We have a great GC question that came in and that is how do you improve resolution in gas chromatography.
That is a great question because resolution means the spacing between the peaks. Remember chromatography is a separation tool.
Step one is to separate. If we can’t separate then we don’t go on to step two. If we cannot quantify. We cannot identify. So separation or resolution is the most important thing in chromatography.
Unfortunately, it’s also a fairly complicated concept. For instance, what causes resolution? What do we need to get resolution? I give a one hour talk on resolution and I think it’s the most powerful talk I give. But let me give you the synopsis of it.
3 Things That Affect Resolution
In order to get resolution in GC there are three things you need. I’ll give you the technical terms. You need the right capacity factor (temperature). You need the correct selectivity (stationary phase). And you need the correct efficiency (column dimensions). So now let me explain what that means.
Capacity Factor (temperature)
The first one, capacity factor. Capacity factor means you have to let the analytes interact with the stationary phase. No interaction, no separation. So the capacity factor is all about the temperature. You need to lower the temperature in your GC enough to make sure the analytes interact with the column.
We have a rule that says the capacity factor should be between one and five. What that means is the first peak should come off twice the time of the solvent front, of the T0. So again, what we’re getting at here is we need to leave the analytes in the stationary phase long enough to get a separation and that means we need to lower the temperature enough for them to interact. So as a general rule of thumb, we drop the temperature by 25 degrees, we double the capacity factor. That’s a rule of thumb. So step one, make sure you’re using the right temperature.
We could tell if it’s the right temperature by how close it’s coming off to your solvent front. If this is a solvent and here’s my peaks of interest. The further we get from T0, the solvent front, the better the separation is. The closer to T0 the worst separation and at T0 by definition we get nothing. So rule number one, move these peaks away from T0. We do that with the temperature. Lower the temperature to make sure they’re retained. That’s the capacity factor.
Selectivity (stationary phase)
The second term is called selectivity. Selectivity is the most complicated term but let me put your minds at ease. There’s a really simple way to address it. Selectivity means, do we have the right chemistry, do I have the right interaction between the stationary phase and the analyte to tell the difference. Can the stationary phase differentiate between my molecules? That’s a chemistry question.
My biggest piece of advice is to either look in a catalog to find the right selectivity, the right stationary phase or let’s just take a guess and let’s use a stationary phase that works for most people. That would be a DB-5, 5% phenyl 95% methyl. Every column company makes one. It’s the go to column in GC. That’s sort of a way to address selectivity.
The alpha term tells us if you have the right stationary phase. If alpha is 1.0 that means you got nothing. Your stationary phase is absolutely wrong. We want to choose a stationary phase that gives us a better alpha.
Again, the easy way to start is to try a DB-5 or look in a column catalog. So that’s the selectivity term.
Efficiency (column dimensions)
The final term is efficiency. Efficiency is a measure of the skinniest of the peak. So if I have two fat peaks that are coming off right next to each other, let’s say these peaks are five minutes wide in a minute apart, well that’s terrible. If I can make the peaks 10 seconds wide and a minute apart that’s fantastic. So the question is how do I make my peaks skinnier. So efficiency is a function of, what I say, the column dimensions.
Long Column – To get more efficiency, which will lead to more resolution, you could use a longer column. Double the column length and you’ll double your efficiency. Resolution goes up by the square root of two. So double the column length, you get 40% more resolution guaranteed. Double column length, get 40% improvement in resolution. It takes longer. It takes twice as long.
Small Diameter – The second thing we do is use a smaller diameter. If we decrease the diameter of the column we increase the efficiency. Peaks are skinnier. Peaks are taller. Better separation.
Thin Film – Along with that we decrease the film thickness. By doing that we also make the peak skinnier.
So the way I remember this is in chromatography I want tall skinny peaks. Tall skinny peaks come from a tall skinny column. Long column, small diameter, thin film.
My go-to column is 30 meters, 0.25, 0.25. That is a fantastic column. You’re talking about 200,000 theoretical plates. A great column to do great separations.
So in a nutshell, there are three things that affect resolution in chromatography.
- Temperature – Lower the temperature to get better separation.
- Selectivity – Choose the right stationary phase.
- Column Dimensions – Use a column with the right dimensions, long column, small diameter, thin film and operate it at the proper flow rate.
Those things will improve resolution hands down guaranteed in all GC separations. Hopefully that helped out. Come back to axion for more tips.