The first research to produce evidence that the lipids of a cell membrane are in two layers (a lipid bilayer) is attributed to Gorter & Grendel (1925). They arrived at this conclusion by measuring how much lipid was present around red blood cells (which they called chromocytes), and determined that it was sufficient to surround the red blood cell twice. The basics of their approach are straightforward: they took red blood cells obtained from various animals (humans, rabbits, dogs, goats, guinea pigs, and sheep) and mixed the cells with acetone. Any lipids in the cells would dissolve in the acetone, leaving the rest of the cell contents behind. Having extracted the lipids from the cells, the acetone could be evaporated off, leaving only the lipids. With a little more work, it’s possible to determine how large a surface those lipids would occupy. If you know how many red blood cells were used to extract the lipids and you know the surface area of an average red blood cell, then you can figure out the total surface area of all the red blood cells. By comparing the surface area of the red blood cells to the surface area covered by the extracted lipids, Gorter & Grendel found that the area covered by the lipids was twice the surface area of the red blood cells. Looking at their original data below, it’s the last three columns on the right that are most important. The third column from the right [“Total surface of the chromocytes (a)”] is the surface area of the red blood cells. The next column is the surface area of the lipids extracted from the red blood cells [“Surface occupied by all the lipoids of the chromocytes (b)”]. The last column is the ratio of the two – and column (b) is approximately twice column (a).
Their conclusion is straightforward: “It is clear that all our results fit in well with the supposition that the chromocytes are covered by a layer of fatty substances that is two molecules thick.” Other work by later scientists would reaffirm this conclusion. However, Gorter & Grendel went beyond this, suggesting the way these two layers of lipids would be oriented: “We therefore suppose that every chromocyte is surrounded by a layer of lipoids, of which the polar groups are directed to the inside and to the outside… On the boundary of two phases, one being the watery solution of hemoglobin [the fluid inside the red blood cell], and the other the plasma [the fluid in which your red blood cells float], such an orientation seems a priori to be the most probable one.”
To understand what Gorter & Grendel are talking about, consider the phrase ‘oil and water don’t mix’. They don’t mix because water is a polar molecule and oils (e.g., olive oil) are non-polar. Polar molecules have an asymmetrical distribution of electrical charge – this is due to an asymmetrical distribution of bonding electrons. As a result, parts of the molecule are more negatively charged than other parts. For non-polar molecules, electrical charge is more or less symmetrically distributed around the molecule. Polar molecules don’t interact very strongly with non-polar molecules, so the two types of substances don’t mix. Since oils are a type of lipid, we can generally conclude that lipids and water don’t interact very well. However, one particular group of lipids, known as phosopholipids, do interact with water. In fact, phospholipids interact with both water and other lipids. Phospholipids are able to interact with water because part of a phospholipid molecule is polar (the head) and part is non-polar (the tails). Because of this property, phospholipids mix well with both water and other lipids. Phospholipids are often represented like the drawing below (with the circle symbolizing the polar head and the two lines representing the two fatty acid – non-polar – tails):
When placed in water, phospholipids tend to form structures that minimize the contact between the molecules of water and the non-polar tails, and maximize the contact between the water molecules and the polar heads. So, Gorter & Grendel are hypothesizing that the two layers of lipids suggested by their data are arranged in the manner shown below.
Gorter, E. and Grendel, F. (1925) “On bimolecular layers of lipoids on the chromocytes of the blood.” Journal of Experimental Medicine 41: 439-443
There's also a nice teaching module designed to help students arrive at the idea of a lipid bilayer on there on, just by considering the data of Gorter & Grendel:
An Emerging Model of Cell Membrane Structure (Laura Martin) – http://cnx.org/content/m15250/latest/ Created October 15, 2007; Connexions web site. Version 1.2.