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Nano-HPLC

It is a relatively new chromatographic technique that has considerable importance in proteomics, where it is important to work on small amounts of sample and it is mandatory to use very small flows and reduced inner diameters of chromatographic columns. The reduction in the volume and diameter of the columns and the diameter of the spheres of chromatographic resins allow the analysis of protein samples from a single spot coming from the 2DE. This samples would be too diluted for normal HPLC analysis.

Lower column volumes and solvent flows allow, also, to interface directly the output of the instrument with a mass spectrometer.

Nano HPLC is also called nanobore or nano-scale HPLC, it is important to note that the definition is not related to the internal diameter of the column used but rather to flows of solvents that are in the order of nanoliters per minute.

The basic principles of nano HPLC are the same as the traditional technique but have different advantages and also some disadvantages:

Pros

  • The sensitivity is greatly increased, especially if nanoHPLC is coupled to a mass spectrometer, it becomes possible to detect peptides in the order of attomoles.
  • It reduces solvent consumption and thus the production of waste.
  • Reducing the diameter of the column and increases sensitivity allows to analyze samples in small volumes.
  • Reducing the diameter of the packing material of columns can increase the peptide coverage thanks to a better peak separation.
  • Allow to use a mass spectrometer as detector, without necessarily using a split-flow systems.

Cons

  • Fluidics system is delicate.
  • The reduced inner diameters increase the likelihood to have a blockage in the tubes and the columns.
  • Chips and columns for the analysis are quite expensive, if not produced in the laboratory.

    • The term nanoHPLC is still not defined clearly, there are also two types of nano HPLC:

    1. Separation system based on microfluidic chip with rectangular section channels.
    2. Capillary columns with circular section and an inner diameter of about 25-100µm

    Classification of HPLC systems:

    Description Inner diameter Flow range
    open tubular LC < 25µm < 25nL/min
    Nano HPLC 25-100 µm 24-4000 nL/min
    Capillary HPLC 100-1000 µm 0.4-200 µL/min
    Micro HPLC 1.0-2.1mm 50-100µL/min
    Standard HPLC 4.0-5.0 mm 1-10ml/min
    Preparative HPLC > 10 mm > 20ml/min
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