Section 4 Extra-Column Volume

Sources of Excessive Extra-Column Volume

Extra-column volume is that volume in an HPLC system between and including the injector and the detector; it does not include the column volume.

The injection volume, detector cell volume, column fitting volume and excessive amounts of connecting tubing between the sample injector and the column and between the column and the detector flow cell are the important sources of extra-column volume (Figure 4-1). Overall extra-column volume should be minimized to maintain chromatographic performance. For example, long lengths of tubing or large diameter tubing (>0.020" i.d.) may create sufficient system volume to cause poor chromatographic performance, (i.e., band broadening).

Determining if Excessive System Extra-Column Volume Exists

Two clues indicate that an HPLC system may have excessive extra-column volume for the column being used. First, the plate count measured on the system is significantly less (20% or more) than what is reported by the column manufacturer. Second, the plate count of early eluting peaks is significantly less (30% or more) than the plate count on late eluting peaks.

Correcting Excessive Extra Column Volume

To get the best performance out of high efficiency or lower volume columns, extra column volume must be minimized. This can be accomplished by doing the following:

	Keep the injector-to-column and column-to-detector tubing length as short as possible.
	Use tubing with 0.010" i.d. or smaller.
	Make sure that all fittings are made correctly and replace worn or damaged fittings.
	Use a detector cell with a volume of <10 無.

Special instrument modifications should be made when using narrow-bore, 2.1 mm i.d. HPLC columns.The HPLC intended for narrow-bore applications should have:

	a detector cell volume of 2 無 or less
	a micro-injection system
	micro-bore tubing (capillary), having a 0.007" i.d. or less
	all zero-dead volume connectors.

Effect of Extra-Column Volume on Small Bore Columns

Many chromatographers are selecting columns with 3.0 mm and 2.1 mm internal diameters to reduce solvent waste. Because columns of smaller diameters have reduced column volume and require use of lower flow rates, their use decreases solvent waste by 60 to 80%. For these same reasons, the sample also elutes from these columns in a smaller volume (see Figure 4-2, assumes same k value).

The smaller the peak volume the more likely the peak will broaden in the extra-column volume of an HPLC system. Peaks eluting early in an isocratic separation are even more susceptible to this broadening,because the volume of early eluting peaks [low k value] is considerably less than for later eluting peaks [larger k value].This observed peak broadening can result in a very significant loss in resolution. In fact,if the extra-column volume of a system is excessive, the performance of any analytical column can be compromised.

An HPLC system with minimal extra-column volume will give similar plates for both the 4.6 x 150 mm, 5 痠 and 3.0 x 150 mm, 5 痠 columns. However, as the extra-column volume of the system increases, the decrease in column efficiency for the narrow-bore column (3.0 mm i.d.) is more dramatic, almost 50% (see Table 4-1).

Effect of Extra-Column Volume on Highly Efficient Columns

In addition, peaks eluting from highly efficient columns, columns with smaller particles (i.e. 3 痠), have smaller peak volumes and are susceptible to extra-column volume as well.

For example, an HPLC column that provides high plate count on an HPLC system with minimal extra-column volume (10 無) may yield less than half that efficiency on another HPLC system with 100 無 in extra-column volume. The higher the efficiency of the column, the more dramatic the effect of extra-column volume on efficiency (Table 4-2).

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