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Ron Majors, editor of "Column Watch" and "Sample Prep Perspectives," has been with LCGC North America for over 26 years. Currently a senior scientist with Agilent Technologies, Wilmington, Delaware, Ron is known industry-wide as one of the premier chromatography experts in the field. He is also a member of LCGC's editorial advisory board.
Method translation software enables conversion and improvement of GC methods.
The benefits of using software to optimize GC methods with practical examples for pesticide analysis are described in this month's "Column Watch."
Everyone wants to improve productivity in their chromatography laboratory. Last month's installment, "Method Translation in Liquid Chromatography," showed how to use a simple software (freeware) package to transfer a method performed on a long high performance liquid chromatography (HPLC) column packed with larger particles to a method using a shorter column packed with smaller particles, to save time and money without sacrificing data quality and without the need to perform time-consuming calculations (1).
The software makes the necessary calculation adjustments to also change other experimental parameters, such as column internal diameter (i.d.), flow rate, solvent viscosity, gradient slope and so on. In this month's "Column Watch," we will show how a similar approach can be used in gas chromatography (GC) to shorten analysis time but preserve the elution order and the resolution. The calculations involved are a bit more complex than the LC examples because the mobile phase in GC is a gas, which is compressible, whereas LC solvents are less so. In both techniques the mobile phase viscosity is temperature-dependent.
There are various ways to reduce analysis time in GC:
Like any chromatographic method, there are trade-offs in any attempt to decrease analysis time. A balance among speed, capacity and resolution must be selected for each analysis to meet the laboratory's goals. Adapting methods for fast GC analysis can be complicated because peak reversals can occur and some fast GC methods may even decrease separation efficiency. Table I compares the benefits and drawbacks when changing to faster GC methods.
Table I: Benefits and drawbacks of changing to fast GC methods (adapted from reference 1)
GC Method Translator
The GC method translation software (2,3) is similar to the LC method translation software (1). This program allows you to transfer a current GC method to another while ensuring that the relative retention order is maintained, that is, the peaks are eluted in the same order. It is free, stand-alone software that runs on a PC and will properly scale factors such as gas velocity and the temperature program to ensure that the elution order is preserved. Users can easily translate to different column dimensions, faster flow rates, different carrier gases, or a combination of all of these. One can do something as simple as change from flame ionization detection (FID) to mass spectrometry (MS) detection (operating under vacuum) or something as complex as changing the column length, internal diameter, detector type, carrier gas type, phase ratio (film thickness) and flow rate (head pressure). The GC method translation software will generate a new method that will recommend a temperature program, head pressure, and other parameter settings. The elution order will remain the same and the new method will attempt to maintain the resolution and selectivity of the original method. Peaks in the translated method do not have to be painstakingly identified. Thus, the GC translation software will reduce method development time or time to validate the new method. Furthermore, it will ensure that the new GC method is compatible with the hardware being used. Experimental parameters can be easily "tweaked" to speed-up the run time.
A couple of caveats should be considered when using the GC method translation software. First, you must not change the stationary phase because the software cannot correct for changes in selectivity. Columns with 100% methylpolysiloxane or 5% phenylmethylpolysiloxane from different manufacturers can often be used interchangeably, but more-polar phases can vary significantly between manufacturers. Note that as a column "ages," the stationary phase may suffer oxygen damage at elevated temperatures or become contaminated with nonvolatiles that affect peak elution order initially and over time. Also, if you are using flow programming, this parameter is not addressed in the software. You could investigate various flow rates using the method translation software and see how changes in flow may affect other parameters. After collecting data at several different flow rates, the best set of conditions can be selected for the application.
The GC method translation software operates in several modes:
Translate Only. Translates the current method to a new one based on a change of column dimensions, carrier gas type, outlet pressure, or phase ratio. The elution order is preserved and the relative efficiency of the current method is maintained. This is useful for converting an established method to a column with different dimensions or a different phase ratio, or to change a detector with a different outlet pressure (for example, a mass spectrometer).
Best Efficiency. Calculates new conditions (using your current column) that correspond to the theoretical optimum gas flow rate for the best separation efficiency. The elution order remains the same but retention times may change.
Fast Analysis. Calculates temperature and pressure for your current column and carrier gas for a run that is twice as fast as the "best efficiency" mode; depending on the pressure drop across the column, run time will decrease by a factor of 1.5–2. The elution order of compounds will stay the same.
None. Allows you to change any parameter of interest; you can play with column dimensions, flowrate, head pressure, temperature program rate and other parameters — even before you actually do any chromatography. You can use it with your current method to see how some parameters affect your results. For example, you can enter a smaller i.d. column to see the impact on run time; if your current method already has plenty of resolution, you can calculate optimum conditions for a shorter column operated at a higher flow rate and a different carrier gas. Thus, you can get a feel for the effect of each parameter adjustment on your chromatography.
Examples of Method Translation
The GC method translation software is easy to implement. It allows one to make simple or complex changes in a GC method to speed-up the analysis, change column dimensions or film thickness, use different detectors, choose a different carrier gas, and make a multitude of other changes. Without running an experiment, one can actually make hypothetical changes and see how they affect the chromatography and help to determine if the proposed changes can be implemented with the current instrumentation. Although the software can be used for method development, it is better served when one wants to improve or change an existing method. A major factor in using this tool is that the elution order remains the same, so one does not have to go back and identify each peak in the chromatogram.
We will illustrate how this works in practice with two examples: one very simple, one a bit more complex. Both screen captures and actual chromatograms will be used to show how to use this tool. The first example involves a complex sample mixture and simply changing the carrier gas. The sample mixture consists of potential residual solvents in pharmaceutical formulations, typical of the United States Pharmacopeia (USP) method 467.(5) Our initial chromatogram and chromatographic conditions are depicted in Figure 1a. A screen capture of the software is shown in Figure 2. The chromatographic parameters under consideration are shown in the left-hand column of the screen and the initial conditions of the original method are shown in the second column. Here we used a DB-624 column (Agilent Technologies, Santa Clara, California) with a stationary phase of 6% cyanophenylpropyl/94% dimethylpolysiloxane. The carrier gas was helium and FID was used.
Figure 1: Capillary GC analysis of solvents in pharmaceuticals. Original chromatographic conditions (a): column: DB-624, 30 m Ã 0.25 mm, 1.4-Âµm d f; carrier gas: helium; injection temperature: 225 Â°C; pressure: 16.2 psi; split ratio: 25; split flow: 37; total flow: 42.2; flow: 1.5 mL/min; mode: constant flow; Oven temperature program: 40 Â°C for 3.84 min, then 40â200 Â°C at 13.01 Â°C/min, hold at 200 Â°C for 1.87 min; detection: FID, hydrogen flow: 40 mL/min; air flow: 400 mL/min; makeup flow:20 mL/min. Translated chromatographic conditions (b): carrier gas: hydrogen; see Figure 2 for calculated method parameters.
Using a temperature-programmed run, a baseline separation was achieved in approximately 15 min on the 30 m × 0.25 mm column with a 1.4-µm film thickness. It was desirable to speed up the separation without having to change the column or affect this nice resolution. Switching to hydrogen carrier gas, the right-hand column of the screen capture in Figure 2 shows the conditions recommended by the method translation software. Making these recommended changes in flow rate and in the temperature program, the chromatogram shown in Figure 1b now provides a 10-min baseline separation of the 30 solvents with little or no change in resolution. The entire new method was set up by the software without the user performing any manual calculations and without a change in the elution order of the solvent peaks. Other changes could be investigated (such as increasing the flow rate or shortening the column) without running another chromatogram.
Figure 2: Screen capture: solvents in pharmaceuticals.
The second example involves systematic method changes looking at several chromatographic parameters. The sample this time is a mixture of 20 organochloro pesticides (OCPs). The determination of OCPs in environmental remediation samples is important, involving high-volume analyses in a competitive contract laboratory marketplace. A standard contract laboratory protocol (CLP) pesticide method was used for these analyses. In many cases a laboratory will analyze large numbers of samples over the course of a given project, adding costs to both the laboratory and its client. It was desirable to decrease the analysis time by changing the carrier gas from helium to hydrogen and also decreasing the column diameter and shortening the column (but keeping the phase ratio the same).
Figure 3a shows the original method accomplished using DB-35ms UI (Ultra Inert) (Agilent Technologies), a 35% phenylmethylpolysiloxane phase designed to determine trace polar compounds, such as pesticides. High-content phenyl phases are known to have high selectivity for OCPs. As a result of its arylene backbone, the phase is also known for its thermal stability and high upper temperature limit (360 °C). The baseline separation of the 20 OCPs was accomplished in just over 18 min using a helium carrier gas and temperature programming conditions. A highly sensitive and halogen-selective electron-capture detector was used. The column dimensions were 30 m × 0.25 mm with a 0.25-µm film thickness. The first change investigated was moving to a shorter, smaller internal diameter column with the same stationary phase. In GC, column efficiency is enhanced by using smaller i.d. columns at the expense of pressure increase. It is necessary to be aware of the column pressure at the high point of the temperature program to avoid exceeding the rated pressure for the GC inlet.
Figure 3: CLP pesticide method translation results. (a) Column: 30 m Ã 0.25 mm, 0.25-Âµm d f DB-35ms UI; carrier: He, constant flow, 45.17 cm/s at 110 Â°C; injector: splitless, 250 Â°C, ultrainert single taper liner with wool; oven: 110 Â°C for 0.638 min, 11.8 Â°C/min to 320 Â°C, hold time: 2.55 min; detection: Âµ-ECD, 330 Â°C, N2 makeup gas at 60 mL/min. (b) Column: 20 m Ã 0.18 mm, 0.18-Âµm d f DB-35ms UI, carrier: He, constant flow, 50.0 cm/s at 110 Â°C; Injector: splitless 250 Â°C, ultrainert single taper liner with wool; oven: 110 Â°C for 0.384 min; 19.54 Â°C/min to 320 Â°C, hold time: 3.0 min; detection: Âµ-ECD, 320 Â°C, N2 makeup gas at 60 mL/min. (c) Column: 20 m Ã 0.18 mm, 0.18-Âµm d f DB-35ms UI; carrier: H2, constant flow, 78.2 cm/s at 110 Â°C; injector: splitless 250 Â°C, ultrainert single taper liner with wool; oven: 110 Â°C for 0.246 min, 30.55 Â°C/min to 320 Â°C, hold time: 2.0 min; detection: Âµ-ECD, 320 Â°C, N2 makeup gas at 60 mL/min.
Typical gas chromatographs are usually pressure rated up to 100 psi. If this pressure is not exceeded, a method can be successfully translated to a smaller i.d. column. In our case, we also wanted to speed up the separation and a new, shorter column dimension was chosen (20 m). To keep the phase ratio the same, the film thickness of the new column was estimated to be 0.18 µm. The GC method translation software determines the optimum film thickness for commonly available in-stock columns for most manufacturers. Figure 4 shows the suggested experimental parameters for a new method, including column head pressure, obtained using the Translate Only mode of the software. The chromatogram in Figure 3b shows that using the suggested conditions and column (keeping He as the carrier gas), the separation time was reduced to just under 11 min: a saving of 7 min compared with the original method.
Figure 4: Screen capture: CLP pesticides method translation from 250-Âµm i.d. to 180-Âµm i.d. column using helium carrier gas.
The second parameter investigated was a change in carrier gas from He to H2. As was shown in the first example, a further decrease in analysis time was determined by the GC method translation software (see Figure 5 for the screen capture showing the original method and the new translated method involving both column dimensional changes and carrier gas changes). The chromatogram for the new method is depicted in Figure 3c. Thus, a further reduction of the separation time to approximately 7 min gave a 60% reduction in time from the original method. Figure 6 shows a direct comparison of the chromatograms, emphasizing the equivalent resolution of the 22 CLP pesticide peaks under all three sets of conditions.
Figure 5: Screen capture: CLP pesticides method translation from 250-Âµm i.d. to 180-Âµm i.d. column using hydrogen carrier gas.
The GC method translation software is a free package available online or to download that helps users make changes to a GC method. Two examples of changes were provided in this installment of "Column Watch" to show how easy it is to use this software to convert, speed up, and improve GC methods by changing the carrier gas and column dimensions. The software can be used to simulate changes to other chromatographic conditions — even without performing any experiments. It also suggests experimental parameters within the scope of the instrumentation being used. For example, if you want to speed up a separation using a 0.05-mm i.d. column at a high flow rate, the method translator would determine if the pressure required would exceed the 100 psi available from the standard GC system.
Figure 6: Comparison the of same three chromatographic conditions as in Figure 4: faster elution, same resolution.
(1) R.E. Majors, LCGC N. Am. 29(6), 476–484 (2011).
(2) L.M. Blumberg and M.S. Klee, Anal. Chem. 70(18), 3828–3839 (1998).
(3) M.S. Klee and V. Giarrocco, "Predictable Translation of Capillary Methods for Fast GC," Agilent Technologies Application Note 5965-7673E, March, 2000.
(5) USP general chapter <467>, Residual Solvents USP 30/NF 25 procedure, 2nd supplement, effective July 2008.
Ken Lynam is a senior applications chemist in the Chemistries and Supplies Division of the Chemical Analysis Group at the Agilent Little Falls site, Wilmington, Delaware. He has worked in the pharmaceutical, environmental and industrial chemical sectors for more than 20 years developing applications using GC, GC–MS, HPLC, and SFC techniques. His current focus is on application support for Agilent's capillary GC columns.
Ronald E. Majors "Column Watch" Editor Ronald E. Majors is Senior Scientist, Columns and Supplies Division, Agilent Technologies, Wilmington, Delaware, and is a member of LCGC's editorial advisory board. Direct correspondence about this column via e-mail to email@example.com
Ronald E. Majors