Column Dead Time as a Diagnostic Tool

Jan 14, 2014

What good is that big, ugly peak at the beginning of the chromatogram?

Often considered a necessary evil, the first peak in a chromatogram can be a useful diagnostic tool for troubleshooting liquid chromatographic (LC) separations. Most people I encounter refer to this as the column dead time peak, abbreviated t 0. However, it has a wide variety of other names: junk peak, garbage peak, solvent front, or hold-up time, with t M as the most common alternative abbreviation. This represents the time it takes something to go through the LC column that does not interact with the column. A corresponding dead volume (or hold-up volume), V M, is the volume of mobile phase inside the column. This volume comprises both the volume of mobile phase between the packing particles (the interstitial volume) and the volume within the particles (the pore volume). We'll see that t 0 can be a useful diagnostic tool to identify potential problems with an LC method.

Measuring t 0

Figure 1: Examples of t0 peaks (arrows). (a) Chromatogram with little unretained material; (b) large peak normally observed at t0.
If we want to use the column dead time as a tool, we need to be able to identify it. Most LC detectors will generate a peak at t 0, the most obvious exception being the mass spectrometric detector (liquid chromatography–mass spectrometry [LC–MS]). Therefore, the chromatogram usually has a peak similar to the first baseline disturbance in Figure 1. If the sample is very clean and has minimal unretained material, a small baseline disturbance as shown in Figure 1a may appear. More commonly, there is sufficient unretained material to generate a large, off-scale peak (Figure 1b). Although there are more-exact measurement techniques for t 0, such as injection of D2O, most of us just use the retention time of the peak. I prefer to pick a measurement that is easy to reproduce, because most of the time an estimate of t 0 is sufficient. For Figure 1a, this is the point the disturbance crosses the baseline, noted by the arrow. Because a large unretained peak usually is off scale so that the top of the peak may be inconvenient to locate, I usually pick the point where the peak rises from the baseline (arrow in Figure 1b). Of course the retention time reported by the data system is another convenient measurement of the dead time.

To confirm a measured value of t 0 or to determine it if there is no corresponding disturbance in the baseline, as with LC–MS, we can estimate the column dead volume, V M, and convert it to t 0. If you are using a 4.6-mm i.d. column, V M can be estimated as follows:

where V M is in milliliters and L is in millimeters. Thus, for a 150 mm × 4.6 mm column, V M = 0.01 × 150 mm = 1.5 mL. For columns of other internal diameters, you can use

where d c is the column internal diameter in millimeters. For a 50 mm × 2.1 mm column, V M ≈ 0.5 × 50 × 2.12/1000 = 0.11 mL. Either of these estimates is good to within approximately ±10% for columns packed with totally porous particles. These estimates are based on the assumption that V M represents ~65% of the volume of an empty column and that about half of this volume is inside the particles and half is between the particles.

The column dead time is simply the column volume divided by the flow rate, F (in milliliters per minute):