This is a question that I have always asked myself, basically where does all the separation and magic happen in liquid chromatography? Does it come from the column and the chemistries within, is it mostly related to the UHPLC instrument, or is it from somewhere such as the mobile phase? I’ve always thought of the magic occurring on the column if I am truthful. However, when attending some of the recent liquid chromatography user meetings in Europe and listening to some of my colleagues and guest speakers present, it got me thinking; was my initial judgement and bias toward the column correct? Let’s look at the evidence and make a decision.
Wizardry Comes in a Column
As I suggested above, I’ve always considered most of the magic to happen on the column. After all, if you look at it simplistically, the instrument is made up of just a pump, an autosampler and a detector, so what can they do to add magic apart from delivering your sample to the column and detecting the output?
The column wizardry is related to the fact that you have so many different chemistries and options available to you to separate compounds. The most commonly employed columns are reverse-phase columns, but you have other choices too, such as HIC, HILIC, affinity, ion-exchange, size-exclusion, and mixed mode columns, which can separate based on two different parameters such as ion-exchange and reverse-phase. Added to this, you have huge variations in the particle size, pore size and column length and diameter. Then you can add in the effects of different eluents. When all of this is put together you have all the components to separate virtually anything, as today’s modern columns allow you the flexibility to adjust the selectivity to get a good separation. Argument over then, all the separation magic happens on the column, whatever compounds you need to separate you will be able to adjust your column chemistry, particle and pore size, and eluent to get clean, sharp, distinct peaks. Is your UHPLC instrument just a magician’s assistant then?
A Bit of Harry Potter in Your UHPLC Instrument
Like what you are learning?
It would appear that not all the magic is related to the column, even if you have the most magical column. If your UHPLC instrument does not have the performance to extract the magic from the column, then the result will be a poor separation. How does the UHPLC instrument add a little bit of magic then? In a surprising number of ways:
- Injecting your sample – With sample pre-compression before injection, there are no pressure shocks, which can damage your column, but more importantly there is less opportunity for dispersion to occur and hence amazing retention time precision.
- Pushing your sample through the column – When using long columns to increase resolution and with the trend towards smaller particles to increase throughput, you need a UHPLC instrument that can handle the backpressures such situations develop, while ensuring a consistent flow.
- Keeping it warm – You can do a lot of magic by altering the temperature and thermostatting of your column compartment. Adjusting the temperature can change selectivity and also alter your peak shape, as can the heating method: forced air or still air.
- Producing a consistent gradient – It may sound simple, but producing a linear and reproducible gradient is beyond some chromatography instruments. A good, linear gradient that matches the programmed gradient is a powerful separation tool in many applications.
- Minimising dispersion – If you have dispersion in your system, then no matter how good your column is, you will have broad and potentially overlapping peaks. To avoid this, your instrument has to be designed to minimise the chances of dispersion through the entire flow path.
- Making sure you see all compounds in your sample – A great separation is useless unless you can actually detect the compounds. The instrument detector can offer that wizardry by being highly sensitive with a wide linear dynamic range to detect compounds at both ends of the concentration spectrum. For mysterious compounds that cannot be detected by UV, then near universal detectors such as charged aerosol detectors can be used.
Where Does the Magic Come From in Liquid Chromatography Then?
I hope that I have shown you in the argument above that the magic in liquid chromatography comes from both the column and the UHPLC instrument, with also some influence from the eluent. Having a good column and a poor UHPLC instrument and vice versa will lead to sub-optimum results and a poorly resolved separation. It’s only when you combine a high-quality column with a state-of-the-art UHPLC instrument and a well-constructed experimental design that you will get the expected results. It’s also important to note that you require the various modules of the UHPLC instrument to work together and in harmony to get the most magic from your system. I’m sure some of my colleagues would like to argue that the magic comes from the mass spectrometer or the chromatography data system software though!
Where do you think the magic lies?