Amicon Ultra-0.

5 ®

Centrifugal Filter Devices

for volumes up to 500 µL

User Guide ���

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For research use only;

not for use in diagnostic procedures

PR02725_RevB.indd 29 7/23/2009 3:01:49 PM

 Notice
The information in this document is subject to change without notice and should not be
construed as a commitment by Millipore Corporation or an affiliated corporation. Neither
Millipore Corporation nor any of its affiliated corporations assumes responsibility for any
errors that may appear in this document. This manual is believed to be complete and
accurate at the time of publication. In no event shall Millipore Corporation be liable for
incidental or consequential damages in connection with or arising from the use of this
manual.
© 2009 MILLIPORE CORPORATION. ALL RIGHTS RESERVED.
PR02725 Rev. B, 07/09

Millipore, Amicon, Centricon, Milli-Q, MultiScreen, and Ultracel are registered trademarks of Millipore Corporation.
The M mark is a trademark of Millipore Corporation.
Alconox and Terg-A-Zyme are registered trademarks of Alconox, Inc.
Lubrol is a registered trademark of Imperial Chemical Industries PLC.
Nonidet is a trademark of Royal Dutch/Shell Group.
Triton is a registered trademark of Union Carbide Corporation.
Tween is a registered trademark of ICI Americas, Inc.

PR02725_RevB.indd 30 7/23/2009 3:01:49 PM

 Contents
Introduction...........................................................................................................................................................1
Applications..........................................................................................................................................................2
Materials Supplied................................................................................................................................................3
Required Equipment.............................................................................................................................................4
Suitability..............................................................................................................................................................4
Device Storage.....................................................................................................................................................4
Specifications.......................................................................................................................................................5
Chemical Compatibility.........................................................................................................................................7
How to Use Amicon Ultra-0.5 Centrifugal Filter Devices.......................................................................................9
How to Quantitate Recoveries............................................................................................................................11
Direct Weighing Procedure.........................................................................................................................11
Performance - DNA Concentration.....................................................................................................................14
Performance - Protein Concentration.................................................................................................................15
Flow Rate...................................................................................................................................................15
Protein Retention and Concentrate Recovery............................................................................................18
Maximizing Sample Recovery............................................................................................................................21
Desalting or Diafiltration......................................................................................................................................22
Centrifugal Product Ordering Information...........................................................................................................23
Technical Assistance..........................................................................................................................................24
Standard Warranty..............................................................................................................................................25

PR02725_RevB.indd 31 7/23/2009 3:01:49 PM

 Introduction
Millipore’s Amicon® Ultra-0.5 centrifugal filter devices provide fast ultrafiltration, with the
capability for high concentration factors and easy concentrate recovery from dilute
and complex sample matrices. The vertical design and available membrane surface
area provide fast sample processing, high sample recovery (typically greater than
90% of dilute starting solution), and the capability for 30-fold concentration. Typical
processing time is 10 to 30 minutes depending on Nominal Molecular Weight Limit
(NMWL). Solute polarization and subsequent fouling of the membrane are minimized
by the vertical design, and a physical deadstop in the filter device prevents spinning
to dryness and potential sample loss. Efficient recovery of the concentrated sample
(retained species) is achieved by a convenient reverse spin step after collecting
the filtrate. Amicon Ultra-0.5 devices are supplied non-sterile and are for single use
only.

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 2 www.millipore.com

Introduction, continued
The Amicon Ultra-0.5 product line includes 5 different cutoffs (Nominal Molecular Weight
Limit, NMWL). These devices are for research use only and not for use in diagnostic
procedures.
■■ Amicon Ultra 3K device — 3,000 NMWL
■■ Amicon Ultra 10K device — 10,000 NMWL
■■ Amicon Ultra 30K device — 30,000 NMWL
■■ Amicon Ultra 50K device — 50,000 NMWL
■■ Amicon Ultra 100K device — 100,000 NMWL
Applications
■■ Concentration of biological samples containing antigens, antibodies, enzymes,
nucleic acids (DNA/RNA samples, either single- or double-stranded), microorgan-
isms, column eluates, and purified samples
■■ Purification of macromolecular components found in tissue culture extracts and
cell lysates, removal of primer, linkers, or molecular labels from a reaction mix, and
protein removal prior to HPLC
■■ Desalting, buffer exchange, or diafiltration

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 Amicon Ultra-0.5 Centrifugal Filter Devices 3

Materials Supplied
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Filtrate Concentrate
collection collection tube
tube

The Amicon Ultra-0.5 device is supplied with two microcentrifuge tubes. During operation,
one tube is used to collect filtrate; the other to recover the concentrated sample.

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 4 www.millipore.com

Required Equipment
■■ Centrifuge with fixed angle rotor that can accommodate 1.5 mL microcentrifuge tubes
CAUTION: To avoid damage to the device during centrifugation, check clearance
before spinning.

Suitability
Preliminary recovery and retention studies are suggested to ensure suitability for intended
use. See the “How to Quantitate Recoveries” section.

Device Storage
Store at room temperature.

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 Amicon Ultra-0.5 Centrifugal Filter Devices 5

Specifications
Maximum initial sample volume 500 µL
Typical final concentrate volume 15–20 µL
Recommended relative centrifugal force 14,000 × g for concentration spin
1,000 × g for reverse spin
Maximum relative centrifugal force 15,000 × g
Active membrane area 1 cm2
Hold-up volume < 5 μL
Dimensions
Filter device and tube
Length (concentration mode; device in tube): 49.9 mm (1.96 in.)
Length (reverse spin; device upside down in tube): 47.4 mm (1.87 in.)
Tube (cap closed) Diameter: 10.8 mm (0.43 in.) Length: 42.1 mm (1.66 in.)
Filter device Diameter: 9.4 mm (0.37 in.) Length: 29.5 mm (1.16 in.)
Materials of Construction
Filter device Copolymer styrene/butadiene
Membrane Millipore Ultracel® low binding regenerated cellulose
Collection tube Polypropylene

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 6 www.millipore.com

Specifications, continued
The ultrafiltration membranes in Amicon Ultra-0.5 devices contain trace amounts of the
following:
■■ Amicon Ultra 3K devices: glycerine
■■ Amicon Ultra 10K device: triethylene glycol (TEG)
■■ Amicon Ultra 30K devices: glycerine
■■ Amicon Ultra 50K devices: glycerine
■■ Amicon Ultra 100K devices: glycerine
If these materials interfere with analysis, pre-rinse the device with buffer or Milli-Q®
water. If interference continues, rinse with 0.1 N NaOH followed by a second spin of buffer
or Milli-Q water.
caution: Do not allow the membrane in Amicon Ultra filter devices to dry out once wet.
If you are not using the device immediately after pre-rinsing, leave fluid on the
membrane until the device is used.

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 Amicon Ultra-0.5 Centrifugal Filter Devices 7

Chemical Compatibility
Amicon Ultra centrifugal devices are intended for use with biological fluids and aqueous
solutions. Before use, check the sample for chemical compatibility with the device.
Table 1. Chemical Compatibility of Amicon Ultra Filter Devices
Acids Concentration Concentration
Acetic acid ≤ 50%* Phosphoric acid ≤ 30%
Formic acid ≤ 5%* Sulfamic acid ≤ 3%
Hydrochloric acid ≤ 1.0 M Sulfuric acid ≤ 3%
Lactic acid ≤ 50% Trichloroacetic acid (TCA) ≤ 10%*
Nitric acid ≤ 10% Trifluoroacetic acid (TFA) ≤ 30%*
Alkalis
Ammonium hydroxide ≤ 10% Sodium hydroxide ≤ 0.5 M
Alcohols
n-Butanol ≤ 70% Isopropanol ≤ 70%
Ethanol ≤ 70% Methanol ≤ 60%
Detergents
Alconox® detergent ≤ 1% Lubrol® PX detergent ≤ 0.1%
CHAPS detergent ≤ 0.1% Nonidet™ P-40 surfactant ≤ 2%
* Contact with this chemical may cause materials to leach out of the component parts. Solvent blanks are
recommended to determine whether leachables represent potential assay interferences.

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 8 www.millipore.com

Chemical Compatibility, continued

Detergents, continued Concentration Concentration
Sodium deoxycholate ≤ 5% Triton® X-100 surfactant ≤ 0.1%
Sodium dodecyl sulfate (SDS) ≤ 0.1% Tween® 20 surfactant ≤ 0.1%
Terg-A-Zyme® detergent ≤ 1%
Organic solvents
Acetone not recommended Ethyl acetate not recommended
Acetonitrile ≤ 20% Formaldehyde ≤ 5%
Benzene not recommended Pyridine not recommended
Carbon tetrachloride not recommended Tetrahydrofuran not recommended
Chloroform not recommended Toluene not recommended
Dimethyl sulfoxide (DMSO) ≤ 5%*
Miscellaneous
Ammonium sulfate Saturated Phenol ≤ 1%
Diethyl pyrocarbonate ≤ 0.2% Phosphate buffer (pH 8.2) ≤1M
Dithiothreitol (DTT) ≤ 0.1 M Polyethylene glycol ≤ 10%
Glycerine ≤ 70% Sodium carbonate ≤ 20%
Guanidine HCl ≤6M Tris buffer (pH 8.2) ≤1M
Imidazole ≤ 100 mM Urea ≤8M
Mercaptoethanol ≤ 0.1 M
* Contact with this chemical may cause materials to leach out of the component parts. Solvent blanks are
recommended to determine whether leachables represent potential assay interferences.

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 Amicon Ultra-0.5 Centrifugal Filter Devices 9

How to Use Amicon Ultra-0.5 Centrifugal Filter Devices

1. Insert the Amicon Ultra-0.5 device into one of the provided microcentrifuge tubes.
2. Add up to 500 µL of sample to the Amicon Ultra filter device and cap it.
3. Place capped filter device into the centrifuge rotor, aligning the cap strap toward the
center of the rotor; counterbalance with a similar device.
4. Spin the device at 14,000 × g for approximately 10–30 minutes depending on the
NMWL of the device used. Refer to Figure 1 and table 3 for typical spin times.

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 10 www.millipore.com

How to Use Amicon Ultra-0.5 Filter Devices, continued

5. Remove the assembled device from the centrifuge and separate the Amicon Ultra filter
device from the microcentrifuge tube.
6. To recover the concentrated solute, place the Amicon Ultra filter device upside down
in a clean micro centrifuge tube. Place in centrifuge, aligning open cap towards the
center of the rotor; counterbalance with a similar device. Spin for 2 minutes at
1,000 × g to transfer the concentrated sample from the device to the tube. The
ultrafiltrate can be stored in the centrifuge tube.
NOTE: For optimal recovery, perform the reverse spin immediately.

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Filtrate Concentrate

Separate device Turn device upside

from tube down in clean tube Spin at 1,000 × g

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 Amicon Ultra-0.5 Centrifugal Filter Devices 11

How to Quantitate Recoveries

Calculate total recovery, percent concentrate, and percent filtrate using the method below.
The procedure provides a close approximation of recoveries for solutions having
concentrations up to roughly 20 mg/mL.
note: Appropriate assay techniques include absorption spectrophotometry, refractive
index, and conductivity.

Direct Weighing Procedure

The density of most dilute proteins is nearly equal to the density of water (i.e., 1 g/mL).
Using this property, the concentrate and filtrate recoveries can be quantitated by weighing
them and converting the units from grams to milliliters. This technique is valid only for
solutions with concentrations of approximately 20 mg/mL or less.
1. Separately weigh the empty filter device, filtrate collection tube, and concentrate
collection tube before use.
2. Fill filter device with solution and reweigh.
3. Assemble device in filtrate collection tube and centrifuge per instructions.

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 12 www.millipore.com

Direct Weighing Procedure, continued

4. Collect the concentrate by reverse spin into the pre-weighed concentrate collection
tube.
5. Remove the device from the concentrate collection tube and weigh the filtrate and
concentrate collection tubes.
6. Subtract weight of empty device/tubes to calculate weights of starting material, filtrate,
and concentrate.
7. Assay the filtrate and concentrate to determine solute concentration.
8. Calculate recoveries using the weight/volume data and the measured concentrations
as follows:
W c × Cc
% concentrate recovery = 100 ×
Wo × Co

Wf × Cf
% filtrate recovery = 100 ×
Wo × Co

% total recovery = % concentrate recovery + % filtrate recovery

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 Amicon Ultra-0.5 Centrifugal Filter Devices 13

Direct Weighing Procedure, continued

Wc= total weight of concentrate before assay

Wo= weight of original starting material
Wf = weight of filtrate
Cc = concentrate concentration
Co = original starting material concentration
Cf = filtrate concentration

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 14 www.millipore.com

Performance - DNA Concentration

The Amicon Ultra-0.5 30K device provides the best balance between recovery and spin
time for double-stranded DNA for base pairs ranging from 137 to 1159. To achieve
maximum PCR product recovery and primer removal with primers greater than 20 bases,
one or two additional spins with Tris-EDTA (TE ) buffer are recommended.
Table 2. Typical recovery of nucleotides from Amicon Ultra-0.5 30K device
PCR Product PCR Primer PCR Recovery PCR Primer TE Washes
(base pairs) (bases) (%) Removal (%) (number)
137 10 ≥ 95 ≥ 90 0
20 ≥ 90 ≥ 85 1
48 ≥ 90 ≥ 75 2
301 10 ≥ 90 ≥ 90 0
20 ≥ 85 ≥ 90 1
48 ≥ 90 ≥ 80 2
648 10 ≥ 95 ≥ 90 0
20 ≥ 90 ≥ 90 1
48 ≥ 95 ≥ 90 2
1159 10 ≥ 90 ≥ 90 0
20 ≥ 90 ≥ 95 1
48 ≥ 95 ≥ 95 2
Spin conditions: 40° fixed angle rotor, 14,000 × g, room temperature, 500 μL starting volume,
20–30 μL final volume, 10 minute spin, n=12.

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 Amicon Ultra-0.5 Centrifugal Filter Devices 15

Performance - Protein Concentration

Flow Rate
Factors affecting flow rate include sample concentration, starting vol­ume, chemical nature of
solute, relative centrifugal force, centrifuge rotor angle, membrane type, and temperature.
Figure 1 and Table 3 can be used to estimate the time required to achieve a given volume of
filtrate or concentrate for a variety of protein markers. A typical spin time for a 500 µL sample in
a fixed angle rotor is approximately 10–30 minutes (depending on device nominal
molecular weight limit). While most of the sample is filtered in the first 5 to 10 minutes of
centrifugation, the lowest concentrate volume (15–20 μL) is reached after spinning for
10–30 minutes.

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 16 www.millipore.com

Flow rate, continued

Figure 1. Typical Filtrate Volume vs. Spin Time
500

400
Filtrate Volume (µL)

300 3K
10K
200 30K
50K
100K
100

0
0 5 10 15 20 25 30 35
Spin Time (min)
Spin conditions: 40° fixed angle rotor, 14,000 × g, room temperature, 500 μL starting volume.
Protein markers used: Cytochrome c for 3K and 10K, BSA for 30K and 50K, and IgG for 100K, n=8.

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 Amicon Ultra-0.5 Centrifugal Filter Devices 17

Flow rate, continued

Table 3. Typical Concentrate Volume / Concentration Factor vs. Spin Time
Concentrate volume / Concentration Factor
3K device 10K device 30K device 50K device 100K device
Conc. Conc. Conc. Conc. Conc. Conc. Conc. Conc. Conc. Conc.
Spin time Volume Factor Volume Factor Volume Factor Volume Factor Volume Factor
(min) (μL) (x) (μL) (x) (μL) (x) (μL) (x) (μL) (x)
5 215 2 66 7 42 12 28 18 58 9
10 114 4 35 14 23 22 20 25 19 26
15 80 6 22 22 19 27 17 30 15 33
20 62 8 20 24 17 30 15 33 13 36
30 48 10 15 31 15 32 15 36 11 41

Spin conditions: 40° fixed angle rotor, 14,000 × g, room temperature, 500 μL starting volume. Protein
markers used: Cytochrome c for 3K and 10K, BSA for 30K and 50K, and IgG for 100K, n=12. Shaded
volumes were used for the calculation of protein recovery in Table 4.

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 18 www.millipore.com

Protein Retention and Concentrate Recovery

The membranes used in Amicon Ultra devices are characterized by a nominal molecular
weight limit (NMWL); that is, their ability to retain molecules above a specified molecular
weight. Solutes with molecular weights close to the NMWL may be only partially retained.
Membrane retention depends on the solute’s molecular size and shape. For most
applications, molecular weight is a convenient parameter to use in assessing retention
characteristics. Millipore recommends using a membrane with a NMWL at least two times
smaller than the molecular weight of the protein solute that one intends to concentrate.
Refer to Table 4 for “Typical Retention of Protein Markers”.

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 Amicon Ultra-0.5 Centrifugal Filter Devices 19

Protein Retention and Concentrate Recovery, continued

Table 4: Typical Retention of Protein Markers
Molecular Device Spin Time
Marker/Concentration Weight NMWL % Retention (min)
α-Chymotrypsinogen (1 mg/mL) 25,000 3K > 95 30
Cytochrome c (0.25 mg/mL) 12,400 > 95 30
Vitamin B-12 (0.2 mg/mL) 1,350 < 42 30
α-Chymotrypsinogen (1 mg/mL) 25,000 10K > 95 15
Cytochrome c (0.25 mg/mL) 12,400 > 95 15
Vitamin B-12 (0.2 mg/mL) 1,350 < 42 15
BSA (1 mg/mL) 67,000 30K > 95 10
Ovalbumin (1 mg/mL) 45,000 > 95 10
Cytochrome c (0.25 mg/mL) 12,400 < 35 10
BSA (1 mg/mL) 67,000 50K > 95 10
Ovalbumin (1 mg/mL) 45,000 ~ 40 10
Cytochrome c (0.25 mg/mL) 12,400 < 20 10
Thyroglobulin (0.5 mg/mL) 677,000 100K > 95 10
IgG (1 mg/mL) 156,000 > 95 10
Ovalbumin (1 mg/mL) 45,000 < 30 10
Spin Conditions: 40° fixed angle rotor, 14,000 × g, room temperature, 500 μL starting volume, n=12.

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 20 www.millipore.com

Protein Retention and Concentrate Recovery, continued

Factors that determine sample recovery include the nature of the protein solute relative to
the device NMWL chosen, starting concentration, and concentration factor. Table 5 provides
typical recoveries for Amicon Ultra-0.5 devices.
Table 5. Typical Concentrate Recovery
Marker/ Molecular Device Spin Time Concentrate Concentration Concentrate
Concentration Weight NMWL (min) Volume (µL) Factor (x) Recovery (%)
Cytochrome c
12,400 3K 30 48 10 98
(0.25 mg/mL)
Cytochrome c
12,400 10K 15 22 22 93
(0.25 mg/mL)
BSA (1 mg/mL) 67,000 30K 10 23 22 97

BSA (1 mg/mL) 67,000 50K 10 20 25 92

IgG (1 mg/mL) 156,000 100K 10 19 26 92

Spin Conditions: 40° fixed angle rotor, 14,000 × g, room temperature, 500 µL starting volume, n=12.
The shaded volumes were taken from Table 3.

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 Amicon Ultra-0.5 Centrifugal Filter Devices 21

Maximizing Sample Recovery

Low sample recovery in the concentrate may be due to adsorptive losses, over-
concentration, or passage of sample through the membrane.
■■ Adsorptive losses depend upon solute concentration, its hydrophobic nature, tempera-
ture and time of contact with filter device surfaces, sample composition, and pH. To
minimize losses, remove concentrated samples immediately after centrifugal spin.
■■ If starting sample concentration is high, monitor the centrifugation process in order to
avoid over-concentration of the sample. Over-concentration can lead to precipitation
and potential sample loss.
■■ If the sample appears to be passing through the membrane, choose a lower NMWL
Amicon Ultra-0.5 device.

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 22 www.millipore.com

Desalting or Diafiltration
Desalting, buffer exchange, or diafiltration are important methods for removing salts or
solvents in solutions containing biomolecules. The removal of salts or the exchange of
buffers can be accomplished in the Amicon Ultra-0.5 device by concentrating the sample,
then reconstituting the concentrate to the original sample volume with any ­desired solvent.
The process of “washing out” can be repeated until the concentration of the contaminating
microsolute has been sufficiently reduced. See example below.

Add 450 µL of
10 mM NaCl or
exchange buffer

Spin to Spin to
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50 µL of 500 µL of � ��
50 µL of
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��
protein in �� protein in �� protein in �� protein in
100 mM NaCl 100 mM NaCl 10.9 mM NaCl 10.9 mM NaCl

100 mM 10.9 mM
NaCl NaCl

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 Amicon Ultra-0.5 Centrifugal Filter Devices 23

Centrifugal Product Ordering Information

Initial Final
volume concentrate Qty / 3,000 10,000 30,000 50,000 100,000
(mL) volume (µL) Product pk NMWL NMWL NMWL NMWL NMWL
Amicon 8 UFC500308 UFC501008 UFC503008 UFC505008 UFC510008
Ultra-0.5 24 UFC500324 UFC501024 UFC503024 UFC505024 UFC510024
0.5 20
device 96 UFC500396 UFC501096 UFC503096 UFC505096 UFC510096
500 UFC5003BK UFC5010BK UFC5030BK UFC5050BK UFC5100BK
Amicon Ultra-0.5 Collection Tubes 96 UFC50VL96
Amicon 8 UFC800308 UFC801008* UFC803008 UFC805008 UFC810008
4 30–70 Ultra-4 24 UFC800324 UFC801024* UFC803024 UFC805024 UFC810024
device 96 UFC800396 UFC801096* UFC803096 UFC805096 UFC810096
Amicon 8 UFC900308 UFC901008* UFC903008 UFC905008 UFC910008
15 150–300 Ultra-15 24 UFC900324 UFC901024* UFC903024 UFC905024 UFC910024
device 96 UFC900396 UFC901096* UFC903096 UFC905096 UFC910096
* Amicon Ultra-4 and -15 10,000 NMWL devices are for in vitro
Related Products diagnostic use. All other devices are for research use only.
Centricon®
70 350 Plus-70 8 UFC700508 UFC701008 UFC703008 N/A UFC710008
device
MultiScreen®
96 well plate/
0.5 20 10 N/A MAUF01010 N/A N/A N/A
Ultracel-10
membrane

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 24 www.millipore.com

Technical Assistance
For more information, contact the Millipore office nearest you. In the U.S., call
1-800-MILLIPORE (1-800-645-5476). Outside the U.S., see your Millipore catalogue for
the phone number of the office nearest you or go to our web site at
www.millipore.com/offices for up-to-date worldwide contact information. You can also visit
the tech service page on our web site at www.millipore.com/techservice.

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 Amicon Ultra-0.5 Centrifugal Filter Devices 25

Standard Warranty
Millipore Corporation (“Millipore”) warrants its products will meet their applicable published
specifications when used in accordance with their applicable instructions for a period of
one year from shipment of the products. MILLIPORE MAKES NO OTHER WARRANTY,
EXPRESSED OR IMPLIED. THERE IS NO WARRANTY OF MERCHANTABILITY OR
FITNESS FOR A PARTICULAR PURPOSE. The warranty provided herein and the data,
specifications, and descriptions of Millipore products appearing in Millipore’s published
catalogues and product literature 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 are not 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
or replace, at its option, the applicable product or part thereof, provided the customer notifies
Millipore promptly of any such breach. If after exercising reasonable efforts, Millipore is
unable to repair or replace the product or part, then Millipore shall refund to the customer all
monies paid for such applicable product or part. MILLIPORE SHALL NOT BE LIABLE FOR
CONSEQUENTIAL, INCIDENTAL, SPECIAL, OR ANY OTHER INDIRECT DAMAGES
RESULTING FROM ECONOMIC LOSS OR PROPERTY DAMAGE SUSTAINED BY ANY
CUSTOMER FROM THE USE OF ITS PRODUCTS.

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