Fluid Handling Solutions for BioPharma Process Improvements Webinar - Thursday, June 11th. Register Now >
|Figure 1. Column|
Column bleed is the normal background signal generated by the column stationary phase. This is illustrated in Figure 1 and it is easy to see from this why low column bleed is a significant factor in making quantitation easier. Also, since column bleed is actually the phase breaking down & leaving the column, columns with low bleed last longer. Because column bleed originates from the phase, the level of bleed is proportional to the amount of phase in the column. Therefore, thicker film columns bleed more than thinner film ones. While column bleed levels may differ from column to column, all columns bleed to a certain extent. The higher the temperature, the higher the level of bleed and, at a constant temperature, the level of true column bleed should remain constant.
|Figure 2. Septum bleed|
Column bleed is not a high baseline at low temperatures. If the baseline is high at a temperature of 100°C for instance, it could be from a number of causes, but it is unlikely it is from the column phase itself. Likely causes are column contamination from material that is then leaching through to the detector. This contamination may originate from the injector, the septum, or even from contaminated carrier gas. It might be that the detector itself is dirty and needs to be cleaned. If the detector is the suspected cause, isolate the detector by removing the column and seal the column connection with a no hole ferrule. Then check the baseline again. Possibly the detector gases are contaminated. If the inlet liner is the suspected cause, replace the liner with a new one, and re-examine the baseline level.
Column bleed is definitely not seen in the form of discreet individual peaks. These will be coming from somewhere else in the chromatographic system, most likely the injector in the form of septum bleed, see figure 2. Any contaminants in the injection system will become trapped at the start of the column, at a low column temperature, and then elute from the column during temperature programming. A wandering baseline is also not a sign of column bleed. This could be caused by semi- to non-volatile material that may have been injected into the chromatographic system several runs before. It may also indicate changes in the either column, or detector, flow rates during the chromatogram. Possibly the injector septum is leaking during the injection of the sample. It may also indicate that some of the sample is decomposing.
Low bleed is better for several reasons. Firstly, low bleed results in better sensitivity due to a better signal-to-noise ratio. The major source of noise in the chromatographic system is chemical noise and this is mostly the result of column bleed. If the noise is reduced, the S/N ratio increases. Also when using Gas chromatography–mass spectrometry, column bleed is a source of non-solute fragment ions. If bleed is minimised, there is a better chance of mass spectral matching against a database of reference spectra. Column bleed inevitably ends up somewhere, and this is on the detector. In a FID it can be seen as a white silica deposit. A black deposit is carbonaceous material from analytes. The silica build-up will result in costly instrument down-time and maintenance and it is especially critical for GC-MS systems. The advantages of low bleed columns are clear:
With all this going for it, the only thing you need to know now is how to achieve it. The answer is careful column conditioning, and good quality carrier gas. At high column temperatures, traces of oxygen can damage column phases leading to increased column bleed. So before conditioning the column, it needs to be thoroughly purged with good quality carrier gas to ensure that no oxygen is present. Then the column can be conditioned. The temperature that the column is taken up to should be either 20°C above your method’s maximum temperature, or the maximum specified temperature for the column, whichever is lower. Using the lowest temperature, rather than the column’s maximum temperature, will give longer column life. Technical article courtesy of SGE - An extensive Range of low bleed SGE columns are available to buy online here.