Critical Evaluation of GC Methods

September 30, 2014
LCGC North America

How to spot weaknesses in methods before problems occur

Gas chromatography (GC) method specifications can tell us a lot about how much thought went into developing a method and how problematic it may be to implement. Further, the parameters and settings that are not included in the specification can tell us a lot about the quality of the method, and how many "assumptions" we need to make to acquire data. A recent ChromAcademy webcast considered some of the more common issues with GC methods and how one might use the method specification to pre-empt issues. Does the method specify flow rate, head pressure, or carrier gas linear velocity? Linear velocity is arguably the best way to work because it is a function of the column geometry and one can quickly identify if the flow rate is at or near the optimum or optimum practical linear velocity for that particular gas type. Flow rate may vary depending on the local pressure conditions, and head pressure settings are meaningless if the incorrect column has been installed. Remember that most systems measure pressure - from which flow and linear velocity are calculated by the instrument based on column dimensions - so it's important to specify the column dimensions correctly. While it isn't absolutely necessary to specify the septum purge flow (some instruments don't allow you to control this parameter), it does remind you to check it manually once in a while. If you don't have an electronic flow meter to check that your instrument digital read out isn't fooling you, then get one as soon as possible! What is the expansion volume of the sample vapor created on injection? At some point this will have been evaluated to check that the sample solvent isn't "backflashing" out of the liner and causing carryover and peak area reproducibility problems. It's difficult to check this without knowing the inlet temperature, pressure, carrier type, and injection solvent. It's also good to specify the type of liner - "straight through, deactivated quartz glass liner 900 mm × 4 mm." The linear geometry just makes it easier to check the available volume (πr 2 × length, all in millimeters) to assess the possibility of the sample vapor overfilling the liner. Don't forget that as the injection is typically made halfway into the liner, then it is usual to halve the calculated liner volume to assess the available volume. It's pretty common to see splitless methods without purge times or with purge times that are just not sensible. The split valve should be turned on as soon as all analytes have been transferred to the column to avoid tailing solvent peaks or raised baselines during temperature-programmed analysis. Sample solvent choice is important in defining initial oven temperature in splitless injection. To achieve good peak shape, one typically would want to start the oven (column) temperature at 20 °C or so below the solvent boiling point - it's worth checking that any of your splitless methods obey this rule otherwise broad and deformed peaks may occur, especially for early eluting analytes. It's important to specify all of the column dimensions in the method - including the stationary-phase film thickness, which plays a critical role in retention. Although the column length may be "nominally" 30 m, one should check the actual length (calculated using the retention time of an unretained compound) and enter the exact value into the data acquisition parameters so that column flow rate and linear velocity can be properly calculated. Most method specifications have a table or several lines that specify the oven temperature program, but few specify the column re-equilibration time. This is the time required for the whole of the cross sectional area of the column and the carrier contained within it to reach the initial oven temperature. Just because the oven reached the required temperature doesn't mean that the column (which has thermal mass and therefore thermal lag) and more importantly the carrier gas contained within also reached the required temperature. Failure to properly equilibrate the GC column within the oven can lead to retention time reproducibility issues. It's important to state within the specification whether the method should be run in constant pressure or constant flow mode. The latter is much more popular with modern instruments using electronic pressure control and avoids problems with detector response characteristics changing as carrier gas flow reduces as the oven temperature is increased. The use of content carrier pressure with gradient temperature programming can lead to unnecessarily long retention times and loss of sensitivity for later eluting analytes. When checking out a method it's worthwhile remembering that a good temperature ramp rate for optimal separation is around 10 °C per hold up time (t 0) of the system. Variation around this is quite acceptable, but ramp rates significantly higher than this value can lead to issues with method robustness. Flame-based detectors should always have the make-up gas flow rate specified, and the fuel and oxidizer flows and the nature of the make-up gas also should be specified (nitrogen, helium, and so on). Stoichiometric ratios of the gases are important and 1:1:10 fuel:make-up:oxidizer is a good guide to the required ratio. Remember to include minor detector settings when using non-ionizing detectors, such as the bead voltage for nitrogen–phosphorus detectors and the nature and flow rate of the make-up gas for electron-capture detectors.