Amicon
®
Ultra-4 Centrifugal Filter Devices
3
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. Figures 1 and 2, and Tables 2 and 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 4 mL sample is approximately 10 to 40 minutes (depending on device molecular weight cutoff).
While most of the sample is filtered in the first 10 to 20 minutes of centrifugation, the lowest concentrate volume
(30–75 μL) is reached after spinning for 20 to 40 minutes.
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.
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
Filtr
ate V
olume
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.
Filtr
ate V
olume
Spin time (min)
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
Table 2. Typical Concentrate Volume vs. Spin Time
(Swinging-bucket rotor)
Concentrate volume (μL)
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: Cytochrome c for 3K and 10K, BSA for 30K and 50K, and IgG for
100K, n=6 (mean value of 3 device 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 (μL)
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: Cytochrome c for 3K and
10K, BSA for 30K and 50K, and IgG for 100K, n=6 (mean value of 3 device lots).
Shaded volumes were used for the calculation of protein recovery in Table 5.
Protein Retention and Concentrate Recovery
The membranes used in Amicon
®
Ultra devices are characterized by a molecular weight cutoff (MWCO); that is, their
ability to retain molecules above a specified molecular weight. Solutes with molecular weights close to the MWCO
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. For best results,
use a membrane with a MWCO at least two times smaller than the molecular weight of the protein solute that one
intends to concentrate. Refer to Table 4.