Concentration, Desalting, and Buffer Exchange with Amicon® Ultra Centrifugal Filters

Amicon® Ultra centrifugal filters are ideal for removal or exchange of salts, sugars, nucleotides, and non-aqueous solvents, as well as other materials of low molecular weight. They also serve to separate free and bound species. One of the most common applications for Amicon® devices is concentration and desalting of column fractions during protein purification by various chromatography methods.

Use this protocol for:

  • Dilute or purified protein solutes, antigens, antibodies, enzymes, or microorganisms
  • Fast, convenient, high-recovery alternatives to dialysis and ethanol precipitation
  • Efficient salt removal independent of biomolecule concentration or size

Amicon® Ultra Method for Concentration, Desalting or Buffer Exchange

  1. Using the Amicon® Ultra Online Selector Tool, select the Amicon® Ultra device with the appropriate nominal molecular weight limit (NMWL) and volume capacity for your application.
  2. (Optional) Pre-rinse device with buffer of choice.
  3. Add the sample to the reservoir of the centrifugal device.
  4. If the sample is smaller than the maximum volume, it can be diluted up to the maximum volume before the first centrifugation step. This will help increase salt removal.
  5. Centrifuge at the specified g-force for the amount of time recommended in the user guide for the selected device [click to browse user guides].
  6. Remove the initial filtrate from the filtrate tube and set aside.
  7. Add enough buffer (for buffer exchange) or water (for desalting) to the device to bring the sample volume up to 4, 15, 2, or 0.5 mL, depending on the volume capacity of the device.
  8. Centrifuge again.
  9. Set aside the filtrate.
  10. Recover the concentrated, desalted, or buffer-exchanged sample.

NOTE: Both of the filtrates should be retained until the concentrated sample has been analyzed.

Principles

During the first centrifugation step, the concentration of macromolecule solute increases; however, filtration removes lower MW solutes, thereby maintaining the salt concentration at 500 mM. For the second spin, another volume of salt-free buffer or water is passed over the retentate, and salt is removed by filtration. This process of “diafiltration” can be repeated to achieve maximum salt removal, or complete buffer exchange.

Amicon® Ultra centrifugal devices allow >90% salt removal in the first centrifugation step. As seen in the table below, one additional centrifugation step generally increases the salt removal to 99% with >90% recovery of the sample.
 

  Cytochrome c
(0.25 mg/mL)
BSA 1 mg/mL BSA 1 mg/mL IgG 1 mg/mL
Device Amicon® Ultra 15 mL filter with 10 kDa NMWL Amicon® Ultra 15 mL filter with 30 kDa NMWL Amicon® Ultra 15 mL filter with 50 kDa NMWL Amicon® Ultra 15 mL filter with 100 kDa NMWL
Spin 1 2 1 2 1 2 1 2
% Protein Recovery 97.3 95.6 96.0 94.4 98.9 92.4 99.9 97.1
% NaCl Removal 97.9 99.9 98.2 99.9 97.1 99.9 97.7 99.5

Table: Removal of sodium chloride and recovery of protein with Amicon® Ultra-15 mL devices.

Three Amicon® Ultra-15 devices of each NMWL indicated were tested with 15 mL of each protein solution containing 500 mM initial concentration of NaCl. Each spin was performed at 4,000 x g for 30 minutes. After the first spin, the retentate was brought up to 15 mL with ultrapure water from a Milli-Q® water system. NaCl removal was calculated by assaying each sample for conductivity (µS/cm) using a standard conductivity meter (Innolab® Conductivity Meter). Protein recovery was calculated by measuring the final amount of protein in the retentate after each spin and comparing to the amount of protein loaded. Amount of protein in the retentate was calculated by measuring the absorbance of the retentate solution at 410 nm (for Cytochrome c) and at 280 nm (for BSA and IgG) and recovery calculated using the following equation:

Where ...

W= total weight of retentate before assay

W= weight of original starting material

C= concentration of protein in retentate

C= original starting material concentration

NOTE: Factors independent of the ultrafiltration device may contribute to low protein recovery. Two examples:

  • Desalting below a certain threshold for a given protein can result in protein precipitation and lower measured protein recovery.
  • Overconcentration of protein can result in lower measured protein recovery.

For single-spin buffer exchange of small samples and with simultaneous protein concentration, refer to the Amicon® Pro system protocol for continuous diafiltration and concentration.