# Separating Lipids from Milk

I am trying to develop a procedure to test the percent mass of lipids in (update: homogenized) milk. It doesn't need to be very accurate, just an interesting application of chemical principles. As I was looking on traditional ways this is tested, I came across the Babcock method, which involves pouring concentrated sulfuric acid into the milk, heating, and centrifuging the sample. According to Wikipedia, "key to this process is that everything in milk except the fat dissolves in sulfuric acid."

The lipids of are surrounded by what is similar to a cell membrane. The fat is an emulsion with these surfacants holding it in the water. The other components of milk are water, colloidal micelles, which hold other proteins (of no interest to me). My question is, how does sulfuric acid dissolve "everything else"? In a biological procedure I know of to break cell membranes, isopropyl alcohol is used. The cell membrane in that case is broken when the alcohol behaves as a surfactant toward individual triglycerides. However, sulfuric acid does not have a nonpolar region, so how does it work to dissolve the phospholipid membrane?

Many Internet resources on this topic are not written from a chemical perspective. In addition, they seem to confuse the lipid bundles with the other colloidal particles.

Also, why not just heat the milk strongly (such as with a Bunsen burner) to break the protein, or add electrolytes to it, as is a common way to separate colloids?

• Do you want to analyze a fresh milk, or homogenized (i.e. almost all milk in stores) ? In principle, the standard method is described at: en.wikipedia.org/wiki/Butyrometer – ssavec May 12 '16 at 6:48

Milk is mostly water, and diluting it (roughly speaking) with sulfuric acid to a concentration of ~50% milk and 50% sulfuric acid will result in a very acidic, but still aqueous, solution. We can examine the likely behavior of other milk components using basic chemical principles.

• Sugars. Milk is rich a variety of di- and oligo-saccharides such as the eponymous lactose, as well as many other more complex molecules:

[Free oligosaccharides are natural constituents of all placental mammals' milk. Human milk contains 7–12 g/L oligosaccharides, making the oligosaccharide fraction a major component of human milk. Compared with human milk, the concentration of oligosaccharides in the milk of the most relevant domestic mammals is smaller by a factor of 10 to 100.]

Fortunately, the effect of sulfuric acid on the sugars is easy to predict: they will simply stay dissolved in aqueous solution. Hydrolysis of the sugars is possible, but will not happen to any great degree without heating or prolonged incubation.

• Proteins. Milk contains thousands of unique proteins. However, by far the most predominant proteins are called caseins. Caseins are relatively hydrophobic, proline-rich molecules and in milk are present as tiny aggregates -- a colloid -- of molecules. Adding sulfuric acid will denature the casein. As a result, the casein molecules are likely to stick to each other and form a centrifugable pellet that goes to the bottom of a centrifuge tube after spinning. Proteins contain amino acids linked via amide bonds that are unlikely to hydrolyze in the acid, unless very high temperatures or very long incubation times are used.

• Phospholipids. Phospholipids in milk include glycerophospholipids and sphingolipids. Polar lipids play an important role in stabilizing the triglyceride (fat) emulsions that keep fat in solution. They do this by forming bilayers and other structures on the surface of triglyceride microdroplets. To do this job, phospholipids have a phosphate head group attached to a fatty acid tail. The fatty part of the phospholipid sticks to the triglyceride, and the polar head group interacts with aqueous solution. The polarity of the head group is tunable by pH. At neutral to mildly acidic pH, the phosphate head groups are (negatively) charged, greatly strengthening their propensity to interact with water and maintain emulsion stability.

Phospholipids may be partially hydrolyzed by the acid, but without very high temperatures or prolonged incubation, most molecules will remain intact.

The strong acid conditions that arise from adding $\ce{H2SO4}$ will protonate these head groups, neutralizing the negative charge and vastly weakening their emulsifying properties.

• Triglycerides. Triglycerides are the molecules that provide most of the fat content of milk. They are triacylglycerols. Unlike phospholipids, they have no polar head groups, and thus they do not dissolve in water. They would "want" to form a separate, oily phase from the water (just like mixing vegetable oil and water), but the fact that they are usually bound in tiny droplets by phospholipids prevents this separation. Because triglyceride oils are less dense than water, if they did ever get the chance to coalesce into large droplets, they would rise to the top of the milk. This happens when cream naturally separates from fresh (raw) milk, but in homogenized milk, the droplets are artificially forced to much smaller sizes, effectively preventing the droplets from coalescing or rising.

Upond acidification, the loss of phospolipid surfactant activity breaks down the stability of the fatty droplets, allowing them to rapidly coalesce, so they rise to the top of the tube, a process that is only accelerated by centrifugation.

Hydrolysis of triglycerides in the acid is possible, but unlikely. In addition to required prolonged incubation or high temperatures, keep in mind that the separated triglyceride phase is actually not exposed to the acidic solution very much at all.

• Are you sure than strong sulphuric acid would not hydrolyse the triglycerides? – matt_black May 13 '16 at 9:37
• So why use sulfuric acid rather than any other acid? If it's dilute, it doesn't exhibit any distinguishable qualities. – Yunfei Ma May 13 '16 at 13:09
• It's probably the cheapest acid, and was almost certainly the cheapest widely available acid when the Babcock method was initially developed. – Curt F. May 18 '16 at 16:05

When you add sulfuric acid, the phospholipid are hydrolysed into glycerophosphoric acid and fatty acid, therefore dissolving the fat globules membrane and freeing the tryacylglycerol.

Furthermore adding H2SO4 to milk is very exothermic (which help with the whole dissolution of the solid non fat) because it react with the Lactose (milk sugar) with a dehydration reaction.

• (i) I don't completely understand what you mean by your first sentence. (ii) Even if I understood it, I'm not sure that it's true. Would you be able to expand your answer, providing citations or links to evidence to back up your statements? In particular, why is the ester bond in phospholipid less stable than the ester bond in triacylglycerol? – Curt F. May 12 '16 at 11:02