Nano Flows

Therefore, fluorescent nano-particles can be used as the tracer in studying micro-fluids. The visualizing approach to nano-flow is not very mature and more experiments should be practiced. Because of its wide connection to modem technologies, the research of the solid-liquid two-phase flow will attract more and more attention. 

Many commercial split flow capillary LC systems incorporate a nano flow sensor mounted online to the capillary channel. The split flow system can be easily modified from a conventional system and performs satisfactorily for capillary LC applications. However, the split flow system may require thermal control and the LC solvent requires continuous degassing. In addition, the system may not work reliably at a high flow split ratios and at pressures above 6000 psi due to technical limitations of the fused silica thermal conductivity flow sensor. The split flow system based on conventional check valve design may not be compatible with splitless nano LC applications. The conventional ball-and-seat check valve is not capable of delivering nano flow rates and is not reliable for 7/24 operation at low flow. 

The development of microbore chromatography and nano-flow HPLC has greatly increased interest in detectors suitable for sub-microliter peak volumes. These include LIE, EC, and MS spectrometry detectors. Zare first described the application of lasers for detection in analytical chemistry in 1984, outlining the potential for the use of lasers in analytical science, especially their application to HPLC [20]. This was followed in 1988 by another review that focused more on HPLC applications and provided examples of the high sensitivity possible with LIE detection [21], Another good review was written by Rahavendran and Karnes outlining the usefulness of LIE in pharmaceutical analysis [22],... 

The detection cell must be designed with its volume small enough to prevent additional peak broadening in the detector in practice, this means the detection cell volume should be at least 10 times smaller than the volume of the first, most narrow, chromatographic peak. For nano-flow HPLC systems, in which the peaks can be sub-microliter, detection cells with a volume on the order of 100 uL or less are appropriate. For conventional HPLC systems, in which the peak volume is tens of microliters, there is no benefit to having a detection volume smaller than a few microliters, and indeed most conventional HPLC flow cells are around 5-12 pL. It is best to ensure that the detection cell is well matched to the sample and peak volumes, as making the detection volume too small will unnecessarily decrease the sensitivity of the detector. 

Although this section provides a brief description of most commonly nsed detectors for HPLC, most of the focus is on a few detection modes. Optical absorbance detectors remain the most widely nsed for HPLC, and are discnssed in some detail. We also focns on flnorescence, condnctivity, and electrochemical detection, as these methods were not widely nsed for HPLC in the past, bnt are especially well suited to micro- and nano-flow instrnments becanse of their high sensitivity in small sample volumes. Mass spectrometry has also come into wide and rontine nse in the last decade, but as it is the subject of another chapter, it will not be fnrther discnssed here. Miniaturization has been particularly important for capillary and chip-based electrophoresis, which often employs sub-nanoliter detection volnmes [36,37]. 

Micro-injections in micro-flow and nano-flow systems are done with injectors in which the external sample loop is replaced with the internal fixed volume within the injector body. HPLC-on-a-chip systems also build the column into the injector body. The internal path within the injector body is abladed with a laser, packed with micro-packing material, and this serves as the separating media. The injector inlet is connected to the pumping system and the outlet to the detector. Sample is loaded into an internal loop in the load position, then injected onto the chip HPLC by turning the injector. Obviously, in a system like this sample size is very limited and the detector is usually a highly sensitive mass spectrometer. 

Chip HPLC—Nano-flow, micro-sample HPLC system in which the packed column resides within the body of the injector. Originally touted as the HPLC of the future for nano-level LC/MS, but its potential has been slow to materialize. 

Syringe Pump—A pulseless pump made up of a motor-driven piston or plunger in a solvent-filled cylinder. Useful only when small solvent volumes are to be pumped often used in micro-flow or nano-flow HPLC systems. 

Basically, no special devices have been developed to handle mobile phases in nano-HPLC. However, our experience dictates that reservoirs small in volume and of high quality glass are preferred for this purpose. Solvent containers should be air tight and free from any contamination. The use of helium gas, through a sparging device, may be beneficial for degasification of solvents in nano-HPLC, which can improve check valve reliabilities, especially at nano flow, and diminish baseline noise in UV detection. Besides, each reservoir should be equipped with a shutoff valve for efficient helium consumption [9]. 

Ideal plug-flow conditions can also be established in so-called nano-flow reactors with catalyst particle sizes from 50 to 200 pm. These reactors were operated in 16-and 64-barrel mode at Avantium for the regression of intrinsic kinetics [4],... 

Figure 2. Workflow of an LC-MS/MS experiment. A mixture of peptides from a protein sample digest is separated by reversed-phase chromatography on a nano-flow HPLC. The peptides elute from the RP column and are ionized by an electrospray source. In the first stage of mass spectrometry, m/z values and charge states for each precursor ion are determined and the most abundant precursor ions are selected for analysis in the second stage. The ions are then fragmented with by collision-induced dissociation (CID) a gas to produce fragment ions which are detected. Using the mass (from MS-1) and sequence information (from MS-2) protein sequence databases are searched to provide peptide identifications and protein matches.

RP-HPLC is also used for separation of peptides deriving from proteolytic digestion of the wine proteins separated on and excided from a electrophoretic gel slab. By this approach and performing nano-flow RP-HPLC coupled with MS/MS analysis, 20 different proteins were identified in a white wine (Kwon, 2004) (see below). 

While the sample is loaded onto the trapping column and desalted by flushing with 0.1% TFA at an increased flow rate, the nano-HPLC branch is equilibrated with 0.1% FA. The UV baseline is set on the FA containing solution. By switching the HPLC valve, the trapping-column loop is then integrated into the nanoflow. Now, the nano-flow pushes the small volume of TFA solution through the... 

As mentioned above, ESI instruments were coupled to liquid chromatography. The biggest impact in ESI MS has been the adaptation to reduced flow capabilities in the 10-500 nl/min range. Wilm and Mann [115] and Emmett and Caprioli [116] developed such improvements in parallel. The combination of nano-flow LC with micro-capillary reversed phase HPLC and nano-ES has i) dramatically improved the sensitivity of ESI-MS/MS and ii) enabled the automation of protein identification by using an auto sampler for loading of samples onto the LC [117]. 

Tsaoulidis D., Dore V., PlechkovaN., Seddon K.R., Angeli P., 2011. Liquid-liquid flows in Microchannels. In proceedings of the 3rd Micro and Nano Flows Conference, 22-24 August, Thessaloniki, Greece... 

Tsaoulidis D., Li Q., Chinaud M., Angeli P., 2014. Two-phase aqueous-ionic liquid flows in small channels of different diameter. 4th Micro and Nano Flows Conference, London, UK, September 7-10... 

Acknowledgements The author would like to acknowledge financial supports of Iranian Elite Foundations for pursing his researches in the field of micro/nano flows under Grant No. 100665. 

Nakagama T, Maeda T, Uchiyama K, Hobo T (2003) Monitoring nano-flow rate of water by atomic emission detection using helium radio-frequency plasma. Analyst 128(6) 543-546... 

Kamiadakis GE, Beskok A, Alum N (2005) Micro flows and nano flows fimdamentals and simulation. Springer, New York... 

Ejtehadi O, Roohi E, Esfahani JA (2012) Investigation of basic molecular gas stmctural effects on hydrodynamic behavior of rarefied shear driven micro/nano flow using DSMC. Int Commun Heat Mass Transf 39 439 48... 

Mobile phase to column Micro or nano flows... 

Fig. 11 Thermal mass flow sensing principle (A) for normal flows and (B) for micro and nano flows (MEMS).

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