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Guard Column or Retention Gap?

Retention gaps are mainly used for focusing sample components when introducing a large (liquid) sample directly onto the column, whereas, guard columns are used to protect the analytical column from contamination.  When using a retention gap system, the retention gap will also act as a guard column, but its primary function is to create a focusing effect.

This article will look at how retention gaps are used with different modes of injection and how to cost effectively use guard columns.

Retention Gap

Cool-On-column Injection

In cool-on-column (COC) injection the sample solvent is injected directly onto the column using a small diameter needle while the capillary column is kept at a temperature 10-15 °C below the boiling point of the sample solvent. During this process the sample components are spread in an unreproducible way over the first 20-100 cm of the capillary column while the solvent is evaporating (Figure 1). 

Without refocusing this may lead to very broad analytical peaks, therefore, several optimization processes may be employed to improve peak shape.  Parameters like injection speed, carrier gas flow, temperature of solvent and column, type of solvent, and pressure will all affect injection band width.  Additionally, when non-bonded stationary phases are used, the direct contact with liquids will result in a distortion of the stationary phase film and very short column lifetime, however, the majority of stationary phases are immobilized by cross- and surface-bonding techniques.

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Figure 1: Cool-on-column (COC) injection.

A retention gap is a few meters of deactivated, non-coated fused silica capillary column connected to the front of the analytical column.  The use of long retention gaps (1-5 m) allows increased injection volumes (2-5 μL) by providing a large surface area for solvent film formation.  Typical lengths of retention gap required are 1.5 m per microliter injected, but there is no issue in using longer than this.  The retention gap is non-retentive towards the analyte (hence the name).  When the oven temperature begins to increase the analytes migrate towards the analytical column, upon reaching the column the analytes will focus on the stationary phase resulting in a narrowing of the injection band width (Figure 2).

In COC injection the entire sample is introduced directly onto the column; the retention gap also serves to protect the analytical column from involatile sample components.  The retention gap may be replaced at less expense than the analytical column when it becomes fouled.  Regardless of the analytical column internal diameter a wide bore (0.53 mm) retention gap may be used which will allow the use of stainless steel needles for direct sample injection.  This increases the robustness of the technique and will allow automation using an autosampler.

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Figure 2: Mechanism of analyte focusing using a retention gap.

Splitless injection

COC injection minimizes sample discrimination and provides the best quantitative data, especially for thermolabile components; however, it can be challenging to perform. For applications which require high sensitivity splitless injection is often the go to as it is a much simpler injection technique in comparison to COC.  In general, splitless injections do not require the use of a retention gap.

In splitless injection the sample is injected into a hot injection port where analyte and sample molecules are vaporized then transported by the carrier gas onto the column.  The amount of solvent vapor that enters the column per unit time is much smaller than with COC injection.  Splitless injections also operate with low initial oven temperatures (20 °C below the boiling point of the sample solvent) to focus the sample at the head of the column.  If a method is used where the initial oven temperature is much lower than the boiling point of the solvent, there is a risk of solvent condensation which can cause unwanted broadening of the injection band.  Under this circumstance a retention gap can be used to mitigate any band broadening effects.

One parameter which must be considered when carrying out splitless injection is the sample solvent; a sample solvent/stationary phase polarity mismatch can have adverse chromatographic effects (Figure 3).  Peak shape problems such as those shown in Figure 3 can easily be remedied by changing sample solvent, however, if the solvent cannot be changed due to, for example, solubility issues then these peak shape issues can be avoided by:

  • Diluting the solvent with a more/less polar solvent
  • Using a pre-column or retention gap with a polarity that is compatible with the solvent
  • Using a retention gap of sufficient length to have the droplets evaporate before they reach the analytical column

Figure 3: Peak splitting from sample solvent/stationary phase polarity mismatch in splitless injection.

Large Volume Injection

Some applications may require the injection of large volumes in order to gain the required sensitivity (e.g. environmental applications).  One approach to achieve this is to use a retention gap. A 1-2 m retention gap would allow injection of 1 μL, whereas, using an 8-10 m retention gap permits injection of 100-200 μL of sample.  Injection must be slow so that the solvent evaporates while passing through the retention gap.  With large volume injection, detection limits can be reduced by a factor of 100.  The technique requires some skill to optimize all the injection parameters. Furthermore, large volume retention gaps pollute relatively quickly due to the large amounts of sample introduced.

Guard Columns

The ideal sample for analysis by GC would be one that is pure and free of any matrix components, however, this is definitely not the case in the real world.  Therefore, guard columns are used to protect the analytical column from contamination from particulates, heavy components, derivatization reagents, ionic residues, acids, bases etc., all of these compounds can interfere with or damage the stationary phase.  Usually the degradation of column performance is a slow process but it will happen.  For very dirty samples proper sample preparation to minimize contamination or interference from matrix components should always be considered; however, the use of sample preparation needs to be carefully balanced to give optimum cleanliness but without adding too much time or cost onto the analytical method. 

As fouling typically occurs at the head of the column it is possible to trim the column and, therefore, restore optimum performance.  Trimming up to 5% of the column length is normally adequate; however, if this does not improve any peak shape problems then a further 5% can be trimmed.  It has been postulated that trimming the column will affect resolution; although it should be remembered that resolution is not directly proportional to column length and doubling column length only provides a 1.4x improvement in resolution, therefore, reducing column length should only reduce resolution by the same factor.  Any concerns can be alleviated by revalidating with a known method.  Using a guard column in front of the analytical column can negate the need for column trimming (Figure 4). 

The guard column is deactivated and can be trimmed when contaminated and eventually replaced (again, as with the analytical column retention times and resolution should be monitored when trimming a guard column).  Depending on the application, guard columns have a lifetime of 1 week up to 6 months.  There are two choices for guard columns; a guard column can consist only of deactivated capillary, or it can be a coated capillary.

Figure 4: Guard column.

Deactivated Capillary Tubing

Deactivated fused silica tubing can be purchased by the meter and then a defined length can be coupled in front of the analytical column. Upon contamination, a section of the guard column is removed. When the whole guard is consumed a new guard column can be coupled. The disadvantage of trimming the guard column is that the column becomes shorter and this may affect retention times. However, if a similar length is always cut from the guard column, the change in retention time becomes very predictable. A deactivated guard column will also result in band focusing. If the injection is not optimal, there will be a focusing effect similar to that of a retention gap.

Coated Capillary Tubing

The guard column needs to prevent contamination of the analytical column; a coated guard column can help as it has both the surface deactivation and also the stationary phase layer. The stationary phase is the same on the guard and analytical column, so there should be no surprises in relation to changes in retention times. A coated guard column provides retention-driven focusing. The stationary phase traps solutes that have retention factors (k) > 5 at the initial oven temperature. This effect is stronger with a coating than without.

The easiest and most economical way of using coated guard columns is to buy two analytical columns. One is used as the analytical column and the second to make coated guard columns. From the second column 2 m sections can be cut and coupled in front of the analytical column. Samples can be run until contamination affects peak shape/response and then the guard column can be replaced with a new 2 m section.  This will yield reproducible retention times as the entire 2 m coated guard column is always being replaced.  Coated guard columns also allow the analysis of more aggressive samples/more contamination before needing replaced. The ability to cut 15 coated guard columns from a full 30 m analytical column makes this a very economical solution.

One disadvantage of using a guard column is the need to make a connection between it and the analytical column, thus increasing the chance of introducing a leak into the system.  To help with this situation manufacturer’s produce integrated guard columns (Figure 5) which contain a deactivated surface within the analytical column prior to the stationary phase.  This all-in-one solution means there ae no connection to make which saves time, means there will not be leaks at connections, or the creation of any dead volumes which could contribute to band broadening.

Figure 5: Integrated guard column.


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Dr. Dawn Watson
 

This article was written by Dr. Dawn Watson.

Dawn received her PhD in synthetic inorganic chemistry from the University of Strathclyde, Glasgow. The focus of her PhD thesis was the synthesis and application of soft scorpionate ligands. As well as synthetic skills, this work relied on the use of a wide variety of analytical techniques, such as, NMR, mass spectrometry (MS), Raman spectroscopy, infrared spectroscopy (IR), UV-visible spectroscopy, electrochemistry, and thermogravimetric analysis.

Following her PhD she spent two years as a postdoctoral research fellow at Princeton University studying the reaction kinetics of small molecule oxidation by catalysts based on Cytochrome P450. In order to monitor these reactions stopped-flow kinetics, NMR, HPLC, GC-MS, and LC-MS techniques were utilized.

Prior to joining the Crawford Scientific and CHROMacademy technical team she worked for Gilson providing sales and support for the entire product range including, HPLC (both analytical and preparative), solid phase extraction, automated liquid handling, mass spec, pipettes, and laboratory consumables.

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