Chromatography Forum

Dear All,

I am trying to decide what is best to do analysis of lipids in general (not necesarily fatty acids).

One option is HT-GC and the other is TLC-FID. I would like to hear any opnions on these two different approaches to the same problem. Information on selectivity, speed of analysis, costs, etc, will be highly appreciated.

I am particularly curios about the performance of the Iatroscan MK6 instrument

Thanks,

josebenjamin

I used the Mk3? Iatroscan for neutral and polar lipids for a few years in the 1990s. It's excellent for the purpose, which is very different to GC.

The advantage of the Iatroscan ( and TLC ), is that material loaded onto the coated rods can be made visible and/or quantified, whereas GC and LC require the loaded material to elute from the column.

For unknown samples, I would trust the Iatroscan over a GC or LC, especially if you are looking at large or reactive lipids. The instrument is very sensitive, and has to be used frequently and consistently to maintain low backgrounds.

The GC is much more useful for characterising non-polar lipids. For example simplae acetylation and a 5m x 0.1mm x 0.1um capillary will provide MAGs, MDAGs, and TAGs. After interesterification, or other derivatisation, FAME profiles can be determined. For more polar materilas, SPE and/or LC may be the preferred option.

Each instrument serves a different purpose, and I would recommend you review what information you want, and that will decide the instrument of choice.

Bruce Hamilton

Dear Bruce,

I would like to hear more details about the good features of the TLC-FID approach. If the only advantage is the possible visualization of the sample loaded on the road, then why not use simple TLC separations?. Also, if the sample is visualized on the road, that implies that the rods are not reused and therefore an increase in cost.

I also wonder about the speed of analysis. I am not familiar with the Iatroscan instrument, and I think that some time is spent developing the separation, then it has to be dried to remove the solvents, and finally it has to be passed through the flame detector to obtain the results. All this implies time that sounds comparable or longer than HT-GC.

If you can add some details I would like to hear them.

Thanks,

josebenjamin

Dear Bruce,

I would like to hear more details about the good features of the TLC-FID approach. If the only advantage is the possible visualization of the sample loaded on the road, then why not use simple TLC separations?. Also, if the sample is visualized on the road, that implies that the rods are not reused and therefore an increase in cost.

I also wonder about the speed of analysis. I am not familiar with the Iatroscan instrument, and I think that some time is spent developing the separation, then it has to be dried to remove the solvents, and finally it has to be passed through the flame detector to obtain the results. All this implies time that sounds comparable or longer than HT-GC.

If you can add some details I would like to hear them.
josebenjamin

Obviously, the main advantage of the Iatroscan is the use of the FID to quantitate the peaks. I've never met an analyst that can visually quantify, or even use a TLC scanning devic, to quantify lipids as accurately as an Iatroscan.

All of the advantages of FID that make it suitable for GC apply to the Iatroscan. The process is simple, spot your sample on the rod, allow any non-volatile solvent to evaporate ( or even warm the rod ) place a rack of 10 rods in the developing tank, allow to ascend about 10cm ( 10 - 20 mins ), remove. oven dry for 5 minutes, scan on Iatroscan, each scan takes 0.5 - 2 mins (adjustable ), so a rack of ten rods takes about 7-8 minutes. Rerun the rack and rods through the flame to condition them, spot the sample, etc etc.

The rods can be reused hundreds - thousands of times, provided the samples are clean. If the samples contain metals etc, then the rods have to be acid cleaned every few weeks/months. They eventually die, but tender loving care ensures a long life. They are much more expensive than TLC plates, but are easily reusable.

The major issues with the Iatroscan occur because of the rod profile ( which is fused silica with a coating of alumina or silica particles ) . You have to ensure the sample spot is consistent around the rod, and that all rods are stored in same humidity and burned in the FID immediately before use.

The precision can be poor in unskilled hands, as the spot is not deposited consistently, or samples are too messy. Typically the cycle time is about two - three racks/hour, as lipids usually use volatile solvents.

As I siad before, the Iatroscan and GC are different, and are used differently, you can't compare, you have to decide what information you require and choose the most suiatble method. The Iatroscan is not easy to use and learn, but it offers a unique method of quantitating coumpounds that are difficult to analyse by other methods.

Maybe it would be a good idea for you to submit samples for analysis by the vendors of instruments using techniques you want to evaluate.

Bruce Hamilton