Question on MS/MS techniques

[Rndirk]

In GC, all else being equal, larger molecules elute after smaller ones, so adding deuterium to a molecule makes it elute later, not earlier. This also applies to breakdown products, metabolites etc.

Peter, I have to correct you on this one. I can show plenty of examples where the deuterated molecule elutes earlier than the non-deuterated component in GCMS.

It would indeed seem logical that the increased molecular mass makes it elute later. But deuterated compounds, compared to the same non-deuterated molecules show less affinity towards the stationary phase. If you combine both effects, it elutes earlier

I'm intrigued. Can you give one example - what kind of compound and the analytical conditions.

Peter

Check this link, it shows a variety of compounds: phenols, pesticides, PAHs,.. where each deuterated compounds elutes before the non-deuterated one.
http://www.restek.com/Technical-Resour ... l/env_A020

For the rest of the discussion: I have serious doubts if the effect you're talking about has anything to do with the mass spectrometer itself. But I can be wrong. I think the difference in retention we observe is due to less affinity between the compound and the stationary phase, if the only difference between 2 molecules is a couple of hydrogens replaced for deuteriums.

Think about this: if there's a difference in retention due to higher mass, why does C-13 labelled components elute at the same time as their C-12 analogues?

One more thing: It wouldn't make sense, but if i'd run a solution with for instance napthalene and napthalene-d8 on GC-FID, i'm sure there would be 2 peaks (hardly seperated; depending on the conditions). No mass spectrometer in the equation.

[Peter Apps]

All of the labelled compounds are aromatic, with the deuteriums on the benzene ring, so I would guess that their presence affects pi-pi bonding to the phenyl in the stationary phase. Above my pay scale I'm afraid.

Peter

[Peter Apps]

Think about this: if there's a difference in retention due to higher mass, why does C-13 labelled components elute at the same time as their C-12 analogues?

One more thing: It wouldn't make sense, but if i'd run a solution with for instance napthalene and napthalene-d8 on GC-FID, i'm sure there would be 2 peaks (hardly seperated; depending on the conditions). No mass spectrometer in the equation.

Do they elute at the same time, or are the peaks simply not resolved ? What would you see if you ran that data through deconvolution to look at the labelled and unlabelled ions separately ? You could even do this by overlaying the the two SIM or extracted ion traces.

On the other hand, if there is no difference in retention due to mass, why does a C11 alkane elute after C10 ? As long as you do not have a phenyl phase, my prediction is that a separation of naphthalene, naphthalene d8 and methylnaphthalene would have the naphthalene d8 about halfway between the other two.

I am also sure you would see two peaks on an FID, which order they come out in is the critical question.

Peter

[Rndirk]

Think about this: if there's a difference in retention due to higher mass, why does C-13 labelled components elute at the same time as their C-12 analogues?

One more thing: It wouldn't make sense, but if i'd run a solution with for instance napthalene and napthalene-d8 on GC-FID, i'm sure there would be 2 peaks (hardly seperated; depending on the conditions). No mass spectrometer in the equation.

Do they elute at the same time, or are the peaks simply not resolved ? What would you see if you ran that data through deconvolution to look at the labelled and unlabelled ions separately ? You could even do this by overlaying the the two SIM or extracted ion traces.

On the other hand, if there is no difference in retention due to mass, why does a C11 alkane elute after C10 ? As long as you do not have a phenyl phase, my prediction is that a separation of naphthalene, naphthalene d8 and methylnaphthalene would have the naphthalene d8 about halfway between the other two.

I am also sure you would see two peaks on an FID, which order they come out in is the critical question.

Peter

It's true the examples in the link i posted are aromatic compounds. But i see the effect for other compounds as well. A nice example (I can't post the data right now) is malathion-d10 vs malathion, an organophosphate pesticide where the d10 is decently separated from the target in retention, but not in MS. The molecule loses the part which contains the deuteriums during EI. It's counter intuitive but it works well as an internal standard.

The point i was trying to make with saying there's no difference in retention due to mass, was about the mass spectrometer itself. If you have a molecule which is non-labelled, a deuterium labelled and a 13C - labelled given that the deuterium and 13C have the same m/z, they are likely to show a difference in retention only due to the GC.

It's an interesting discussion, it would be nice to here some other insights as well.

[Peter Apps]

This is one of those problems that looks different according to whether you think that an MS is a fancy detector for a GC or HPLC, or that a GC or HPLC is a fancy inlet for an MS.

Retention time in chromatography clearly refers to the time a peak takes to elute from the column, and this is nearly always much longer than any of the processes in a detector. In an MS the peaks leave the column as they enter the ion source, so anything that happens in the MS is strictly not part of chromatographic retention - although as MAForensics observes and as is well known in GC-MS where peaks are sharp compared to scan times, it can affect the retention time that is shown in the results.

Although scan skew is still the most likely and most parsimonious explanation for the perfectly repeatable detection time differences that MAForensics sees it occurs to me that an electrospray interface is another place where particles of different masses might move at different speeds, but the distances involved are so short and the speeds are so high that I doubt the difference would be measurable on the timescales that we are talking about.

As you say, we could do with some other inputs.

Peter

[Rndirk]

I agree with you, but not on the part where the answer to the OP's question is MS related.

I have googled around a bit now and it surprises me that the effect this topic and discussion is about is not very well documented. A number of studies however, refer to this as the "isotope effect in chromatography".

In the abstract of this article you can read that the difference in retention has to do with (small) differences in inter- and intramolecular forces. The bond length of C-D is shorter than C-H. I think this effect has a similar explanation/mechanism in apolar GC columns and RPLC. Polar columns might show the opposite.

You'll see more difference in GC compared to RPLC because there's generally speaking a higher plate count.

In chemistry we learn that isotope-labelled compounds show the same chemical behavior. However, the closer you look ...

This is one of those problems that looks different according to whether you think that an MS is a fancy detector for a GC or HPLC, or that a GC or HPLC is a fancy inlet for an MS.

I belong to the first group. Is that a good or a bad thing ?

[pepterpol]

I am just a rookie and don't want to interfere in the conversation of professionals but...isn't best way to check influence of detector on the retention time just by disconnecting column and direct injection of deuterated and non-deuterated analyte.

Great discussion.

[Peter Apps]

I am just a rookie and don't want to interfere in the conversation of professionals but...isn't best way to check influence of detector on the retention time just by disconnecting column and direct injection of deuterated and non-deuterated analyte.

Great discussion.

Brilliant ! with no chromatographic retention and a flat-topped profile to analyte concentration the only effects are those due to the MS, and since a quadrupole MS has constant ion production and analysis the only time-modulated process that could give rise to a difference in detection time is the scan.

How about it MAForensics ? - you are the one with the LC-MS. Do a driect infusion of analye and labelled standard and see if you get the 0.01 min time difference.

Peter

[Rndirk]

I'm skeptical about which data you could produce by infusing that proves or disproves theories launched in this topic.

How about doing the same chromatographic run but with a UV detector (for example). I expect to see 2 (hardly separated) peaks.

Edit: the difference in retention time in this particular analysis will not be enough to see 2 peaks in HPLC-UV..

[pepterpol]

If not direct infusion than maybe simple connect autosampler with nebulizer using capillary (peek/stainless steel), 30-40 cm of tubing shouldn't give you chromatographic separation but you will see something like peak.

[Peter Apps]

Edit: the difference in retention time in this particular analysis will not be enough to see 2 peaks in HPLC-UV..

Got it in one; chromatography, especially liquid chromatography, does not run at the time scales of the differences in "retention" we are discussing.

Peter

[lmh]

... and in any case, when you're talking about a quadrupole instrument, what do you mean by the time the ion arrived at the detector anyway? There is no magic connection between the time that's displayed as the retention time and the time a bunch of electrons finally made it down an EM horn. It is up to the manufacturer of the instrument to supply that time.

Important bit: at this time-scale, in a quadrupole, The values you are looking at are not measured, they're made up.

Say you ask for scans from m/z 100-1000 over 0.1sec.
The instrument starts scanning at 1.0min and does its stuff. The software produces a single spectrum from this scan. What's the time associated with the spectrum? Some manufacturers would simply report a single time when the spectrum was completed (or started, or something). Others might try to estimate the time that ions of a particular mass were arriving. If the instrument scans high to low, then m/z 1000 arrived at 1.0min, while m/z 100 arrived 0.1sec later. That has absolutely nothing to do with the masses of the ions, or their deuteration or anything else. It's down to what scan time you asked for. If the instrument scans low to high, it's the other way round.

To be honest, this is all completely irrelevant provided you've chosen a sensible scan-speed for your chromatography. If retention-time differences shorter than the scan speed are of interest, then you are stuffed - you have an inadequate idea of your peak shape if this has happened.

The influence of isotopic labelling on retention time is a whole different area. Yes, it exists. But it's usually a very small effect. If it weren't, then isotopically-labelled compounds would be bad internal standards. If you're going to worry about it, you should also worry about the relative rates of different fragmentations as influenced by isotopes.

[Peter Apps]

viewtopic.php?t=5162

The ions that are enhanced on the rising front of the chromatographic peak reach their maximum before those that are enhanced as the peak drops. Consequently the ions that are enhanced on the upslope show earlier retentions than those on the downslopes.

Peter

[GOM]

What a fascinating discussion

IMH (like this one), Rndirk and Peter have made some interesting comments

Totally outside my area of expertise but ---

Always enjoy reading forum replies and learning new things with interest.

Regards

Ralph

[James_Ball]

Yeah, I am strictly talking about quadrupole and triple quad MS here.

As I've always understood it, chromatographic separation should be the same for any analyte and it's deuterated analogue because they chemical properties of the two molecules are the same, they only differ in mass.

.

Running volatile organics I have a retention time of 8.553 minutes for Chlorobenzene-d5 and 8.588 for Chlorobenzene. It is not always true that a deuterated molecule will elute at the same time as the non-deuterated analog. The difference here is the difference in hydrogen bonding forces. Deuterium has weaker bonding forces so the deuterated analog elutes first. The difference in time here can not be explained by quadrupole residence time as the peaks are actually chromatographically separated. I don't have any of my deuterated/non-deuterated pairs which elute at the same time. 1,4-Dichlorobenzene-d4/1,4-Dichlorobenzene is 12.049/12.085 minutes 1,2-Dichloroethane-d4/1,2-Dichloroethane is 4.448/4.534 minutes. Hydrogen versus Deuterium cause differences in bonding, physical size, boiling point and many other things which affect chromatography.

If you inject the two without a column then depending on scan direction you would have a lag to favor either heavier or lighter.

MS/MS can depend on how you have the experiment setup. Does it always perform the analysis in mass order? If you tell it to perform a transition of 357/150, then 200/57, then 400/310, you will always see the results in that order since it will send only one mass at a time through the first quad, into the cell then through the second quad to the detector. Then it will process the second set and then the third. If you make the dwell 1 second, then all of those ion pairs will arrive at the detector one second apart which is 0.0167 minutes difference. If you scan for 20 ion pairs with such settings then the first pair will elute 0.33 minutes before the last pair, if you use 100ms dwell then it will be 0.033 minutes separation, so it will definitely not hold true for MS/MS systems.