Nano LC: Principles, Evolution, and State-of-the-Art of the Technique

Nano LC: Principles, Evolution, and State-of-the-Art of the Technique
Oct 01, 2011

In genomics, polymerase chain reaction (PCR) is used to amplify the amount of DNA and increase the signal above the limit of detection. This approach is not applicable to proteomics, however. Therefore, the intrinsic sensitivity of the analytical method applied to the analysis of biomolecules, generally liquid chromatography–mass spectrometry (LC–MS), needs to be enhanced. The miniaturization of the LC system and its interfacing with MS results in the necessary increase in sensitivity. Nano LC is at the heart of this gain in sensitivity. This column installment reflects on the principles, development, and current state of nano LC instrumentation.

History has a tendency to repeat itself. In the 1990s, genomics led to the development of an array of dedicated analytical techniques to solve the challenges in DNA identification. During the past decade, the identification and quantification of proteins and peptides in biological fluids, also known as proteomics, has necessitated similar developments. Although polymerase chain reaction (PCR) is used in genomics to amplify the amount of DNA and increase the signal above the limit of detection, it is not applicable to proteomics. Therefore, the intrinsic sensitivity of the analytical method applied to the analysis of biomolecules, generally liquid chromatography–mass spectrometry (LC–MS), needs to be enhanced. The miniaturization of LC systems that led to the development of nano LC (Table I) and its interfacing with MS is at the heart of this gain in sensitivity.


Table I: LC columns for various applicatons
The analysis of samples as complex as those in proteomics consists of multiple steps (sampling, sample pretreatment, separation and detection, and data analysis) in which nano LC is applied as a routine part before tandem MS detection. This installment of "Innovations in HPLC" reflects on the principles, development, and current state of nano LC instrumentation.

Miniaturization of LC Systems

Theory

A reduction in column internal diameter results in less chromatographic dilution and, consequently, increased concentration of the injected sample on the high performance liquid chromatography (HPLC) system. The chromatographic dilution (D) of the sample, when injected on a LC system, is expressed by the following equation (1):



where C o is the initial compound concentration in a sample (before injection into the LC system); C max is the final compound concentration at the peak maximum; ε is the column porosity; r is the column radius; k is the retention factor; L is the column length; H is the column plate height; and V inj is the sample volume injected.

D increases proportionally with the square of the column radius and with the square root of the length of the column. Thus, a reduction in column diameter results in a significantly lower dilution factor, thereby increasing the concentration in the eluted peak.

Though this formula applies to isocratic elution conditions, its consequences are commonly extrapolated to gradient elution conditions. Under gradient elution conditions, dilution is partly counteracted by increasing the strength of the mobile phase over time. However, the gain in sensitivity of this effect is far smaller than what is gained by decreasing the column internal diameter. The gain in sensitivity (f) resulting from the use of a LC column with a smaller internal diameter can be approximated by the following relation (1,2):



where d 1 and d 2 are the diameters of the conventional and nano LC columns, respectively.

Therefore, downscaling the column used in an analytical method from 4.6 mm i.d. to 75 µm i.d. should result in an almost 4000-fold gain in sensitivity. However, such an increase in sensitivity is not readily achieved because reducing the column internal diameter has practical consequences for the entire setup. The influence of the different system parameters is discussed below.