1 Amicon Ultra-4 Centrifugal Filter Devices

English
User Guide
Amicon

Ultra-4
Centrifugal Filter Devices
for volumes up to 4 mL
Amicon Ultra-4 10K device for in vitro diagnostic use
Amicon Ultra-4 3K, 30K, 50K, and 100K devices for
research use only; not for use in diagnostic procedures
Introduction
Amicon Ultra-4 centrifugal lter devices provide fast ultraltration, 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 80-fold concentration.
Typical processing time is 10 to 40 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 lter device prevents spinning to dryness and potential sample loss. The concentrate is
collected from the lter device sample reservoir using a pipettor, while the ultraltrate is collected in the
provided centrifuge tube. The device can be spun in a swinging bucket (for optimal performance) or xed
angle rotor. Amicon Ultra-4 devices are supplied non-sterile and are for single use only.
The Amicon Ultra-4 product line includes 5 different cutoffs (Nominal Molecular Weight Limit, NMWL, or
Molecular Weight Cutoff, MWCO):
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
Amicon Ultra-4 10K lter devices are for in vitro diagnostic use and can be used to
concentrate serum, urine, cerebrospinal uid, and other body uids prior to analysis.
Amicon Ultra-4 3K, 30K, 50K, and 100K lter devices are for research use only and not for use in
diagnostic procedures.
2 Amicon Ultra-4 Centrifugal Filter Devices
Materials Supplied
The Amicon Ultra-4 device is supplied with a cap, a lter device, and a centrifuge tube.
Filter device
Centrifuge tube
Cap
30,000
MWCO
ul
500
250
100
50
4
3
2
Applications
Concentration of biological samples containing antigens, antibodies, enzymes, nucleic acids (DNA/RNA
samples, either single- or double-stranded), microorganisms, column eluates, and puried samples
Purication 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 dialtration
Required Equipment
Centrifuge with swinging bucket (preferred) or xed angle rotor with wells/carriers that can
accommodate 17 mm 124 mm 15 mL conical-bottomed tubes
CAUTION: To avoid damage to the device during centrifugation, check clearance before spinning.
Pipettor with 200 microliter (L) tip for concentrate recovery
Suitability
Preliminary recovery and retention studies are suggested to ensure suitability for intended use. See the
How to Quantify Recoveries section.
Device Storage and Shelf Life
Store at 1530 C. Shelf life is 3 years from date of manufacture. For cat. nos. UFC801008, UFC801024, and
UFC801096, refer to expiration date on package label.
3 Amicon Ultra-4 Centrifugal Filter Devices
Prerinsing
The ultraltration membranes in Amicon Ultra-4 devices contain trace amounts of glycerine. If this
material interferes with analysis, prerinse 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 lter devices to dry out once wet. If you are not
using the device immediately after prerinsing, leave uid on the membrane until the device is
used.
How to Use Amicon Ultra-4 Centrifugal Filter Devices
1. Add up to 4 mL of sample (3.5 mL if using a 23 xed angle rotor) to the Amicon Ultra lter device.
2. Place capped lter device into centrifuge rotor (swinging bucket preferred); counterbalance with a
similar device.
3. When using a swinging bucket rotor, spin the device at 4,000 g maximum for approximately
1040 minutes.
When using a xed angle rotor, orient the device with the membrane panel facing up.
For Amicon Ultra 3K, 10K, 30K, and 50K devices, spin at 7,500 g maximum for approximately
1040 minutes.
For Amicon Ultra 100K devices, spin at 5,000 g maximum for approximately 1020 minutes.
NOTE: Refer to Figures 1 and 2, and Tables 2 and 3 for typical spin times.
4. To recover the concentrated solute, insert a pipettor into the bottom of the lter device and withdraw
the sample using a side-to-side sweeping motion to ensure total recovery. The ultraltrate can be
stored in the centrifuge tube.
NOTE: For optimal recovery, remove concentrated sample immediately after centrifugation.
Desalting or Dialtration
Desalting, buffer exchange, or dialtration 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-4 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 sufciently reduced. See example below.
30,000
MWCO
4
3
2
ul
500
250
100
50
30,000
MWCO
4
3
2
ul
500
250
100
50
Spin to
concentrate
Spin to
concentrate
4 mL of
1 mg/mL
protein in
100 mM NaCl
30,000
MWCO
ul
500
250
100
50
4
3
2
30,000
MWCO
ul
500
250
100
50
4
3
2
50 L of
80 mg/mL
protein in
100 mM NaCl
100 mM
NaCl
50 L of
80 mg/mL
protein in
11.1 mM NaCl
11.1 mM
NaCl
Add 3.95 mL of
10 mM NaCl
or exchange
buffer
4 mL of
1 mg/mL
protein in
11.1 mM NaCl
4 Amicon Ultra-4 Centrifugal Filter Devices
Performance - Protein Concentration
Flow Rate
Factors affecting ow rate include sample concentration, starting vol ume, chemical nature of solute,
relative centrifugal force, centrifuge rotor angle, membrane type, and temperature. Figures 1 and 2,
and Tables 2 and 3 can be used to estimate the time required to achieve a given volume of ltrate or
concentrate for a variety of protein markers. A typical spin time for a 4 mL sample is approximately 10 to
40minutes (depending on device nominal molecular weight limit). While most of the sample is ltered in
the rst 10 to 20 minutes of centrifugation, the lowest concentrate volume (3075 L) is reached after
spinning for 20 to 40 minutes.
Performance - DNA Concentration
Merck Millipore Ltd. has determined that the Amicon Ultra-4 30K device provides the best balance
between recovery and spin time for double-stranded DNA for base pairs ranging from 137 to 1,159.
Table 1. Typical Recovery of Nucleotides from Amicon Ultra-4 30K Device
Double-stranded
DNA Base Pair Size Spin Time (min)
Concentrate
Volume (L) Recovery (%)
1371,159 10 5070 > 85
Spin conditions: Fixed angle rotor, 5,000 g, room temperature, 2 mL starting volume.
Figure 1. Typical Filtrate Volume vs. Spin Time (Swinging bucket rotor)
Spin conditions: 4,000 g, room temperature, 4 mL starting volume.
Protein markers used: Cytochrome c for 3K and 10K, BSA for 30K and 50K, and IgG for 100K, n=6.
Spin time (min)
F
i
l
t
r
a
t
e

V
o
l
u
m
e

(
m
L
)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0 5 10 15 20 25 30 35 40
3K
10K
30K
50K
100K
5 Amicon Ultra-4 Centrifugal Filter Devices
Flowrate, continued
F
i
l
t
r
a
t
e

V
o
l
u
m
e

(
m
L
)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0 5 10 15 20 25 30 35 40
3K
10K
30K
50K
100K
Spin time (min)
Figure 2. Typical Filtrate Volume vs. Spin Time (Fixed angle rotor)
Spin conditions: 7,500 g for 3K, 10K, 30K, and 50K, 5,000 g for 100K, room temperature, 4 mL starting
volume. Protein markers used: Cytochrome c for 3K and 10K, BSA for 30K and 50K, and IgG for 100K, n=6.
Table 2. Typical Concentrate Volume vs. Spin Time (Swinging bucket rotor)
Concentrate volume (L)
Spin time (min) 3K device 10K device 30K device 50K device 100K device
10 1,369 176 73 32 264
15 76 46 36
20 478 58 37 30 33
25 228
30 159
40 94
Spin conditions: 4,000 g, room temperature, 4 mL starting volume.
Protein markers used: Cytochromec for 3K and 10K, BSA for 30K and 50K, and IgG for 100K, n=6 (mean
value of 3 membrane lots). Shaded volumes were used for the calculation of protein recovery in Table 5.
Table 3. Typical Concentrate Volume vs. Spin Time (35 Fixed angle rotor)
Concentrate volume (L)
Spin time (min) 3K device 10K device 30K device 50K device 100K device
10 613 97 42 23 53
15 54 30 30
20 170 35 22 15 26
25 118
30 92
40 62
Spin conditions: 7,500 g for 3K, 10K, 30K, and 50K, 5,000 g for 100K, room temperature, 4 mL starting
volume. Protein markers used: Cytochromec for 3K and 10K, BSA for 30K and 50K, and IgG for 100K, n=6
(mean value of 3 membrane lots). Shaded volumes were used for the calculation of protein recovery in Table 5.
6 Amicon Ultra-4 Centrifugal Filter Devices
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 specied molecular weight. Solutes with
molecular weights close to the NMWL may be only partially retained. Membrane retention depends on
the solutes molecular size and shape. For most applications, molecular weight is a convenient parameter
to use in assessing retention characteristics. Merck Millipore Ltd. 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.
Table 4. Typical Retention of Protein Markers
Marker/Concentration
Molecular
Weight
Device
NMWL
% Retention
Swinging Bucket
% Retention
Fixed Angle
Spin Time
(min)
-Chymotrypsinogen (1 mg/mL) 25,000 3K > 95 > 95 40
Cytochrome c (0.25 mg/mL) 12,400 > 95 > 95 40
Vitamin B-12 (0.2 mg/mL) 1,350 < 35 < 35 40
-Chymotrypsinogen (1 mg/mL) 25,000 10K > 95 > 95 15
Cytochrome c (0.25 mg/mL) 12,400 > 95 > 95 15
Vitamin B-12 (0.2 mg/mL) 1,350 < 15 < 15 15
BSA (1 mg/mL) 67,000 30K > 95 > 95 10
Ovalbumin (1 mg/mL) 45,000 > 90 > 90 10
Cytochrome c (0.25 mg/mL) 12,400 < 20 < 20 10
Vitamin B-12 (0.2 mg/mL) 1,350 < 10 < 10 10
BSA (1 mg/mL) 67,000 50K > 95 > 95 10 (SB), 5 (FA)
Ovalbumin (1 mg/mL) 45,000 ~ 60 ~ 65 10 (SB), 5 (FA)
Cytochrome c (0.25 mg/mL) 12,400 < 10 < 10 10 (SB), 5 (FA)
Thyroglobulin (0.5 mg/mL) 677,000 100K > 95 > 95 15
IgG (1 mg/mL) 156,000 > 90 > 90 15
Ovalbumin (1 mg/mL) 45,000 < 25 < 20 15
Spin Conditions: Swinging bucket (SB) rotor, 4,000 g, or 35 xed angle (FA) rotor, 7,500 g for 3K, 10K, 30K,
and 50K, 5,000 g for 100K, 4 mL starting volume, room temperature, n=6 (mean value of 3 membrane lots). The
shaded volumes were taken from Tables 2 and 3.
7 Amicon Ultra-4 Centrifugal Filter Devices
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, temperature and time
of contact with lter 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-4
device.
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-4 devices.
Table 5. Typical Concentrate Recovery
Concentrate
Volume (L)
Concentration
Factor (x)
Concentrate
Recovery (%)
Marker/
Concentration
Device
NMWL
Spin Time
(min)
Swinging
Bucket
Fixed
Angle
Swinging
Bucket
Fixed
Angle
Swinging
Bucket
Fixed
Angle
Cytochrome c
(0.25 mg/mL)
3K 40 94 62 43.5 65.0 98.2 96.7
Cytochrome c
(0.25 mg/mL)
10K 15 76 54 52.3 76.6 97.3 98.5
BSA (1 mg/mL) 30K 10 73 42 56.1 98.6 95.8 95.0
BSA (1 mg/mL) 50K 10 32 23 137.0 177.4 98.8 92.8
IgG (1 mg/mL) 100K
15 (SB),
10 (FA)
36 53 115.9 56.8 92.2 91.3
Spin Conditions: Swinging bucket (SB) rotor, 4,000 g, or 35 xed angle (FA) rotor, 7,500 g for 3K, 10K, 30K,
and 50K, 5,000 g for 100K, 4 mL starting volume, room temperature, n=6 (mean value of 3 membrane lots). The
shaded volumes were taken from Tables 2 and 3.
Protein Retention and Concentrate Recovery, continued
8 Amicon Ultra-4 Centrifugal Filter Devices
How to Quantify Recoveries
Calculate total recovery, percent concentrate recovery, and percent ltrate recovery 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, radioimmunoassay, 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 ltrate volumes can be quantied 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. Before use, separately weigh the empty lter device, the centrifuge tube, and an empty tube for
concentrate collection.
2. Fill lter device with solution and reweigh.
3. Assemble device and centrifuge per instructions.
4. Collect the concentrate with a pipettor and dispense it into the preweighed concentrate collection
tube.
5. Remove the device from the centrifuge tube and weigh the centrifuge tube and concentrate collection
tube.
6. Subtract weight of empty device/tubes to calculate weights of starting material, ltrate, and
concentrate.
7. Assay the starting material, ltrate, and concentrate to determine solute concentration.
8. Calculate recoveries using the weight/volume data and the measured concentrations as follows:
% concentrate recovery=100
% ltrate recovery=100
% total recovery=% concentrate recovery + % ltrate recovery
W
c
= total weight of concentrate before assay
W
o
= weight of original starting material
W
f
= weight of ltrate
C
c
= concentrate concentration
C
o
= original starting material concentration
C
f
= ltrate concentration
W
c
C
c
W
o
C
o
W
f
C
f
W
o
C
o
9 Amicon Ultra-4 Centrifugal Filter Devices
Specications
Maximum initial sample volume
Swinging bucket and
xed angle rotors (45 and 35)
4.0 mL
Fixed angle rotor (23) 3.5 mL
Typical nal concentrate volume 50100 L
Maximum relative centrifugal force
Swinging bucket rotor 4,000 g
Fixed angle rotor 7,500 g for 3K, 10K, 30K, and 50K NMWL
5,000 x g for 100K NMWL
Active membrane area 3 cm
2
Dimensions
Filter device in tube (capped)
Length: 124 mm (4.9 in.) Diameter: 17.3 mm (0.7 in.)
Filter device
Length: 73.4 mm (2.9 in.) Diameter: 17.2 mm (0.7 in.)
Materials of Construction
Filter device Copolymer styrene/butadiene
Membrane Ultracel low binding regenerated cellulose
Filtrate tube Polypropylene
Filtrate cap and liner Polyethylene
10 Amicon Ultra-4 Centrifugal Filter Devices
Chemical Compatibility
Amicon Ultra centrifugal devices are intended for use with biological uids and aqueous solutions. Before
use, check the sample for chemical compatibility with the device.
Table 5. 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% Triuoroacetic 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% Sodium dodecyl sulfate (SDS) 0.1%
CHAPS detergent 0.1% Tergazyme

detergent 1%
Lubrol PX detergent 0.1% Triton X-100 surfactant 0.1%
Nonidet P-40 surfactant 2% Tween 20 surfactant 0.1%
Sodium deoxycholate 5%
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) 1 M
Dithiothreitol (DTT) 0.1 M Polyethylene glycol 10%
Glycerine 70% Sodium carbonate 20%
Guanidine HCl 6 M Tris buffer (pH 8.2) 1 M
Imidazole 100 mM Urea 8 M
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.
11 Amicon Ultra-4 Centrifugal Filter Devices
Product Ordering Information
This section lists the catalogue numbers for Amicon Ultra Ultraltration Devices. See the Technical
Assistance section for contact information. You can purchase these products on-line at
www.millipore.com/products.
Initial
volume
(mL)
Final
concentrate
volume (L) Product
Qty /
pk 3K 10K 30K 50K 100K
0.5 1520 Amicon
Ultra-0.5
device
8
24
96
500
UFC500308
UFC500324
UFC500396
UFC5003BK
UFC501008
UFC501024
UFC501096
UFC5010BK
UFC503008
UFC503024
UFC503096
UFC5030BK
UFC505008
UFC505024
UFC505096
UFC5050BK
UFC510008
UFC510024
UFC510096
UFC5100BK
Amicon Ultra-0.5 Collection Tubes 96 UFC50VL96
2 1570 Amicon
Ultra-2
pre-launch
device
24 UFC200324PL UFC201024PL UFC203024PL UFC205024PL UFC210024PL
4 50100 Amicon
Ultra-4
device
8
24
96
UFC800308
UFC800324
UFC800396
UFC801008*
UFC801024*
UFC801096*
UFC803008
UFC803024
UFC803096
UFC805008
UFC805024
UFC805096
UFC810008
UFC810024
UFC810096
15 150300 Amicon
Ultra-15
device
8
24
96
UFC900308
UFC900324
UFC900396
UFC901008*
UFC901024*
UFC901096*
UFC903008
UFC903024
UFC903096
UFC905008
UFC905024
UFC905096
UFC910008
UFC910024
UFC910096
* Amicon Ultra-4 and -15 10K devices are for in vitro
diagnostic use. All other devices are for research use only.
In Vitro Diagnostic Product Labeling
The following table denes the symbols found on Amicon Ultra-4 10K device labels.
Symbol Denition Symbol Denition
V
In vitro diagnostic medical device
N
Date of manufacture
h
Catalogue number
M
Manufacturer
D
Do not reuse
l
Temperature limitation
H
Use by
d
Non-sterile product
g
Batch code
C
CE conformity marking
12 Amicon Ultra-4 Centrifugal Filter Devices
Notice
The information in this document is subject to change without notice and should not be construed as a
commitment by Merck Millipore Ltd. (Millipore) or an afliate. Neither Merck Millipore Ltd. nor any of its
afliates assumes responsibility for any errors that may appear in this document.
Technical Assistance
For more information, contact the ofce nearest you. Up-to-date world-wide contact information is
available on our web site at www.millipore.com/ofces. You can also visit the tech service page on our web
site at www.millipore.com/techservice.
Standard Warranty
The applicable warranty for the products listed in this publication may be found at
www.millipore.com/terms (within the Terms and Conditions of Sale applicable to your purchase
transaction).
Made in Ireland
Merck Millipore Ltd.
Tullagreen,
Carrigtwohill,
Co. Cork, IRL
The M logo is a trademark of Merck KGaA, Darmstadt, Germany.
Millipore, Amicon, Milli-Q, and Ultracel are registered trademarks of Merck KGaA.
Alconox and Tergazyme 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 Corp.
Tween is a registered trademark of ICI Americas Inc.
2012 EMD Millipore Corporation. All rights reserved.
PR03521TR, Rev. A, English, 03/12
Contents
Introduction 1
Applications 2
Materials Supplied 2
Required Equipment 2
Suitability 2
Device Storage and Shelf Life 2
Prerinsing 3
How to Use Amicon Ultra-4 Centrifugal Filter Devices 3
Desalting or Dialtration 3
Performance - DNA Concentration 4
Performance - Protein Concentration 4
Maximizing Sample Recovery 7
How to Quantify Recoveries 8
Specications 9
Chemical Compatibility 10
In Vitro Diagnostic Product Labeling 11
Product Ordering Information 11
Notice 12
Technical Assistance 12
Standard Warranty 12