Guide to GC Column Selection and Optimizing Separations

• Learn how to choose the right column the first time.
• Optimize separations for the best balance of resolution and speed.
• Troubleshoot quickly and effectively based on chromatographic symptoms.

You can improve lab productivity by assuring that speed and resolution are optimized. One of the best ways to do this is to use the resolution equation (Figure 1) as the key to controlling your separations. This fundamental equation helps you choose the best column stationary phase, length, inner diameter (ID), and film thickness for your specific applications. Once you understand the basics of how resolution is related to column characteristics, optimizing your analysis for both separation and speed becomes easier. This GC column selection guide discusses the basics of separation and teaches you how to choose the right GC column!

Resolution is the goal of every chromatographer, but how much resolution is enough? Practically speaking, we need enough retention to get sharp symmetrical peaks that are baseline resolved from each other, but not so much retention, that retention times are too long, and peaks start to broaden. To achieve this goal, we must consider the column and non-column factors that affect our “perfect separation.” Only then can we work towards selecting the right column and optimizing GC separations and analysis speed. Now, let’s consider the separation factor (α), retention factor (k), and efficiency (N) in turn and how they can help you select the right column and optimize your separation.

Use Separation Factor (α) to Choose the Best Stationary Phase

Choosing the right stationary phase is the first step toward optimizing your GC separation. It is the most important decision you will make because the separation factor (α) has the greatest impact on resolution, and it is strongly affected by stationary phase polarity and selectivity.

Stationary phase polarity is determined by the type and amount of functional groups in the stationary phase. When choosing a column, consider the polarity of both the stationary phase and your target analytes. If the stationary phase and analyte polarities are similar, then the attractive forces are strong, and more retention will result. Greater retention often results in increased resolution. Stationary phase polarity strongly influences column selectivity and the separation factor, making it a useful consideration when selecting a column.

Stationary phase selectivity is defined by IUPAC as the extent to which other substances interfere with the determination of a given substance. Selectivity is directly related to stationary phase composition and how it interacts with target compounds through intermolecular forces (e.g., hydrogen bonding, dispersion, dipole-dipole interactions, and shape selectivity). As methyl groups in the stationary phase are replaced by different functionalities, such as phenyl or cyanopropyl pendant groups, compounds that are more soluble with those functional groups (e.g., aromatics or polar compounds, respectively) will inter- act more and be retained longer, often leading to better resolution and increased selectivity. In another example of the effect of stationary phase-analyte interactions, an Rtx-200 stationary phase is highly selective for analytes containing lone pair electrons, such as halogen, nitrogen, or carbonyl groups, due to interactions with the fluorine pendant group in this phase. Selectivity can be approximated using existing applications or retention indices (Table I), making these useful tools for comparing phases and deciding which is most appropriate for a specific analysis.

Due to their influence on the separation factor, polarity and selectivity are primary considerations when selecting a column. However, temperature limits must also be considered. In general, highly polar stationary phases have lower maximum operating temperatures, so choosing a column with the appropriate maximum operating temperature, as well as optimal polarity and selectivity for the type of compounds being analyzed, is crucial. Use Table II and Figure 2 to determine which general-purpose column is most appropriate based on the selectivity, polarity, and the temperature requirements of your analysis. See Table III for a list of specialty stationary phases designed for specific applications.

 

Select Column Film Thickness and Column ID Based on Retention Factor

Once you have chosen the stationary phase, you need to determine which column film thickness and inner diameter combination will give the retention factor (k) needed for optimal resolution and speed. Retention factor is sometimes referred to as “capacity factor,” which should not be confused with sample loading capacity.

The retention factor (k) of a column is based on the time an analyte spends in the stationary phase rela- tive to the time it spends in the carrier gas. As a general rule, the thicker the film and the smaller the inner diameter, the more an analyte will be retained. Note that as temperature increases k decreases, so at higher temperatures analytes stay in the carrier gas longer and are less retained.

In practice, if the value of k is too large, the peak will broaden, which can reduce resolution by causing peaks to overlap or coelute. Narrow, symmetrical peaks are important to maximizing resolution, so the goal is to select a column with a sufficient retention factor, such that resolution occurs, and peak shape does not suffer. Once the proper stationary phase is selected, column film thickness, column inner diam- eter, and elution temperature should be optimized to produce an acceptable retention factor.

Film Thickness

Film thickness (μm) has a direct effect on both the retention of each sample component and the maximum operating temperature of the column. When analyzing extremely volatile compounds, a thick film column should be used to increase retention; more separation is achieved because the compounds spend more time in the stationary phase. If analyzing high molecular weight compounds, a thinner film column should be used as this reduces the length of time that the analytes stay in the column and minimizes phase bleed at higher elution temperatures. Use Figure 3 to select the best film thickness for your application. Note that as a general rule, the thicker the film, the lower the maximum temperature; exceeding the maximum temperature can result in column bleed and should be avoided.

 

Inner Diameter (ID)

Column ID does not have as great an effect on retention factor as film thickness does. However, when selecting column ID with retention factor (k) in mind, a general rule of thumb applies; smaller ID columns produce higher retention factors compared to larger ID columns. This is due to less available mobile phase (carrier gas) volume in the column. Because smaller ID columns produce higher k values, they are more suited towards complex sample analysis where a range of low to high molecular weight compounds may exist in the sample (Figure 4). Keep in mind that both ID and film thickness should be optimized together to produce the best resolution and peak shape.

 

Phase Ratio (β)

The relationship between column inner diameter and stationary phase film thickness is expressed as phase ratio (β). If a good separation has been achieved on a larger diameter column, and a faster anal- ysis is desired, this can often be accomplished by reducing the inner diameter of the column without sacrificing, and sometimes even improving, separation efficiency. To maintain a similar compound elution pattern when narrowing column inner diameter, film thickness must also be changed. By choosing a column with a similar phase ratio, it will be easier to translate your application to the new column. Phase ratios for common column dimensions are given in Table IV. As shown here, an analyst wanting to decrease analysis time could switch from a 0.32 mm x 0.50 µm column (β = 160) to a 0.25 mm x 0.25 μm column (β = 250) and obtain a very similar separation upon proper method translation. Importantly, column inner diameter and stationary phase film thickness show a combined effect when it comes to sample loading capacity, which is decreased as column inner diameter and film thickness are reduced. It may be necessary to inject a lower sample amount in this case.

Consider Efficiency when Choosing Column Length, Column ID, and Carrier Gas

Column Length

Capillary GC columns are made in various lengths, typically 10, 15, 30, 60, and 105 meters, depending on the inner diameter. Longer columns provide more resolving power than shorter columns of the same inner diameter, but they also increase analysis time and should be used only for applications demanding the utmost in separation power. Column length should only be considered once the stationary phase has been determined. This is because the separation factor has the greatest effect on resolution and it is maximized through proper stationary phase choice for the compounds of interest. Doubling the column length (e.g., 30 m to 60 m) increases resolution by approximately 40%, while analysis time can be twice as long. In addition, longer columns cost more. Conversely, if a separation can be performed on a shorter column (e.g., 15 m versus 30 m), then both analysis time and column cost will be less. Figure 5 summarizes the characteristics and general application parameters for a range of typical column lengths.

 

Inner Diameter (ID)

Compared to larger ID columns, smaller ID columns generate more plates per meter and sharper peaks, leading to better separation efficiencies. When more complex samples need to be analyzed, smaller ID columns can produce better separation of closely eluting peaks than larger ID columns. However, sample loading capacities are lower for smaller ID columns. Smaller ID columns, especially those at 0.18 mm and less, demand highly efficient injection techniques so that the column efficiency is not lost at the point of sample introduction. Column characteristics based on ID are presented in Table V.

Generally speaking, a 0.25 mm column will produce the most efficient sample analysis while simultaneously considering analysis time and sample loading capacity. For these reasons, in combination with its relatively low outlet flow, it is also the best column choice for GC-MS work.

Carrier Gas Type and Linear Velocity

Carrier gas choice and linear velocity significantly affect column separation efficiency, which is best illustrated using van Deemter plots (Figure 6). The optimum linear velocity for each gas is at the lowest point on the curve, where plate height (H) is minimized, and efficiency is maximized. As seen in Figure 6, the optimum linear velocities differ among common carrier gases.

Nitrogen provides the best efficiency; however, the steepness of its van Deemter plot on each side of optimum means that small changes in linear velocity can result in large negative changes in efficiency. Compared to nitrogen, helium has a wider range for optimal linear velocity, but offers slightly less efficiency. In addition, because of its optimum velocity being faster, analysis times with helium are about half those when using nitrogen, and there is only a small sacrifice in efficiency when velocity changes slightly. Of the three common carrier gases, hydrogen has the flattest van Deemter curve, which results in the shortest analysis times and the widest range of average linear velocity over which high efficiency is obtained.

Regardless of the type of gas used, the carrier gas head pressure is constant during column temperature programming, whereas the average linear velocity decreases during the run. For constant pressure work then, the optimal linear velocity should be set for the most critical separations. More common today, electronic pneumatic control (EPC) of carrier gas allows for constant flow or even constant linear velocity, which helps maintain high efficiency throughout a temperature programmed run.

Another consideration for carrier gas type that is important, even if not directly related to column efficiency, is whether a mass spectrometer (MS) is used as a vacuum-outlet detector for GC. In almost all cases, helium is the carrier gas of choice, not only for its chromatographic efficiency but also because it is easier to pump than hydrogen. Hydrogen can be reactive in MS sources, leading to undesirable spectrum changes for some compounds. Nitrogen is typically not a carrier gas option for GC-MS, as it severely reduces sensitivity.

GC Troubleshooting Tips

Basic Steps

Follow these basic troubleshooting steps to isolate problems related to the sample, injector, detector, and column. Check the obvious explanations first and change only one thing at a time until you identify and resolve the problem.

Check the Obvious:

Identify the Cause:

• Define the problem clearly; for example, “Over the last 4 days, only the phenols in my sample have been tailing.”
• Review sample and maintenance records to identify trends in the data or problem indicators, such as area counts decreasing over time or injector maintenance not being performed as scheduled.
• Use a logical sequence of steps to isolate possible causes.

Document Work and Verify System Performance:

• Document all troubleshooting steps and results; this may help you identify and solve the next problem faster.
• Always inject a test mix and compare it to previous data to ensure restored performance.

Example Troubleshooting Sequence

An analyst observed that no peaks appeared during a GC-FID analysis. The flowchart below shows a logical progression of steps that can be used to identify the cause and correct the problem.

Symptoms and Solutions

Good chromatography is critical to obtaining accurate, reproducible results. Coelutions, asymmetric peaks, baseline noise, and other issues are common challenges in the GC laboratory. These analytical problems and others can be overcome by troubleshooting your separations using the tips below.

Poor Resolution
	Causes	Solutions
	Non selective stationary phase	Choose appropriate stationary phase and column dimensions
	Poor efficiency	Optimize carrier gas linear velocity and GC oven temperature program.
	Sample overload	Adjust sample concentration or amount on column.
	Incorrect analytical conditions used	Verify temperature program, flow rates, and column parameters.

Fronting Peaks
	Causes	Solutions
	Incompatible stationary phase	Choose appropriate stationary phase
	Column overloading	Reduce amount injected, dilute sample. Increase column inner diameter and/or film thickness.

Carryover/Ghost Peaks
	Causes	Solutions
	Contaminated syringe or rinse solvent	Replace rinse solvent. Rinse or replace syringe.
	Backflash (sample volume exceeds liner volume)	Inject a smaller amount. Use a liner with a large internal diameter. Increase head pressure (i.e., flowrate) to contain the vapor cloud. Use slower injection rate. Lower inlet temperature. Increase split flow. Use liner with packing. Use pressure-pulse injection.
	Last analysis ended too soon	Extend analysis time to allow all components and/or matrix interferences to elute.

High Bleed
	Causes	Solutions
	Improper column conditioning	Increase conditioning time and/or temperature.
	Contamination	Trim column and/or heat to maximum temperature to remove contaminants. Replace carrier gas and/or detector gas filters. Clean injector and detector.
	Leak in system and oxidation of stationary phase	Check for oxygen leaks across the entire system and replace seals and/or filters. Replace column.

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Pro EZGC Chromatogram Modeler YOU NEED: To develop a method from scratch, including the column and conditions. YOU HAVE: An analyte list (and you may have a column in mind, too). YOU GET: Customized, interactive model chromatograms that provide a specific phase, column dimensions and conditions. You can change columns, modify conditions, zoom in, view chemical structures, and even overlay mass spectra of coeluting compounds. Watch our instructional videos and get started today at www.restek.com/proezgc

Modify Methods Quickly and with Confidence Using the EZGC Method Translator and Flow Calculator

The EZGC method translator and flow calculator tool makes it simple to switch carrier gases, change column dimensions or control parameters, or to optimize a method for speed or efficiency. Simply enter your method specifications, and the program will return a full set of calculated method conditions that will provide similar chromatography. Use the EZGC method translator and flow calculator tool to optimize your analysis for speed so you can increase sample throughput!

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