Chromatography Forum

Hello,
We have a Triplus liquid autosampler hooked up to a Thermo GC Ultra, which we use for fast GC analyses of FAMEs. Unfortunately, about 10% of the samples run in any given sequence do not inject properly. After extensive (and tedious!) testing involving watching autoinjections and adjusting parameters, we have identified the problem as cavitation/bubble formation during the final sample draw-up prior to injection. What we actually see is an air bubble lengthening out from the end of the plunger in the syringe barrel during the sample-draw up step. We have optimized the system as far as possible to prevent this, using the following optimized injection procedure:

Syringe: 10uL 80 mm fixed-needle syringe from SGE with cone tip (23 gauge)

Sample: dissolved in hexane; 50-200uL volume in 1-2mL glass vials closed with 11mm crimp-caps (seal = PTFE/blue silicone).

Injection procedure:
1) 3 x wash with 5uL solvent A (hexane) with draw-up at 10uL/s
2) 2 x rinse with 2uL sample with draw-up at 10uL/s
3) 5 x bubble elimination pumping; 5uL/s strokes with 0.1s delay between strokes
4) Sample draw-up. 1.5uL drawn up at 1uL/s, then 0.5uL expelled at same rate back into sample vial to give final volume of 1uL in syringe. 2s delay before syringe is removed from vial for injection.
5) Inject using rapid injection technique (100uL/s, no delays; using Focusliner containing glass wool).
6) 3 x wash with 5uL solvent B (hexane) with draw-up at 10uL/s to end injection cycle.

We have tried slowing down all the sample and rinse solvent aspiration speeds to 0.1uL/s, but this hasn't helped. We have also changed syringes 4 times, thinking the needle may have been partially blocked or the plunger poorly fitted to the barrel, with no improvement. In all cases the end of the needle is several mm away from the bottom of the vials, so sealing against the bottom of the vial is unlikely. All samples are also particle-free in hexane; we get the same problem with pure standards. Vacuum formation in the vials shouldn't be happening as the solvent volume is 10% or less of the total vial volume.

Why this cavitation is occuring is a complete mystery to me; especially as it only happens on sample draw up, even when the solvent AND sample rinses jsust prior to sample draw-up are fine! And yes, the end of the needle is well below the sample level in all cases; in all cases, re-injection of a suspect sample gives a normal injection and trace.

I would really appreciate any suggestions on how to eliminate this problem. The only solution I can think of is to use a slightly larger gauge needle and/or a needle with a gas-tight design (PTFE in plunger) to minimize possible blockages. Any suggestions would be most welcome.

thanks
Tony

Hi Tony

I have not used a Triplus, but this is probably a generic autosampler problem.

There are two ways to get a bubble up against the plunger tip; cavitation and a leak around the plunger. If the bubble is due to cavitation it usually shrinks quickly and may even disappear at the top of the plunger stroke. A cavitation bubble would almost certainly disappear when the 0.5 ul is injected back into the vial.

A metal syringe plunger is sealed to the glass barrel by a film of solvent held in place by surface tension and viscosity. After the solvent rinse the film is hexane, after the bubble elimination pumps it is sample. Although the slow uptake rate of sample gives a higher pressure in the syringe, it also gives more time for the sealing film to creep out of the way of the air leaking down past the plunger. The change in composition of the sealing film and the slow uptake rate are why you only get a bubble on the final fill.

The easiest solution to the problem is the one that you suggest - a teflon tipped plunger.

Peter

Dear Peter,
Thanks for you reply. Yes, it does appear to me to be a metal plunger/glass barrel sealing problem that would be solved by a gastight teflon-type seal. These types of syringes are available, but I was wondering what their lifetime is like compared to the metal/glass varieties - I would assume shorter, but how short?


thanks
Tony

One other thing - the sample and rinse solvents I use are all hexane, so there is no solvent change. Someone suggested to me that I could use a more "wettable" solvent like methanol in place of hexane for the pre-injection rinse. I think I might try this before I go investing in diffferent syringes!

Hi Tony

I'm not sure that teflon tipped plungers wear out any quicker than metal ones. The teflon is softer, but it slides more easily on glass, and can take up some wear, which metal cannot. In my experience bent needles, blocked needles and jammed plungers all kick in before teflon needle seals start to leak.

Although you do not change solvent, your samples have FAMES in them which probably change the mechanical properties of the film just enough for it to start leaking.

If you are going to try a different solvent, and you are not interested in short-chain FAMES that elute early in the chromatogram , I would go to heptane or octane which are much more viscous than hexane, and would give a better seal.

Peter

Tony,

Do you have other GC injector such as Agilent 7683? I have been using Agilent injector for years and do not have problems on bubble formation and the syringe used is only metal plunger without Telfon tip. If you have one, you may simulate the default injection sequences (injection speed, sample pumping, etc) on your Triplus sampler and see if any improvement can be made. Also I experienced carryover problem by using Telfon tipped syringe for injecting some compounds.

Strange problem
From your description this does not occur on sample washes only on actual injections. The formation of a bubble near the plunger indicates either air ingress past the plunger or evaporation of the Hexane.
Possible cures/suggestions
You do not mention use of an air gap after taking in the sample ? In my opinion taking an air gap into the syringe of about 1ul should solve your problem and would of course also ensure that if the sample is coming out of the bottom of the needle that this loss would not occur Use of a "Hot Empty Needle injection technique should give better results too Although the recommended syringe length for Splitless injections is 80mm I know of plenty of customers who have not noticed any adverse effects of changing to a 50mm needle. Again recommended with Hot empty needle injections.
Regards Richard
CE Instruments Ltd

Richard,
Wonder if you could elaborate more on how "hot empty needle injection technqiue" works. Thanks.
chhubert

Re-posted from the GC post listed as manual injection
I was always taught the Hot empty needle technique was best for both manual and automated injection

Filling the syringe is nicely covered however I would always draw the whole of the sample into the barrel after pushing out the excess sample. The sample plug will now be in the barrel of the syringe and so you should not get evaporation from the needle. I would then insert the syringe needle through the septa, wait 3 seconds for the needle to reach injector temperature and push the plunger down. Note keep your finger over the plunger so the head pressure in the injector cannot blow it out of the barrel

The theory. Discrimination occurs due to poor transfer of the sample from the syringe and reactions of compounds to poorly designed injectors and to the metal of the syringe. Having taken the sample into the syringe barrel pre evaporation does not occur. When you push down the plunger the samle reaches the hot needle and starts to evaporate. The gas formed cannot go back up the syringe as you are pushing it down so it is forced downwards. As liquids evaporate from the surface a solvent barrier of evaporated solvent shields the compounds as they are forced rapidly out of the needle into your injector stopping most disrimination in the needle and hopefully driving heavy compounds out of the syringe into the injector before all the solvent has gone and they can stick in the needle. Always works for me and can be duplicated by most autosamplers. Just my $0.02

Hot empty needle technique does not work with fast GC, because the peak widths are so narrow (1-3s). Thus a 3s delay in the injection port whilst waiting for the needle to heat up means residual sample in the needle vaporizes onto the column forming early-eluting ghost peaks. This is why I use the rapid injection technique into a liner containing glass wool for fast GC. Using an airgap after the sample is taken up has no advantage in this case.
I have tried using an airgap with the rapid injection technique in case sample was "falling out" of the bottom of the syringe after sample draw-up, but this didn't fix the failure to draw up sample, which still occured in about 10% of cases. Filling with a rinse solvent first (hexane), then the sample, also did not improve this statistic.
I spoke to one of the Thermo service engineers who deal with GC autosamplers, and his experience was they always use methanol as the wash solvent because this keeps the working parts wetted (and thus sealed) in metal plunger-in-barrel syringes. It is this wetting that is crucial in preventing leakage around the barrel during sample draw-up. This is a problem for us, since our samples are in hexane. I've since tried replacing the wash solvents with methanol, but because it is still necessary to rinse the syringe with sample (in hexane) prior to an injection, the methanol in the syringe is replaced by hexane and we are thus still seeing a 10% failure rate in injections due to air bubbles during sample draw-up. I am therefore now going to test a gastight (teflon plunger seal) syringe to see if this fixes the problem.

Hot empty needle injections work with all GC methods. Why wait 3 seconds when 1 is enough ? Any trace of sample left in an empty needle should not be enough to cause problems.

Why are you not following the FAMES application note
Fames

For this application, the TRACE GC Ultra is configured
with a SSL injector, the Ultra Fast option (including the
analytical column), and a Fast Flame Ionization Detector
(FFID) featuring 6 ms as the detector’s response time to
signal and acquisition frequencies up to 300 Hz. Such a
high speed is, in fact, a compulsory requirement for the
correct acquisition (15-20 points/peak) of the extremely
narrow peaks (approx. 100ms PW1/2h) typical for this
type of chromatography [2].
The column module, connected to the Split-Splitless
injector and the FID detector as a removable accessory, is
completely and directly controlled by the instrument local
user interface and electronics. The same Gas
Chromatograph has been used to perform the same
application in conventional mode with the easy removal of
the Ultra Fast accessory.
All the samples have been injected through the
TriPlusâ„¢ Autosampler (Figure 2) using the liquid band
formation technique [3,4], automatically selectable on the
control software. This technique implies the presence of a
plug of glass wool in the middle of the liner, in order to
retain all the liquid and enable the beginning of the
evaporation process from a single droplet.
The wool also plays an important role in preserving
the column’s efficiency: the non-evaporating by-products,
eventually present, remain trapped on the wool layer
without entering the column. The use of glass wool is
strongly suggested only for samples that do not contain
thermo-labile components, which could undergo
degradation catalyzed by the wool itself.

References3. F. Munari, S. Trestianu in Proc. 4th Int. Symp.Capillary Chromatography,
Hindelang Germany, R. E. Kaiser (ed), Hüthig, Heidelberg, 1981, p. 349.
4. K. Grob, M. Biedermann, J.Chromatogr. A, 2000, 897, 237-246.

Dear CE Instruments,
I was not aware of that application note; perhaps you could give me an html link or send me the PDF? In any case, it applies to ultrafast GC, which uses the special heated module, not fast GC as we are using, which uses a narrow bore column in the normal GC oven. However, despite this, I accept that the injection paramenters suggested will be similar. I am using glass wool, but not using the recommended air-gap in the syringe. In theory, there should be no difference between the hot-empty needle technique and rapid injection into glass wool as ways to get the sample on column in a split injection. Both techniques promote full sample vaporization and so minimize discrimination. It follows that cavitation will be a problem with both techniques as it occurs during sample draw-up, so subsequent air-gap draw-up is irrelevent if the sample has not be taken up correctly.
I can assure you that using an air gap results in ghost peaks early in the chromatogram, and not using an air gap removes this problem. I have tried many, many variations of injection techniques over the last month (hundreds of runs) and in all cases rapid injection without an air-gap gives the cleanest chromatograms. It might be possible to get around the ghosting problem by using a lower start temperature on the column, which would promote a degree of analyte focussing prior to separation. However, this would negate the point of fast GC; at the moment I am achieving 5 min run times with full resolution of all 37 FAMEs in the Supelco 37 FAME mix using a 10m x 0.1mm BPX70 column. This requires a start temperature of 150 degrees; lowering it to between 50-100 degrees to promote focusing doubles the run times and so negates the advantage of fast GC. I'm sure this might be posible with ultrafast GC which has much higher possible temperature ramp rates, enabling a lower starting temperature. However, this won't work on my system.
I have found one other piece of information that may shed more light on the original issue of 10% missed injections due to air bubble formation/cavitation. It seems the default syringes and needles we are using and recommended by Thermo (80 mm fixed needle from SGE) have quite a narrow ID - 0.11mm according to SGE. However, the removeable needle variety of the same syringe comes with 0.15mm ID needles, which should help prevent cavitation on sample draw-up. We could also move to shorter needles. For split injections, as we are doing, the recommended setup is a 50mm needle and glass wool in the liner that surrounds and wipes the needle on entry/withdrawal. However, I think there has been a design fault somewhere in the manufacture of Focusliners from SGE for Thermo instruments, because the centre position of the glass wool is 50mm from the injector end of the liner. However, the actual maximum depth that a 50mm needle can travel into the liner is only 43 mm because of the extra height of the septum and septum mounting materials!! Thus a longer needle is required to actually get to a 50mm depth in the liner, which is why we use 80 mm needles.

Tony

Hi Tony

Back to the original problem.

If the lab with the GC in it is cooler than the lab where the samples were prepared and put into the vials it is possible that you have a slight vacuum in your sample vials which makes the syringe more prone to leak during draw up.

It is a bit of a long shot, but it has happened to me.

Peter

Peter's reply made me think of an even longer long shot - try cooling some of your samples in the fridge before injecting. If the samples are warm due to a hot lab or positioned over a very hot oven I have seen vapourisation of the solvent in the syringe barrel during filling.

Regards,

Ralph