Application Brief: Using Narrow-Bore HPLC Columns

Solvent consumption is directly related to column volume
Mobile phase solvents add considerable expense to the cost of an HPLC analysis, often more than the chromatographic column. Not only are the solvents costly to buy, but their disposal is becoming a considerable expense, as well as an increasingly important environmental issue.

One way to reduce solvent consumption in HPLC laboratories is to use chromatographic columns with small internal volume. Narrow-bore HPLC columns (2.1 mm ID) have significantly lower column volume than standard HPLC columns and can, therefore, reduce solvent consumption by almost 80%.

Table 1
Solvent consumption is directly related to the volume of the HPLC column.

Column Dimensions	Column Volume	Solvent Consumption
9.4 x 250 mm	10.5 mL	79.1 mL
4.6 x 250 mm	2.5 mL	18.9 mL
4.6 x 150 mm	1.5 mL	11.4 mL
4.0 x 80 mm	0.6 mL	4.5 mL
2.1 x 150 mm	0.3 mL	2.4 mL

Solvent consumption was calculated from the elution volume of toluene at a k' value of 6.5 on ZORBAX^® Rx-C8 stationary phase.

Narrow-bore columns often perform poorly because of excessive extra column volume.
Reducing solvent consumption in the HPLC laboratory is not just a simple matter of choosing to use narrow-bore columns. Most HPLC systems in use today are designed for standard-bore columns and cannot effectively use columns with internal volumes less than 0.5 mL. The reason for this is the extra column volume, the system volume measured from the injector through the detector (see Figure 1), that reduces the achievable efficiency of the column. If the calculated value for theoretical plates is less than 90% of the manufacturer's reported value, there is probably an excessive amount of extra column volume in the system for the column being used. Also, if early eluting peaks have more "tailing" than later eluting peaks, there may be a problem with extra column volume. Early eluting peaks are less diluted and have smaller volumes than late eluting peaks and will consequently be more susceptible to the detrimental effects of excessive extra column volume.

FIGURE 1
Extra column volume

91061-ab-fig1.gif (26890 bytes)

The system extra column volume is equal to the sample volume plus the connecting tubing volume plus the detector volume plus the volume from any fittings and connectors.

FIGURE 2
HPLC systems must be optimized to obtain the best performance from narrow-bore columns

Typical HPLC System
Standard-Bore Column, 4.6 x 150 mm
91061-ab-fig2a.gif (5288 bytes)

Typical HPLC System
Narrow-Bore Column, 2.l x 150 mm

Optimized HPLC System
Narrow-Bore Column, 2.l x 150 mm

Chromatographic Conditions

Stationary Phase:	ZORBAX Rx-C8
Mobile Phase:	36% CH3CN, 64% 0.025M KH2PO4 + 0.2% TFA
Flow Rate:	1.0 mL/min for 4.6 x 150 mm column and 0.2 mL/min for 2.1 x 150 mm column
Sample:	Tricyclic antidepressants l. Doxepin 2. Nortriptyline 3. Amitriptyline 4. Trimipramine

Narrow-bore columns often perform poorly in typical HPLC systems. To obtain the best performance from these columns, the HPLC system must be "optimized" by reducing the amount of extra column volume.

Figure 2 provides an example of what can happen when a narrow-bore column is used with a typical HPLC system. The narrow-bore column is unable to achieve the baseline separation provided by the standard bore column. However, when the narrow-bore column is used in an "optimized" HPLC system that has less than 10 microliters of extra column volume, excellent resolution and peak shape is obtained.

To obtain the best performance from narrow-bore columns, extra column volume must be minimized.
If excessive extra column volume is a problem, several things can be done to improve the performance of the system.

Table 2
Volume estimates for connecting tubing

Tubing ID (inches)	Volume per Length (µL/cm)
0.006 (0.152mm)	0.18
0.010 (0.254mm)	0.50
0.020 (0.508mm)	2.02
0.030 (0.762mm)	4.56

Narrow-bore columns normally require detector flow cells with less than 2 microliter volumes. The design of the flow cell, however, can be just as important as the cell volume. Some low volume flow cells perform worse than larger volume flow cells because of their inadequate design.