What is chromatography?
Chromatography is one of the analytical techniques that has been most successfully diversified and applied to different needs; it allows the separation of compounds by their differences in properties such as size, charge, affinity for a particular compound, and affinity for a particular substance. molecule or other property.
Mikhail Tsvet was a Russian botanist who carried out the first deliberate chromatography; separating pigments from plants on a simple column of carbonate of calcium carbonate fine (chalk dust). Chromatography is not, in fact, an analytical technique, but a separation technique.which can be coupled to a detector to carry out an analysis. In theory, this technique consists of 3 elements; mixture of compounds to be separated; the mobile phase which will consist of a fluid that circulates the sample in solution through the third element and; the stationary phasewhich can be a solid or a liquid adsorbed on the surface of a solid. Up to this point, we may not fully appreciate how this is able to separate compounds; or why it has become a prevalent technique in chemical analysis.
Well, let’s look at a street where a group of people, all starting from the same point and at the same time, are of different ages; and let’s say that in this street there are many sportswear shops; technology and pubs, as the group moves down the street, it will be less and less uniform because the younger ones will be amused by looking at shop windows and prices; because they have affinity for that environmentand yet they don’t stop walking or go in to buy anything, whereas older people are more passive and this is not a distraction; they just move on. with little interaction with the environment.
If the street is long enough, we will see that, at the end of it, the original group of people who started out at the beginning has split into groups of people who take more or less time to cross the street according to their age. We will see that first the older ones pass by, then some adults who may have been window-shopping and finally the younger ones who have been talking all afternoon about what they would like to buy.
So this is basically chromatography. The sample, which would be the initial group, is dragged by the mobile phase through the stationary phase, which would be the street. The first chromatographic techniques consisted of separating compounds by their capacity to be dragged by organic solvents; through stationary phases consisting of porous beds of substances with a certain polarity; (this technique would later be called normal phase) thus establishing an environment in which there are two distinct zones; that of the apolar solvent that advances and that of the static porous bed.
Here a strongly soluble substance in the solvent would barely interact with the stationary phase and would be dragged along at the same speed as the solvent advances; and a strongly polar substance would have a strong affinity for the stationary phase and would strongly adhere to the surface of the stationary phase, being barely dragged along by the solvent and taking a long time to advance. Through this, complex parameters such as the polarity or hydrophobicity of a substance can be simplified a priori into a measure of time, which we call, retention time.
Other characteristics of the molecules can also be exploited, such as their size (in the case of size exclusion chromatography) or affinity for a very selective compound (in the case of affinity chromatography).
Example of chromatography
Let us now take an example to imagine how size exclusion works, we can imagine a country road, dirt and very stony. If at the beginning of this path a group of adults and children start walking, we will see that, along the path, the children, smaller in size and smaller in size, will be more likely to be able to foothave more difficulty in moving forward because their shoes get stuck on their feet. get between the stones and get stuck in crevices. between the earth; while the adults, being larger in stature, tread in a surface area greater than that of the hollows in the surface, and therefore do not get stuckand thus progress with less difficulty.
Size exclusion chromatography works in this way, there is a bed of solid, porous particles, like many microscopic sponges, through which a mixture of substances passes; of these the larger substances will not fit into the porosities of the particles and will flow easily between them, while the smaller ones will flow erratically in and out of the porosities of the particles. This technique is used both to analyse and purify a molecule and to estimate the size of the molecule.
Techniques such as electrophoresis, in which the mobile phase is not pumped, but the compounds are propelled by an electric field that makes them migrate from one pole to the other according to the sign and magnitude of their charge, work just the opposite, they are not beds of particles, but gels; continuous mayas of polymers such as polyacrylamide or agarose, which allow smaller molecules to pass through their pores more easily and make it more difficult for larger ones.
In the case of affinity chromatography, we could imagine another group of people starting at the same point at the beginning of the street. In this street there are gentlemen collecting signatures to defend different causes and they ask everyone to support them. These gentlemen will not have much collaboration unless they find someone who is very committed to their cause; and is willing to make regular donations or support a manifesto, then they will be able to support them. they will stop at this point because they fit in so well and they won’t give up on it.until they have effectively helped this cause. After a while there will be no one left on the street of those who started at the beginning except those who are trying to help the cause advocates.
In this type of chromatography, the stationary phase is coated with substances that are able to bind quite strongly to specific molecules, as for example with His-Tag affinity chromatography, biotin or antibody-binding, which is a very effective method of chromatography. do not delay the substances for which they have an affinity, but retain themunder certain conditions of acidity and salinity, but these forces weaken when the environment changes in these parameters, allowing them to leave the stationary phase.
Biotin for example is a vitamin that is strongly anchored by the protein avidin; proteins with 6His fragments bind strongly to the nickel bound in stationary phases and unligates when imidazole is present in the environment; and antibodies can bind molecules against which they have specific affinity. In this way a compound can be isolated with high purity, as we allow any substance other than the one for which the stationary phase is designed to pass through the column and we can unbind it at the appropriate time.
Affinity chromatography
What does Pink Floyd have to do with all this? Well…
Now that we have a good technique for separating substances, we need another good technique for detecting when these substances leave the column; the most common detectors are based on a light source passing through a chamber, through which the flow out of the column passes, and a detector that measures the light received from the source. Thus, when the column flow carries a substance that absorbs a particular type of light, the detector senses this drop in light intensity and registers it.
How it is detected
There are many different types of electromagnetic waves, including visible light, which is made up of colours.
A priori, white light contains all the colours that human sight can detect, well, each material has the property of transmitting the light of certain colours and absorbing that of others; transparent materials transmit most colours, the chlorophyllchlorophyll, the green pigment in plants; it absorbs blue and red light well and converts it into energy, but transmits a lot of green light, which is why we see plants in this colour. All of the above could be very nuanced and some terms could be elaborated on, but this would be enough to understand the detector.
This is important to understand these detectors as they do not consist of a beam of white light as such, but rather, from this beam of light, through a chromator, the colours are separated and the colour to be detected is selected; the one that best absorbs the material we wish to analyse. We also have detectors for “the light we cannot see”, ultraviolet and infrared, which are in fact much more widely used than visible light detectors.
Types of chromatography
Although the main focus here has been on high performance liquid chromatography (High Performance Liquid Chromatography or simply HPLC) there are many variants of this, such as the gas chromatographythe liquid-liquid chromatographythe centrifugal chromatography or the thin layer chromatography. The latter is really simple and accessible and is still valid for some applications. To make it at home, as we said at the beginning, we need 3 elements, a mixture of compounds to be separated, stationary phase y mobile phase.
Home Chromatography
Our stationary phase will be a piece of paper about 5 cm wide and 20 cm long; the mobile phase shall be 96º alcohol and our mixture of compounds will be a dot on the paper which we will impregnate with at least 2 different types of marker pen in different colours (sometimes blue or black felt-tip pens come with 2 different compounds); to improve the result it is advisable to make several quick applications of ink and let the dot dry between them; this way we will have a smaller and more defined dot that will not widen too much in the process. This dot should be made about 2 cm from one of the short sides of the strip of paper.
We take a jar with a height of at least 10 cm, but better if it is 20 cm, we put alcohol in the jar until there is “a finger” of liquid. Now, with adhesive tape, we join the short side opposite to the side where we have put the dot to a pencil, for example. This way we can put the pencil in the mouth of the jar so that our stationary phase hangs from it, although before this we have to check a key detail, that the dot we have applied the ink will not be below the level of the alcohol when we introduce it in the jar, otherwise our sample would be diluted in the alcohol and would not rise by capillarity along the paper.
When the alcohol reaches the point of the sample, we will start to see how it drags the sample through the paper; and a few centimetres later we will see how areas of different colour and intensity start to appear. A trick to make this technique work better is to cut off the peaks of the part to be dipped in alcohol; removing only two small triangles from the ends, as this will help the alcohol to flow more evenly throughout the paper. We can also experiment with different proportions of alcohol and water to see how different polarities result in different forms of entrainment of the compounds in the sample.
And we would have our own chromatography!