The optimization of chromatographic methodology starts with the column selection. In this guide, discover the latest advice on how to choose the best GC column for your experiments. Based on four significant factors, column selection is different for every experiment. Download the guide today, and keep the advice to hand.
Gas chromatography is an analytical separating technique used to separate volatile and semivolatile substances from complex mixtures. The method consists of a gaseous mobile phase, usually an inert carrier gas, such as helium, and a liquid stationary phase adsorbed onto an inert cylindrical solid support called a column. Once the sample to be analyzed is injected into the GC system, an isothermal oven temperature or temperature gradient is applied to the column. The compounds in the mixture interact with the stationary phase. Depending on the type of interaction and the boiling points of the compounds, separation occurs.
GC columns are broadly classified into two types: packed and capillary (Figure 1). The packed columns are filled with inert solid material coated with the liquid stationary phase, while the capillary columns maintain a hollow interior with the stationary phase coated along its inner walls. Packed columns are preferred for analysis of gas samples, but for most analytical separations, capillary columns are more efficient and provide good peak separation and consistent results.
Figure 1: Packed GC columns (left) and capillary GC columns (right). Stationary phase appears in blue.
Compound Separation in Gas Chromatography
The analytical separation of volatile and semivolatile substances from complex mixtures can be carried out using gas chromatography. In gas chromatography, separation of compounds is largely based on their boiling points, as well as intermolecular reactions. Whilst elution generally follows the boiling point of a compound, with a higher boiling point indicating a higher retention time in the column, intermolecular reactions between the solute and stationary phase also play a part in separation. As such, two analytes with a similar boiling points can be separated on the basis that they have a different chemical structure.
The Cross Section of a Capillary GC Column
There are three distinct layers that make up the cross section of a capillary GC column (Figure 2):
Figure 2: The cross-section of a capillary GC column. Image courtesy of Phenomenex.
The structural characteristics of the stationary phase divide GC columns into three categories (Figure 3): (i) Wall-coated open tubular column (WCOT), (ii) Porous-layer open tubular column (PLOT), and (iii) Support-coated open tubular column (SCOT). A wall-coated open tubular column (WCOT) consists of a thin film coated on the inner wall. The porous-layer open tubular column (PLOT) is a porous solid layer on the capillary’s inner wall, while the support-coated open tubular column (SCOT) includes a liquid stationary phase in addition to a porous support.
Figure 3: There are three categories of GC columns, depending on the structure of the stationary phase: (i) Wall-coated open tubular column (WCOT), (ii) Porous-layer open tubular column (PLOT), and (iii) Support-coated open tubular column (SCOT).
Once you’ve identified the analytes that you want to separate, there are a number of additional considerations in a GC column to ensure a high-quality separation and a clean chromatogram.
Figure 4: When choosing a column there are four factors you need to consider. Image provided courtesy of Restek.
Listed below are the factors to consider, in the order of importance, when choosing a GC column (Figure 4):
Selecting the stationary phase
The master resolution equation (Figure 5) defines that the best resolution obtained for two solutes in a GC separation comprises three terms: efficiency, selectivity and retention. Of these, the biggest impact on resolution is made by selectivity (α) which, in turn, directly relates to the stationary phase.
Figure 5: The master resolution question breaks down the relation between column length, phase, I.D. and film thickness.
Your choice of a stationary phase in the GC column has the highest impact on the separation, and eventually, your data. Following the principle of ‘like dissolves like,’ the interaction between the analytes of interest with the stationary phase forms the core of chromatographic separations. The physical and chemical properties of the analyte and the stationary phase govern the analyte-phase interactions. In separating two compounds, if the interaction between one analyte and the stationary phase is stronger, it is retained longer in the column, and helps achieve a good separation. Alternatively, with certain stationary phases, when two analytes do not separate (i.e. they coelute), changing the stationary phase by altering its chemistry can help achieve a separation. GC column manufacturers provide a wide range of capillary column phases to choose from, with each phase providing a specific chemical property.
The first step in selecting a stationary phase is to review the large volume of scientific publications in your area of application. Chances are that you’ll find a stationary phase well-suited for your experiment. Additionally, GC column manufacturers provide phase selection charts, compiled from the numerous GC successes in different industries and applications, that can aid your selection.
If, however, your application is unique and has no reference, it is important to know the physical and chemical properties of the analyte of interest. Listed below are some factors that can help with your method development.
A stationary phase is selected on the following criteria:
Phase selectivity is the ability of the stationary phase to differentiate between two analytes by identifying the differences in their chemical or physical properties. It is directly related to the composition of the stationary phase, and how it reacts with the targeted compounds intermolecular forces.
Phase polarity is determined by the structure of the stationary phase. The polarity of the substituted groups in the stationary phase and their abundance affects the overall polarity of the column. Phase polarity is often mistaken to be the most influential factor in solute resolution, which, in fact, is phase selectivity. Polarity is, however, one of the many factors that affects retention and, therefore, peak separation. Phase polarity follows the ‘like dissolves like’ principle. Non-polar compounds are better retained and separated by non-polar columns, and vice versa.
Selecting column length
The efficiency of a column is directly proportional to the column length. Longer columns, therefore, are meant to yield a higher resolution. However, column resolution is related to the square root of the column length. This means increasing the length will only have a limited improvement in terms of resolution. Column length is directly related to run time. Increasing the column length will, therefore, also increase the time taken for analyte separation (Table 1).
|√L ~ R||Length of a column (L) has a square root relationship with resolution (R). Increasing the resolution by increasing column length is not a customary practice due to the inconvenience of very long columns and due to the increase in run times.|
|L ~ tR||Length of a column (L) is directly related to the run time (tR). Increasing column length will increase the time for analysis.|
Table 1: The relationship between length of the column, resolution and run time.
In summary, longer columns yield a greater resolution but increase back pressure and run times, while shorter columns are preferred for applications where resolution is not the criteria, for example, when screening for analytes. In general, the column I.D. is often changed along with column length to yield the desired separation (Figure 6).
Figure 6: Considering length of a GC column.
Selecting column internal diameter
The internal diameter (I.D.) of a column directly affects the column efficiency (i.e. the number of theoretical plates) and the sample capacity (i.e. the amount of sample that can be injected into the column without causing an overload). The column efficiency, measured in plates (N), is inversely proportional to column I.D.; it increases as column I.D. decreases. Whereas the sample capacity is directly proportional to column I.D.; it decreases as the column I.D. decreases (Figure 7).
Figure 7: Considering the internal diameter of a GC column.
Selecting film thickness
The thickness of the stationary phase on the inner wall of the capillary column determines solute resolution and sample capacity. The thinner the film coating, the higher the resolution, but lower the sample capacity, and vice versa. Decreasing the film thickness yields sharper peaks, reduces column bleeds and increases signal-to-noise ratio. Whereas increasing the film thickness increases sample capacity and reduces maximum operating temperatures, but decreases analyte-phase interaction (Figure 8).
Figure 8: Considering the film thickness of a GC column.
GC Accelerator Kits: If you need to speed up your sample analysis, consider purchasing a GC Accelerator Kit. These kits work by reducing the volume of the oven, allowing for faster ramp rates to be attained – reducing the oven cycle time and enabling increased sample throughput.
One such kit from Restek, is designed specifically for Agilent 6890 and 7890 instruments. You can simply apply an existing method, scaled down using Restek’s EZGC Method Translator, allowing you to obtain the same chromatographic separation in a fraction of the time.
Column contamination: A contaminated column is one of the most common causes of performance degradation in GC systems. The contaminants typically originate from injected samples. Over time, semivolatile and nonvolatile sample components, upon injection, accumulate in the injector and on the column surface. The semivolatile contaminants accumulate in the column, but eventually elute out, however, the nonvolatile ones do not elute and continue accumulating. Loss of peak size and shape, and peak tailing, are usual symptoms of a contaminated column. Additionally, the stubborn nonvolatile residues can reflect on the baseline of the chromatogram, causing instability, wander, drift and ghost peaks.
To tackle column contamination, consider heating the column to a high temperature, known as 'baking out' at the end of your run. This helps remove high boiling contaminants from the columns. Care should be taken to control the temperature and time of such a bake-out so as to prevent thermal damage. Alternatively, in some GC columns, soluble contaminants can be removed with solvent rinsing, starting with the most polar solvent first. It is advisable to check whether your column’s stationary phase is compatible with a solvent rinse before proceeding as it can cause swelling of the stationary phase.
Finally, consider cutting off contaminated parts of the column, usually 1-2 loops from the column's inlet, to retain the remaining functional parts of the column, as featured in this video from Restek. Note that each GC column is different. Please read the manufacturer's instructions carefully before proceeding towards troubleshooting.
Guard Columns and retention gap: The use of guard columns, especially when samples injected are dirty, can increase the life of the analytical column. A short, deactivated fused silica tubing (0.5 – 1 meter) installed between the injection port and the analytical column serves as the guard column. It catches contaminant residues injected with the sample and prevents it from accumulating on the analytical column. The guard column, when longer (between 3 – 10 meters long), can serve as a retention gap, which allows for an improvement in peak shapes for early eluting analytes, stationary phase and GC conditions.
In an interview with a technical specialist at Phenomenex, this article highlights the most common issues with GC and a recommended troubleshooting process for researchers
Always buy from a reliable source: When investing in GC columns, it’s important to choose a reliable source. Manufacturers perform multiple tests on the column before it finds its way to the customer. These tests include resolution of critical pairs, bleed test, retention factor of probe analytes, efficiency of the column and so on. Choose a reliable source that performs testing on individual columns instead of batch testing. To aid your decision, you can read what other scientists think of their GC columns by viewing their reviews under GC products at the SelectScience® product directory.
GC column manufacturers offer application-specific searches to aid your selection of GC columns, as well as the relevant system considerations and experimental conditions. Listed below are a few application search programs:
Accurate analysis of pesticides, volatile organic compounds (VOCs), persistent organic pollutants (POPs) and industrial chemicals is becoming increasingly important as they creep further into the environment. Effective sampling is vital to ensure the safety and quality of our water, soil and air – and a range of chromatographic methods are used.
Application and technical notes are available for many types of contamination analysis, and can be downloaded from SelectScience's extensive library. In this compendium, you can see three novel ways scientists are using chromatography to keep environmental contaminants at bay.
In this application note, you can learn how the Rxi®-5Sil MS (Fused Silica) columns by Restek Corp. are used to measure volatile organic compounds in contaminated ambient air. Another application method from Phenomenex outlines fast separation of chlorinated pesticides using their ZB-MultiResidue GC columns.
Organochlorines from pesticides enter ecosystems and food chains and remain in them for years. There is a need for robust methods in their detection. An application note from Thermo Fisher Scientific highlights the detection of organochlorine pesticides by GC using an electrochemical detector, employing the TRACE™ 1310 Gas Chromatograph. In another application note by Restek Corp., different techniques used to analyze pesticides in strawberries and spinach grown by commercial and Amish growers are compared.
A presentation by Anne Hazelden and John Adey of the National Laboratory Service, UK, explains the method development for analysis of low-level contaminants and pesticides in water.
Figure 9: Watch a lecture presentation explaining the analysis of low-level contaminants and pesticides in water.
Food, Flavors and Fragrances
Chromatographic techniques are employed when testing whether food and beverages are safe for consumption and analyzing their components.
The Zebron™ ZB-5MSplus GC column by Phenomenex Inc., designed with a rigorous fused silica deactivation process for improved inertness, is recommended for polycyclic aromatic hydrocarbons (PAHs) testing in drinking water.
A technical application guide by Restek Corp. lists methods for the analysis of alcohols and aldehydes in alcoholic beverages, as well as flavor compounds in distilled liquor products.
In a SelectScience video interview, Dr. Robert Trengove, of Murdoch University, Australia, highlights his team’s crucial work in screening for pesticide residues in human breast milk. By optimizing GC-MS/MS analysis for human breast milk, the team has successfully increased the number of pesticides that can be simultaneously screened to 88. The presence of pesticide residues in breast milk is considered a matter of huge concern for the health of the child.
Figure 10: Dr. Robert Trengove, Murdoch University, Australia, discusses his work on detecting pesticide residues in food, and his overarching goal of healthy food for human consumption.
Chromatographic techniques have long supported anti-doping examinations. Watch the video below to learn about how GC, coupled with isotope-ratio mass spectrometry (IRMS), was used for steroid confirmation in the London Olympics anti-doping labs. The technique detects endogenous vs. exogenous testosterone by measuring ratio of C12 to C13 in athlete samples.
Figure 11: Dr. Alan Brailsford, King’s College London, UK, discusses how gas chromatography combustion isotope ratio mass spectrometry (GC-IRMS) was used in the London Olympics Anti-Doping Labs.
Drug testing by employers has also become a common practice so as to reduce absenteeism, accident rates, workers’ compensation expenses, and turnover costs. For those looking to identify employees with repeat or chronic drug abuse, hair follicle testing provides a wider window of detection. In this interview with Lisa Thomas, Thermo Fisher Scientific, learn how GC coupled with mass spectrometry (MS) is used in hair testing for recreational drug use.
In the video below, Fabien Bévalot, one of the directors at the LAT LUMTOX Forensic Toxicology Laboratory, France, explains the laboratory’s post-mortem forensic toxicology capabilities using Agilent’s GC-MS systems.
Figure 12: Mr. Fabien Bevalot, director at the LAT LUMTOX forensic laboratory in France reviews forensic toxicology applications using the GC/MS system.
Petroleum and Petrol Chemicals
Petrochemical analysis needs to be carried out at all stages of production, from tanker, to refinery and on to the plant. It is also a demanding area of analytical chemistry due to the huge costs involved.
Whilst testing is routinely carried out during the production process, analysis in the event of petrochemical spill is also important. In this webinar, find out how one environmental lab is analyzing VOCs in soil after a petrochemical spill, using US EPA Method 8260.
The world of gas chromatography is ever growing and with increasing regulation across the sector, the development of simple and automated gas chromatography will be seen over the years to come. As you look to purchase your next GC column, be sure to consider its environmental impacts, we expect to see a rise in greener chromatography, with many labs looking to reduce the carbon footprint of their lab activity.
The current trend towards online resources and tools is only going to grow, making gas chromatography increasingly accessible to those who do not consider themselves to be experts. Find out one manufacturer’s perspective on the future trends for GC in this interview on SelectScience.
Many factors, ranging from dimensions to analyte properties, can influence your choice of a GC column. By reviewing the library of published findings and applying a few simple considerations, it’s easier to select the correct column and get reproducible data.
With the growth in application areas for GC, there will be a rise in ready-to-use GC columns for your experiments. Visit the SelectScience product library to find out about the latest GC systems and columns from leading manufacturers. When making a decision to purchase, be sure to explore our user reviews and request more information about a product of interest. Stay up to date with GC methods and free application notes by visiting the SelectScience application note library.