Column bleed is a simple term, but it is worth making sure we know what we are talking about. 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.
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 column bleed should remain constant.
Now that we have defined what column bleed is, a robust method of determining this value is needed to allow consistent and reproducible results. Measurements of column bleed are determined at a specific temperature but differences in instruments can alter the measured value of the column bleed. It is normally reported as a value in pA, or mV, but this is simply an indication of the detector response, and it is dependent on a number of instrumental parameters. Results may vary from instrument to instrument. What is needed is a method for determining actual bleed in ng/s. This can then be reproduced on any instrument.
Determination of absolute bleed is measured using a standard solution of Octamethylcyclotetrasiloxane (D4) which is one of the components of column bleed, polydimethylsiloxane (PDMS). The standard solution is injected onto the column and the peak height is measured at a temperature that is sufficiently low that there is no significant contribution to the signal from column bleed. The difference between the signal at 50°C and maximum operating temperature of the column can there be calibrated against the standard to give the actual bleed measurement.
The bleed levels can then be expressed in either of two ways. The first, which is of most use to the analyst, is as ng bleed/sec. This allows the estimation of detection limit at the upper temperature limit of the column. The second is the percentage rate of loss of the total stationary phase volume, which is a more meaningful indicator of the rate of column degradation.
Column bleed is, as expected, not an artifact that appears at a particular temperature but increases exponentially. Figure 2 shows the increase in bleed rate of a column at different temperatures. The oven was held constant until a stable detector signal was attained.
Having determined the most accurate way of measuring column bleed we will quickly look at the benefits of why low bleed is better. Low bleed results in better sensitivity, and more accurate quantitation, 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 GC-MS, column bleed is a source of non-solute fragment ions. If bleed is minimized, there is a better chance of mass spectral matching against a database of reference spectra (Figure 3). Another benefit is that less bleed means less contamination of the detector, so less frequent cleaning is required.
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