Your supplement on vitamin purification

Vitamins might be micronutrients, but they are of macro importance when it comes to our health. Because they are so essential to nearly any bodily function, research and processing of vitamins has always been and remains a very popular field in pharmaceuticals and biotechnology. It is time that we also supplement our blog with vitamins. I hope a discussion on vitamin purification is not a pill that is too tough to swallow for you! But see for yourself and read on.

My sister called me the other day to tell me about a successful sleepover birthday party for my nephew. She did mention one child brought along a variety of vitamins he had to drink in the morning. She wanted to consult with me if my nephew should also take some supplements. Now, typically most of us get all the vitamins we need from a balanced diet and the environment. But there are many cases where vitamin supplements can be beneficial. From the top of my head, I could list picky eaters, those living through dark winters where vitamin D deficiency could be a problem, people with medical conditions or those on stringent diets.

After all, we need vitamins to heal wounds, boost our immune system, regulate our metabolism, repair cellular damage and much more.

Due to their importance, the research of vitamins is a very popular field. Vitamins can be negatively affected by heat, water, other liquids, or sunlight, causing them to degrade and lose their function. Because of this, we investigate derivatives of vitamins to overcome these stability hurdles. These derivates are produced via chemical synthesis, and are subsequently purified (separated), concentrated and formulated for further analysis:

vitamin; HILIC; chromatography; purification

The vitamin purification step is typically performed either by ion exchange chromatograph or by adsorption chromatography.

Ion exchange chromatography involves the separation of ions by their adsorption on a stationary phase that contains fixed charges on its surface. The ions in the sample interact with the ionic groups of the opposite charge of the stationary phase. Salt or pH gradients are used to elute and isolate the vitamins. In terms of vitamin purification, ion chromatography is a fast method, but it is only suitable for water soluble vitamins. The method also requires use of expensive consumables.

In adsorption chromatography, the molecules get separated by different polarities. The compounds of the sample mixture dissolve in a mobile phase and move with different speed through the stationary phase, which is either polar (normal phase) or non-polar (reversed phase). The elution is governed by the changing polarity of the mobile phase. In terms of vitamin purification, adsorption chromatography is cost-efficient, easy to set up and suitable for separation of both water- and fat-soluble vitamins. However, the method is time-intensive and requires pH control for ionized samples.

Because of its suitability for more types of vitamins, I would like to focus this post on adsorption chromatography as a method for vitamin purification.

Adsorption chromatography can be either flash chromatography or prep HPLC. I’ve discussed the differences between these chromatography types in a different post. Both flash and prep HPLC can be either normal phase or reversed phase. A comparison table between normal phase and reversed phase can be found below:

 Normal phaseReversed phase
Stationary phasePolarNon-polar
Mobile phaseNon-polarPolar
Substances eluted firstLow polarity substances (the elution of more hydrophilic components requires use of more polar solvents in the mobile phase)Hydrophilic molecules (the elution of more hydrophobic components requires a decrease in the polarity of the mobile phase through use of organic, non-polar solvent)
Solvent polarityTypically increases during runTypically decreases during run
Common stationary phasesSilica, amino, diolC4, C8, C18, amino, diol
Common mobile phasesHexane / ethyl acetate
Dichloromethane / methanol
Water / methanol
Water / acetonitrile
Water / ethanol
Methanol / acetonitrile
Methanol / dichloromethane

For vitamin purification, you can use either normal or reversed phase conditions. The choice depends mainly on the solubility of the vitamins. Solubility refers to the property of how well a sample dissolves in a solvent.

The ideal sample loading technique for vitamins is liquid loading onto your chromatography system, so vitamins should be soluble at the start of the vitamin purification. But remember to consider the retention of the compounds on the stationary phase. Retention is needed to separate the target compound from impurities. In the case of no retention, the compound simply runs with the solvent front through the stationary phase of your column. Since the polarity of the stationary phase and the starting solvent condition cannot be the same, the choice between normal phase and reversed phase in vitamin purification is often a compromise.

Let me try to simplify your vitamin purification further with the following table of vitamin solubility I generated based on Thermo Fisher’s technical note: Determination of water- and fat-soluble vitamins by HPLC:

Vitamin A (Retinol)Solubility in ethanol, methanol, chloroform, ethyl-ether, and oil; insoluble in water and glycerol
Vitamin B1 (Thiamine)Soluble in water, slightly soluble in ethanol, insoluble in ether and benzene
Vitamin B2 (Riboflavin)Soluble in basic aqueous solution; slightly soluble in water and ethanol; insoluble in chloroform and ether
Vitamin B3 (Nicotinic acid)Soluble in water
Vitamin B5 (Pantothenic acid)Soluble in water, ethanol, alkali carbonate hydroxide solution and alkali solution; insoluble in ether
Vitamin B6 (Pyridoxine/pyridoxal hydrochloride)Soluble in water, ethanol, methanol, and acetone; insoluble in ether and chloroform
Vitamin B9 (Folic acid)Soluble in alkali solution; slightly soluble in methanol; insoluble in water and ethanol
Vitamin B12 (Cyanocobalamine)Soluble in water and ethanol; insoluble in ether, acetone, and chloroform
Vitamin C (Ascorbic Acid)Soluble in water; slightly soluble in ethanol; insoluble in ether
Vitamin D2 (Ergocalciferol)Soluble in alcohol, ether, and chloroform; insoluble in water
Vitamin D3 (Cholecalciferol)Soluble in alcohol, ether, acetone, chloroform, and vegetable oil; insoluble in water
Vitamin E (Tocopherol)Soluble in alcohol, ether, acetone, chloroform and oil; insoluble in water
Vitamin K (Phylloquinone)Soluble in ether, acetone, and chloroform; slightly soluble in oil and methanol; insoluble in water

Let us examine these vitamins based on their solubility to find the optimal vitamin purification conditions.

Vitamin purification of water-soluble vitamins

Water soluble vitamins can dissolve in water. Most of these types of vitamins are not stored in the body, so they must be eaten daily. Any excess of absorbed vitamins is normally disposed of via the urine. Water-soluble vitamins are usually purified via reversed phase chromatography using water and low concentrations of an organic solvent, such as acetonitrile or methanol. You can use common buffers such as phosphate, formic acid and acetic acid for pH control. If you are using solvent with water concentrations higher than 95%, I recommend using a C18 phase with polar end-capping in your cartridge to prevent the collapse of the alkyl chains.

Vitamin purification of fat-soluble vitamins

Fat-soluble vitamins can be dissolve in fat/lipids. These types of vitamins are absorbed in fat globules which travel in the general blood circulation through the lymphatic system of the small intestine. Once inside the body, fat-soluble vitamins are usually stored in the liver and fatty tissues, where they remain until further use. Fat-soluble vitamins can be purified via reversed-phase chromatography using less polar solvents, such as dichloromethane (DCM), acetonitrile and methanol. If you are working with very non-polar vitamins, you could try normal phase chromatography with very non-polar organic solvents, such as hexane and a medium polar solvent such as ethyl acetate.

Vitamin purification of highly polar vitamins

Usually, you cannot separate highly polar vitamins by normal phase or reversed phase chromatography. In normal phase conditions, the compounds are too sticky on the polar stationary phase, so elution would take too long. Polar compounds also show good solubility in aqueous mobile phases and are therefore not very compatible with typical normal phase solvents. If you try reversed phase instead, you will run into different problems. The highly polar compounds do not interact at all with the non-polar stationary phase of your column. Instead, your highly polar vitamins will likely elute with the solvent front and no separation will occur. I would recommend trying an alternative chromatography method, namely hydrophilic interaction chromatography (HILIC) on your chromatography system. And you’re in luck, because I just wrote a blog post on HILIC. You are more than welcome to read about this method there.

This is all the vital information on vitamin purification I have for you in a nutshell. But if you are interested in digging deeper and supplementing this blog post further, feel free to check out a nice free resource on using chromatography for vitamin purification or a great vitamin research roadmap for something slightly more visual or a vitamin webinar for something super visual. It might just be the micro-knowledge you need to get yourself a macro-boost in the separation of your vitamins!

Till next time,

The Signature of Bart Denoulet at Bart's Blog