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HPLC Extra Column Volumes

It is of particular importance to reduce extra column volumes when using small volume columns or UHPLC.  However, where do these extra column volumes come from, how can they be minimized, and what effect do they have on chromatography?

Question:  
If the HPLC system contains a large extra-column volume should it not be that the later eluting peaks are broader and have a higher plate count than the early eluting peaks?  

Answer:
Firstly, I thought I would detail a couple of definitions in relation to the subject of efficiency and extra column volumes and their effects.

Definitions:
Extra column volume – the volume between the injection point and the detection point, excluding the part of the column containing the stationary phase.  It is composed of the injector, connecting lines, frits, and the detector; and it is this that determines the extra column effects.
Extra column effects – the total band broadening effects of all parts of the chromatographic system outside of the column itself.  Extra column effects must be minimized to maintain the efficiency of the column.  Areas of band broadening can include the injector, injection volume, connecting tubing, end fittings, frits, detector cell volume, and internal detector tubing.  The variances of all these contributions are additive.

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W = bandwidth

The following are bandwidth contributions from

Wc = column

Ws = injector/autosampler

Wlc = lines and connectors

Wfc = detector flow cell

As long as the bandwidth contributions Ws, Wlc, and Wfc are each less than 1/3W their effect on W can be neglected.

The Effect of Extra Column Volumes

A band of analyte molecules contained in the injection solvent will tend to disperse in every direction due to the concentration gradient at the outer edges of the band. 

This broadening effect is called longitudinal diffusion because inside tubes, the greatest scope for broadening is along the axis of flow (Figure 1). 
The band will broaden in all system tubing but the worst effects will be encountered in the column itself. 

 

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Figure 1: Longitudinal diffusion.

 
 

For an isocratic separation all peaks broaden as they move through the column, and the longer the retention time the broader the peak, therefore, the later eluting peaks will be broader in a system where the extra column volumes are minimized.

If there are extra column volumes in your system, which will lead to distortion of your peaks, then a decrease in N for earlier eluting peaks will be seen (i.e. broader, less efficient peaks).  Extra column effects typically only produce a small increase in retention time but a comprehensive increase in peak dispersion/band broadening (Figure 2).

 

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Figure 2: Effect of extra column volume.

 

For example, the separation of six nitroaromatic compounds under identical analytical conditions but on two HPLC systems with differing extra system volumes exhibit peaks with markedly different efficiencies (Table 1).  The greater effect of the extra column volumes can be clearly seen on the early eluting peaks.

 

Peak

Efficiency (N)

ΔN

Extra Column Volume 20 μL

Extra Column Volume 80 μL

1

19,651

14,355

5,296

2

19,769

16,404

3,365

3

19,749

16,618

3,131

4

19,461

17,463

1,998

5

19,339

17,578

1,761

6

19,139

17,667

1,472

7

17,100

16,689

411

8

16,967

16,584

383

Table 1: Peak efficiencies for the separation of six nitroaromatic compounds on HPLC systems with different extra column volumes.  The greater the value of N the more efficient (narrower) the peak. 
Column: C18, 250 x 4.6 mm 5 μm; Flow rate: 2 mL/min.; Mobile phase: MeCN:H20 46:54 v/v.

 
 

Band broadening/efficiency (N) in the column is given by Equation 1.

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Where:

tr = retention time

Wb = baseline peak width

W1/2 = peak width at half height

Which, can be expressed in volume units V (baseline band volume, Equation 2).

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Where, VR is the retention (peak) volume (Equation 3).

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Therefore, substituting VR (Equation 3) into Equation 2, gives Equation 4.

 

Vm is the column dead volume and is proportional to the internal volume of the column (i.e. as the column volume increases so will the dead volume).  Bandwidths will be smaller for shorter, narrower columns (small Vm) packed with smaller particles (larger N per unit length).  V is also smaller for bands that are less retained (smaller k values).  When V is small extra column effects will have a greater impact, hence, greater band broadening will be seen for early eluting peaks as they have the smallest volume (i.e. narrowest peaks).  This also means that when working with small volume columns or UHPLC equipment, extra volume effects must be minimized in order to maintain the efficiency gained from using smaller column volumes or UHPLC equipment.

The use of optimized lengths and internal diameters of capillary tubing is important to retain the highest efficiency in LC systems.  Tubing length is important but the internal diameter is of much greater importance, which can be demonstrated by the Aris-Taylor equation (Equation 5).

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Where:

F = flow through the tubing

L = tubing length

DM = analyte diffusion coefficient

i.d. = tubing internal diameter

The overall dispersion of the analyte band is proportional to the length of the capillary tubing but is proportional to the capillary tubing internal diameter raised to the 4th power, thus, producing a profound effect upon analyte peak dispersion.

 
 
 
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