerosene ● ▲ ●




Durapore Polysulfone/Biomax Regenerated celluloseMEK ▲ ❍ ●MIBK ● ❍ ●Nitrobenzene ● ▲ ●Paraldehyde ● ● ●Ozone (10 ppm in water) ● ● ●Pet Base Oils ● ▲ ●Pentane ● ● ●Perchloroethylene ● ● ●Petroleum Ether ● ● ●Phenol (5.0%) ● ❍ ●Pyridine ● ❍ ●Silicone Oils ● ● ●Toluene ● ❍ ●Trichloroethylene/ethane ● ❍ ●THF ● ❍ ●Xylene ● ▲ ●



Flow Rate

Flow rate is affected by several parameters, including the NMWL, solute concentration,viscosity, centrifugal force, and temperature. For maximum recovery in protein concentra-tion or buffer exchange applications, select a UF membrane with a NMWL at least threetimes lower than the molecular weight of your target molecule. Expect longer spin times forstarting solutions with very high solids. Flow rates are approximately 1.5 times slower whenthe device is operated at 4ºC than when operated at 25ºC. Viscous solutions, such as 50%glycerol, will take up to five times longer to concentrate than very dilute solutions.

Biomax PB UF membranes, particularly those in the 5,000 NMWL and 10,000 NMWLUltrafree-CL centrifugal filter units, have exceptionally high flow rates when compared tomembranes of the same NMWL, and will typically flow at 2× the flowtime of the PLmembranes. A PL-10 membrane will require 60 minutes to concentrate a 2 mL 0.25 mg/mLCytochrome C sample at 30×.


Flow Rate Comparison (0.25 mg/mL BSA in PBS [pH=7.4])uhttp://www.millipore.com/ultrafree 13

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

0 15 30 45 60 75 90 105

Time (min) jj

PB (5K)

PL (5K)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

0 5 10 15 20 25 30

Time (min) jj

PB (10K)

PL (10K)

PT (10K)

5,000 NMWL Membranes at 5,000 g 10,000 NMWL Membranes at 5,000 g

u All tests were performed at 25°C.

How to Use the Ultrafree-CL Centrifugal Filter UnitThis section provides steps for general use.

General Use

1. Remove the top cap from the filtrate collection tube. Then pour or pipette your sampleinto the filter cup. Hold the Ultrafree-CL vertically, with the filter cup opening facingup.

2. Replace the top cap onto the filtrate collection tube. Then insert the Ultrafree-CL filterinto the centrifuge fixed-angle rotor head.

3. Centrifuge the sample until required retentate or filtrate volume is achieved.

Filter Type Typical Spin TimeMicroporous 1–4 minutesUltrafiltration* 30–60 minutes

* There is no deadstop with ultrafiltration devices and concentrate volume must be monitored carefully to prevent running to dryness.


SpecificationsMaterials Molded polypropylene filter cup and microcentrifuge tube for filtrate collection,

silicone rubber O-ring in filter cupDimensions Filtrate collection tube 76.5 mm length, 16.2 mm inside diameter

Filter cup 41 mm length, 11.8 mm inside diameterCapacity Filter cup 2.0 mL

Collection tube 5 mLHold-up volume 0.03 mL

Effective filtration area 0.8 cm2

Maximum temperature 50°C (122°F)Microcentrifuge rotors Centrifuge with fixed-angle rotors capable of holding

15 mL microcentrifuge tubesMaximum g Force Rating 5,000 g (ultrafiltration devices)

12,000 g (microporous devices)


Ordering InformationUltrafree-CL filters are dry and nonsterile. Each contains a preassembled filter with collectiontube and cap.

Biomax UF Membranes 25/box

5,000 NMWL Biomax-5 high-flux UFC4 BCC 2510,000 NMWL Biomax-10 high-flux UFC4 BGC 2530,000 NMWL Biomax-30 high-flux UFC4 BTK 2550,000 NMWL Biomax-50 high-flux UFC4 BQK 25100,000 NMWL Biomax-100 high-flux UFC4 BHK 25

Regenerated Cellulose UF Membranes 25/box5,000 NMWL regenerated cellulose UFC4 LCC 2510,000 NMWL regenerated cellulose UFC4 LGC 2530,000 NMWL regenerated cellulose UFC4 LTK 25


Ordering Information, continued

Polysulfone UF Membranes 25/box

300,000 NMWL polysulfone UFC4 TMK 25

Microporous Membranes 25/box 100/box Presterile* 50/box0.1 µm pore size Durapore PVDF UFC4 0VV 25 UFC4 0VV 00 —

0.22 µm pore size Durapore PVDF UFC4 0GV 25 UFC4 0GV 00 UFC4 0GV 0S

0.45 µm pore size Durapore PVDF UFC4 0HV 25 UFC4 0HV 00 UFC4 0HV 0S

0.65 µm pore size Durapore PVDF UFC4 0DV 25 — —

5.0 µm pore size Durapore PVDF UFC4 0SV 25 — —

0.2 µm pore size PTFE UFC4 0LG 25 — —

0.5 µm pore size PTFE UFC4 0LH 25 — —* Presterile packages consist of 10 devices in each of five ETO-sterilized Tyvek® pouches.


Technical AssistanceCall the office in your country to get additional product information, technical assistance, orto order parts. In the U.S., call 1-800-MILLIPORE (800-645-5476). Elsewhere, see ourlaboratory products catalogue for the office nearest you. To receive the current edition ofour catalogue, contact Technical Service at the Millipore office closest to you. You can alsolook us up on the Internet at our World Wide Web site (http://www.millipore.com). Up-to-date information on the Ultrafree product line can be found athttp://www.millipore.com/ultrafree. Our e-mail address is [email protected].


Standard WarrantyMillipore Corporation ("Millipore") warrants its products will meet their applicable publishedspecifications when used in accordance with their applicable instructions for a period of oneyear from shipment of the products. MILLIPORE MAKES NO OTHER WARRANTY, EXPRESSEDOR IMPLIED. THERE IS NO WARRANTY OF MERCHANTABILITY OR FITNESS FOR APARTICULAR PURPOSE. The warranty provided herein and the data, specifications anddescriptions of Millipore products appearing in Millipore's published catalogues and productliterature may not be altered except by express written agreement signed by an officer of Millipore.Representations, oral or written, which are inconsistent with this warranty or such publications arenot authorized and if given, should not be relied upon.

In the event of a breach of the foregoing warranty, Millipore's sole obligation shall be to repair orreplace, at its option, the applicable product or part thereof, provided the customer notifiesMillipore promptly of any such breach. If after exercising reasonable efforts, Millipore is unable torepair or replace the product or part, then Millipore shall refund to the customer all monies paid forsuch applicable product or part. MILLIPORE SHALL NOT BE LIABLE FOR CONSEQUENTIAL,INCIDENTAL, SPECIAL OR ANY OTHER INDIRECT DAMAGES RESULTING FROM ECONOMICLOSS OR PROPERTY DAMAGE SUSTAINED BY ANY CUSTOMER FROM THE USE OF ITSPRODUCTS.


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Ultrafree-CL Centrifugal Filter Units User Guideaix1.uottawa.ca/~sgee/protocols/Millipore Ultrafilters Manual.pdfanalysis (HPLC, MS, IC, GC) ... Xylene KEY: ... Viscous solutions,