New to Chromatography - Manual Injection Technique

Basic questions from students; resources for projects and reports.

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Hello,

I am new to chromatography, and just started working on a 5890 Series ii with FID / ECD. I'm not sure what to expect as far as deviation based on injection technique.

I found a Hamilton Digital Syringe (with series 700 syringes) on ebay new, but cheap to try to minimize variation in my sample introduction. It measures the fluid to .05ul with a 0.038% accuracy (based on the calibration certificate) and has a plunger brake to help smooth sample introduction. I'm injecting 1ul of sample and have been able to get within <1% deviation when injecting a known reference. Then, out of the blue, 100%, or even 1000% deviation. When I'm injecting and re-injecting actual samples, I can't seem to get a straight answer out of the machine.

The technique that seemed to provide to most reproducibility was to start with a completely evacuated syringe and needle. draw 1ul of sample, leaving the air that was in the barrel as a plunger cushion, then draw up 1ul of air to sandwich the sample in an attempt to minimize discrimination. I am so careful to try to be precise, but my results still seem to vary so widely. I feel that I have eliminated paralaxing errors, am certain that there is no bubble in my measurement, and try to time everything exactly the same each time.

Sorry for noob'ing up the place, but I really am so new that I don't really even know how much technique will sku my quantification, or really even how to tell if my GC in operating within intended parameters. Any tips or advice that is offered will be greatly appreciated. If I end up saying things that don't even make sense, I apologize.. it is because I am just now starting to learn terminology.

Warm regards,

Mike
Presumably you are testing cannabis ?

Are the deviations high or low ?

What are you instrument operating conditions - inlet temp, flow, split, column etc etc ?

Peter
Peter Apps
Its very difficult or impossible to manual injekt completly without deviation.

If its possible you should injekt more than 1µl cause the mistake you can have with one is very high.
How much is your split ?
What kind of liner do you use ?
Is your sample homogenized, clean up ?
What is your extraktion method ?
An error of 100% or 1000% is not likely to be an injection technique error. Unless you are not putting the syringe needle far enough into the inlet. For a manual injection, I tend to insert the syringe needle to the point that the end of the syringe barrel is about 2-3 mm from the septum nut. If it were a volume issue, for a 1000% error, you would have to be injecting 10 uL - and I think that plunger displacement would be noticed.

As long as you are inserting the needle far enough into the inlet - I'd look at the samples. You can have interfering compunds which will elute at the same retention time as the analytes in your standard. In which case, they can be very large. These are common in complex matricies (like plants) when no cleanup or insufficient cleanup has been performed on the samples. Do you know that the compunds in your samples are not interferences?

If you use an internal standard, you have a compund that is not found in your sample, added at a fixed level, and a good indicator of what is going on in an injection. (A recovery standard is better - it is added after extraction so none can be lost in extraction.)
When doing manual injections I usually just use a normal 10ul syringe designed for an Agilent Autosampler, the one without the button on top. I was the syringe with the standard or sample a few times and then just pump the syringe plunger with the needle in the solution to make sure there are no air bubbles( about 4-5ul is good for the pumps), then pull up to 1ul with no air gaps. I normally put the needle into the injection port until the barrel touched then inject quickly and remove.

I used the air gap method back in college when we ran packed columns but not since I began using capillary columns, since air is not so good to put through a hot column. Consistency in technique is key, practice until you can do it over and over without thinking about it. Just be glad you only have to inject and hit one start button consistently, back in college doing pyrolisys I had to put the sample in, hit start on the pyro, start on the gc and start on the integrator for every sample. :mrgreen:
The past is there to guide us into the future, not to dwell in.
Sounds like the good old days. You actually felt like you did somethign by the end of the day! :-)
Peter Apps wrote:
Presumably you are testing cannabis ?

Are the deviations high or low ?

What are you instrument operating conditions - inlet temp, flow, split, column etc etc ?

Peter


Hi Peter,

Thank you for the time. Yes, I am in deed testing Medical Cannabis. Inlet temp is 225C, flow is 7psi i think, split (unsure on split flow as it was set up for me, and don't have my flowmeter yet), column is a 15m x .32mmID (Restek cat # 10251). I have dual injector ports, and swapped over the column to the back port just to rule out an issue there.

It seems like the wild swings are under control, but still seeing deviation to the tune of 30%. I am totally open to the problem solely existing between the machine and the chair. I have been experimenting on different injection techniques, and did notice my area counts up by an order of magnitude when I don't do the air sandwich. I think I will refine the technique Jame_Ball suggested, and perhaps order my dual tower auto-sapler. I think it's a good way to go anyway as I can run the ECD and FID simultaneously.
adler_LAB wrote:
Its very difficult or impossible to manual injekt completly without deviation.

If its possible you should injekt more than 1µl cause the mistake you can have with one is very high.
How much is your split ?
What kind of liner do you use ?
Is your sample homogenized, clean up ?
What is your extraktion method ?


Hi,

I will find out about my split flow. The liner is a Agilent Blue sky (pn:5181-8818) direct 2mm ID. My extraction method is sonicate aprox. 100mg of raw cannabis in 10 ml HPLC grade methanol for 480 sec..

Thanks for replying to my post.

Warm regards,

Mike
Are the solutions clear or hazy as you shoot them? With your taking 100 mg, I assume that you have taken a larger sampel and ground it to allow for 100 mg to be a representative sample? Ground samples have the potential for small particles, which if enriched in analyte will show an increase in signal if they are included in an injection.

Try a few injections as you are making them now from a sample - and then let the vial stand over night so that it becoems totally clear. Sample without distrubign the sedement and make a few injections and see if the variabiltiy is down.

I used to take a gram of cut tobacco - cut as for cigarettes - and extract with 20 mL methanol and got good results. We would draw the liquid off the sample and put it in a GC vial and it was clear enough to use.
Methanol is one of the trickier solvents to get repeatable results with. Its main drawback is that it produces much larger volumes of vapour than non-polar solvents like hexane. You need to use the largest inlet liner diameter that you can get - this provides more volume for the vapour to expand into.

Peter
Peter Apps
Following on Peter's observation. If your GC oven temperature is too low at injection, your peak shapes can be ugly (and hard to integrate). You need to be above the boiling point of methanol - you don't want methanol to condense in a DB-5 type column. The methanol does not wet the column, forming a narrow band and focusing the peak; rather it forms beads along the column and defocuses the peaks.
Both the impact of vapour volume, and the chances of condensation on the column depend on the split ratio - higher will be better in both cases, but will make the peaks smaller of course.

Your inlet pressure is a bit high for the column dimensions - as long as you are getting the separations you need don't worry about it, and I doubt very much that it could affect your injection repeatability.

Do you have any glass wool or similar packing in the inlet liner ?

Peter
Peter Apps
Peter Apps wrote:
Methanol is one of the trickier solvents to get repeatable results with. Its main drawback is that it produces much larger volumes of vapour than non-polar solvents like hexane. You need to use the largest inlet liner diameter that you can get - this provides more volume for the vapour to expand into.

Peter

Hi Peter,

Thanks for the tip. It makes sense. I will look into that. The 4mm was the recommended. Would you expect I would need larger?

And, yes.. there is glass wool in the inlet liner. I would be interested in hearing opinions about the use of wool as the fellow that set it up seemed to not have one. He indicated that it didn't matter, so I just went 'with' to hopefully minimize the opportunity for debris winding up on my column

Mike
Also an update to you kind folks that were interested in taking the time to provide tips and insights. I moved the column to the back injector port and it seems that things are looking a lot better. There appears to be less noise, and less deviation in my results from shoot to shoot. I still have some deviation, but now it tends to stay within about 10-15%. I still am not certain if that (the injector port) was actually an issue, or I am just becoming a better chromatographer after the 125th injection or so. I guess I will know if the inlet port was having an issue as soon as I get my UHP N2 delivered, and start running my ECD again.

I am also considering using an internal standard until I can get my auto-sampler. Hopefully, this will serve as a 'de-noobification' for my sample. It is probably a silly question, but why would it be a bad idea to have my internal standard in my methanol already (on the front end) as opposed to adding it each time? It seems (to a non-chemist) that adding it to each sample would create variation as it is impossible to have the exact conditions each time.

I also don't know if the methanol spike itself can be used as an indicator as to the volume/quality of the shoot.

Thanks again guys.
Glass wool in the inlet can either help or hinder (with methanol I would expect it to help), and the only way to find out is to try both ways. One thing to be wary of is that the position of the wool in the liner, and how much of it there is can make a surprisingly large difference to performance. Focus liners have the wool between two ridges on the inlet walls to stop it moving around.

I think that 4 mm is the largest i.d. you can get - ultimately you are up against geometry.

You probably are getting better with practise, but it would be a good use of 10 minutes or so to compare the liners and the column installation between the two inlets.

I have often spiked my internal standard into the bulk solvent so that the concentration is the same in every sample, but if you do that you still have to be very repeatable in the amount of solvent that you use for each sample - usually I use an autopipette, check the weight and then use that as a correction factor.

Peter
Peter Apps
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