Chromatography is a technique used by scientists throughout all disciplines; many different separation techniques exist, each catered to a specific type of analyte. That’s because the principle behind it is simple, separate a mixture of compounds based on their physical or chemical properties. These techniques can be very broadly classified by their ‘mobile phase’, either liquid or gas.
So you’ve synthesized your molecule, done your work-up to obtain your (somewhat maybe) pure substance, and a quick FTIR scan shows that yup, the functional groups that you tried so hard to incorporate into your structure are actually (somewhat maybe) there, hooray! What’s next?
Separation is achieved by passing a mixture through the an inert adsorbent known as the stationary phase, which separates the individual compounds based on their physical interactions with it. Remember thin layer chromatography in high school (it’s not been that long!) when you spotted marker ink and watched as its components rose up a silica plate immersed in ethanol? It’s the general principle applied to all chromatography techniques!
Chromatography of a given substance will usually provide reasonable evidence of purity if uniform separation results, that is, if it doesn’t decide to elute with other random bits and pieces (adsorbents, solvents, by-products that should have been removed during work-up). It is also possible to identify molecules based on its rate of migration, as this is based on its interaction with both the mobile phase and the stationary phase. Sometimes the random bits and pieces that aren’t your analyte interferes with the separation and causes the whole chromatogram to misbehave, such as if it adsorbs more strongly to the stationary phase.
This is just one of the reasons why choosing the right technique for your separation is crucial. Without any direction as to where to begin, it can be difficult to choose parameters that will work for you the first time round. If you’re designing a method from scratch, be prepared for lots and lots of trial and error!
As the name suggests, liquid chromatography (LC) gets its name from the mobile phase that is the solvent and carrier of your compounds. Anything that dissolves can therefore be studied in a LC system, but endless possibilities optimizing the makeup of your mobile phase can really mess with your head (gradient flow, pH buffering, polarity calculations, detergents and stabilizers if you’re working with large proteins…).
LC employs the use of a column containing a solid stationary phase that interact with your sample and mobile phase. These columns can be packed with polar (normal phase) or non-polar (revered phase) material, usually chosen to be the opposite of mobile phase. More specialized columns exist, such as size exclusion and ion exchange columns which – coupled with the ability to collect eluted fractions – make LC systems extremely versatile.
LC systems are usually fitted with a refractive index (RI) detector, which measures the difference between the RI of the solvent with whatever it is that is passing through. Higher sensitivities can be attained using a UV-Visible detector, which can analyze the absorbance of the elutions at a certain wavelengths (very useful for proteins due to aromatic amino acids – tyrosine and tryptophan – absorbing UV light at a wavelength of 280 nm). There are a whole range of detectors available in the market but if you’ve got the budget, a mass spectrometer detector coupled with the versatility of LC systems are able to separate and analyze just about anything!
Gas chromatography (GC) relies on the use of an inert gas mobile phase to ‘carry’ compounds through the system. This means that ionization of the sample to its gaseous phase is required, which immediately rules out larger, less stable molecules from being analyzed using GC techniques as they are more prone to fragmentation (read: proteins). Having an inert carrier gas means not having to worry too much about mobile phase interactions and flushing the system afterward, and compared to solvents used in LC, is much cheaper in the long run.
GC systems also use columns as stationary phases, but these are not ‘packed’ but rather consists of a liquid film either chemically bonded or coated on the inner wall of a column3, and should be selected based on the application to be performed. Similar to choosing the mobile phase in LC, column selection is based on the general chemical principle that “likes dissolves like.” A non-polar column for separation of non-polar compounds and polar columns for polar compounds.
Just like with LC systems, you can attach any piece of detector equipment at the back of your GC system (most systems nowadays even allow for two!) But what’s this? Already popular with the analysis of small, organic molecules, GC scores even more points with the flame ionization detector (FID) – basically a cheaper mass spectrometer that can pick up hydrocarbon signals by burning whatever comes out end other end of a column in a fireball of hydrogen. Of course, you could just go and buy that mass spectrometer detector that you’ve always wanted… Or not.
There are a whole range of combinations available out there, but it all boils down to what you are trying to analyze. Because at the end of the day, all you really need is separation and identification, just like how you managed to visualize the components of ink on that silica plate in a jar not TOO long ago…
Featured image of a packed column credited to Andra Mihall (Flickr)
- Peck, R. L. (1950). Characterization of organic compounds. Analytical Chemistry, 22(1), 121-126.
- Snyder, L. R., Kirkland, J. J., & Dolan, J. W. (2011). Introduction to modern liquid chromatography. John Wiley & Sons.
- How to Choose a Capillary GC Column. (n.d.). Retrieved March 31, 2018, from https://www.sigmaaldrich.com/analytical-chromatography/gas-chromatography/column-selection.html