The Ins and Outs of Chromatography

What is chromatography?

Chromatography is a scientific process in separation science, the part of chemistry concerned with the separation of compounds and mixtures. Generally speaking the two significant categories are preparative and analytical.

Analytical chromatography uses smaller sample sizes and the primary objective of separating compounds is to identify their components. A good example is in a lab environment where you are searching for toxins or pollutants in a sample.

Meanwhile preparative work uses large quantities with the aim of removing impurities from samples to prepare them for use outside the lab. Pharmaceutical production is a good example of preparative chromatography.

Who discovered chromatography?

A Russian botanist named Mikhail Tswett unearthed Chromatography in the early 1900s after discovering that ground-up plant material extracts produced different coloured solutions based on the type of solvent used in the extraction process.

Tswett’s breakthrough came through experiments involving plant extracts poured through a glass tube packed with calcium carbonate powder. Tswett discovered that as the liquid extract passed through the powdered calcium carbonate bands of colour appeared in the tube. Individual compounds had separated from each other as the powder (a solid) interacted with the liquid flowing through the tube.

The word itself is derived from the Greek words for colour (chroma) and writing (graphe).

What is chromatography used for?

Chromatography is actually one of the most valuable processes available to chemists. It can be used for anything from crime scene investigation to identifying biological matter. Advanced chromatography is used in bomb detection. It can be used to determine how much pesticide is on your fruit skin, or if an athlete has been using a banned substance. Here are some other uses for this this important chemical process:

• Determining the’ heat’ of a chilli pepper.
• Ebola Immunisation – while no immunisation yet exists, chromatography was important in creating Zmapp, and continues to play a role in the investigation of this disease.
• Quality assurance – alcohol brands like Jaegermeister use chromatography to ensure sugar levels remain consistent throughout the product cycle.
• Meat testing – remember Britain’s horsemeat scandal back in 2013? Chromatography was instrumental in determining fact from fiction after other tests proved inconclusive.

What can’t chromatography do?

Like all experiments, there are limits to what chromatography can tell you. You won’t find the secret recipe to Coca Cola through chromatography. Just like the components of wine don’t tell you the process by which it was made, recipes and mixtures go through a number of preparation processes that can’t be reverse engineered through chromatography with a high degree of accuracy. You can find out the basic components of a cola soft drink, but not necessarily a specific recipe. In the case of coca cola, caramelisation of the sugars plays a huge part. Still, chromatography is one of the best methods for discovering compound components within the separation sciences.

Essentials of chromatography

At its most basic the chromatographic process consists of a stationary phase and mobile phase (also called an eluent). As in the early example above, the stationary phase consists of a solid, thick liquid or bonded coating that remains fixed in place. The eluent moves through the stationary phase, and is comprised of either a liquid or gas.

A sample compound (called an analyte) is then added to the process. As the eluent travels through the stationary phase the analyte should react with both the mobile and stationary phases.

Chromatography is essentially like a running race. The chemical compounds are the runners and stationary phase is what separates the runners. At the beginning, they’re all at the starting line. As they cross (elute) the finish line one after the other we can identify each runner (chemical).

Why should the analyte react with both phases in a chromatographic process?

If the analyte doesn’t react with the stationary phase then it will travel at the same speed as the eluent and leave the process (elute) at the same rate as the eluent. Similarly, if the sample doesn’t react with the mobile phase, it will remain in the process with the stationary phase. Neither is ideal.

Different types of chromatography

While the type of chromatography described above most closely resembles liquid column chromatography, there are actually many ways to attempt a chromatographic process.

Paper chromatography

You might remember this experiment from school science class – what happens to ink on paper when you make it damp? The process is reasonably simple, take some filter paper and place a spot of ink close to the edge. Hang the paper with the blotted side facing down. Now dip in a solvent like alcohol or water.

The solvent will travel up the paper via capillary action and dissolve the ink. The solvent is the mobile phase, the paper the stationary phase. The ink is the analyte and as it travels up the paper with the mobile phase it separates into different components. Depending on the type of ink these components might be visible. If they are not, you can use a developing fluid to bringing out the separate colours.

Column chromatography

Column chromatography uses a vertical glass cylinder for the stationary phase, packed with silica gel or some other highly absorbent solid. Mobile phase (eluent) is pumped at high pressure into the stationary phase.

Thin-film chromatography is a type of column chromatography where the column is a film of plastic, metal or glass.

Gas chromatography

Gas chromatography uses gases for the mobile phase. Components of the sample are heated and vaporised in the machine, with a neutral gas used for the eluent (hydrogen or helium). As each component elutes, it pases an electronic detector which identifies it and prints a peak on the chart. Gas Chromatography is sometimes called vapor-phase chromatography (VPC).

Gel permeation chromatography (GPC) or Size exclusion chromatography (SEC)

ATA Scientific offers  a number of GPC/SEC  systems for determining molecular weight, where multi-detection is the key method of measurement. Multi detection is capable of collecting more comprehensive data in a single experiment by using a series of detectors which includes light scattering, RI and UV detectors, and viscometer detectors.

The OMNISEC Advanced Multi Detection SEC/GPC is a gel permeation and size exclusion chromatography system designed for discovering characteristics of natural and synthetic polymers, copolymers, proteins, nanoparticles and other macromolecules. It combines Refractive index, UV VIS absorbance, light scattering and viscometer detectors to measure concentration, molecular weight and intrinsic viscosity with unparalleled accuracy. The OMNISEC can also be connected to any third party GPC/SEC system to enhance existing capabilities.

The Viscotek SEC MALS 20 Detector is a multi angle light scattering detector used for measuring absolute molecular weight and  oligomeric state of proteins, independent of retention times. The Viscotek SEC MALS 20 offers up to 20 angles and a unique vertical flow cell to ensure optimum performance and accuracy.

Find out more

As you can see chromatography can be extremely valuable and is used for a variety of applications.

You can find out more about ATA Scientific’s Chromatography Products here.

To view a recent webinar, visit this site here.