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It can help to carry out a little detective work prior to starting any experimentation; spotting potential issues with a separation or changing the method (where applicable) can all save time in the long run.
In my January blog (1) I introduced the concept of critical evaluation of chromatographic methods as a skill that every analytical chemist should possess. The ability to predict issues and highlight unusual method parameters, before ever entering the laboratory, allows you to be alert for potential issues with a separation, or to change the method (where applicable) prior to initiating any experimentation. These detective skills, when applied in retrospect, are also invaluable for troubleshooting problems with the chromatographic separation or quantitative results.
As with any profession, there really is no substitute for a lifetime of experience and the “school of hard knocks” is a successful school indeed, however, the intention with this series is to condense some of these invaluable lessons into bite-sized articles, which will help you to quickly gain the ability to critically evaluate your methods and link practical issues with methodological “quirks”.
This time we turn our attention to a gas chromatography (GC) method that I recently came across that was producing less than ideal results. I asked not to know anything of the problems, rather that I was given the method to study in the hope that I could predict some of the issues and hopefully guide the analyst to a better-performing method.
The method, outlined below, is for the analysis of a suite of barbiturate compounds in an extract from physiological matrices. While there were a number of issues with the extraction method, we will consider only the chromatographic aspects here, leaving the evaluation of extraction methods for a future issue.
Method: column: 5% phenyl polydimethylsiloxane, 30 m × 0.25 mm, 0.25‑µm; carrier: hydrogen at 60 cm/s, measured at 60 °C; oven: 60 °C, 60–150 °C at 25 °C/min, 170–300 °C at 10 °C/min; injection: splitless, 250 °C, 1 min purge time; sample: 2 µL of extract (isopropyl alcohol); detector: MS, 250 °C transfer line full scan at m/z 40–270 flame ionization detection (FID), 300 °C, air 450 mL/min, hydrogen 30 mL/min, helium makeup 50 mL/min; capillary flow splitter with makeup to balance retention times between both detectors.
So, where to start with the critical evaluation of a GC method such as this?
I always like to know the chemistry of the analytes, so Figure 1 shows analyte structures for some of the target analytes in this method.
The barbiturate structures, with their multiple aldehyde and amine groups, would indicate that these relatively small analyte molecules would be reasonably polar in nature. The Log D value of barbital, for example, is 0.6 at pH 7, falling to 0.1 at pH 8. The GC column stationary-phase choice is 5% phenyl polydimethylsiloxane, however, if separation problems occur, a more polar phase such as the 35% phenyl polydimethylsiloxane could be considered.
The analysis of structurally related target compounds often requires the use of longer and narrower bore GC columns, especially when using apolar phases, which tend to separate on analyte volatility. Where boiling points do not differ greatly, a stationary phase that can chemically discriminate between analytes should be considered. This seems to be the case for our analytes above. Once in the laboratory, if we discover that there is poor resolution between analytes, apart from selecting a more highly polar stationary phase as discussed above, we may need to move to a longer (60 m) column, or to reducing the column internal diameter to drive the separation through improved efficiency rather than chemical means. Of course, a longer column will extend the analysis time, and the use of 0.18 mm internal diameter columns requires careful planning and reduced extracolumn volumes within the GC system. Because the barbiturate analytes are not highly volatile, the relatively thin stationary phase film should be satisfactory.
For more information on GC column phase selection see the following links: https://www.crawfordscientific.com/chromatography-blog/post/gc-column-selection; https://www.chromatographyonline.com/view/lcgc-blog-chromatography-technical-tips-pragmatic-gc-column-selection-principles
Using hydrogen carrier gas with mass spectrometry (MS) detection should always be considered as a potential issue because of the vacuum at the outlet of the GC column, which can lead to very low injector head pressure requirements. One should consider the gas chromatograph head pressure requirements because very low values may lead to irreproducible retention times, separation variability, or even an inability for the instrument to attain or maintain a pressure set point. While 60 cm/s is a reasonable linear velocity for hydrogen as a carrier gas with the column dimensions used in the method, due to the fact that we have a vacuum at the column outlet (meaning the MS detector), the head pressure associated with this linear velocity under the method conditions is only around 2 psi, which may be difficult for the instrument gas pressure control systems to deliver in a reliable fashion, even though the quality and reliability of gas pressure or flow control units are very high in modern instruments.
I used a manufacturer’s pressure/flow calculator to calculate the pressure required to achieve the desired linear velocity of the carrier gas under the method conditions ahead of even venturing into the laboratory. These tools are excellent for our critical evaluation toolkit, and help to evaluate and anticipate problems such as those described above. The use of wider-bore capillary columns with low linear velocities, the use of hydrogen as a carrier gas, and the fact that MS detectors present us with a vacuum at the column outlet can all give rise to potential problems with low carrier gas outlet pressures, all of which can be investigated ahead of any practical work, using the type or pressure/flow calculator shown in Figure 2. One further parameter worthy of note is the outlet flow, which is shown in Figure 2. In this case, the flow is actually split between the mass spectrometer and a flame ionization detector (FID) using a capillary flow splitting device, so we cannot assume that the full 1.1 mL/min of carrier gas reaches the spectrometer. However, this is not always the case and it is worthwhile to “predict” the outlet flow coming from the analytical column to ensure that it does not exceed the maximum for the spectrometer in use. Even though modern vacuum pumps can often withstand gas flows of 2 or even 4 mL/min of carrier gas flow (check the manufacturer’s flow or pressure limits for your particular instrument), it is always good practice to check the column outlet flow rate under your particular experimental combination of carrier gas type, pressure, and column dimensions. Exceeding the carrier gas flow limits will cause a noticeable reduction in the performance of the detector, especially in terms of the analyte ionization characteristics (spectral appearance or analytical sensitivity), and may even cause the instrument to shut down. One should also bear in mind that the use of hydrogen carrier may require adjustments to the MS instrument hardware and may also result in changes to the spectra of certain analyte types. For further information on the use of hydrogen with MS detectors, see the following links: https://www.crawfordscientific.com/chromatography-blog/post/hydrogen-carrier-gas-ms-detection; https://www.chromatographyonline.com/view/lcgc-blog-using-hydrogen-carrier-gas-mass-spectrometric-detection
When using splitless injection, it is typical to start the oven temperature program at around 20 ºC below the boiling point of the sample solvent to ensure good thermal focusing of the analyte at the head of the analytical column, and therefore sharp, rather than broad or split peaks. In this case, the solvent is isopropyl alcohol with a boiling point of around 82 ºC and an oven starting temperature of 60 ºC seems appropriate; we would assume that the earlier eluting peaks within the chromatogram should be “sharp” from a chromatographic perspective. This so-called “solvent-focusing” phenomenon applies mainly to earlier eluting species because it is assumed that the more volatile analytes will remain associated with the injection solvent vapour when transitioning from the inlet onto the analytical column. If the starting oven temperature were any higher, the solvent containing the analyte molecules would be unable to condense onto the inner walls of the capillary column, which is the basis of the solvent-focusing effect, and peak shape broadening or deformation could occur. It should also be noted that it is usual practice to hold the initial oven temperature to allow optimum peak focusing through both thermal and solvent effects. In this method there is no initial hold time, and this can potentially lead to peak shape deformation (typically peak splitting/tailing) and issues with peak area reproducibility. One should consider the introduction of an isothermal period that initially matches the purge time (see below) and which can be empirically optimized by extending or shortening the hold time to overcome any peak shape issues.
There are two further important considerations when investigating potential issues with a new method that uses splitless injection: the injection volume and the splitless or purge time associated with the injection phase. Splitless injection, as you may be aware, is used when we need to introduce all, or the vast majority, of our sample onto the GC column to obtain the required sensitivity when analytes are present at very low concentrations. To achieve this, during the first part of the injection phase, the split valve is closed and all of the sample vapour produced in the inlet is passed to the column. After a predefined time, the split valve is opened, and any residual solvent/sample vapours are vented from the inlet to avoid large or tailing solvent peaks and rising baselines, as well as to protect the inlet from contamination. It is important that during the splitless phase of the analysis, the volume of sample vapours generated does not exceed the available volume within the inlet (predominantly defined by the internal volume of the inlet liner). If the inlet volume is exceeded, a process colloquially known as “backflash” can occur. Backflash may lead to deposition of analyte within the gas supply and outlet lines leading into and out of the inlet, ultimately risking carryover and problems with quantitative reproducibility.
Again, I have used a helpful tool to predict the vapour volume created under the conditions used in our method. Figure 3 shows that the 2-mL injection under the inlet conditions selected is dangerously close to the total volume of the liner chosen. If issues with carryover are encountered when implementing the method, one might consider reducing the injected volume, as long as sensitivity requirements can still be met. Alternatively, one could also consider a pressure-pulsed injection, where the inlet pressure is raised during the injection to constrict the expansion of the sample vapour. This will avoid backflashing the solvent vapour, while retaining the 2-mL sample injection size.
For a more comprehensive treatment of optimizing splitless injection conditions with pressure pulsed injection, see these links: https://www.chromatographyonline.com/view/lcgc-blog-improve-sensitivity-and-reproducibility-using-pulsed-pressure-splitless-gc-injection; https://www.crawfordscientific.com/chromatography-blog/post/pulsed-pressure-splitless-gc-inject
The purge or spiltless time corresponds to the time at which the split vent is opened to release any lingering vapours and needs to be optimized empirically. Typically, one would begin with a shorter purge time (30 s would be typical), and the purge time lengthened until the peak area of analytes stabilizes. Too short a purge time leads to analytes that have not passed onto the column being lost from the split line. Typically, the analyte peak area will stabilize and remain constant, and the purge time should be increased in 10–15 s increments until the peak area stabilizes and the purge time is set to the value that corresponds to the second time increment where peak area remains stable. The 1-min purge time within the method seems reasonable. However, if the solvent peak tails or the baseline is noisy and rises dramatically throughout the analysis, one may consider reducing the purge time.
Without seeing a chromatogram, it is very difficult to predict the suitability of the temperature program gradient; however, when analytes are very similar in chemistry (homologs and such), the rule of thumb is that lower ramp rates tend to work better. The 25 ºC ramp rate at the start of the analysis should be carefully considered if problems with selectivity or resolution are encountered. However, as the gradient program in our method slows to 10 ºC per min from 170 ºC, then I suspect that any problematic analyte peak pairs occur from 4 min onwards within the chromatogram, and the rapid initial program is merely used to reduce overall analysis time. If one finds a need to further optimize the oven temperature gradient, many good tips can be found at these links: https://www.crawfordscientific.com/chromatography-blog/post/gc-temperature-program-development; https://www.chromatographyonline.com/view/lcgc-blog-heat-insights-more-logical-approach-gc-temperature-program-development
In terms of the FID parameters, the absolute values for temperature and gas-flow rates will vary by manufacturer, though we can highlight some general principles. It is usual to keep the detector temperature elevated above the final oven temperature to prevent condensation effects. One should consider raising the detector temperature from 300 ºC to 325 ºC or 350 ºC to prevent water condensation in the detector, which reduces sensitivity, and to reduce the risk of analyte deposition within the detector body. The gas-flow settings within the method seem reasonable, although most methods tend to have similar gas flow for the fuel (hydrogen) and makeup (helium) gas flows, typically because they have not been optimized to obtain the best sensitivity. In our example, the makeup flow is higher than the fuel gas flow, but this may well be because the method has been optimized. If not, we should consider reducing the makeup gas flow. Typically, the air-to-fuel gas ratio would be around 10:1, and in the method cited the ratio is a little higher than this. Again, if sensitivity needs to be improved, one might consider lowering the airflow rate or slightly increasing the fuel gas flow; however, these recommended settings are somewhat manufacturer-dependant.
The MSD transfer line is a heated line that carries the column effluent from the oven to the ion source within the detector. The temperature of the transfer line should be set to avoid condensation of analytes within the heated region, which may result in reduced method sensitivity and carryover. It is recommended that the transfer line temperature should not be more than 20 ºC below the oven temperature; therefore, in our method, we should consider raising the transfer line temperature to at least 280 ºC. No further details are given regarding the other MS parameter settings, which is an omission in the method specification. For brevity, we will consider critical evaluation of MS detector settings in a future blog entry.
Who would have thought that there was so much to discuss from a few simple GC method settings! The skill in critical evaluation of methods is to be prepared, and almost to predict issues before they occur. In reality, this method may work perfectly well in practice; however, undertaking this short evaluation exercise will allow us to prepare for any problems, and to prioritize the adjustments we might need to make if specific issues arise.
Tony Taylor is Chief Scientific Officer of Arch Sciences Group and Technical Director of CHROMacademy. His background is in pharmaceutical R&D and polymer chemistry, but he has spent the past 20 years in training and consulting, working with Crawford Scientific Group clients to ensure they attain the very best analytical science possible. He has trained and consulted with thousands of analytical chemists globally and is passionate about professional development in separation science, developing CHROMacademy as a means to provide high-quality online education to analytical chemists. His current research interests include HPLC column selectivity codification, advanced automated sample preparation, and LC–MS and GC–MS for materials characterization.