Dialysis Buffer Exchange Calculator (Depletion & Diafiltration)
Calculate small-molecule contaminant depletion (imidazole, urea, salts) across successive dialysis buffer exchanges or centrifugal ultrafiltration spin columns, with automatic MWCO membrane pore-size retention checks.
Dialysis Chamber Parameters
Change 1: 3–4 hours at 4°C with magnetic stirring. Change 2: 3–4 hours. Change 3: Overnight (12–16 hours).
Dialysis Depletion Results
| Buffer Change | Residual Conc | % Depleted |
|---|---|---|
| Change #1 | 2.488 mM | 99.50% |
| Change #2 | 0.012 mM | 100.00% |
| Change #3 | 6.16e-5 mM | 100.00% |
Dialysis Buffer Exchange Calculator Formulas & Equilibrium Physics
Removing salts (e.g. NaCl, ammonium sulfate), denaturants (urea, guanidine-HCl), and elution ligands (imidazole, glutathione) after affinity chromatography is essential to preserve biological activity and prepare samples for downstream assays, crystallization, or cryo-EM.
Equilibrium Dialysis
Solutes diffuse across a semipermeable regenerated cellulose membrane driven by concentration gradients. At equilibrium, remaining concentration is Cn = C0 · [Vs / (Vs + Vb)]^n. Multiple changes with fresh buffer drive clearance exponentially.
Centrifugal Diafiltration
Hydraulic pressure forces solvent and micro-solutes through an ultrafiltration polyethersulfone (PES) membrane, concentrating macromolecules in the retentate. Successive dilution and re-concentration clears contaminants rapidly in hours rather than days.
Dialysis Membrane MWCO Selection & Protein Retention Guidelines
The Molecular Weight Cut-Off (MWCO) designates the pore size where ≥ 90% of a spherical globular solute is retained.
MWCO Selection Heuristic
An MWCO at or below roughly one-third of the target protein molecular weight can be used as an initial screening heuristic (for example, consider 10 kDa for a 35–45 kDa protein). This does not predict a specific recovery or guarantee zero leakage; validate it for the membrane and sample.
Non-Globular Conformations
Denatured, unfolded, or extended fibrous proteins possess larger hydrodynamic radii than globular proteins and are retained more readily by larger pores.
Glycerol Pre-wetting
Dialysis membranes are stored in trace sodium azide or glycerol. Rinse tubing in deionized water for 15–30 minutes prior to loading protein to prevent contamination.
Dialysis Buffer Exchange Calculator Worked Lab Example: Imidazole Removal
Scenario: Desalting 5 mL His-Tag Eluate (500 mM Imidazole)
Dialyzing 5 mL sample (10 kDa MWCO) against 1.0 L fresh buffer at 4°C with 3 sequential buffer exchanges.
| Exchange Step | Buffer Vol | Dilution Factor | Residual Imidazole | Cumulative Clearance |
|---|---|---|---|---|
| Initial Stock | — | 1.0 | 500 mM | 0.00% |
| Change #1 (4 h) | 1.0 L | 1 / 201 (0.004975) | 2.49 mM | 99.50% |
| Change #2 (4 h) | 1.0 L | 1 / 201 (0.004975) | 12.4 µM | 99.9975% |
| Change #3 (Overnight) | 1.0 L | 1 / 201 (0.004975) | 0.062 µM (62 nM) | > 99.9999% |
832 µM (0.83 mM)
High residual imidazole risks protein precipitation
0.062 µM (62 nM)
13,400-fold higher purity using the exact same volume of buffer!
Frequently Asked Questions (FAQ)
Why is multiple smaller buffer changes more effective than a single large volume?
Dialysis is an exponential depletion process: Cn = C0 * [Vs / (Vs + Vb)]^n. Three sequential 1 L buffer changes deplete a 5 mL sample by ~8,000,000-fold, whereas a single 3 L change only dilutes it by 600-fold.
How should I choose the membrane MWCO relative to my protein size?
Follow the 3-fold rule: Choose an MWCO that is at least 3-fold smaller than your target protein's molecular weight (e.g. use a 10 kDa MWCO for a 30–50 kDa protein) to prevent protein leakage through pores.
How does temperature affect dialysis rate?
Diffusion coefficient is proportional to absolute temperature and inversely proportional to viscosity (Stokes-Einstein equation). Dialysis at 4°C is ~25% slower than at room temperature, requiring gentle magnetic stirring.
What causes severe protein precipitation during dialysis?
Sudden removal of stabilizing salts or co-solvents (e.g. abruptly dropping NaCl from 500 mM to 0, or dropping urea from 6 M to 0) causes aggregation. Perform step-wise dialysis with intermediate salt/denaturant concentrations, or add 5–10% glycerol.
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