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GC Column Maintenance - Prevention is Better than Cure

Using proper procedures for capillary GC column storage and conditioning can have a major impact on column lifetime and the quality of results obtained. This ‘Tips and Tricks’ instalment covers everything you wanted to know but were never told about proper GC column maintenance.

Column Storage
Correct column storage is necessary to prevent two major occurrences – the ingress of atmospheric oxygen and moisture into the column and the oxidative degradation of the bonded stationary phase through UV catalyzed mechanisms. The following guidelines will help ensure longer column lifetime:

  • Remember to seal the column ends when they are not in use to exclude atmospheric oxygen and moisture. The easiest way is to seal the column using silicone septa (cut them in half – it’s less expensive!!) or column sealing caps.
  • Don’t leave the column out on the bench where it can be damaged. Store the column so it will not be scratched. If scratched, the stress to the column may cause it to crack during operation.
  • Store the column boxed with the test chromatogram in a dark place. Exposure to high levels of ultra-violet light can initiate oxidization of the stationary phase.

If the column is to remain on the instrument a constant low flow of carrier gas should be maintained with the split flow on.  If the split flow is switched off back diffusion of air into the column can occur; this air can then cause damage.  In order to prevent a build-up of moisture and air in the oven it should be left on at a
temperature of 60 °C.

Oxygen rapidly degrades the stationary phase by cleaving bonds along the back-bone of the column. This is known as a “cyclic backbiting reaction” where the siloxane chain breaks into more thermodynamically stable, but also more volatile, cyclic siloxanes (Figure 1). It is the elution and detection of these cyclic siloxanes which constitutes column bleed. This damage is irreversible.  The cyclic structures which are formed during this process have characteristic mass spectra at m/z 207, 281, and 355.

 

figure 1

Figure 1: Typical capillary GC column bonded phase ‘backbiting’ bleed reaction.

 

The increase in the number of Si-OH groups within the oxidized phase will lead to an increase in the number of secondary silanol (Si-OH) interactions and peak tailing may be observed (Figure 2). This effect is most noticeable with polar and basic compounds.


Figure 2: Peak tailing of polar compounds due to
secondary interactions with exposed silanol groups
in a column showing bleed symptoms. »

figure 2


Column Bleed 
Column bleed happens with all columns, all the time. It is the continuous elution of compounds produced from the degradation of the stationary phase as described above (Figure 1). It is important to ensure that the amount of column bleed is minimal and constant, i.e. a flat baseline is achievable at low detector response.

In general, polar stationary phases and thicker films bleed to a greater extent. Bleed is normally seen as an increase in signal at increasingly higher temperatures when operating the GC in constant flow mode and when conditioning a newly installed column (Figure 3). Bleed may appear to be worse when using detectors that are particularly sensitive to the cyclic siloxane bleed products. Examples are; cyanopropyl phases with NPD detection and polyethylene glycol phases with electron capture detectors. 

Bleed is best measured as the difference or change in signal at two temperatures, usually around 100°C and at the column’s upper isothermal temperature limit. Of course, the signal at both temperatures will have a contribution from background generated from system components that must be subtracted to determine bleed values accurately.

 

 

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Figure 3: Measurement of relative column bleed

Excessive column bleed appears as a larger rise in the baseline at the higher temperature regions. There is no absolute measurement to indicate when column bleed is excessive.

Column bleed is best measured as the difference or change in the background signal at two temperatures - Relative Bleed.

Usually the column’s upper temperature limit and a lower value around 100oC are used. The absolute background signal is a composite of the background generated by the entire GC system. It is not possible to determine the contribution of column bleed to this total background signal. By measuring the relative amount of column bleed, the other contributors to the background signal are subtracted out.

Most columns are tested using FIDs - the output signal for which is measured is in picoamps (pA). Bleed levels are usually reported as the difference (DpA) in the FID signal at two temperatures (4.3 or 8.6 DpA in the example shown here).

 

 

Figure 4: Excessive column bleed

 

The bleed profile should be continuous, with no discrete peaks and should begin at around 30-40 °C below the column upper temperature limit, with the profile flattening to a constant signal level. The column will normally have two upper temperature limit values – the lower temperature is the isothermal limit and the column may be operated at this temperature indefinitely without significant degradation of column performance (Figure 5). The upper temperature limit of the column is the gradient limit and this temperature may be maintained for 10-15 minutes before significant degradation of the column performance. The upper temperature limits of the column should not be exceeded. Column bleed will increase with column use, exposure to oxygen (usually due to poor conditioning practice or air leaks from loose fittings, compromised septa or exhausted gas traps), using the column at or near the upper gradient temperature for prolonged periods, or through the repeated use of aggressive solvents such as tetrahydrofuran, water, or acetonitrile.

 

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Figure 5: GC column temperature limits

 

Low bleed phases and ‘MS’ designated phases are now available from many manufacturers that show reduced bleed at elevated temperatures and are especially useful for high sensitivity applications with Mass Spectrometric Detectors. Many phases use an altered 'phenylene' chemistry with phenyl phases which have the functional moiety included in the polymeric backbone rather than as a silicon substituent, resulting in reduced de-polymerization and oxidation of the phase (Figure 6).

Figure 6: 'Phenylene' type low bleed phase chemistry (left) and a ‘standard’ polysiloxane backbone (right).
Note - selectivity between traditional 5% phenylmethylpolysiloxane phases and the 'phenylene' equivalent may vary slightly.

It is important to regularly check the condition of self-indicating gas traps or calculate the usage of non-indicating traps (6 bottles of gas maximum prior to a trap change is recommended). It is important to protect the columns from both oxygen and moisture using the correct gas trap.  Column fittings should also be regularly tested for leaks using a highly sensitive leak detector.

One major source of column contamination comes from the carrier stream just prior to the bottle running out of gas. Even in gas bottles containing dip tubes there will be excess moisture and other dissolved components significantly above the gas specification levels (typically 0.99999% purity) in the ‘dregs’ of the carrier. Most manufacturers (and bottled gas suppliers) recommend that cylinders are changed when the bottle pressure (stage 1 of the two stage regulator) falls to about 10% of the original fill value (about 200-250 psi on a ‘Size K’ cylinder). 


Column Conditioning 
The column should be conditioned at 20-30 °C above the final temperature of the gradient program or the isothermal temperature in the intended method of use, but the upper gradient programming temperature limit of the column should not be exceeded. After installing the column purge with carrier at room temperature for around 10-15 minutes at the flow rate required by the analytical method prior to raising the temperature – this ensures the removal of dissolved air within the stationary phase preventing unnecessary oxidation. For most columns a conditioning time of around one hour is more than sufficient (even for polar and thick film columns). Once the signal plateaus (usually following a sharp increase and shallow decrease), at the conditioning temperature the column may be considered as being conditioned, however for applications that require very high sensitivity (good signal to noise performance), the column should be held at the conditioning temperature for up to three hours (Figure 7).
  figure 6

Figure 7:
Typical chromatogram before and after column conditioning.
Note the lower bleed at high temperatures and the reduction in background
noise throughout the chromatogram. 

Phase Fouling and Contamination
Analyte, matrix components, and solvent impurities can all contaminate the column. The contaminants may either be involatile, in which case they will be deposited onto the stationary phase, or semi-volatile, and will elute over extended periods causing baseline disturbances. Involatile impurities will tend to accumulate at the head of the analytical column giving rise to many chromatographic problems including, broad, tailing, and split peaks especially when using splitless injection (Figure 8). Peak shape problems usually arise due to an interference with the gross separation and focusing processes that occur at the head of the analytical column.
As fouling normally 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.  Any concerns can be alleviated by revalidating with a known method.

 

Distorted peak shapes

Figure 8: Distorted peak shapes resulting from contaminated stationary phase (disubstitued aromatic amines, 14% cyanopropylphenyl stationary phase).


Chemical Damage
Most GC columns will have good chemical resistance except towards inorganic acids and bases; H2SO4, HCl, NH3, KOH, NaOH, H3PO4, HF etc.  When a GC column has been exposed to these types of compounds it will exhibit column bleed, a lack of inertness (evidenced by peak tailing), and a loss of retention and resolution. 

The use of perfluoroacids, such as trifluoroacetic, pentafluoropropanoic and heptafluorbutyric acid can damage the stationary phase, however, they have to be present in high levels, 1% or higher.

Cutting 0.5-1 m from the front of the column can remedy the effects of damage.  To prolong the column lifetime a guard column or retention gap can also be used, although these may also be damaged by harmful substances and trimming or replacement will be necessary; this, however, is a lot more cost effective than replacing a GC column. 


Peak Fronting caused by column overload
An old or highly oxidized column is likely to show symptoms of ‘overloading’ due to the loss of phase at the head of the column resulting in lowered capacity and efficiency. Fronting peaks may be observed (Figure 9).

Figure 9: Peak fronting due to column overload
ultimately caused by loss/occlusion of stationary
phase. »

 


 

 
 
 

Retention Gaps
Up to 5 meters of deactivated silica tubing known as a Retention Gap may be connected via a simple push-fit union to the analytical column. Retention gaps are used to:

  • Trap the non-volatile residues from samples that may potentially contaminate the analytical column. The retention gap acts to retain the non-volatile materials but does not interact with the analyte species. Regular trimming of the column inlet may take place to avoid peak shape problems caused by contaminant build up.
  • Alleviate the problems associated with polarity mismatch, as well as some of the peak shape problems associated with on-column and large volume injection. The deactivated uncoated retention gap material allows the formation of contiguous films with most solvents that will tend to focus the analyte on the stationary phase at the head of the analytical column. Solutes eluting closest to the solvent front or those that most closely match the polarity of the solvent will show the greatest peak shape improvements when a retention gap is used.
 

 

Leak Checking
It is important to check for leaks to minimize the ingress of air and moisture which can damage the GC column.  Regular leak checking should be part of the routine maintenance schedule, especially after any new connections have been made (i.e. changing the column, routine maintenance etc.).  Leak detectors can be used or if a mass spectrometer is connected as the detector the leak detection function can be employed.  A leak will produce a distinctive mass spectrum with peaks at m/z 18, 28, 32, 44 or 14, 16 which correspond to H2O, N2, O2, CO2 or N, O.  Usually if m/z 28 is larger than m/z 18 there is a leak.

 

 

Column Rinsing
A capillary GC column must have a bonded and cross-linked stationary phase to be compatible with solvent rinsing.

Solvent rinsing kits can be purchased from any column manufacturer (Figure 10).  The process of rinsing involves passing millilitres of solvent through the column. 
An injection of a large volume of solvent will NOT have the same effect and contaminants will not be removed from the column.

 

Before rinsing 50 cm should be cut from the inlet end of the column.  The inlet end of the column is then inserted into the vial of the rinse kit which is attached to a pressurized gas source (N2, He).  Successive solvents are then passed through the column by applying 10-15 psi of pressure from the pressurized gas source (a flow of 1 mL/min is desirable).  If viscous solvents are being used longer rinse times may be required.  After the last solvent has been passed through the column the pressurizing gas is allowed to flow for 5-10 minutes before the column is properly re-installed into the GC system and conditioned as normal.
Solvents which are appropriate for column rinsing and will provide good results are methanol, dichloromethane, and hexane which are applied in series.  Other solvents will also work (and may be required depending on the samples which have been analysed), however, the following criteria should be met:

  • A polar and non-polar solvent should be used. 
  • The most polar solvent should be used first followed by the other solvents in order of decreasing polarity.
  • Using the injection solvent is advisable as the sample components should be soluble in it.
  • Each successive solvent should be miscible with its predecessor.
  • Water followed by methanol should be used if aqueous-based samples have been analysed (i.e. biological extracts, waste water, soil etc.).
  • Halogenated solvents should be avoided as the final rinse solvents if the detector installed is an electron capture detector (ECD).  Acetonitrile should not be used if a nitrogen phosphorous detector (NPD) is installed.
 

GC-column-rinsing-kit

Figure 10: GC column rinsing kit.

 

Suggested solvent volumes for different column diameters are detailed in Table 1.

Table 1: Solvent volumes for rinsing columns.  Using larger volumes will not damage the column. »

 
Column I.D. (mm) Solvent Volume (mL)
0.18-0.2 3-4
0.25 4-5
0.32 6-7
0.45 7-8
0.53 10-12
 

 
Extending GC column lifetime
Once the GC column has been correctly fitted it is desirable to achieve the best possible results for as long as possible, so here are some final tips for extending column lifetime.
  1. Fit carrier gas traps close to the instrument (to remove oxygen and moisture) this is a great way to improve column lifetime and detection limits.
  2. Use the plate count of a test analyte to monitor column efficiency and set a column discard limit based on your knowledge of required plate counts for your types of analysis.
  3. Columns can often be miraculously restored to life by trimming 5% of the total column length from the inlet end — note that retention times may shift slightly after this operation, but efficiency may increase and peak shapes will improve. Retention time changes can also be mitigated by using the instrument software to accurately calculate the length of the cut column. This typically involves inputing the column dimensions, as denoted on the column ID tag, and the retention time of an unretained peak - volatile gases such as propane or butane or volatile solvents such as methanol at higher temperatures with an FID.
  4. Consider using a guard column if your samples are particularly dirty.
 

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