Video transcript: Extra Column Volume

Hi, my name is Conner McHale from Advanced Materials Technology, and today we’re going to talk about extra column volume.

Extra column volume is the volume in an HPLC system external to the column that contributes to the total peak volume. Most new HPLC chromatographic systems have been optimized and, therefore, are able to use high-efficiency SPP-infused core columns like HALO. But many older chromatographs are still in active use.

Older chromatographs were used with longer columns packed with larger particle phases where extra column volume in the injector and connecting tubing lines had less effect on resolution and peak width. Now in switching to shorter high-efficiency SPP columns, this extra column volume needs to be reduced to avoid peak broadening that can reduce the resolution and reduce the accuracy of quantitative results. Now let’s have a brief overview of an HPLC system.

So this is a typical HPLC system. It consists of a degasser, a pump, in this case, we have two: an auto-sampler where your injection is going to be placed, a column oven, which is where the column goes in, and a detector. Let’s take a closer look inside.

Extra column volume typically comes from the sample injector, including the volume of the sample injector, the tubing connecting the sample injector to the column, the tubing connecting the column to the detector, the detector flow cell, plus any volume added by heat exchangers, fittings, connectors, and in light filters.

Extra column volume should be minimized in order to maximize the performance of the column by obtaining the best separations that the column is capable of. In general, you want to keep the extra column volume to less than one half of the total peak volume to get 90% of the maximum resolution from a column. Let’s take a closer look.

First, we need to calculate the peak volume of a given peak. In order to do this, we multiply the flow rate, in this case, one-and-a-half ml a minute, by the peak width, 0.04 minutes. This gives us a total peak volume of 0.06 milliliters, or 60 microliters. The total extra column volume in this example is 13.98 microliters, well below half of the total peak volume.

How to minimize extra column volume

There are several ways to minimize extra column volume.

Number one, your injection volume. You want to make sure that you keep your injection volume to a minimum.

Number two, your sample solvent composition. Keep your sample solvent strength equal to or less than the initial mobile phase if possible. If a hundred percent organic must be used as your sample solvent, use minimum injection volume, if possible.

Number three, your precolumn tubing volume. You want to make sure you use minimum lengths of ID tubing. Note that using multiple tubing IDs in the sample flow path can cause additional dispersion. For gradient separations, one can usually ignore the precolumn contribution to the extra column volume, except, of course, that this volume does contribute to the gradient delay volume.

Number four, your heat exchanger volume. Some instruments have column compartments with different heat exchanger volume options. This helps with thermal mismatch between the mobile phase and the column when elevated temperatures are used. You want to make sure you use the smallest volume consistent with flow rates, especially for 2.1 millimeter ID columns.

Number five, your post column tubing volume. Use minimum lengths of ID tubing. Different tubing IDs in the sample flow path can produce additional dispersion. For 20 to 50 millimeter 2.1 millimeter ID columns, 75 or 100 micron ID tubing may provide better results at the expense of higher back pressure.

Number six, your detector flow cell volume. Use flow cell volumes 1 to 5 microliters for most 2.1 and 3 millimeter ID columns. Larger cell volumes can often be used for 4.6 millimeter ID columns in longer lengths.

Chromatographic Example

So let’s take a look at a chromatographic example. In the figure, the performance of 150 millimeter 2 micron HALO column is compared using an isocratic separation with an HPLC in its standard configuration and a UHPLC system. In this example, the extra column volume from the standard HPLC configuration is 64 microliters and 16 microliters with the UHPLC system. With the HPLC in its standard configuration, the ultra-fast HALO column provides slightly longer analysis time and low plates due to the peak broadening. In addition, the resolution is less than one would predict on the HALO column. After moving to a UHPLC with lower extra column volume, the HALO column delivers a high-speed separation with increased efficiency in baseline resolution.

Modifying conventional LC equipment

If you don’t have a UHPLC, do not worry. There are several things you can do to get high performance from your system.

We recommend taking consecutive steps in modifying conventional LC equipment for use with high-efficiency SPP columns, such as HALO, beginning with the easiest and cheapest modifications to make and progress to more difficult and more expensive modifications as needed. Once you have achieved acceptable results, you may wish to forego any further equipment modifications.

So these are a lot of changes to make, and you might be wondering what’s the quickest way to reduce your extra column volume.

Number one, decrease your detector response time and increase the data collection rate. A good place to begin is to set the detector response time less than 0.1 seconds and the data collection rate greater than 20 hertz. For example, in this chromatographic overlay, you can clearly see that as you increase your data collection rate, your plate efficiency also increases from 11,000 to over 38,000.

Number two, reduce sample injection volume to a minimum amount necessary to achieve an acceptable peak response.

Number three, you want to make sure that you keep your sample solvent weaker than the mobile phase. If this is not possible, it will place more restrictions on the volume of the sample that you can inject. If you are running gradient conditions, you will likely be able to inject considerably more sample volume than you would under isocratic conditions. However, it is still important to keep the sample solvent weaker than the initial strength of the mobile phase.

Number four, replace the capillary connector tubing with smaller volume tubing of shorter lengths.

And finally, number five, replace the detector flow cell with a smaller volume flow cell.

I hope this video will be able to help you reduce your extra column volume on your HPLC system. If you have any additional questions, please visit our website at fused-core.com. Thank you.