Peak area reproducibilty problems with EtOH and H2O solvents

[sirpatty79]

Greetings everyone, I'm new to the board and quite new to GC also. I've been doing a lot of reading lately on this site but have had some difficulty finding all the info I need. I am anxious to find out why my areas are not reproducible. Using EtOH as the solvent, my RSD averages about 4-5% and using H2O it is around 2%. The experiment requires higher precision for the EtOH solution at least. I have noticed that when I get a high deviation in area the retention time is usually quite a bit higher as well.

There is an outside possibility that my injection technique (or the low injection volume?) is causing the problem but I don't think so.

I'm doing a analysis of the amount of an organophosphate absorbed by a porous solid in both water and 95% Ethanol/H2O. No internal standard. My questions are is my method improvable and what RSD would it be realistic to achieve?

The instrument, column, and method info:

Shimadzu GC-17A chromatograph w/ JW Scientific DB-5 column (30m, .25 mm, .25 um). I am using a plunger-in-barrel 10uL syringe with standard (NOT on-column) manual injection.

Injector: 250 C; 100 kPa; total flow rate 104 mL/min; column flow rate 1 mL/min; linear velocity 26.9 cm/sec; split ratio of 100:1 w/ a 1 uL injection volume.

Temp. Program: 100 C for 1 min, then increase by 20 C/min for 5 min, then hold at 200 C for .5 min. My analyte elutes out just after 5 minutes.

I know it could be blowback or uneven vaporization in the liner. But I also figured that my pressure or temp or type of injection may be responsible. Wouldn't ethanol analysis benefit from a lower injector temp or higher pressure, etc. than water?

Thanks so much for your help and sorry for the huge post!!

P.S. On another note, I am having a hard time getting tiny air bubbles off of the white cap (gas-seal?) on the tip of the plunger while working with the water solution. I invert the syringe and flick it over and over but the bubble won't come free. How can I fix this?

[Consumer Products Guy]

Water expands a lot, try 0.5 microliters, 1.0 is too large for your split liner volume.

[Peter Apps]

Thank you for posting enough detail !

I presume that this all refers to repeat injections from a single solution in each solvent ?

I would have expected the repeatability to be better with ethanol than with water, which is the most intractable of all solvents for flash vaporization injections.

With the high split ratio your inlet liner volume is not likely to be a problem, and I would not expect that you would get any solvent recondensing on the column becuase the column initial temperature is close to the BP of both solvents.

I ul from a 10 ul syringe should be enough volume.

How are you doing your manual injections ? - is there a pause after sticking the needle through the inlet septum before you depress the plunger, and if there is, is the length of the pause always the same ? Try counting slowly to three after you insert the needle before you depress the plunger - this gives the needle a chance to heat up repeatably.

How do you trigger the start of the run on the GC, and are you sure that it is not going to a splitless standby state between injections ? - although if it was I would expect way higher rsds.

An internal standard would help a lot, you can add it to the solution that you inject from, so it need not be a problem with the adsorption that you are studying.

What type of inlet liner are you using, and do you have glass wool or other packing in the inlet liner ? - a little bit of deactivated glass wool would give more repeatable evaporation, and help to prevent aerosol droplets getting onto the column.

What else is there in the samples that you need to separate from the organophosphate ? the temperature programme makes me think that you have a chromatogram with only one peak on it, which allows all sorts of changes to the conditions without compromising the separation.

Peter

[AICMM]

Sirpatty79,

Peter is right about your split flow and liner volume unless you are using a narrow bore inlet liner. I would also strongly concur about the use of a bit of wool if it does not degrade your target compound. I too am puzzled by the better RSD for water over ethanol. Not what I normally hear. Finally, the shift in retention times is usually associated with overloading which is unusual considering your split ratio.

Can you do a solvent extraction on your water to pull out the organophosphate? If doing 100:1 you could probably solvent extract to a small volume in an organic and then shoot splitless. You might also try raising your initial column temp 20C so it is not sitting right at the BP of the water.

Having said all that 2-5% RSD for a manual injection is not all that bad. For an autosampler, perhaps, but not for a manual injection.

Best regards.

[sirpatty79]

Thanks for your replies everybody.

Yes Peter, I am using two separate solvent systems and am talking about replicate injections from the same sample vial.

As soon as the needle is fully inserted I depress the plunger and immediately withdraw the needle... I'm definitely consistent with this.

To start the run, I just initialize the method in the software, then do the injection and press start on the machine. No more than 3-5 minutes elapse between injections.

I'm not sure about the silica wool but I'll check the liner.

As far as other substances to separate from the organophosphate, there are just the denaturants in the ethanol (I know, we just ordered some pure analytical grade), which elute in the 2.5 minute range. The water contains a peak (from a contaminant I assume) at 2.6 minutes. Throughout the 6.5 minute program, there are no peaks closer to my analyte's retention time than these.

I'm not sure what method alterations might help here. Also the tiny bubble(s) sticking to the end of my plunger are really frustrating me. I have tried everything I know to clear them but they are really stuck on there. The plunger tip has a Teflon fitting that the bubble is sticking to. With ethanol as the solvent, clearing the bubbles from this tip is no problem. Anyone run into this type of thing?

[Peter Apps]

Hi Sirpatty79

No matter what you think, no human can do a fast cold needle injection (which is what you are attempting) consistently enough to give repeatable results. Try putting in the three-count pause before you depress the plunger. I predict that you will get better repeatability, and an increase in peak area becuase more sample will be evaporating out of the hotter needle.

It sounds as if the inlet might not have been cleaned for a while.

The bubbles onthe plunger tip are a more or less inevitable consequence of the very high surface tension of water on Teflon. Things to try: overfill the syringe and then put it in an ultrasonic bath, or rinse the syringe with isopropanol than with a couple of fills of clean water - the idea being to leave just enough isopropanol to wet the plunger, or overfill the syringe, stick the needle into a spare septum, then pull the plunger back further - this will expand the bubble and it might then float free but it might also produce more bubbles, or degas the sample with ultrasound or vacuum before you fill the syringe.

Peter

[sirpatty79]

Thanks again for your replies guys. I've switched to a syringe without the Teflon and it took care of the bubble problem. I will let you know how my next set of RSD's turn out.