Amicon® Ultra Centrifugal Filter Units are useful for a variety of purposes. They can be used to concentrate samples into smaller volumes, but can also be adopted to separate compounds based on their molecular weight, or even desalt samples. The filter units reside within 50 ml tubes that are compatible with most laboratory centrifuges and are fitted with the Millipore Ultracel® regenerated cellulose low binding membrane which facilitates sample separation. Devices with molecular cut offs of 5K, 10K, 30K, 50K, 100K Daltons are available, although my own experiences are with the 5K units.
To use the filter units, you first place your sample in the filter unit sample reservoir. The tube is then centrifuged and the liquid remaining (retentate) in the reservoir is composed primarily of molecules larger than the cut-off while molecules below the molecular cut-off pass through the filter. There is ample clearance between the bottom of the filter unit and the bottom of the tube to allow for ultrafiltrate collection. Successive washes of the sample with buffer will help to reduce contamination. Whilst these devices are compatible with a large range of solvents, it is best to check the Millipore online catalogue to determine the compatibility of your solvent with the filter.
The maximum loading capacity of samples is 15 ml per centrifugal device. Samples larger than this volume will therefore need to be split across several tubes. For sample volumes smaller than 4 ml, it may be more appropriate to concentrate your sample using the smaller, 15 ml tube version of this product (Amicon® Ultra-4 Centrifugal Filter Units), which has a maximum loading capacity of 4 ml.
Each column is a single use device. Once a particular sample has been used with a device, the device cannot be “washed” to be used with another separate sample. However, aliquots of the same sample may be passed through the column up to an additional 3 times or the initial sample washed with buffer to increase purity. Through this method, it is also possible to desalt samples or separate labeled larger molecules from unlabelled molecules in certain preparations. For example, radioisotope labels incorporated into proteins in cell culture-conditioned media can be separated from the unincorporated radioisotopes still present in the media.
A word of caution: In my hands, several of the filter units have not withstood repeated spins and fractured within the centrifuge, leading to sample loss. This problem only occurred with a particular batch of product, and I have since come to believe that it was a problem with that particular batch. As a precaution, never exceed the maximum centrifugation speed and never spin each tube with loaded sample more than what is recommended (usually 4 spins). It is best to seek the advice of a qualified Millipore technician regarding the suitability of this procedure with your particular sample and solvent. You should also exercise caution if your sample has a high debris content capable of blocking the membrane. Pelleting cell debris before passing your sample through the device may avoid any potential problems.
Users may find that precious samples are better concentrated using alternative concentration methods to maintain the highest possible yield, although the manufacturer claims that these devices maintain a high sample yield if used as recommended. In-house testing by Millipore (as indicated in the user guide supplied with the product) indicates that typical recovery is at best 97.94% with the 30K units, and 91.08%, 93.03%, 93.32%, 89.03% with the 5, 10, 50 and 100 units, respectively. Similarly, maintaining maximum yield when separating incorporated and unincorporated labels in biological samples may be more accurately achieved using conventional chromatographic techniques. However, these devices are a useful alternative when processing high numbers of samples as chromatographic techniques would normally be extremely time consuming. This is particularly true if you have access to a large centrifuge with buckets capable of spinning many 50 ml tubes at once.
PhD Student
School of Biomedical Sciences
University of Queensland