Explorative experiments on separation of Steviol Glycosides with Ion-Exchange Chromatography and Pulsed Amperometric Detector

Abstract

Stevia is a natural sweetener with increasing use in known products, for instance Coca Cola Company launched a new brand called "Life" were they use Stevia as a sweetener. In this work, we have attempted to separate and analyze the different compounds that give the sweet taste, known as steviol glycosides, by ion-exchange chromatography with pulsed amperometric detection. The known methods for separating rebaudiosides often use HPLC and thereby often use polar interaction to separate the compounds. No research has been found where High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detector (HPAEC-PAD) is used to separate the steviol glycosides, or as some of them are called, rebaudiosides. In this work the sugar moieties, attached to a non-polar diterpene as aglycone, were ionized in highly alkaline mobile phase and separated on a CarboPac PA20 column. This use of the sugar moieties for separation have not been used before and proved to give a good separation. The resin of this column is non-polar and interacted with the diterpene part of the rebaudiosides, there for acetonitrile was added to the mobile phase to counteract the hydrophobic interaction between these two parts. The exploratory experiments showed that acetonitrile does reduce the hydrophobic interaction and that the rebaudioside A, used as standard, does also have ionic interaction with the column and could be eluted with 200 mM NaOH, gradient of 0-200 mM NaOAc and 10 % ACN. A commercial product with stevioside and rebaudioside A are run under same conditions and achieves a good separation, thereby opening for further investigation of the separation of steviol glycosides with anion exchange chromatography.

Popular Abstract

Good taste, bad taste - The quest for the prefect natural sweetener

Have you ever noticed how light products differ in taste from the real thing? Well, I have noticed that sometimes there is an off taste or maybe a strange after taste. Not too long ago the Coca Cola Company launched a new brand called "Life". With the green logo they want to make us aware of its natural contains. It is actually a plant called Stevia and Cane sugar they use. Cane sugar is, well normal sugar, however Stevia has a very sweet taste, actually 30 - 300 times sweeter, but low on calories. Great, so let us just use Stevia then! Well it of course it is not that simple, nothing ever is... You see, when you extract the sweet compound you basically make a cup of tea. Just imagine your water heater being a machine large as a moving box, but costing a lot more, and your teabag being a metal cylinder build to withstand high pressures. So you add your dried Stevia leafs into the cylinder and add hot water under high pressure and let it sit for a while. Then you get something that looks a lot like tea, however I call it Stevia extract. So in this extract I have a lot of things that I don't want like colour, small particle from the leaves and much more. But I only want to have the sweet thing! These contamination, as I call them, can be removed with organic solvents such as ethanol, you know Vodka, Gin, hangover, and filters. Ok so now the contaminations are gone, now I can add it to the Coca Cola? Well, you see there is not one but several compounds that are sweet in Stevia. They are called steviol glycosides, which you actually can see if you look at product containing Stevia or maybe rebaudiosides which is another name for some of them (The plants Latin name is Stevia Rebaudiana). So why can I not take all of them? sweet is sweet right? Not really, actually these compound are sweet but some of them have bitter taste and some have liquorice taste. So what I need to do is to separate them from each other, because I only want the one with the best resemblance to normal sugar. Now we are at the tricky bit, these compounds are very similar which is a problem when trying to separate them. What I have tried to do is to use ion-exchange chromatography. So what is that, well ions are basically molecules that have a charge, positive + or negative - and as you know + likes - . I can make the steviol glycosides negative by adding them to a very basic solution. Ok so now I have negative steviol glycosides and the thing is that they have different amount of negative charge depending on their small differences in structure. That's great but how does ion-exchange work? I use a cylinder with small positively charged particles that are permanently bond to the sides of the cylinder. Then I add my negatively charged rebaudiosides to this cylinder, or column as I call it, and guess what the steviol glycosides find a positive match in the column! So now they are stuck! But now I want them come out and not all together. Then I introduce a new negatively charged ion, let us call them bullies, that the positive parts in the column likes much better. I start sending them in small numbers and gradually increase their numbers. This will make the rebaudiosides with the weakest charge break up, how sad, with their positive charge and as the number of bullies increase even the stronger rebaudiosides have to give up. But I am happy the rebaudiosides didn't just give up, because now they left the column one at a time so I could pick which one I wanted and needed to get the best sweet taste.
SWEET!

• author: Brorström, Fredrik
• supervisor: Irene Rodriguez Meizoso
• organization: Department of Chemistry
• course: KEMX11 20161
• year: 2016
• type: M2 - Bachelor Degree
• subject: Chemistry
• keywords: Ion-Exchange Chromatography, Steviol Glycosides, KEMX11, Stevia, tillämpningskurs, Pulsed Amperometric Detector
• language: English
• id: 8892918
• date added to LUP: 2016-11-06 11:34:13
• date last changed: 2016-11-06 11:34:13