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Extracolumn Volume and its Effects in Gradient HPLC

To Maintain Efficiency and Resolution, Use Short Lengths of Narrow-Bore Tubing

✔

Amount

and

location

of extracolumn volume affect efficiency and resolution.

✔

Extracolumn volume

after

the column has greater effect than extracolumn volume

before

the column.

✔

150µL of extra volume can cut efficiency by almost 50%.

Effects of extracolumn volume on band broadening,

and the resulting chromatography, have been well

studied and documented. These investigations, how-

ever, have primarily explored effects in isocratic sep-

arations. In this investigation, we have taken a prac-

tical look at extracolumn volume in gradient analy-

ses, and studied the effects on actual separations.

The data we have obtained show that the location of

extracolumn volume in the sample flow path, as well

as the amount of extracolumn volume, has a nega-

tive effect on theoretical plates (efficiency) and reso-

lution. These extracolumn effects, in combination

with the variables of column dimension and analyte

retention, play important roles in the resulting chro-

matography.

To establish baseline chromatographic perform-

ance, we analyzed a homologous series of com-

pounds consisting of toluene, ethylbenzene,

propylbenzene, butylbenzene, and pentylbenzene

on an optimized Agilent series 1100 chromato-

graph, using a 150 x 4.6mm Pinnacle II

™

C18

column (5µm packing) and a methanol gradient

(80-100% in 10 min.) or a 50 x 4.6mm

Pinnacle II

™

C18 column and methanol gradient

(80-100% in 3.3 min.). After establishing per-

formance baselines, we added PEEK

tubing of a

known internal volume to the sample flow path,

ahead of the column or after the column, and

repeated the analysis.

Effect of Extracolumn Volume and Location:

15cm Column

Figures 1a and 1b show the effect on efficiency

(plates/meter, N/m) and resolution caused by

increased extracolumn volume when using a 150 x

4.6mm C18 column under gradient conditions.

Chromatographic performance deteriorates, as

expected. Unlike observations from isocratic sepa-

rations, however, extracolumn volume in the por-

tion of the sample path between the column and the

detector has a more significant effect than extracol-

umn volume in the tubing, connections, guard col-

umn, etc. located before the column inlet. In fact,

for the later-eluting compounds in the test mix,

150µL of extra volume after the column had the

same effect as 500µL of extra volume before the

column.

Effect of Extracolumn Volume and Location:

5cm Column

Figures 1c and 1d show the effect on efficiency and

resolution caused by adding extracolumn volume

when using a 50 x 4.6mm C18 column. The effects

are, overall, equivalent to those observed with the

150 x 4.6mm column. Since the peak volume is

much smaller for this shorter column, however,

equal amounts of extracolumn volume have greater

effect than on a 150mm column. In this system,

150µL of extra volume before the column reduced

efficiency by 46%, whereas with the 150mm column

the loss in efficiency was only 20%.

Conclusions

In a gradient analysis, the location of extracolumn

volume in the sample flow path can be equally

important to the amount of extracolumn volume in

its effects on chromatographic performance. In par-

ticular, extra volume after the column should be

reduced. This is especially important for fast analy-

ses on short columns.

For any HPLC separation, it is best to keep tubing as

short and the ID as narrow as practical. Additionally

it is wise to use precut stainless steel tubing, or

PEEK

tubing cut with a guillotine-style cutter, to

ensure square, burr-free ends for minimal dead vol-

ume at connections.

For an extensive selection of tubing, low-volume fit-

tings, and related tools, request our HPLC catalog

(lit. cat.# 59241A).

Figure 1

— Extracolumn volume after the column has greatest negative impact on efficiency and resolution in gradient analysis, as shown by

consistently lowest values for plates/meter and resolution versus k.

15cm column

a) efficiency

b) resolution

5cm column

c) efficiency

d) resolution

N/m

resolution

N/m

Gradient: 80–100%

methanol in 10 min.

150µL, before the column

150µL, after the column

500µL, before the column

500µL, after the column

Gradient: 80–100%

methanol in 3.3 min.

150µL, before the column

150µL, after the column

By Randy Romesberg, HPLC Applications Chemist