by Dr Imran Ali
With the ability to achieve nano-level sensitivities and requiring only tiny sample volumes, Nano-liquid chromatography (NLC) and Nano-capillary electrophoresis (NCE) are emerging techniques that have applications in many research areas, including genomics, proteomics and drug discovery. However the full potential of the techniques can only be achieved by the prior coupling (hyphenation) of a sample preparation step. Hyphenation approaches that have been used for conventional chromatography systems are not suitable for nano techniques and specially developed nano hyphenation techniques (NHTs) are necessary. This article lists the most frequently used NHTs.
Only a decade ago, microanalysis was considered to be the state of the art technology that was suitable for both research and routine purposes in analytical laboratories. However, over the past decade, thanks to the remarkable development of microfluidics, there has been a steady development in techniques capable of reaching sensitivities in the nano range or even lower, with the result that the once dominant microanalytical techniques are progressively being left behind. The principal new nano scale techniques are Nano Liquid Chromatography (NLC) and Nano Capillary Electrophoresis (NCE), both of which are suitable for samples in which either the analyte concentration is very low or the sample volume itself is extremely limited [1]. Such samples are increasingly being found in many application fields including proteomics, genomics and metabolic research. In addition, more and more regulatory or governmental authorities are requiring analyses to be carried with ever higher sensitivities.
While the use of ultra-pure test samples shows that the techniques of NLC and NCE can easily achieve the required sensitivity, in real life samples frequently contain many impurities, of which even trace amounts can interfere with the analysis. Thus, the full potential of NLC and NCE can only be achieved if, prior to the analysis itself, there is a suitable sample preparation step. Conventional sample preparation methods, including on-line micro solid phase extraction or any other micro level techniques, are not suitable for use with NLC and NCE. Instead, special sample preparation techniques based on microchips are needed; these are collectively known as Nano-Hyphenation Technologies (NHTs).
Although up until now there have only been relatively few research publications devoted to NHTs, their development is an extremely rapidly developing field. This article summarises the principles behind NHTs and their advantages.
Sample preparation techniques
Hyphenation is of course the coupling of a sample preparation process with a core analytical instrument. Several methods that have been used for hyphenation with conventional analytical techniques have also been tried with NLC and NCE. Such methods include micro solid phase extraction, liquid chromatography, immuno-affinity, matrix solid phase dispersion, membrane filtration and mole mass filtration. Combining these methods with NLC and NCE is time-consuming and technically demanding and requires a skilled operator. It is, therefore, not surprising that none of the methods is suitable (or sufficiently developed) for hyphenation with NLC and NCE. Special NHTs are needed; these are described below.
Hyphenation in nano liquid chromatography
If sample contamination is to be avoided and reproducible results are to be generated efficiently at the nano levels, specially designed NHT methods are the only possible approach. An on-line desalting of macromolecules (betaine-type amphoteric or zwitterionic surfactants solutions) using a two-layered laminar flow system and based on the differential diffusion of analytes has been reported [2]. Likewise an on-line sample preparation method based on electro-wetting on dielectrics has been developed for MALDI-MS [3]. Hyphenated nano-SPE with micellar electro kinetic chromatography (MEKC) has been shown capable of the analysis (in less than five minutes) of rhodamine B with a detection limit of 60 femto mol [4]. Figure 1 is a schematic representation of this hyphenation process and shows the sample preparation and separation components. Other NHTs are a chip-based on-line solid-phase extraction (SPE) used with NLC for the DNA polymerase chain reaction (PCR) [5]; a novel method for the extraction of aromatic amines coupled with NLC with overall sensitivity of 20.0 ng/L has also been described.
Hyphenation in nano-capillary electrophoresis (NCE)
Since the principles of separation are different in NLC and NCE, the latter technique is frequently used with samples which cannot easily be separated by NLC. Given the generally charged nature of samples found in proteomic or genomic research, NCE is mostly the technique of choice in these fields. Overall there are more published papers on NHT-NCE than on NHT-NLC. Some of the more significant NHT-NCE systems are listed below. An integrated nano on-line coupling of SPE with NCE has been described for use with Rhodamine 123 and FITC-labelled ephedrine [7] [Figure 2], while a poly dimethyl siloxane chip system has been used for SPE-NCE hyphenation followed by ESI-TOF-MS detection for the analysis of peptides [8]. Another chip, made from multiple layers of SU-8 polymers, has been used for SPE-NCE [9]. A microchip-based on-line SPE-NCE has been developed for the analysis of λ-DNA from human hepatocellular carcinoma (HepG2) cells and human whole blood samples [10]; while hyphenated micro-dialysis combined with NCE has been used for monitoring the reaction of fluorescein-labelled mono-β-D-galactopyranoside (FMG) with β-D-galactosidase [11] and also the analysis of amino acid and peptides [12]. A chip based on a built-in membrane has been used with NCE for the analysis of glutathione in human plasma and red blood cells [13].
Sample stacking is the best pre-concentration technique in conventional CE, and this approach has also been utilised in NCE, albeit with some modifications such as large-volume, field-amplified, micellar electrokinetic chromatography, and pH-mediated sample stackings for use in NLC and NCE. One example of the sample stacking approach is a microchip-based field amplification sample stacking (FASS) system, which allows the formation of comparatively long, volumetrically defined sample plugs [14]. A FASS-NCE chip was produced using a photo-initiated porous polymer and has been succesfully applied for the analysis of samples containing fluorescein and BODIPY dyes [15]. The hyphenation with NCE of an on-line sample pre-concentration system using field amplified stacking through pressure driven flows has been shown to give a 94, 108 and 160 fold increase in detection sensitivity for various fluorescein molecules [16]. Finally, a multi-T microchipintegrated FASS system has been hyphenated with NCE, for use with rhodamine123 and fluorescein sodium salts.
Conclusion
Successful hyphenation of sample preparation techniques with NLC and NCE is vital; it is virtually impossible to analyse accurately and precisely a chemical species using NLC and NCE without hyphenation of the sample preparation step. Still in their evolutionary stages, NHTs will undoubtedly undergo extensive further development in the future.
References
1. Ali I et al. Nano Chromatography and Capillary Electrophoresis: Pharmaceutical and Environmental Analyses, Wiley & Sons, Hoboken, USA (2009).
2. Ali I et al. J Sepn Sci 2008; 31: 2040.
3. Wheeler AR et al. Anal Chem 2005; 77: 534.
4. Ramsey JD et al. Anal Chem 2005; 77: 6664.
5. Legendre LA et al. Anal Chem 2006; 78: 1444.
6. Yazdi AS et al. Chromatogr A 2005; 1082: 136.
7. Long Z et al. Lab Chip 2007; 7: 1819.
8. Dahlin AP et al. Anal Chem 2005; 77: 5356.
9. Tuomikoski S et al. Chromatogr A 2006; 1111: 258
10. Zhong R et al. Electrophoresis 2007; 28: 2920.
11. Huynh BH et al. Anal Chem 2004; 76: 6440.
12. Huynh BH et al. J Pharm & Biomed Anal 2006; 42: 529.
13. Long Z et al. Electrophoresis 2006; 27: 4927.
14. Lichtenberg J et al. Electrophoresis 2001; 22: 258.
15. Jung B et al. Electrophoresis 2003; 24: 3476.
16.Gong M et al. Anal Chem 2006; 78, 3730-3737.
17. Zhang L et al. Chromatogr A 2006; 1137: 243.
The author
Dr Imran Ali
Email: drimran_ali@yahoo.com
Dr Ali is currently Associate Professor, Department of Chemistry, Jamia Millia Islamia (Central University) New Delhi 110025, India. His principal research interest is Analytical and Environmental Chemistries, in particular chromatographic and capillary electrophoretic techniques of pharmaceutical compounds (simple and chiral) and environmental-derived samples. He has also expertise in water quality and advanced wastewater treatment technology. With 200 publications in his bibliography including four full books and several chapters, reviews, research papers, technical reports and conference presentations, Dr Ali sits on the editorial boards of several renowned academic journals.
