Non reproducibility when injections are manuals

[oliverC]

Hello to all,
when I am injecting manually (we haven't got an autosampler and will not have one in the near future), I have different response ratio responses for my compound of interest and my internal standard. This with the same syringe (eg 1.05, 1.16, 0.94).
If I take a different syringe and inject different volumes the same pattern is obtained but with different values and in any order.
I am injecting in split mode at 50ml/min (on a Perkin Elmer Clarus 500).
Temperature of injector 260C, Detector 240C, column temperature 65C for 5min and program to 205C, run time 40min.
The product injected is cineole Oil and the internal standard is Decanol, The program is almost similar to the Pharmacopeia.

We put some glass wool in the injector.
I know the method appear not to be robust, but how can I improve it?
My main concern is the variation in response, that affect the sample concentration result and and fail sometimes the acceptable criterias.
My colleague, has generally some acceptable results arguing the fact that she injects standards and samples with different pressure on the syringe plunger.
If I am lucky I can have acceptable results.
Any suggestion would be appreciated.
O

[zokitano]

Hello Oliver,

I'm also working on PE Clarus 500 and when I'm using manual injection of the sample I get different peak areas for both the analyte and internal standard, but the response ratio between the analyte and internal standard varies within a small range (RSD of response ratios <2%)
You might have problems with retaining of your analyte or internal standard along the GC system because you obtain different response ratios for every injection.
Try to inject blank sample and see if there is any peak on the Rt of your compoments of interest. If that is a case, than you'll be sure that those components probably retain in your GC.

Best regards

[chromatographer1]

The most obvious answer is the problem is variable discrimination in the injector. Are you aware that most manual syringes have a tapered tip?

You spray the sample solution at an angle to the direction of the needle. Think about the implications.

Also, the placement of the column tip within the injector has a great effect on the injector's discrimination pattern.

Lastly, the high injection port temperature combined with active sites in your injection liner or in the head of your column may be causing variable decomposition of your analytes.

best wishes,

Rod

[Consumer Products Guy]

I consider the #1 advantage to autosamplers to be the consistency and uniformity of injection, enabling better repeatability than most operators can achieve manually. Advantage #2 is the increased throughput (working through lunch, after hours, etc.). In that order.

[AICMM]

Oliver,

What liner are you using. Is the decanol an internal standard or a surrogate.

Best regards.

[Bruce Hamilton]

The fact that your colleague is more consistent suggests you have a technique problem. You haven't said what the solvent is, but I suspect it's quite volatile, or whether you are using plunger-in-needle or plunger-in-barrel type of syringe.

In my experience, the critical parameter for all syringes is consistent motion. You must quickly push the syringe in fully and firmly, push the needle down consistently quickly, allow the contents to volatilize, and withdraw the needle rapidly after counting "1, 2", although some people prefer to wait longer, and then withdraw the needle.

Although speed is important, espectally for plunger-in-barrel syringes, don't try to emulate an autosampler, as most of those use rapid, cold needle techniques. Also, don't punch the plunger, or bounce it off the needle at the bottom, as the needle is spring-loaded, and will cause variation. "More haste, less speed" is good advice for working with chromatographs.

I try to have the whole injection completed in 3-4 seconds. If you use a plunger-in-barrel syringe, you could try using the four segment technique to fill the syringe needle volume with clean solvent, then an air gap, then the sample volume, finally another air gap - all visible in the barrel before injection. I prefer that technique for plunger-in- barrel syringes.

The only way you will improve precision is to practice different techniques, and standardise on one that works for you. The best way is to make up a simple standard in the same solvent and practice.

There have been earlier threads that discuss manual injection techniques as well, so a search and review may be helpful.

Please keep having fun,

Bruce Hamilton

[oliverC]

Thank you all for your answers.
Some precisions:
Decanol is the internal standard, diethyl ether is the solvent, 0.6 micl is the injection volume, the liner is fill with glass wool and is for split injections.
My syringe is a Hamilton #801.
Hope these informations are usefull to give me other good advice.
Regards,
O

[chromatographer1]

A Chaney adapter might be helpful in improving your reproducibility.

Is your glass wool deactivated or not?

I would also reduce the injection port temperature. 200°C or even less. 260°C is for steroids, not C8-C10 oxygenated hydrocarbons!

You can try injecting a test solution of toluene and cumene with decanol in ether and see if your injections are reproducible. Are the three peaks stable in relation to each other?

You need to determine if your problems are physical or chemical in getting your analytes to your detector reproducibly. Are you releasing sample as you remove the needle from the septum ? Is your septum leaking after the needle is removed?

best wishes,

Rod