Today there was a question posted by scholar on Yahoo! Answers:

Hi, I want some basic information on, what is nano Liquid Chromatography??? & how it is different form normally used Liquid Chromatography (like HPLC or other LC Techniques)? What are its applications compared to HPLC? Or please provide me some reference web site or book on this topic.

I would like to answer this question in a more elaborate way.

Nano HPLC is a relatively new development in chromatography world driven by recent advancements in proteomics that would require decreasing of inner diameter (ID) of liquid chromatography (LC) column to allow for a smaller sample amount and to increase sensitivity. The other demand for nano LC comes from the new field referred as “lab-on-a-chip” that would require “on-chip” liquid chromatography separation as part of other chip-based chemical analysis.

Presently the term “nano HPLC” is not well defined in analytical chemistry community and there are two types of nano HPLC:

  1. Most “on- chip” microfluidic columns, which are actually rectangular in cross-section.
  2. LC that has the flow rate on the scale of nano liters per minute (nL/min).

Classification of HPLC systems.

Desciption ID Flow Rate
Open Tubular LC <25 μm <25 nL/min
Nano HPLC 25-100 μm 24-4000 nL/min
Capillary HPLC 100-100 μm 0.4-200 μL/min
Micro HPLC 1.0-2.1 mm 50-1000 μL/min
Normal HPLC 4.0-5.0 mm 1.0 -10.0 mL/min
Preparative HPLC >10 mm > 20 mL/min

Nano HPLC is sometimes also referred to as “nanobore HPLC” or “nano-scale HPLC” in the literature, but it is also worth noting that “nano HPLC” does not imply that the column ID (inner diameter) is on the nanometer scale.

The basic principles of nano HPLC are the same as in the traditional one; however, miniaturization gives a lot of advantage over the conventional LC:

  • Significantly reduces solvent consumption and subsequent waste production.
  • ID diameter reduction increases sensitivity and/or less sample requirement.
  • Decrease in column bead size (packing) narrowers the peak width of chromatogram due to better separation efficiency.
  • Does not increase system pressure.
  • Significantly cheaper than its conventional counterpart.
  • Potential portability due to a system size reduction.
  • Allows for simultaneous mass separation if LC chips are used.

Nano HPLC ChipsNano HPLC Chip

So far there are only two commercial manufacturers of LC chips: Nanostream Inc and Agilent.

Nanostream’s “Brio Cartridge” made of polymeric material and has 24 bead-packed parallel microfluidic LC columns that allows simultaneous analysis of 24 samples. The actual sample injection and detection happens “off-chip”, just like in conventional LC.

Agilent has a chip-based fritless LC column with electrospray ionization (ESI) nozzle The chip is made of two polyimide layers bonded together, while the ESI nozzle is fabricated by laser ablative trimming of the bonded chip. The column is packed with C18 beads using a tapered outlet rather than frit, utilizing a so-called “keystone effect”. Samples are injected from off-chip using an injection valve attached to the back of the chip. A mass spectrometer is used as the detector.

So far the most complete LC chip is a device from a research group at California Institute of Technology that has on-chip gradient pumping, sample injection, column, and electrospray nozzle. Its 1 cm long column is packed after chip fabrication and a mass spectrometer is used as the detector.

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