General (very general) guidelines to help meet GC detection limits.

Sometimes we (tech service) receive requests from customers who cannot meet detection limits when developing a new method or when trying to follow a current method. They ask for advice on how to meet these limits.  As a result, I decided to provide some (very) general guidelines that I would follow back-in-the-day when I would develop GC methods.

In the context of this blog post, the topic of detection limits is limited to being able to achieve a certain signal/noise (S/N) ratio* for a particular compound, usually ranging from S/N of at least 2.5 to around S/N of 10. Some refer to this as Instrument Detection Limit (IDL).  The topic of detection limits can be very complex, so when reviewing the following, don’t think of it in terms of Quality Assurance, but in terms of optimizing your instrument’s sensitivity.

* Some consider the signal/noise ratio an outdated industry standard, especially when using a mass spectrometer (MS) or MS/MS for detection.

 

As with most procedures performed in a laboratory, there are usually multiple ways to accomplish the same task. These were the steps I followed to optimize my GC’s sensitivity.

 

1.  Make sure all gas flows are measured and adjusted according to the instrument manufacturer’s recommendations. You may need all the sensitivity your instrument is capable of to meet the required limits.

Keep in mind that older instrumentation may not be as sensitive as newer instrumentation, so if you are having difficulty meeting detection limits, ask the instrument manufacturer and/or detector manufacturer if your particular instrument has the necessary sensitivity.

Injection technique is important. You will get the most sensitivity by injecting in splitless mode, or cool-on-column. Large volume injections may be possible with certain types of injection ports, but do not simply inject more to overcome the steps needed to optimize your instrument.

For maximum sensitivity, make sure that your detector is clean and if needed, replace any worn or malfunctioning parts. If developing a method, make sure the most appropriate detector is chosen.

 

2.  When following pre-existing methods, make sure every step listed in the method is followed. Do not skip steps, do not modify steps, and do not rush through the steps. They are there for a reason.  If developing a method, someone with experience is an invaluable asset.  Make sure the appropriate person is assigned the task.

 

3.  If the method already exists, one or more column choices should be listed within the method. If you are developing a new method, have you performed your due diligence to choose the best column, or at least one capable of performing the necessary separations?

If you only are looking for one or just a few compounds, a 15-meter (or shorter) GC column may be all that is needed. However, as the number of compounds increase, or the matrix becomes more complex, a longer column is usually preferred.  Keep in mind that (generally speaking) the shortest GC column with the smallest ID (internal diameter) and thinnest liquid film/phase will usually provide the best signal/noise ratio for each peak, and therefore the most sensitivity, because (in theory) this column should provide the lowest bleed and sharpest peak shape.   But, sometimes a longer column with a thicker film is necessary such as to aid in separation, especially when there are early eluting compounds and/or when extra column capacity is needed.

 

4.  Once the instrument has been optimized and an appropriate column has been chosen, begin your preliminary testing. I would first prepare a (relatively) highly-concentrated chemical reference standard to make sure the instrument would detect the peak(s) so I could determine the retention time(s).  Injecting a high concentration should also help “prime” the surfaces (column, injection port liner, etc.) the compound will contact, minimizing activity for future injections and helping to stabilize the system. Just do not forget to inject a blank (usually pure solvent) to make sure carry-over is not an issue.

As a general rule, if possible, you would want the compound(s) to elute where the baseline is the lowest in order to achieve the best signal/noise. This area is usually after the solvent peak has completely eluted from the column and while the GC oven is at a relatively low temperature (which minimizes column bleed).

During this step, no changes to the carrier gas flow rate should be made as this parameter should have already been optimized. However, you may need to optimize the GC oven temperature ramp for compound separation, or other GC temperatures to prevent issues such as compound/sample matrix condensation (which may lead to ghost peaks) or to minimize the break-down of thermally liable compounds.  Sometimes this will not be known until actual samples are analyzed.

 

5.  Once you have decided on the (presumed) best GC temperatures and ramp rate, decrease the concentration of the chemical reference standard with each injection until you have reached the minimum signal/noise ratio allowed for each compound. This will likely be the minimum limit possible with your particular instrument. You may even want to consider using only higher concentration standards which provide a S/N of at least 10 because if the instrument loses sensitivity after analyzing samples, you don’t want to have backed yourself into a corner.  In other words, leave some wiggle room.

 

6.  After performing Steps #1 through #5, it’s time to determine if you can achieve the limits listed in a pre-existing method. Keep in mind, these limits usually include sample size and sample preparation/concentration and not simply the limit of the chemical reference standard injected as described in Step #5. For example, let’s say you were able to detect 1ppm (parts-per-million) on-column of compound xyz.  Through sample preparation, whether it is via extraction and/or concentration or some other means, you are able to concentrate the sample 100x, your new limit would be 0.01ppm, or 10ppb (parts-per-billion).

 

7.  Perform any changes/modifications to optimize the instrument and/or sample preparation (prep) and/or analysis once sample matrix has been injected. Hopefully this won’t be necessary, but with dirty matrices, usually some sort of clean-up step needs to be added. In extreme situations, the sample prep may need to be changed completely, such as going from extraction/concentration to SPME or headspace (compound boiling point will be an important determining factor).

To elaborate a little more on this topic, consider the three things that may happen to a compound’s response once sample matrix comes into play:

a. No change in compound response. This is the best-case scenario.

b. Compound response decreases and/or the compound may disappear altogether. This is the worst-case scenario. Unless corrected through sample clean-up or changing the sample prep, analysis is going to be difficult or impossible.  If this happens even when only injecting reference standards, you may need to consider more extreme measures like cool-on-column injections or even switching to HPLC or some other method of analysis.

c. Compound response increases. Not an ideal situation, but usually not an insurmountable issue. Some may refer to this as “matrix enhancement” or “matrix effect”. I always thought this effect was caused either by the matrix covering compound active sites, or possibly adding to the baseline which may improve a compound's peak area, but I really don’t know for certain.  To deal with this, I would keep injecting samples (containing matrix) until compound responses stabilized.

 

8.  The information described above is to help you get started developing a method, or to assist you achieve the limits in a pre-existing method, but by no means is this the end of the story. As a matter of fact, sometimes it is only the beginning, and may actually have been the easiest part.

 

I hope you all have found this helpful. Let me know if you have any questions.  Thank you.