One problem we found in large volume injection is the significant vacuum being generated.  If one cap seals better than the other you will have this random jumps in injection volume.

You can try using pre-slit caps, or don't use the septum at all.

Also you can try lower draw speed. 

Hi Marcus,

I suggest you perform a full maintainance of the autosampler, i.. e. replacing the parts you already mentioned, maintaining the injection valve andt the metering device. I have attached some relvant Pdfs that you can download from Agilent so that you can find the relvant par numbers. The  metering device ("sample syringe)" is responsble for the correct sample volume. It uses a piston that is the same as the ones in the pump. In the manual from page 98 there is the procedure how to maintain it.

Viel Glück!

Holger

Did you try reinstall the software program.  The software may not work well. 

Thanks Holger for your valuable answer, I guess I'll include changing the piston from the injection valve. I'll post if the steps worked out later on so that everyone can use this as help for the future.

 Buy PM kit and consult engineer concerned. Let him fix everything. Do not open anything, it may hamper your system.

One problem we found in large volume injection is the significant vacuum being generated.  If one cap seals better than the other you will have this random jumps in injection volume.

You can try using pre-slit caps, or don't use the septum at all.

Also you can try lower draw speed. 

Thanks for that information Gang, really appreciate that. I'll try that additionally.

I still need to maintain the unit, since in the past we never had problems with huge injections. We use the same vials and cap types since years (we ordered a huge batch, so the caps are from the same).

We have had the problem described by Gang on a LaChrom machine with rather soft polymeric tubing in the sample line, but never on an Agilent 1100 which has stainless steel on the inside (unless you have changed tubing). And as Marcus says, they never had these problems before.

I would say that the most important choice regards whether to do maintenance yourself of have a skilled technician come service your instrument; This choice depends more than anything on your own confidence in being able to accurately perform maintenance. If you're confident in your own skills, then Holger's suggestions are good. If you're not, then you should be aware that you may be causing more harm than good by messing with a delicate piece of equipment.

That being said, I'd probably recommend you to go at it yourself anyway - It's a rewarding learning experience even if you should mess things up ;) And then you can call in the service engineer ;)

           The problem is most likely that the syringe loses its seal during the time it is drawing sample into the loop. The fact that the thing works well at low volumes makes me suspect the syringe of a slight misalignment of the syringe in its drive, or that the plunger and syringe barrel dont seal at high extents of withdrawl.  The needle seems to still be in place, so that is an unlikely suspect. Most software makes the same mistake over and over, so that is less likely the root of the problem. Connections at the syringe to needle and the syringe itself are the most likely suspects. If there is a way to observe it in operation, that may reveal the problem immediately.

This entire approach to a precision sampling  device is flawed by the assumption that the metering step is a precise operation, and by the fact that there are a lot of parts wetted by the sample which must be cleaned to prevent sample carryover problems.. 

    I have designed autosamplers for HPLC and GPC, and found that the best precision is not made in this style design. It is not an appropriate design to be used with samples of significant viscosity, with very volatile solvents, or short  sample cycle  times. 

ps Why such large sample injection volumes? Sensitivity limitation? 

Hi Scot,

thanks for your points. The sample I analyze is in a very low concnetration, it is a drug which was uptake by a hydrogel (20 µl) and then degraded in 1 ml collagenase to release the drug. I freeze-dry the sample and dissolve it again in 1/10th of the volume to upconcentrate the sample. The injected amount is still in the range 5-10 ng (~ 0.02 nmol), so I need to inject a lot to get a signal (luckily it has a strong absorption at a certain wavelength, so this is enough). Because I knew that there was an issue with the autosampler, I decided not to inject the whole 100 µl or 95, but 80. Still, it was a problem...

I mean, some samples were injected with the right volume, some not. Randomly.

Hi Marcus: A few comments as I have been designing analytical instruments for decades and servicing the G1313A and G1329A/B series since they were introduced in the 1990's. These A/S are some of the best and most reliable on the market. Most any issues with inj volumes not being reproducible are always attributed to poor training OR lack of system maintenance.

Maintenance should always be addressed first by someone properly trained to perform the services. The Rheodyne brand injection valve's rotary seal (and associated parts) is the weak point here and should be changed regularly. Needle seats eventually wear out too (only replace if they are damaged or leaking), but needles should last many years (unless abused or bent). The loop is fine and should never need service. The metering system ("syringe") is a high performance pump unit rarely needing service (seals on rare occasion and a new piston even more rarely, unless high concentrations of buffers are used and the system poorly cared for). BTW: This autoinjector design is completely different than the type Scott describes above. No glass syringe is used. No piston is in contact with the "syringe". In fact, this SS high pressure injector is a pump with a floating piston (no contact) and is part of a fully flushed flow path so carryover is almost impossible. It is a unique design to Agilent. Once the autosampler/injector has been fully evaluated and repaired (if needed), it needs to be formally tested to verify it operates to meet or exceed the original manufacturer's specification. This is critical to verify and also make note of how the injection volume effects the accuracy too (normal for all A/I).

Correct operation of the hardware/software and sound training in the fundamentals of chromatography are the next areas to address. Highest accuracy is usually found within the middle of the 0.1 - 100 ul injection range (~10-90ul), not the extremes (0-10, 90-100ul). *This optimum range varies per manufacturer. Liquid viscosity also has an effect on the accuracy too as thick samples require very slow aspiration rates. The draw rate is something that is adjustable directly through the software menu, but the default values often work well for 99% of samples.

One statement that you made which concerns me is: "For example, if I have 83 µl in my microvolume insert and it is doing 80 µl injections, some of the vials are nearly empty after analysis (that's how it should be!), but some are not (10-50 µl left in the insert)!" I hope that I have misinterpreted your initial statement, because it implies that you are trying to inject 80ul of sample from a vial containing only 83ul of total volume. If this is true, then we have found the problem. You can not accurately aspirate nearly the same amount of liquid that the container (vial insert) contains. You need to have at least 10% or more extra volume in the vial to insure a good filling each time. You should NEVER have an empty or near empty vial after injection. Always fill the vial with an excess of liquid. Choose the vial type and form based on the available volume of liquid and the injection volume. Next, for large volume injections, always leave the vial cap a bit loose on the vial and not tightly secured. If the cap is too tight, then a vacuum may be created when the sample is aspirated. This results in a lower volume of liquid being aspirated into the needle/loop. Leave the cap a bit loose and this will not happen. This is something that I have taught in my training courses for decades and has solved a lot of problems. One caution with this. If the vial contains volatile organic solvent(s), then the liquid could evaporate over time resulting in samples (or stds) appearing to be more concentrated with each injection. Samples should be freshly prepared and vials kept closed when not being used.

Attention to the above details will eliminate many injection reproducibility problems.

Here is a link to an article which may be of interest to anyone servicing or maintaining an HPLC injector: "Troubleshooting HPLC Injectors (Manual and Automated) "; http://hplctips.blogspot.com/2013/06/troubleshooting-hplc-injectors-manual.html

Hi Bill,

thanks a lot for that really great answer, I will keep that in mind. I usually close the vials quiet tight (we sometimes use 5 or up to 30% AcN/water as solvent), so this may be the main problem, not the injector as initially expected. This would also explain the problem that the injection volume changed randomly, some vials may were not so tightly closed as others.

Thanks again to all researchers givining such great input! I'll try the suggestions as soon as I have a bit more time and post some results.

Hi all,

sorry for the late reply. It took me some time to find out what really was the problem with the injector and I had some other lab issues in between...

First of all: The injected volumes are right again!

Now what did I do:

A) changed sapphire piston of the injector, changed rotorseal of the injector and stator face assembly. I guess changing any of the parts was not really necessary at the end, but I'll come to that in the next few sentences.

B) Tried different "cap screw tightness" from tight, to medium, to lose (tested 5 vials each) for the maintained injector unit. For all tested conditions, injection volume varied again, not improvement.

C) By accident, a collegue found a recommendation from Agilent, how to set up the sampler (unfortunately I can not find it anymore but maybe someone could add this for complete information). Because we use microvolume inserts, the recommended draw position for that is 3 mm. The needle will not get in touch with any plastic of the insert, preventing from forming a vacuum (but 4-6 µl will not be in reach of the needle then; e.g. for 1 µl injection there need to be 8 µl in the insert at least).

D) I checked our gradient programs. The draw position was always set to 0.0 mm - the needle may (nearly) touch the bottom of the insert and depending on the tolerance of the plastic insert, sometimes a vacuum is formed, sometimes not.

E) Did injector test with 3 mm draw position for different "cap screw tightness" from tight, to medium, to lose for 80 µl injection (100 µl in insert). For all conditions, injection volume was identical!

Conclusion: it may not have been the hardware parts, a simple check of the draw position may be enough. Problem solved, I guess ^^'

Anyway, in the future I will keep the vials closed not so tightly to be on the safe side.

Thanks again for all your valuable answers!

Thank you so much for updating us on your progress and glad to hear things are working again. :)

Yes, keeping the caps a bit loose is usually a good idea for all samples. Esp when large volumes are aspirated.

Based on your observations, I suspect that you may have had a couple of different issues cause the variability seen. Inserts can be very problematic because of variation in dimensions and also the fact that they MOVE during the injection process. This movement can be responsible for errors, but normally is not an issue when the draw amount is small relative to the amount contained in the vial (not the case in your example). We have used the solid glass inserts with flat bottom with great success vs the conical shaped plastic vials (this may or may not be an option for you). The flat bottomed glass vials move much less during the injection process keeping the needle in the same relative position.

Hi Bill,

as always, very good answer. I will think about the glass inserts when the plastic ones are gone, we still have plenty of them. I guess the pricing will be much greater for the glass inserts... could you provide a link for that inserts anyway, please?

And yes, the inserts move during injection, that's right. I never thought that they have such huge variability creating that error, but I'm happy I learned this now :)

Marcus: We use wide mouth 2ml vials (12 x 32mm, 9mm diameter) in our A/S. The inserts come from a number of different manufacturers. We usually have flat bottomed glass inserts with 250ul or 400ul volumes (sometimes the 400's are actually 440ul volume). Agilent sells these same glass inserts in conical and flat bottomed versions, but of course their pricing is about 2 or 3x what it is from other manufacturers so I suggest you compare pricing from your local laboratory supply house first.

Thank you Dr Binner and Dr Letter, your solution is of great help to my analysis.

I've been having issue with getting reproducible intensity of my OPA Amino Acids analysis, turn out it was because of incorrect drawn volume from the needle due to the use of conical polymer inserter.

Just this afternoon, as soon as I changed my injector program of "DRAW" command with a needle offset of 3 mm, it effectively produced the intensity I intended for my analysis. I can't believe what have been bothering me for so long (wasted so much precious time and chemical) was due to the use of inserter.

Following Dr Letter's advice, I did also unscrew the capped vials a bit to improve the drawn volume accuracy. Will test again soon for reproducibility of the results.

Thank you all. I wish I had discovered this post soon!

Hi Chee,

Great to hear that we could help you, it's very rare these days that people come back to say 'thank you'. It was pretty much only William's experience which also helped me out of the misery. I can guarantee you that with unscrewing the caps slightly, this will further improve the reproducibility especially when you do kinetics measurements from only one vial and several injections, there will be no vacuum. However, I always do that even if I only take few µl from only one vial.

Marcus