Experiment: Essential Glasswares

In preparation for our upcoming experiments, I’m going to introduce you to four laboratory glassware that are absolutely essential and indispensable. They are designed by early scientists to facilitate extraction and chemical separation of a mixture, and I can’t imagine life as a chemist without them. As I’ve mentioned earlier, I will try to adapt laboratory experiments into a DIY home friendly setup. In fact, there’s not much difference between laboratory apparatus and everyday cooking or baking utensils. You can substitute a Bunsen burner with a camping gas stove, a beaker with a Pyrex jar, an electronic balance with a scale, a magnetic stirrer with an egg blender whatnot. However, here are the fantastic four that you’ll have to no alternatives for. 

 

Three major components of the Soxhlet extractor, from left to right, reflux condenser, percolator, extraction flask.

Number 1, the Soxhlet extractor. This fancy piece of apparatus allows us to extract a plant material with relatively little monitoring. We can leave it alone to do its job and obtain a concentrated crude extract overnight. The Soxhlet extractor was invented by a French chemist called Franz Von Soxhlet in the 1800s to extract fats out a solid substance. Extraction means the selective separation of a desired component out a mixture, which is usually a solid or liquid. In solid-liquid extraction like Soxhlet, we will be using a specific solvent to dissolve our plant toxins or any substances to be extracted. This solvent will be introduced into a flask at the bottom of the Soxhlet extractor, which is heated to boil the solvent. The solvent vapour is condensed into a liquid by a condenser located at the top of the Soxhlet apparatus. The condensed solvent then drips into the middle component called a percolator, which soaks (percolate) plant materials in solvent to facilitate extraction. Once the solvent reaches a certain volume in the percolator, a siphon mechanism empties the solvent extract back into the bottom flask. The solvent is then boiled and recycled again to percolate the plant material, producing an extract that becomes more and more concentrated. We usually leave the Soxhlet extractor to run for about 12-24 hours, and I will show you how it works in our future experiments. In fact, we will be using the Soxhlet extractor to remove fats, oils, chlorophyll, or any unwanted materials out of plant material. We will then re-Soxhlet extract the cleaned up matrix to obtain a crude extract containing alkaloids or cardiac glycosides of interest. 

 

Separatory funnel

 

Number 2, the separatory funnel. We all know that oil and water don’t mix, and thanks to this phenomenon we can achieve something called liquid-liquid extraction. Different solutes have different solubility in different solvents depending on the universal rule of ‘like dissolves like’. For example, salt and sugar preferentially dissolve in water because they contain water-loving or water-like properties such as hydroxyl group or charged ions. Conversely, paint preferentially dissolves in paint thinner because both contain ‘oil-based’ property. Thus, if we have a mixture of solutes in a liquid, we can shake this liquid with another solvent of opposite solubility (like mixing oil into water) to separate the solutes according to their preferential solubility in the two solvents. That also means, we will end up with two solvents that cannot mix with one another (immiscible), each containing solutes that  are ‘like-minded’. The separatory funnel was invented just for this purpose, so that chemists can shake two immiscible solvents together to achieve liquid-liquid separation, and drain/separate the two immiscible solvents (phase) with ease. In the lab, that’s usually water  (aqueous phase) versus a hydrophobic organic solvent (organic phase) like petrol, ether, chloroform etc. Unlike the Soxhlet extractor, no one really knows who invented the separatory funnel, but it is thought that its design gradually evolved and improved over the 1800s. Another aspect of liquid-liquid extraction is that it gets more and more effective via subsequent re-extractions, so it’s common practise for chemists to shake their separatory funnels twice or thrice with fresh solvent to achieve more efficient extractions. In our experiments, we will be using the separatory funnel to separate alkaloids or  acidic plant toxins like carboxyatractyloside by acid-base induced liquid-liquid separation. Here in the demonstration below, I will show you how I separated chlorophyll out of a water extract using n-butanol (oil-based solvent) as the immiscible phase. We will definitely be seeing the separatory funnel frequently in the future.

           

Glass column for column chromatography

Full setup of preparative, vacuum assisted column chromatography I performed in the lab.

Number 3, the column chromatography. Our previous two methods allow us to obtain a crude extract from plants, but they can hardly go any further. It is this most powerful method called chromatography that allows us to purify and isolate an individual component out of hundreds if not thousands in a plant. Chromatography deserves its own article, but for now you’ll think of it as a kind of molecular racing competition. Imagine we have a crowd of people, how do we separate them quickly? By chromatography, we make the crowd run on a racing track. Different people have different sizes, strength and shoes, so everyone runs at a different pace. If we control the cut-off point or racing time carefully, we can separate different people out of a crowd. We can even tailor the racing track to retain certain people more slowly than others, or allow some to run faster. It is this interaction between different molecules (c.f., people) with a chromatography medium containing a solvent (c.f., timer) and a stationary matrix (c.f., running track) that enables separation of a mixture down to individual components. The column chromatography is one that uses a glass column to hold its chromatography racing tract. Column chromatography was first invented by a Russian botanist Mikhail Tsvet in 1906 to separate plant pigments, but it now used by chemists all over the world to separate all sorts of chemical mixtures. The glass column itself can be used to hold various stationary matrix (racing tracks) like silica-gel, alumina, Sephadex, ion-exchange resins etc, and it can be as thin as a pencil or as thick as a human thigh. Occasionally, extra air-pressure or vacuum can be applied at either ends of the glass column to make compounds separate faster. The separated compounds drip out of the glass column tap and we call the process elution. The elution is controlled by different solvent mixtures and/or cut-off times, which means that different fractions of eluents are collected at different time intervals. Some of which may still be a mixture, others pure compounds. We can re-chromatograph impure fractions under different conditions to further isolate pure compounds. In the real laboratory, I conduct column chromatography almost on a monthly basis, and as you can see it’s pretty neat and sophisticated (picture above). However, in our lay-people centered experiments, we will only be doing simple column chromatography containing simple stuff. Hence, we will make do with a tiny glass column and if it’s too short, we will be using a long graduated burette stuffed with cotton at the tip. We will explore its full procedure in the future. For now, just bear in mind that we will need a long glass tube to carry out molecular running competition. 

 

Simple distillation glassware, from left to right, receiving flask adaptor, condenser, and distillation flask with thermometer.

Number 4, the distillation setup. This is one of the apparatus that you may think you can get away with, but soon realise it’s all too important when needed. Regardless if your extract or fraction comes from the Soxhlet, separatory funnel, or column chromatography, it will always be dissolved in a solvent. We will have to remove this solvent to obtain a solid solute. Sometimes the solute can even react with solvent, causing its degradation. We can let the solvent evaporate overtime, or over a large surface but that risks oxidation or further degradation of our precious extract. Furthermore, many chemical solvents are expensive and hazardous, so you will want to recycle them and avoid releasing them into the air if possible. In the lab, we achieve this by using a very efficient equipment called the rotary evaporator (see video below). But now that we are doing it kitchen chemistry style, we will have to distill our solvent away drop by drop. In a way, it’s satisfying and meditating to watch.  A simple distillation setup is all that’s needed, a thermometer, two flasks, a condenser and some adaptor joints to connect things up. In fact, the process of distillation is one of the first chemical separation method that was discovered in the age of alchemy. What’s most remarkably is that it is still performed today, perhaps by you and me later!

That’s all for today, and next time we will be looking at some of the most important laboratory techniques before we start our experiments.