Benefits

• High sensitivity
• Low sample mass and volume applications
• LC-MS and LC-MS/MS applications
• Very low solvent consumption

Capillary and nano HPLC are gaining acceptance in applications where limited sample amounts lead to problems in detection sensitivity. High sensitivity and high resolution separations can now be achieved for small sample volumes. This is relevant in the areas of pharmacokinetics, trace analysis and particularly the rapidly expanding field of bioanalytical and proteomic analysis. Figure 1 shows the analysis of 7.5pmol/µl from a tryptic digest of horse skeletal muscle myoglobin.

Capillary and nano columns are ideal for use with detectors requiring the lowest flow rates, such as the electrospray LC-MS detector. Indeed, the on-line coupling with a mass spectrometer has been a major driving force behind the development of capillary chromatography.

Sensitivity

The introduction of capillary and nano columns has made possible high sensitivity and high resolution separations for small sample volumes. Table 1 shows the theoretical sensitivity increase of various i.d. capillary columns compared with a 1mm i.d. microbore column. The use of a  0.075mm (75µm) i.d. column, for instance, can decrease detection limits by a factor of >3500 relative to a 4.6mm i.d. column 0.075 when the same sample size is used, due to lower chromatographic dilution of the sample.

Instrument Modifications

In order to fully exploit the benefits of using capillary  dimensions, the HPLC system must be capable of handling , sample volumes in the sub-microlitre range. The use of  columns of <1mm i.d. requires either a specially designed micro-LC instrument or extensive modifications of a standard HPLC instrument. The major principles in the conversion are flow rate reduction and elimination of dead volume.
Flow rate reduction can be achieved using a high performance, low µl/minute pump or by incorporating a flow splitting tee between a standard HPLC pump and the injector. The majority of the flow can be split to waste or recycled.
 

To ensure that band spreading is kept to a minimum, low dispersion column hardware must be used throughout the system. Connecting capillaries must be dead volume free and as short as possible. A micro-scale injector allowing injection of sub-microlitre sample volumes should be used. Micro flow cells with appropriate internal volume (<1µl) and path length should be used for UV detection.

Column Availability

Capillary and nano columns, ranging in i.d. from 0. 05mm to 0.8mm, are available in the ACE , Inertsil , Thermo Scientific Hypersil GOLD , Vydac , YMC , ZIC-HILIC and Zorbax ranges.

Table 1