Optimal Conditions for CSR_LVSI

Discussions about GC-MS, LC-MS, LC-FTIR, and other "coupled" analytical techniques.

33 posts Page 2 of 3
LALman wrote:
I am dealing with it here trying to switch 8270/625 analysis to 100ml extractions and LVI, we have to make sure the the whole process is cleaner since any source of contamination makes a 10x larger problem than before. It isn't as simple as extract less, inject more, which is what the accounting people want. When I went from 1uL to 5uL that began to be apparent. But as you say it really came home when I went up to 25uL.

I'm having a problem with spit peaks with this 25uL injection. My solvent peak goes from 1 min to 3.25 min. The void time for my column is 32 seconds. So I am injecting at 60C, purging the inlet at 1 minute, also ramping 60C/sec to 80C. Then holding 80C until 2.75 minutes.

After a few tries its apparent...
Its because my column flow is 2.5mL/min and the pressure pulse at 24psi makes a flow of 5.4mL/min during the pulse. With 1uL and 5uL injections under these conditions, peak splitting was not apparent. But with 25uL the peaks for C18-C25 are splitting. So, I have tried the whole run with the same 24psi pressure pulse and then a flow of 5.4mL/min. This shrank my solvent peak down to 2 minutes so I made appropriate timing adjustments. The peaks are no longer split and are almost exactly 5X the area of the 5uL peaks.


Peak splitting after LVI points towards your solvent boiling dry on your (pre)column in different spots. This can be due to bad wetting behavior of the solvent film on your column, or (too much) solvent being evaporated just too fast to consistently boil down nicely into one spot.

Personally I use solvent vent when working with LVI to reduce the amount of solvent in the inlet down to a couple of µL to avoid this. But if I understand correctly, you work in splitless mode. The drawback of solvent vent is that you have more parameters to optimize (injection time, vent time, vent flow), and you risk losing low boiling point analytes.
What do you mean "solvent vent"? I'm injecting splitless for 1 minute, then purge flow to split vent is 100mL/min for 4 minutes then gas saver to 50mL/min.
Solvent vent is injecting in split mode at a temperature around the boiling point of the solvent. The aim is to blow off solvent while keeping analytes in the inlet, after which the inlet is heated and it turns into splitless. You need a PTV inlet for that.
Solvent venting is done with a normal LVI injection port like the Agilent MMI(multi mode inlet) or the Gerstel CIS inlets, which have temperature ramp rates of up to 700C/min.

With solvent vent you cool the inlet, inject slowly in vent mode, then go splitless and rapidly heat the inlet to transfer the analytes to the column, then go to split mode to clean out high boiling junk and excess solvent. It works good once you have all the parameters optimized.

For CSR-LVSI, you want a large enough guard column to contain the solvent in a thin film with the oven set below the boiling point of the solvent, then rapidly heat to above the solvent boiling point and hold until the solvent passes through the column then resume normal temperature ramp to separate analytes. You may need longer or larger diameter guard ( some use 0.53 guard with 0.25 analytical column for this). Also you need to make sure the polarity of the deactivation layer on the guard column matches the polarity of your solvent. Polar deactivated for methanol or water solvent, intermediate polarity for things like ethyl acetate and non-polar deactivation for solvents like hexane or DCM. This allows the solvent front to form a film instead of droplets inside the guard column for better solvent effect.
The past is there to guide us into the future, not to dwell in.
These are my parameters, while injecting 25uL. Trying to get even height/area for all 16 peaks C10-C25 peaks in my test mix. I'm getting C10,C11,C12 rather high then level for the remaining peaks. Not sure how long to hold purge vent closed and whether to hold pressure after purge vent opened or not or how long.

Carrier Gas Hydrogen, constant flow 5.4mL/min (except during pressure pulse)
guard/pre-column: 5m of 0.53 deactivated silica Rxi guard column
analytical column: HP-5ms 30m x 0.25um x 0.25um column
Inlet Temperature: 280C
FID Temp: 350C
Samples are in n-hexane B.P. 69C
Syringe: 50uL SGE 50R-HP-0.63
Liner:Topaz 4.0 mm ID Precision Inlet Liner w/ Wool

Pressure Pulse: Trying 24, 48, and 60 psig.
Inlet Mode: Splitless for 0.5-1.0 min then purge vent at 100 mL/min for 4 minutes

Flow Program: 2.5mL/min (hold 3.25min) to 5.4 mL/min at 4 mL/min/min then hold 5.4mL/min till end of program.
Oven Temperature 60C (hold 1.0 min) to 80C at 60C/min (hold 2 min) ~3.33min total. Continue Program: to 320 @ 20C/min (hold 3-4 min) total time ~19 min

Edit: The liner volume is 986uL. So, when I do a 24 psig pressure pulse (5.4mL/min) for one minute, the liner is swept ~5 times.
....
You are getting solvent condensing as a film on the wide-bore precolumn. This gives a focussing effect that can deliver razor sharp peaks and very good repeatability if the solvent film is stable and it evaporates steadily from its upstream edge, Unfortunately you are doing everything you can to break up the film and makes it formation and evaporation eratic.

Reduce the carrier gas flow to the optimum for the column, and leave out the pressure pulse, it is rapid gas flows that shear your solvent film into separate liquid lenses that get blown along by the gas. You can also reduce the inlet temperature, you do not need to follow flash evaporation in the inlet with condensation and re-evaporation on the precolumn at only 60C, and you do not need to a rapid transfer of your high-boiling analytes to the column because they will be focussed by the solvent film and the stationary phase anyway. You can increase your splitless time to 2 or 3 min to better stabilize gas flows while the film is evaporating and to get all the high boilers onto the column at the lower inlet temperature.

Solvent film focussing can work very well indeed, but it is very temperamental and vulnerable to small changes in conditions, so you need you method to be as simple as possible.

Peter
Peter Apps
I normally end the pulse if I use one about 0.1minutes before I open the split vent, and the initial oven hold is about 0.1 or 0.2 minutes after the split vent opens.

I look at it a little differently in that those high boilers are going to have trouble getting into the column so I adjust the temp up until I get good response on the high boilers. Then adjust split vent timing sooner until you see loss of analytes. This helps optimize temperature and timings. Of course adjust pulse and oven hold to match when adjusting the split vent timing. It can take a while to get it dialed in and it will still work even when not at optimum, but I would watch the peak shapes more than the absolute peak heights overall.
The past is there to guide us into the future, not to dwell in.
James_Ball wrote:
I normally end the pulse if I use one about 0.1minutes before I open the split vent, and the initial oven hold is about 0.1 or 0.2 minutes after the split vent opens.

I look at it a little differently in that those high boilers are going to have trouble getting into the column so I adjust the temp up until I get good response on the high boilers. Then adjust split vent timing sooner until you see loss of analytes. This helps optimize temperature and timings. Of course adjust pulse and oven hold to match when adjusting the split vent timing. It can take a while to get it dialed in and it will still work even when not at optimum, but I would watch the peak shapes more than the absolute peak heights overall.


Thank you for the pulse vs split vent timing suggestion, I will try that. I'm especially interested in making sure the result is evenly balanced from C10 to C28 in light of finding out my UV/Pest hexane has a cluster of volatile peaks just preceeding the C10 peak. I don't want the results to be weighted to the lighter end of the DRO spectrum where I have this blank interference.

Peter:

I was just re-examining the CSR_LVSI post that I was using as a starting point. I was using the RESTEK EZGC Method Translator to compare that setup to mine. That is how I originally came up with the pulse pressure of 24 psi because it corresponds to the 5.08 mL/min initial flow in the CSR_LVSI post if you have a 60C inlet temperature. The method translator from the CSR_LVSI suggests that the time optimal flow be 2.5mL/min. 5 mL/min on the EZGC flow calculator corresponds to 24psig at 60C. My 6890 however, says that 24 psig pulse is 5.4mL/min and that is where I got that number for my runs.

So, its a choice between ramping flow down after 0.5 min in the original method or a short 24psig pulse for 0.5 min. Ramping the flow might be less disturbing of the condensed phase as it evaporates. So, I will give that a try.
Don't do any flow, ramping, pulsing or any other fancy stuff. Set the linear flow to the column optimum and leave it there. The more changes you have in conditions the more there is that is not going to be precisely repeatable from run to run, and repeatability is critical to using solvent effects on condensed films in smooth-walled pre-columns.

Peter
Peter Apps
James_Ball wrote:
That is the syringe I am using, except with a 23gauge needle to be compatible with a Merlin Microseal (can't use the dual taper needles with Merlin or Gerstel/Agilent septumless head). They seem to work well, but you will want to take the plunger out ever once in a while and wipe it down with a kemwipe and solvent as it gets a little sticky over time, probably from dust getting into it at the top.

I have to replace a needle because I wasn't paying attention and installed a cyclo liner upside down :roll: The needle didn't bend surprisingly but it did rough up the tip enough that it will cut the septum or Merlin if I try to use it.

That needle really does get sticky easily. A couple of days running and I came in to find it frozen in the barrel. Took quite a bit of gentle sonication of the barrel and wiping of the needle to make it smooth again.

I'm using the red high temperature septums and the vials have the red PTFE/rubber lining. I am still tracking this signal that shows up after a few punctures of either the autosampler vials or the silicon/PTFE septum 40mL VOA's that I used to hold my standards.

At the moment, I am thinking maybe it is the silicon rubber from the VOA vials getting into the standards after I puncture the vial to make dilutions.

I got an excellent cal curve from 10 ppm to 10,000 ppm DRO yesterday. But the surrogate peaks are fronting very strongly for 50 ppm. I think my column does not have sufficient stationary phase to handle the concentrations this syringe can put on the column.
LALman wrote:
James_Ball wrote:
That is the syringe I am using, except with a 23gauge needle to be compatible with a Merlin Microseal (can't use the dual taper needles with Merlin or Gerstel/Agilent septumless head). They seem to work well, but you will want to take the plunger out ever once in a while and wipe it down with a kemwipe and solvent as it gets a little sticky over time, probably from dust getting into it at the top.

I have to replace a needle because I wasn't paying attention and installed a cyclo liner upside down :roll: The needle didn't bend surprisingly but it did rough up the tip enough that it will cut the septum or Merlin if I try to use it.

That needle really does get sticky easily. A couple of days running and I came in to find it frozen in the barrel. Took quite a bit of gentle sonication of the barrel and wiping of the needle to make it smooth again.

I'm using the red high temperature septums and the vials have the red PTFE/rubber lining. I am still tracking this signal that shows up after a few punctures of either the autosampler vials or the silicon/PTFE septum 40mL VOA's that I used to hold my standards.

At the moment, I am thinking maybe it is the silicon rubber from the VOA vials getting into the standards after I puncture the vial to make dilutions.

I got an excellent cal curve from 10 ppm to 10,000 ppm DRO yesterday. But the surrogate peaks are fronting very strongly for 50 ppm. I think my column does not have sufficient stationary phase to handle the concentrations this syringe can put on the column.


What is the film thickness of the column? You may need to go thicker to improve loading.

Have you tried using vials with the Mini-inert caps? I use them on either 12ml amber vials or the 5ml conical bottom reaction vials to store my standards. The combination of septa and teflon slide valve reduce evaporation and I haven't had trouble with contamination.

If the inlet septa are a problem you could try using the straight 23 gauge needle with cone tip and a Merlin Microseal. I was using that with mine until I inserted the cyclo liner upsidedown and the rough tip tore the microseal :( I need to get another microseal. If it ever begins to leak just use a syringe and flush it with methanol by placing the syringe tip just against the top of the seal and pushing the solvent through the duckbill seal. Be sure to use a flat or conical tipped needle.
The past is there to guide us into the future, not to dwell in.
LALman wrote:
I tried the new syringe and with 25uL injections, I see about 5X the trash peak intensity that I had with 5uL. Suspecting solvent...

I put a 2" square of Parafilm in 4mL of hexanes and sonicated then centrifuged the result. Then I diluted that another 80X (50uL/4mL) and shot it for reference. The peaks I am seeing are not parafilm. The worst two peaks are at C22 and C24 decreasing from there. But Parafilm rises to a peak at C29 and C30.

Pest grade acetone gives a clean run! So, now I know this it a solvent contamination issue. I suspect my dispenser but I am checking my reagent bottles now also. It appears that UV/Pest grade Hexane fresh from bottle, has baseline trash before C10 and between C11 to C12 but higher peaks are nearly clean. So, dispenser is contaminated, darn it to heck.

Fisher Pest Grade Hexanes used for O&G and redistilled gives a clean baseline run with a single very very small peak between C25 and C26. I used a steric acid and hexadecane spike for O&G. Neither of those could be a fit. But this is clearly clean enough to do DRO work! Cleaner than the UV/Pest grade right out of the bottle.

So, what is happening is apparently septum bleed. This... Example of Vial Cap or Injection Port Septum Bleed is what I am seeing. I think its the vial cap material contaminating the contents of the vial if the vial is reanalyzed a few hours later. I am using standard 11 mm crimp seals with Teflon/red rubber septa. If I wait a few days and check again, the pattern is stronger.

I've been reusing std vials during method development and this bleed ruins the response of everything below about 250 ppm.
James_Ball wrote:
What is the film thickness of the column? You may need to go thicker to improve loading.

Have you tried using vials with the Mini-inert caps? I use them on either 12ml amber vials or the 5ml conical bottom reaction vials to store my standards. The combination of septa and teflon slide valve reduce evaporation and I haven't had trouble with contamination.

If the inlet septa are a problem you could try using the straight 23 gauge needle with cone tip and a Merlin Microseal. I was using that with mine until I inserted the cyclo liner upsidedown and the rough tip tore the microseal :( I need to get another microseal. If it ever begins to leak just use a syringe and flush it with methanol by placing the syringe tip just against the top of the seal and pushing the solvent through the duckbill seal. Be sure to use a flat or conical tipped needle.
I'm using a 30m x 0.25mm with 0.25um film thickness. I recall the RESTEK guy doing CSR_LVSI is using a 1.0um film.

I am using 5mL conical vials with Mini-inert caps for my 100K, 10K stocks but a standard 40mL VOA for the 1000. Then I was diluting directly from those into 2mL of hexane in precapped vials. Apparently puncturing the precapped vials to withdraw the appropriate volume and add the appropriate volume is sufficient to create bleed spectrum. That or its the VOA vial septum contaminating the 1000 standard that I used to make my 50, 25, and 10 ppm standards. So, I'll make up a special 1000 ppm in Mini-Inert capped vial just for making standards. Not sure what can be done about autosampler vial caps.
I remember about 20 years ago we dealt with bad vial caps on our autosampler vials and did a study using the red rubber, red rubber with teflon and also silicon rubber septa. I just don't remember now which one came out the best. I think Agilent makes a low bleed septum cap for vials but I believe they are rather expensive. Might be something to look into.

I am dealing with the same things. Management wants to move to low volume extraction/large volume injection to save costs, but don't like it when I tell them it might involve using more expensive consumables/solvents to make it practical. No one thinks about background interferences scaling up with the scaling down of the process :)
The past is there to guide us into the future, not to dwell in.
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