Salting out is the process of driving a dissolved substance out of solution by adding a high concentration of salt. The salt competes for water molecules so aggressively that the dissolved substance, whether a protein, a gas, or an organic compound, loses its hydration shell and either precipitates, forms a separate liquid phase, or escapes as a gas. The phenomenon has been exploited for well over a century in fields ranging from biochemistry to soap manufacturing, and its underlying physics connects to broader questions about how ions reshape the behavior of water itself.
Why Adding Salt Pushes Things Out of Solution
When you dissolve a salt like sodium chloride or ammonium sulfate in water, the ions that form pull water molecules toward themselves. Each ion surrounds itself with a tight shell of oriented water molecules. At low salt concentrations this barely matters to other dissolved substances. But as you keep adding salt, there is progressively less “free” water available to hydrate anything else in solution. A protein, for instance, normally stays dissolved because water molecules coat its surface, especially the hydrophobic patches that would otherwise clump together. Pile in enough salt and those water molecules get stripped away, exposing hydrophobic regions that then stick to one another, causing the protein to aggregate and drop out of solution.
Research into the thermodynamics of this process shows that when the dominant interaction between a protein and the added salt is preferential exclusion of the salt from the protein’s surface, the protein is effectively squeezed out of solution. The increase in the surface tension of water caused by the dissolved salt makes it energetically costly to maintain the large solvent-exposed surface area of the dissolved protein, so the system minimizes that area by forcing the protein to aggregate and precipitate.1PubMed. Preferential interactions determine protein solubility in three-component solutions: the MgCl2 system In simpler terms, the salt raises the energy cost of keeping the protein wet, and precipitation is the system’s way of lowering that cost.
Not All Salts Are Equal
Different salts drive salting out to very different degrees, a pattern that scientists have organized into the Hofmeister series. Salts with strongly hydrated ions, sometimes called kosmotropes, such as sulfate and phosphate, are powerful salting-out agents. They increase the surface tension of water substantially and are strongly excluded from protein surfaces. At the other end of the spectrum sit weakly hydrated or chaotropic ions like thiocyanate and iodide, which interact more directly with the protein surface and can actually increase solubility, an effect called salting in.
Molecular dynamics simulations have shed light on why the Hofmeister ranking holds so consistently. In simulations of hydrophobic molecules in various salt solutions, the degree to which each salt strengthened hydrophobic interactions correlated tightly with experimental solubility data. A strong connection was found between salt-induced changes to water’s hydrogen-bonding network and the observed salting-out strength, suggesting that much of the Hofmeister effect comes from how salts restructure the water around them rather than from direct salt-protein contact.2PubMed Central. Protein stabilization and the Hofmeister effect: the role of hydrophobic solvation Kosmotropic anions produce stability changes that scale with how much protein surface area is exposed, while chaotropic anions produce effects that are more independent of the protein’s surface characteristics.
This ion-specific behavior also explains why picking the right salt matters so much in practice. Ammonium sulfate dominates protein purification because sulfate is one of the strongest kosmotropes, it is cheap, and it is highly soluble in water, letting you reach the high concentrations needed without running into the salt’s own solubility limit.
Ammonium Sulfate Precipitation in the Lab
If you have ever worked in a biochemistry lab, you have almost certainly done an “ammonium sulfate cut.” The idea is straightforward: different proteins precipitate at different salt concentrations, so by raising the ammonium sulfate level in steps, you can selectively knock out groups of proteins one at a time. You spin down the precipitate at each step, keep the fraction that contains your target protein, and discard the rest. It is not the most refined purification method, but it is fast, gentle on proteins, and works at large volumes, which makes it a natural first step before moving on to chromatography or other higher-resolution techniques.3PubMed Central. Protein precipitation using ammonium sulfate
The relationship between salt concentration and protein solubility follows a well-known empirical pattern. For many proteins, the logarithm of solubility decreases linearly with increasing salt concentration once you are above a certain threshold. Work on lysozyme, for example, found that the protein concentration in the supernatant was independent of how much protein you started with, meaning solubility at a given salt concentration behaves like a true thermodynamic quantity that can be described predictably.4Biotechnology and Bioengineering. Some characteristics of protein precipitation by salts That predictability is what makes the technique so reliable: once you know the salt concentration at which your protein precipitates, you can reproduce the purification consistently.
Ammonium sulfate precipitation has also been scaled up well beyond the benchtop. Researchers have used it to fractionate plasma proteins from porcine blood at industrial scale, isolating functional protein ingredients that were then concentrated by membrane filtration and spray-dried into stable powders.5PubMed. Scale-up of the process to obtain functional ingredients based in plasma protein concentrates from porcine blood The fact that salted-out proteins generally retain their biological activity after redissolving is a major advantage over harsher precipitation methods that use organic solvents or extreme pH.
When the Hofmeister Series Flips
One of the more confusing wrinkles in salting-out science is that the order of effectiveness of different salts can reverse depending on conditions, particularly the pH of the solution relative to the protein’s isoelectric point (the pH at which the protein carries no net charge). When the solution pH is below the isoelectric point, the protein carries a net positive charge and anions act as counterions. Under these conditions, chaotropic anions like thiocyanate and iodide, which normally promote solubility, actually become more effective at driving crystallization than kosmotropes like chloride. The ranking of anions flips to a reversed Hofmeister sequence.6PubMed. Why forces between proteins follow different Hofmeister series for pH above and below pI
Above the isoelectric point, where anions are co-ions (same charge as the protein surface), the standard Hofmeister order holds and kosmotropes win. This reversal has practical consequences: if you are trying to crystallize a protein for structural studies and you are working at a pH below its isoelectric point, following the textbook Hofmeister ranking could steer you to the wrong salt. The reversal was confirmed both by solubility experiments and by small-angle X-ray scattering measurements of inter-protein forces, giving it a solid experimental footing.
Low Salt Versus High Salt and the Curve in Between
Salting out only dominates at high salt concentrations. At low salt, the opposite often happens: adding a little salt increases protein solubility. This salting-in effect occurs because the added ions screen electrostatic attractions between protein molecules, reducing their tendency to aggregate. As more salt is added, the screening effect saturates and the salting-out mechanism, driven by preferential exclusion and surface tension, takes over.
Theoretical work has captured both regimes in a single framework. At low salt, the dominant contribution comes from the cost of confining ions within protein aggregates, which makes aggregation unfavorable and keeps proteins dissolved. At high salt, the ions drive a depletion-like attraction between proteins that favors aggregation.7PubMed Central. Ion Specificity and Nonmonotonic Protein Solubility from Salt Entropy The crossover between these two regimes produces the characteristic U-shaped or non-monotonic solubility curve that biochemists observe: solubility first rises, then falls as salt concentration climbs. The exact crossover point depends on the specific salt used and the surface chemistry of the protein, which is why there is no universal ammonium sulfate concentration that precipitates everything.
This competition between salting in and salting out has been documented in food science as well. Soy protein particles in solutions of sodium chloride show both behaviors as the salt concentration changes, with aggregation first decreasing and then increasing, directly affecting how well the particles can stabilize oil-in-water emulsions.8PubMed. Salting-out and salting-in: competitive effects of salt on the aggregation behavior of soy protein particles and their emulsifying properties For food manufacturers formulating products with plant proteins, getting the salt level right is a balancing act between these opposing forces.
Salting Out Beyond Proteins
Proteins get most of the attention, but salting out applies to any dissolved substance whose solubility depends on interactions with water. Dissolved gases are a classic case. As salt concentration rises, the gas molecules are effectively squeezed out. Measurements in sodium chloride solutions up to about 260 parts per thousand show that dissolved oxygen concentrations drop steadily with increasing salinity at any given temperature.9Limnology and Oceanography. Dissolved oxygen concentrations in hypersaline waters This has real ecological significance: hypersaline lakes and estuaries hold less oxygen than freshwater at the same temperature, limiting the kinds of organisms that can survive there. The Dead Sea, for instance, is almost lifeless in part because its extreme salinity crushes dissolved gas levels.
In soap manufacturing, salting out is used to separate glycerol from spent soap lye. During traditional soap-making, fats react with an alkali to form soap and glycerol. Adding common salt causes the soap to precipitate and float, leaving the glycerol in solution for recovery. Studies of this process report glycerol recovery rates above 80 percent from various soap lye sources.10Hindawi / Journal of Chemistry. Recovery of Glycerol from Spent Soap Lye By‐Product of Soap Manufacture The salt here is acting on the soap (forcing it out of the aqueous phase) rather than on the glycerol, which stays dissolved. It is the same physical phenomenon applied to a very different system.
Salting-Out Assisted Liquid-Liquid Extraction
One of the fastest-growing applications of salting out is in analytical chemistry, specifically a technique known as salting-out assisted liquid-liquid extraction, or SALLE. The idea is to mix a water-miscible organic solvent like acetonitrile with an aqueous sample, then add salt. The salt forces the organic solvent and water to separate into two distinct phases, carrying dissolved analytes preferentially into the organic layer. You then analyze the organic phase directly.
SALLE has become popular in clinical and forensic laboratories for analyzing drugs and metabolites in blood and plasma because it is simple, fast, cheap, and avoids the toxic organic solvents traditionally used in liquid-liquid extraction.11PubMed. Salting-out assisted liquid-liquid extraction (SALLE): Principle, optimization, and applications in blood sample analysis The salt does the heavy lifting of phase separation, and the technique can be optimized by choosing the right salt and concentration for a particular analyte. One study optimizing SALLE for the heart medication carvedilol in human plasma achieved about 93 percent recovery of both enantiomers using acetonitrile and ammonium acetate, numbers competitive with far more elaborate extraction methods.12PubMed. Chemometric optimization of salting-out assisted liquid-liquid extraction (SALLE) combined with LC-MS/MS for the analysis of carvedilol enantiomers in human plasma: Application to clinical pharmacokinetics
A related approach uses polymer-salt aqueous two-phase systems, where adding salt to a polymer solution creates two water-rich layers with different compositions. Biological molecules partition between the layers based on their size, charge, and surface properties. This has been used to separate DNA from proteins with striking efficiency: adding a small amount of sodium chloride to a polyethylene glycol/phosphate system recovered 88 percent of DNA in the salt-rich phase while removing over 99 percent of contaminating protein.13Biotechnology Journal. Partitioning behavior of short DNA fragments in polymer/salt aqueous two‐phase systems The salting-out effect here acts selectively on different biomolecules, making it a purification tool rather than just a precipitation hammer.
Hemoglobin, Phosphate Buffer, and a Disease Connection
Salting out shows up in unexpected biomedical contexts as well. Sickle cell disease involves a mutant form of hemoglobin that polymerizes under low-oxygen conditions, distorting red blood cells into their characteristic crescent shape. Researchers studying this polymerization often use high-concentration phosphate buffer, typically 1.5 to 1.8 molar, as a model system to drive hemoglobin aggregation without needing to manipulate oxygen levels. But work using light scattering and microscopy has shown that in these concentrated phosphate solutions, both sickle hemoglobin and normal hemoglobin aggregate even at protein concentrations below the threshold usually associated with polymerization.14PubMed Central. Aggregation of normal and sickle hemoglobin in high concentration phosphate buffer
The aggregates appear to result from salting out rather than from the specific polymerization mechanism of sickle hemoglobin, consistent with much older observations of horse hemoglobin precipitating in concentrated phosphate.15Biophysical Journal. Aggregation of Normal and Sickle Hemoglobin in High Concentration Phosphate Buffer – Section: Discussion This matters for interpreting experiments: if researchers assume that aggregation in high-phosphate buffer mimics the disease-specific polymerization, they could be confusing a generic salting-out artifact with a pathological mechanism. It is a useful reminder that salting out is not just a laboratory tool but a background physical phenomenon that can quietly influence any system where ion concentrations climb high enough.
Common Misconceptions About How Salting Out Works
A widespread oversimplification is that salt “steals” water away from the protein, as if there were a fixed pool of water molecules being literally removed. The reality is subtler. The ions do not remove water from the system; they change the thermodynamic cost of having water in contact with the protein surface. The protein is still surrounded by water, but maintaining that hydration layer becomes energetically unfavorable relative to the alternative of protein-protein contact and aggregation. Framing salting out as “competition for water” is not wrong as an intuition, but it misses the central role of surface tension and the way different ions reshape the hydrogen-bonding structure of the entire solution.
Another misconception is that salting out is always irreversible or damaging. In many cases, the precipitated protein can be fully redissolved by lowering the salt concentration, and it returns to its native, functional conformation. This is precisely why ammonium sulfate precipitation is so popular as a first purification step: the protein does not denature. Contrast this with precipitation by heat or strong acid, which typically unfolds the protein permanently. The gentleness of salting out stems from the fact that the protein is being driven out of solution by changes in solvent properties rather than by forces that disrupt the protein’s internal structure.
A third common error is assuming that a higher salt concentration always means more precipitation. As noted earlier, the solubility curve is non-monotonic for many proteins, with salting in dominating at low concentrations. Jumping straight to a very high salt concentration can sometimes co-precipitate contaminants that would have stayed in solution at a more moderate level. Good practice in protein purification involves stepwise increases, checking each fraction, rather than a single massive salt addition.
Choosing the Right Salt for the Job
Ammonium sulfate is the workhorse, but it is not the only option and it is not ideal for every situation. Sodium sulfate is another strong salting-out agent, but it is much less soluble in water, especially at lower temperatures, limiting how high a concentration you can reach. Sodium chloride is gentler and is often the salt of choice in food processing, where taste and regulatory considerations matter. Magnesium chloride can work, though it introduces divalent cations that may bind to proteins and complicate things.
For SALLE, the choice of salt depends on compatibility with the downstream analytical instrument. Ammonium salts like ammonium acetate and ammonium formate are favorites in mass spectrometry workflows because they are volatile and do not leave residues that interfere with ionization. Sodium and potassium salts, while perfectly good at inducing phase separation, can suppress signal in mass spectrometry and clog the instrument. The choice of salt, in other words, is never just about the salting-out strength but also about what happens after the precipitation or phase separation step.
Temperature matters too. Most salting-out protocols are done at 4 degrees Celsius, both to protect sensitive proteins and because protein solubility in concentrated salt is generally lower at lower temperatures. But this is not universal, and some proteins actually salt out more readily at room temperature. Checking the temperature dependence for your specific system, rather than blindly defaulting to the cold room, can save time and improve yields.