How do you do manual injection?

[zimanli]

Dear pros,
I am doing manual injection and have very bad reproducibility. Could you please tell me your precious experience and tips in doing this. Is fast the most important?

ziman

[skunked_once]

ziman,

One key to reproducible results using manual injection is to practice your technique and do the injections exactly the same each time. Other factors such as solvent, type of syringe, GC setup (split or splitless), etc. are also important. If you click on the LC-GC logo on the top of the page you will find an article titled "Syringes for Gas Chromatography" under "GC Connections" which will give you much useful information. Good luck!

[chromatographer1]

Review the availability of a Chaney Adapter. This inexpensive tool will improve most analysts technique.

best wishes,

Rod

[Peter Apps]

Hi Ziman

No, fast is nowhere near the most important. Do not try to copy the action of an autoinjector.

Assuming that you have all you GC conditions set right, here is an extract of the notes for a course that I give on Basic Capillary GC:

Loading the syringe with sample

There are three practically useful ways of loading a microsyringe with sample:

1. Sample fill:

rinse the syringe with solvent by drawing up and ejecting to waste at least 6 times.

draw more than the required volume of sample into the syringe and eject to waste at least three times.

keeping the tip of the needle in the sample, draw up sample and expel it quickly to get rid of air bubbles. If the bubbles do not go away, you probably have a leak between the needle and the syringe barrel.

move the tip of the plunger to the mark, take the needle out of the sample, wipe the outside of the needle, draw the sample up into the barrel, inject.

Because some sample evaporates from the needle the volume injected is always more than was drawn into the barrel. If you really want to know how much was injected draw the sample back into the barrel with the tip of the plunger on a mark, read where the sample meniscus is (if you have an air cushion read both ends of the sample plug), inject and then draw the residue into the barrel, put the tip of the plunger at the same mark and read again.

2. Solvent flush

If the actual volume of sample injected has to be a particular measured amount, then use solvent flush injection:

draw 1 l of solvent into the syringe with no bubbles,

draw the plunger back 1 l to form an air gap

draw in the required volume of sample with no air bubbles

draw the plunger back to pull the sample up into the barrel

inject

If anything goes wrong you have to start all over again.

3. Air cushion:

rinse the syringe with solvent and return the plunger to the zero mark.

draw the plunger back to the 2 l mark to draw a plug of air into the barrel.

put the needle into the sample and draw the plunger back to the 3 l mark, withdraw the needle from the sample, draw the sample up into the syringe barrel, wipe the needle.

inject

Note that this is actually a solvent flush injection with less solvent and more air, the pressure in the inlet compresses the air cushion so it is very difficult to be certain how much sample gets injected. This technique is not recommended.

Injection

The most robust manual injection technique is:

insert the needle through the septum

when the syringe barrel touches the injector nut start counting 1,2,3

depress the plunger quickly (not sharply)

count 1,2,3

remove the needle from the inlet.

Remember to rinse the syringe with solvent immediately after each injection.

Do not bother to try a manual version of a fast, cold needle autosampler injection.

As long as samples and standards are injected the same, the actual injection volume does not matter. This means that samples and standards must be in the same solvent and must be injected by the same operator. Never inject different volumes of standards and samples.

Consistency is the key to repeatable injections.

Enjoy !!!!

Peter

[zimanli]

Thank you so much guys. I printed your responses for referrence. They are very useful!

best wishes
ziman

[CE Instruments]

Puzzled, 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

[Peter Apps]

Hi CE

Depending somewhat on the solvent and analytes I would agree that a hot needle injection with the sample drawn up into the syringe barrel(which is what I was trying to describe) is the most likely to work well.

Nonetheless, Agilent disagrees - the default on their autosamplers is a fast cold needle injection. This often works fine, but it is impossible to do reliably by hand, and when people try to copy it they get very poor results.

Peter

[Victor]

A distinction needs to be made between injection with a fast autosampler and manual injection.

With a fast autosampler, the residence time of the needle in the injector is extremely short, and the needle does not have time to heat up. Thus the sample is expelled into the injector as a liquid plug, and there is (in theory) no possibility of discrimination against high boiling compounds.

With a manual injection, it is not possible to replicate the situation in an autosampler. The needle heats up, and at least some of the sample exits the syringe needle by boiling out of it, causing possible discrimation against less volatile constituents. In the case of manual injection, it may be better in this case to pull the sample into the syringe barrel (so that the needle is empty), stick the needle in and wait for it to heat up, then push the plunger. The theory behind this operation is that if the sample is going to distil out of the syringe needle, it is better if it distils out of a hot needle.

These instructions are guides, but of course there are a lot of variable-e.g. how hot the syringe needle gets in the injector, what the exact temperature is in the injector (could vary from instrument to instrument).

It may be possible to get round these things by the use of an internal standard, if you can find one that replicates the sample behavior-not always easy.

[CE Instruments]

Nonetheless, Agilent disagrees - the default on their autosamplers is a fast cold needle injection. This often works fine, but it is impossible to do reliably by hand, and when people try to copy it they get very poor results.

Why is it that some people believe these people
I have seen customers get better results by changing to a decent autosampler that does support "hot empty needle" type injections
The orginal 7673 sampler only did fast injections, a marketing man said this is the best because this is how we do it. I understand that the newer samplers also offer slow injection as fast is not always good. Those who bought CTC A200s samplers and other better samplers could choose the best injection method.

[chromatographer1]

How true it is that fast injection autosamplers are not the best, marketing to the contrary.

Often putting a solvent plug behind the sample is useful.

Slow injection, timed injection, and air/solvent plugs, timed entry and exit of syringe, plunger speeds .... these are factors which determine the best injection procedure for the application at hand.


And an air plug or solvent plug in a manual syringe fitted with a Chaney adapter is rarely bested.

Just my two cents worth.

Rod

[Russ]

One person I knew would "pump" the syringe several times in the injection port after making the injection. It must have been correct, he was made the lab manager. Fortunately, we were using packed columns.

[fisio625]

Hi Zimanli

If you are using with high split ratios, you have to use split liner. It is a very important parameter for the analysis. Check your liner.

Thanks

Savas
SEM Lab. Co.
Ankara/Turkey

[Bruce Hamilton]

I agree that the solvent flush technique, as described by Peter Apps, is preferred for most situations. I also agree that consistency is the most important aspect for precision. It's important to practice and obtain an idea of your actual precision on each instrument, and with different solvents.

I prefer to have a reasonably quick insertion of the syringe and only 1-2 seconds before depressing the plunger, and I usually wait only 1 second to windraw the syringe when analysing samples in volatile solvents. I increase the times with solvent boiling point. Works for me, but ymmv.

I would emphasize that, for removable needle syringes, be very careful not to force the plunger down too quickly or too hard, as it's possible to overwhelm the spring that provides the seal on some models of syringes. That very brief vent will give very variable results, sometimes obvious by slight changes in retention times.

Please keep having fun,

Bruce Hamilton