Let’s try to understand the functioning and chemistry of GC columns and how to change and manipulate the resolution equation and column chemistry to maximize our own analyses.
Gas chromatography (GC) resolution and analyses depend on the functionality of the chromatography phases. In the past columns, we looked at gases and the choices surrounding the gas phase. In this edition of “Navigating the Labyrinth,” we will seek to understand the functioning and chemistry of GC columns and how to change and manipulate the resolution equation and column chemistry to maximize our own analyses. We will take a deeper look into the chemistry, physics, and methodology of GC columns to see what changes can be made to increase resolution, efficiency, capacity, and change retention.
The first thing most analysts decide upon when developing a gas chromatography (GC) or gas chromatography–mass spectrometry (GC–MS) method is the chromatography column that will be used for analysis. There are two basic configurations for columns: packed or capillary columns (Figure 1). The earliest GC columns were packed columns composed of either glass or stainless-steel loops or coils between 1 m and more than 12 m in length with diameters in the mm range, which are filled with a stationary phase or packing material.
In the early days of GC, many analysts had to load or pack their columns with their selected solid phases. The packing or phase can be a solid, liquid, or mix of materials to facilitate chromatography. Packing materials are broken down into either coated or porous particles. Coated particles have an internal structural particle made of some type of inert compound or matrix such as diatomaceous earth and are coated with the active liquid stationary phase. Porous particles are materials like polymers or carbons (sieves, traps, and so on). Packed columns have shorter lengths than capillary columns and are very efficient at separating very volatile gases and other low-boiling compounds.
The second general type of column is a capillary column. The capillary column is composed of a very long tube of thin hollow silicate glass anywhere from 10–100 m long and diameters in the micron range. This small diameter and long length dramatically can increase pressure in a GC system, but produce more theoretical plates from the efficiency equations we looked at in previous columns where efficiency can be measured as a relationship between separation factor (α), column length (L), and particle size or film thickness (dp). So, more length or a thinner film equates to more theoretical plates.
The interior of the capillary column is coated with a stationary phase composed of either a solid or a liquid material. Capillary columns are able to achieve better separation, but can become overloaded by excess sample volumes. Several different physical configurations exist for capillary columns including some configurations that contain solid particle stationary phases instead of just liquid stationary phase.
The most common type of capillary column is the wall coated open tubular (WCOT) columns or the fused silica wall coated (FSWC) columns. The WCOT column is usually composed of a silica tube coated with a liquid stationary phase. Most modern open tubular capillary columns are FSWC columns that are extremely thin-walled silica columns with open active site surfaces on the interior of the column where stationary phase not only coats the column but bonds to active, open sites.
Specialty open tube configurations such as the support coated open tubular (SCOT) and the porous layer open tubular columns (PLOT) are a blend of solid and liquid phases and supports, in some ways similar to both capillary and packed columns. The SCOT column has a solid framework or support upon which the liquid layer coats the support structures and the silica column. PLOT are a hybrid of an open tubular column and a packed column that contains a solid porous layer inside the column structure unlike packed columns which are completely filled or “packed” with a porous stationary phase (Figure 2).
There are two primary modes for chromatography: partition and adsorption chromatography. Partition or gas-liquid (GLC) chromatography occurs in the transitioning of an analyte from the gas phase to liquid phase and back to gas phase along the column (that is, partitions). GLC is found in the capillary columns with a liquid stationary phase. Adsorption or gas-solid chromatography occurs (GSC) when targets in the gas phase are absorbed into a solid material stationary phase. GSC is found in both packed columns and in the PLOT columns. The materials of the solid support structures found in packed, PLOT, and SCOT columns can be made from a variety of inert materials like diatomaceous earth coated with a liquid stationary phase. Other stationary phase particles are created from porous materials such as carbon compounds and polymers.
For capillary columns, the most common stationary phase is composed of polysiloxanes with added phenyls and functional groups to the polarity and affinity of the column for a wide variety of compounds. The least polar stationary phases are polydimethyl siloxane (PDS). The most polar phase is composed of polyethylene glycols (PEG columns, carbowax or “wax” columns) (Table I) (Figure 3).
Polarity increases with the number of phenyl groups or other functional groups used to create separation (Figure 3). The amount of these functional groups in some cases translates to the column name reflecting the percentage or composition of the siloxane groups. Table II lists the most commonly used capillary column phases and their naming convention, composition, and bonding characteristics. Most companies that produce columns use the same naming conventions in regard to common columns; with the first two or three letters being the company or brand with a dash followed by the composition. So, an RTX-5 is a Restek 5% DP, 95% PDS column and is equivalent in theory to another XX-5, like an Agilent StableBond SB-5.
Once a stationary phase or polymer is added to coat a column, a process of cross-linking may be used to stabilize the polymer over a wide range of temperature and reduce column bleeding. Manufacturers can also produce their own polymers and cross-linking agents, which can impact the column’s function. In all columns, there are potential active sites within the column material and open functional groups that are not deactivated fully even with crosslinking that could cause difference between different versions or brands of the same phase columns.
There are a number of factors that can affect resolution including: chemistries of solute, gas phase, and stationary phase plus a number of physical parameters such as flow rate, temperature, and column length, diameter, and film thickness.
If we think back to the resolution equation discussed in previous columns on chromatography, resolution is a product of retention, separation, and efficiency. Changing or influencing any of these parameters will change resolution (Equation 1).
One of the first changes a chromatographer can make to a system without changing the column is often temperature. Changes in temperature can influence the resolution equation by changing retention and selectivity. Increases in temperature change the amount of time the solute or target spends in the stationary phase, usually increasing retention times or by losing retention. Temperature increases can also influence or change the chemistry or physics of the interactions between the phases and thereby reduce selectivity. Another problem with raising temperature is that it will change flow rates of the system.
The next column variable to change is often the column length (L), which can then change resolution by altering the efficiency (N). While increasing length can change efficiency and resolution, large increases in length only fractionally change the resolution. Increasing a column length by four times only increases resolution about two times; but often comes with an exponential cost increase for that column (Equation 2).
A better way to increase retention is to decrease the column diameter (d) or alter both the film thickness and the diameter since they are interrelated. Film thickness (df) is a measure of the amount of stationary phase that can interact with the target solutes. To understand this relationship, first we must look at the distribution constant (KD) which is the ratio of the concentration of the solute in the stationary phase versus the concentration of the solute in the mobile phase (Equation 3).
This distribution constant is also equal to the capacity factor (k) multiplied by the phase ratio (b), meaning it will affect the k retention variable. The true importance and influence of k occurs when the ratio between the phases is small (<5), increases to the film thickness will then increase resolution but, when k is greater than five, then resolution decreases as film thickness increases.
The phase ratio is the relationship between the column inner diameter and the stationary phase film thickness, where the phase ratio is equal to the inner diameter divided by four times the film thickness (Equation 4).
The phase ratio can help narrow down the best choice of column ID and film thickness or can help change from one column dimension to another. Each phase ratio is best suited for particular classes of compounds. Very volatile compounds do best with a phase ratio less than 100, while most other compounds fall in a phase ratio between 100 and 400. The highest boiling or molecular weight compounds will do best at phase ratios greater than 400 (Table III).
Thicker films allow for more solute capacity on the column and decreases interaction with any open sites on the tubing, but thicker films increase column bleed and reduce the operating temperature for the column. Increasing film thickness can increase retention since peaks spend more time in the stationary phase, but they lose height and broaden. Increasing peak width and reducing peak height reduces efficiency as can be seen in the efficiency equations where large widths or shorter heights adversely affect efficiency (Table IV) (Equation 5).
A number of factors surrounding the GC column can influence your chromatographic resolution depending upon which component of the resolution equation is affected. Selectivity is mostly influenced by the type of stationary phase selected. The more substituted siloxanes of the phase are, the more polar the column, and the more polar the analytes it will detect. Selectivity can also be influenced by increases in temperature but often at a loss of other variables. Efficiency is most affected by column radius and film thickness, but can be increased with increasing column length, however, often at a monetary cost. Retention can be changed by manipulating the temperature of the system or changing the film thickness or column diameter. Decreases in column diameter can increase resolution but will also increase pressure and reduce sample sizes therefore reducing sensitivity. There is an old adage among chromatographers that you can want resolution, sensitivity, speed, and low cost but you cannot have them all at once. Often choosing one goal means sacrificing in another area. By understanding the function of each of the variables and how these variables can be manipulated in the chromatographic system, analysts can begin to make better choices to get the best response from their system.
Patricia Atkins is a Senior Applications Scientist with Spex, an Antylia Scientific company and has been a member of many cannabis advisory committees and working groups for cannabis including NACRW, AOAC, and ASTM.
P. Atkins, Cannabis Science and Technology 5(3), 20-24 (2022).