We’ve talked about purification of proteins, spray drying of nucleic acids before, but how could I have left lipids out until now? Deservedly, the spotlight this time is on giving you a few tips on how to purify lipids. Let us go over mobile phases and stationary phases, sample loading and detection of lipids together, so you can obtain a lipid purification that can make you as happy as a warm cheeseburger.

I don’t know about you, but I am constantly hungry these days. And not just for any food, but for super fatty food. I’m constantly dreaming about fries, pizza, chips, chocolate cake. I am trying to reason out that with winter approaching, my body just really wants to stock up on fat content. It’s just its way to make sure I stay nice and warm. Although, I would rather do like a big bear and hibernate through the cold times instead!

I mean, let’s face it, fats are not as bad as their rep suggests. Of course, anything in excess is undesirable, but lipids are physiologically important and play an essential role in many bodily processes. Some of these include intermediary metabolism as both energy storage and energy molecules, in membrane structures, in signaling and in protection as antioxidants, thermal insulation and shock absorption.

Biochemically, lipids are a diverse group of molecules which can be categorized as:

• Fatty acids: fatty alcohols and acids
• Glycerolipids: mono-, di-, and triacylglycerids
• Glycerophospholipids: phosphatidyl choline, phosphatidyl serine
• Sphingolipids
• Sterol lipids. cholesterol, bile acids, vitamin D
• Prenol lipids: vitamin E, vitamin K
• Saccharolipids
• Polyketides: aflatoxin B1

Impressive list of categories, isn’t it?

Now you can do all kinds of things if you work on lipid research, but many projects require you to first purify lipids. So, I would like to offer you a few tips on how to do just that: use chromatography to purify lipids.

Firstly, lipids are not soluble in water as they are non-polar. They are, however, soluble in organic solvents. There are two ways you can purify lipids: using normal-phase chromatography or reversed phase chromatography with organic solvents. Let us now explore how you can choose the most appropriate solvents and stationary phases, sample loading techniques and of course, how to detect your lipid compounds.

Selecting mobile and stationary phases to purify lipids

In normal phase chromatography, you can use common solvents such as hexane, ethyl acetate or DCM. Bear in mind that the highly polar stationary phases typically exhibit lower retention, because lipids have fewer polar groups that can interact with the highly polar silica surface. Lower retention could also lead to poor resolution when you purify lipids.

I would recommend using reversed phase (C18) as the stationary phase. Combine this with hydrophobic, non-polar solvents such as DCM, acetonitrile or methanol when you purify lipids. This non-aqueous reversed -phase technique allows for both solubility and retention of lipophilic compounds.

If you cannot purify lipids with silica or C18 in your column, good alternatives you could consider are medium polar phases such as amino or diol. Both phases can be used under reversed phase or normal phase conditions and are suitable for purification of highly lipophilic compounds. Here, it is important to note that due to their different polarities, amino and diol can have a substantial effect on the elution order of the compounds.

Lipid sample loading

Due to their hydrophobicity, you can inject lipids as a liquid only if you use organic solvents to purify lipids. If you are facing solubility issues, you could consider performing solid loading, followed by flash chromatography. Here, the lipophilic mixture is added to support material, such as silica, and solvents are removed by evaporation. The solid sample is then placed in front of the separation cartridges where the compounds are gradually eluted from the support material.

Lipid detection

I’ve covered various detection methods used in chromatography with you before in a previous blog post. If you remember, UV is the most common method used for compound detection. Unfortunately, it is difficult to use a UV detector to detect lipids, since they have little UV absorbance. The traditional approaches to lipid detection are refractive index (RI) or low-wavelength UV (190 – 205 nm). If you want to detect lipids, keep in mind that the RI is a poor choice, due to low sensitivity and gradient incompatibility. Similarly, low-wavelength UV lacks sensitivity and presents a problem with the absorption characteristics of mobile phases. Additionally, both techniques show substantial baseline shifts and long equilibration times, as the detector responds to spectroscopic changes in the solvent.

I would suggest performing collection by volume with post-purification analysis by TLC or an alternative detection technique, such as ELSD.

As lipids are a structurally diverse group, working with ELSD is most likely to get you a separation of all lipophilic compounds you would like to monitor. If you want more information on ELSD, check out my two previous blog posts on ELSD as a detection method.

So there you have a short and sweet post on how to purify lipids. Hope I cut the fat and provided you with the most essential information you need to achieve proper lipid separations. But in case you need more grease, check out these free literature insights on lipid purification. Any other compounds you find challenging to purify and you’d like me to cover on the blog? Don’t hesitate, leave me a comment below!

Till next time,