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.
There are three distinct layers that make up the cross section of a capillary GC column:
1. Polyimide coating: This is a protective coating applied to the outer surface of the column. Polyimide is most commonly used as a coating. It not only gives the column its distinctive brownish appearance but also makes the column flexible and resistant to temperature.
2. Fused silica: The main material with which the column is built needs to be inert, with the compounds being separated. Fused silica, which is synthetic quartz of high purity, has been a reliable and routinely used material for GC columns. It is supported with an outer layer of polyimide coating to add strength and an inner coating of stationary phase that performs the separation.
3. Stationary phase: A thin film of stationary phase coated on the inner wall of the capillary column serves as the most important factor in selecting a GC column. The fundamental rule in choosing the stationary phase for your application is ‘like dissolves like’. Analytes interact better with stationary phases of similar chemical nature, yielding a better separation.
The structural characteristics of the stationary phase divide them 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.
In gas chromatography, compounds are separated primarily based on their boiling points, although intermolecular interactions also have an influence on resolution. Elution of solutes generally follows the boiling point of the compounds; the higher the boiling point, the more the retention time in the column. Intermolecular interactions between the stationary phase and the solutes can also aid compound separation. For example, for two analytes with similar boiling points but different chemical structure, the strength of their dipole-dipole interactions with the column can help achieve separation.
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.
Listed below are the factors to consider when choosing a GC column:
• Stationary phase
• Column length
• Column internal diameter
• 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 a certain stationary phase, 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, a result of compiling 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
• Phase polarity
Phase selectivity is the ability of the stationary phase to differentiate between two analytes by identifying the differences in their chemical or physical properties.
The master resolution equation that defines the best resolution obtained for two solutes in a GC separation comprises of 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.
Retention mechanisms: Stationary phase-analyte interactions
Analytes are retained in the stationary phase using the following interactions:
– Dispersive forces (Van der Waals interactions)
– Dipole-dipole interactions
– Hydrogen bonding
Dispersive forces are the weakest of all interactions and increase with the size of the analyte (i.e., larger compounds with dispersive interactions with the stationary phase have longer retention times). Dipole-dipole interactions, on the other hand, are stronger and these unique chemical interactions with analytes can aid the elution of two compounds with very similar boiling points. Hydrogen bonding between an analyte and the stationary phase causes a poor peak shape or irreversible binding to the column.
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 nonpolar columns, and vice versa.
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.
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 4).
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 5).
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 6).
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 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. 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.
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.