Open Access is an initiative that aims to make scientific research freely available to all. To date our community has made over 100 million downloads. It’s based on principles of collaboration, unobstructed discovery, and, most importantly, scientific progression. As PhD students, we found it difficult to access the research we needed, so we decided to create a new Open Access publisher that levels the playing field for scientists across the world. How? By making research easy to access, and puts the academic needs of the researchers before the business interests of publishers.
We are a community of more than 103,000 authors and editors from 3,291 institutions spanning 160 countries, including Nobel Prize winners and some of the world’s most-cited researchers. Publishing on IntechOpen allows authors to earn citations and find new collaborators, meaning more people see your work not only from your own field of study, but from other related fields too.
The trend toward instrument miniaturization in recent years has made it possible to develop new and sophisticated analytical techniques, such as nano-liquid chromatography (nano-LC). This has made it possible to improve the sensitivity and resolution of chromatography. Nano HPLC is essential for both qualitative and quantitative analytical methods. The growing interest in nano-LC methods has led to the development of several fascinating and inventive applications. This chapter will cover the theoretical aspects of the nano-LC method and its current practical uses in the analysis of pharmaceutical and biological molecules. Furthermore, the future prospects regarding the development of nano-LC techniques will be examined.
Department of Pharmacy, College of Health and Medical Science, Dilla University, Dilla, Ethiopia
Ayenew Ashenef
Department of Pharmaceutical Chemistry, School of Pharmacy, College of Health Science, Addis Ababa University, Addis Ababa, Ethiopia
Center for Innovative Drug Development and Therapeutic Trials for Africa (CDT-Africa), College of Health Science, Addis Ababa University, Addis Ababa, Ethiopia
Ariaya Hymete
Department of Pharmaceutical Chemistry, School of Pharmacy, College of Health Science, Addis Ababa University, Addis Ababa, Ethiopia
Tadios Niguss
Department of Research and Training, Eka Kotebe General Hospital, Addis Ababa, Ethiopia
*Address all correspondence to: esa.tesfaye7@gmail.com
1. Introduction
It is not novel to see systems becoming smaller. Miniaturization is the current trend. According to statistics published in the literature in the 1978–1990s, miniaturization was introduced by researchers in the field of liquid chromatography (LC) approximately 30 years ago. Early research, for example, by the groups of Novotny and Knox, examined theoretical as well as practical aspects of lowering the column internal diameter (i.d.) that is used in chromatography [1, 2, 3, 4]. More recently, biological applications in the proteomics research field have been the main driving force behind the expansion of miniaturized LC devices. The developments in micro-column liquid chromatography will be covered in this chapter, with particular emphasis on column development, instrumentation, analyte detection, and application areas.
The analytical discipline is the primary application for miniature techniques including nano-LC, which use stationary phases (SPs) housed within capillaries (fused silica). Typical example of this, capillary electrophoresis (CE), one of these methods with several modes, which has been extensively researched and used [5, 6, 7, 8, 9, 10, 11]. However, only nano-liquid chromatography, or nano-LC, will be covered in this chapter.
2. Theory and principle of nano-liquid chromatography
2.1 Theoretical aspects of nano-LC
The foundation principle of a tiny chromatographic system is the same as that of normal chromatography. The injected analytes may dilute in the column during the chromatographic process, changing the effectiveness of the separation process:
D=CoCmax=Πdc2ε(1+k)2LHΠVinjE1
The expression for this dilution event, known as chromatographic dilution (D), is as follows: where Co represents the starting concentration, Cmax denotes the analyte’s final concentration during the chromatography process, dc denotes the inner diameter of the column, ε denotes column porosity, L denotes column length, 4Vinj denotes sample injection volume, k and H are the chromatographic parameters retention factor and plate height, respectively. The formula states that as the square of the column diameter decreases, D reduces correspondingly. A considerable drop in D-values is made possible by lower i.d. nano-LC as opposed to traditional HPLC. Therefore, less chromatographic dilution is required with a smaller chromatographic system, improving the separation’s mass detectability [12]. Miniaturizing LC systems should theoretically be very advantageous for liquid phase separations. However, because they lead to decreased separation efficiency, a few real-world separation challenges need to be taken into account.
2.2 Analytical instrumentation of nano-LC systems
2.2.1 Pump
In order to facilitate nanoscale gradient elution, pumps for nano-LC should have stable, repeatable nanoflow rates throughout separations. For the Nano-LC, there are two primary systems available: split pumps and splitless pumps, the latter of which are offered for sale and are commercially available. Split systems distinguish high flow rates (mL/min) from traditional HPLC pumps by using a flow restrictor between the pump and a smaller column. With a straightforward nanoflow restrictor design, these devices can utilize conventional HPLC pumps [13]. Split systems, however, have the potential to result in inconsistent split ratios, inconsistent nanoflow, and inconsistent separation. It is quite challenging to achieve reproducible gradient elution, particularly when using DIY (Do it yourself) split devices. This manner of elution may be limited by differences in backpressure caused by different mixed solvent viscosities [14]. Splitless systems are currently commonly employed in nano-LC. These technologies provide extremely reproducible nanoflow rates while eliminating solvent loss. Split systems are inferior to syringe pumps with a single limited-volume reservoir; however, continuous-flow pumps, which resemble traditional piston pumps with two pistons per channel, are now the most widely used type. Both isocratic and gradient nanoflow elutions can be performed using continuous-flow pumps, and adjusting to the required nanoflow rate is simple.
2.2.2 Injection
Another crucial factor in chromatography that requires careful consideration is the volume of the injected sample. Inadequate injection volume can lead to major issues and worsen the analytes’ separation by broadening the column band. As a result, the sample must be injected into any chromatographic system as narrowly as feasible; the issue becomes more apparent when the column’s i.d. decreases. The maximum sample volume that can be injected into a column depends on several parameters such as the length of the column (L), the particle diameter (dp), the column diameter (dc), and the retention factor (k):
Vmax=0.36Ldpdc(K+1)E2
As a result, specific, commercially accessible valves with internal loop capacities of 40–60 nL are utilized. As an alternative, loops with larger volumes are used; however, in this instance, the time is controlled by injecting only a portion of the sample solution [15]. Therefore, an LC injector with minor changes would be required for a laboratory-made nano-LC system (see Figure 1) in addition to a split pump system and a UV-visible detector. Lastly, automated sample injection techniques are used in current nano-LC instrumentation, where volumes—typically μL—are split into nL.
Figure 1.
Schematic diagram of assembled nano-LC instrumentation.
2.2.3 Nano columns
While 10 μm i.d. columns are also an option, 75 μm i.d. nano-LC columns are the most commonly utilized. In nano-LC separations, this i.d. column offers a reasonable balance between robustness, loadability, and detectability.
Since there were no commercially available columns in the early days of nano-LC, researchers had to make their own. Thanks to effective packing techniques, the first nano-LC columns were made commercially available in the 1990s. The selection of columns has expanded significantly over the past 20 years, and nano-LC columns are also offered. Research laboratories utilize self-packed nano-LC columns frequently for cost-effective purposes on a periodic basis. There are several variants and formats of nano-LC columns, monoliths, open tubular columns, and the pillar array format. Most applications are done with packed columns, while the monolith and open tubular columns are still less established as routine tools. The two types of nano-LC columns are explained below.
Packed columns
Fused silicon capillaries covered with polyimide are utilized to create the packed columns used in the nano-LC columns. In addition to their current flexibility, strong mechanical resistance, and range of internal dimensions, nanocolumns can also be made of titanium and stainless steel tubes. They can be filled with a monolithic bed of silica-based particles, packed with silica-based particles, or, much less frequently, wall-coated with the proper inorganic or organic materials. Particle sizes between 3 and 5 μm are most frequently used in packed nanocolumns. Particle-filled tiny i.d. columns are challenging to prepare, nevertheless.
Monolithic columns
In biospecific analysis, biocompatible compounds present an intriguing alternative to conventional synthesis methods for processing monoliths. Because the stationary phase is fixed to the column wall in this sort of column, a porous (silica or polymer) structure forms throughout the column, obviating the requirement for frits. Single rods of organic or inorganic material that can form in the capillary column are known as monolithic stationary phases. Monolithic columns do not require frits, and their high porosity permits faster mobile phase flow rates, which shortens the separation time. Modern nano-LC columns with the separation power needed for these complicated proteomics samples are now commercially available in lengths as long as 50 cm.
2.2.4 Analyte detection
The kinds of detection used in HPLC separations are also used in nano-LC. Nano-LC frequently uses diode array detection (DAD) due to its low cost, broad applicability, and usage of online detection. Nevertheless, when using on-column detection, detectability is restricted because of the nano-column’s small path length. Longer light routes are provided by specially designed detecting cells, which solve this problem [16]. Although they are employed in nano-LC detection, laser-induced fluorescence [17] and inductively coupled plasma MS [18] are not reliable enough to be used in routine analysis. Applications in biomedicine and pharmacology typically need for universal detection techniques with high detectability, like MS detection. For MS coupling across different nanospray interfaces, particularly electrospray ionization (ESI), which only needs a tiny quantity of eluent from the LC column to be successful, the nanoflow from the column (often 100–500 nL/min) is sufficient [19].
Numerous studies and reviews on miniaturized nano-LC, covering theoretical issues as well as practical applications, support these methods as effective tools that are also more environmentally friendly than the existing separation techniques, particularly when combined with MS.
3.1 Pharmaceutical application
Pharmaceutical analysis is a significant subject with a wide range of applications in various domains, including drug quality control, pharmacokinetic research, and examination of the chiral purity and quality of pharmaceutical formulations. For chiral analysis, nano-LC and capillary LC are favored methods of quality control; however HPLC is still the method of choice for industrial quality control. Actually, a lot of research is being done on novel chiral stationary phases (CSPs), which are frequently synthesized in little quantities or with low yields yet sufficient to build a capillary column.
One of the applications of Nano-LC on pharmaceutical analysis is the enantiomeric separation of amlodipine and its two chiral impurities by nano-liquid chromatography and capillary elecrochromatography using a chiral stationary phase based on cellulose tris. In this work, a novel polysaccharide-based chiral stationary phase (CSP), cellulose tris (4-chloro-3 methylph enylcarbamate), also called Sepapak 4 has been evaluated for the chiral separation of amlodipine, and its two impurities. Capillary columns of 100 μm id packed with the chiral stationary phase were used for both nano-liquid chromatography (nano-LC) and capillary elecrochromatography (CEC) experiments. To fully resolve the mixture of six enantiomers, parameters such as buffer pH and concentration sample injection have been then investigated. A mixture of ACN/water (90/10, v/v) containing 5 mM ammonium borate buffer pH 9.0 enabled the complete separation of the three couples of enantiomers in less than 30 minutes [20].
Another study shows a new nano-liquid chromatographic method for b-blocker enantiomers’ separation. This method uses a capillary column packed with silica particles, which were chemically modified with vancomycin. On-column focusing allowed the inject of relatively high sample volumes (1500 nL), increasing method sensitivity. The studied racemic compounds, namely atenolol, propranolol, oxprenolol, and metoprolol, were dissolved in methanol and injected for chromatographic separation. The effect of injected sample volume was studied in the range of 50–2100 nL. Under optimal experimental conditions, LODs and LOQs (LOD and LOQ for each alprenolol enantiomer) were 9.0 and 15.6 ng/mL, respectively. Calibration curves were linear in the studied range (9–250 ng/mL). The optimized method was applied to the analysis of a human plasma sample spiked with racemic alprenolol [21].
The other application focuses on the development of a microfluidic confocal fluorescence detection system for the hyphenation of nano-LC to online biochemical assays. Here, they worked toward minimization of sample and reagent consumption by coupling nano-LC online to a light emitting diode (LED) based capillary confocal fluorescence detection system capable of online BCD with low-flow rates. In this fluorescence detection system, a capillary with an extended light path (bubble cell) was used as a detection cell in order to enhance sensitivity. The current setup uses 50 nL as injection volume with a carrier flow rate of 400 nL/min. Finally, coupling of the detection system to gradient reversed-phase nano-LC allowed analysis of mixtures in order to identify the bioactive compounds present by injecting 10 nL of each mixture [22]. Some applications of nano-LC in pharmaceutical analysis are explained in Table 1.
Analytes
Column
Detector
Mobile phase
Comments
References
Racemic mixtures of 50 multiclass pharmaceutical
20 cm × 150 μm i.d. S.P.: CSP- single-walled carbon nanotubes (SWCNTs) in polymer monolithic backbone
Applications of nano-LC in pharmaceutical analysis.
3.2 Biomedical application
It is essential to quickly and highly accurately identify molecules of biological interest. Recent advancements in analytical instrumentation and sample preparation techniques have facilitated biological investigations aimed at identifying these remarkable compounds. Nowadays, medicinal and veterinary medications, doping management, disease diagnosis, and the quantitative identification of biomarkers and proteomes are the main application sectors for nano-LC analysis; the latter is mainly due to the extremely small sample size needed. HPLC-based methods overcome the classical problems of protein analysis, such as gel electrophoresis and immunoanalysis, which are both limited by multiple steps before analyses. The introduction of nano-LC coupled to MS and MS-MS has increased the need for quick and reliable identification methodologies due to the complexity of the proteome diversity. With the use of a comprehensive identification database, they have made it possible to precisely determine the amino acid sequences from proteins or peptides. Nevertheless, because a combination of two or more identification methodologies yields a wealth of information about protein sequencing and peptide mapping, classical approaches are still employed with nano-LC-MS. Using nano-LC–MS-MS, proteomic investigations have been carried out on synovial fluid from rheumatoid arthritis patients. Destructive articular illnesses including osteoarthritis and rheumatoid arthritis are marked by inflammatory abnormalities when defense cells gradually break down the cartilage tissues. Peptides associated with both articular disorders and additional peptides unique to each were found in one investigation. Understanding the synovial fluid proteome was crucial for identifying protein fractions that served as biomarkers and facilitated effective clinical management of patient care [28].
One biomedical application was to identify epithelial and stromal proteins that exhibit up- or down-regulation in keratoconus (KC) versus normal human corneas. Because previous proteomic studies utilized whole human corneas or epithelium alone, thereby diluting the specificity of the proteome of each tissue, they selectively analyzed the epithelium and stromal proteins. Individual preparations of epithelial and stromal proteins from KC and age-matched normal corneas were analyzed by two independent methods, that is, a shotgun proteomic using a Nano-Electrospray Ionization Liquid Chromatography Tandem Mass Spectrometry [Nano-ESI-LC-MS (MS)2] and two-dimensional-difference gel electrophoresis (2D-DIGE) coupled with mass spectrometric methods. The label-free Nano-ESI-LC-MS (MS)2 method identified 104 epithelial and 44 stromal proteins from both normal and KC corneas, and also quantified relative changes in levels of selected proteins, in both the tissues using spectral counts in a proteomic dataset [29].
A similar relative analysis of stroma by this method also showed upregulation of aldehyde dehydrogenase 3A1 (ALDH3A1), keratin 12, apolipoprotein A-IV precursor, haptoglobin precursor, prolipoprotein and lipoprotein Gln in keratoconus corneas. Together, the results suggested that the nano-ESI-LC-MS (MS) 2 method was superior to the 2D-DIGE method as it identified a greater number of proteins with altered levels in KC corneas. Further, the epithelial and stromal structural proteins of KC corneas exhibited altered levels compared to normal corneas, suggesting that they are affected due to structural remodeling during KC development and progression. Additionally, because several epithelial and stromal enzymes exhibited up- or down-regulation in the KC corneas relative to normal corneas, the two layers of KC corneas were under metabolic stress to adjust their remodeling [29].
In another biomedical application of nano-LC, the therapeutic monoclonal antibodies (mAbs) constitute a group of highly effective agents for treating various refractory diseases. Nonetheless, it is challenging to achieve selective and accurate quantification of mAb in pharmaceutical matrices, which is required by PK studies. In this study, they employed a suite of technical advances to overcome these difficulties, which include: (i) a nano-LC/SRM-MS approach to achieve high analytical sensitivity, (ii) a high-resolution nano-LC/LTQ/Orbitrap for confident identification of candidate peptides, (iii) an on-the-fly orthogonal array optimization (OAO) method for the high-throughput, accurate and reproducible optimization for numerous candidate peptides in a single LC/MS run without using synthesized peptides, (iv) a comprehensive evaluation of stability of candidates in matrix using the optimized SRM parameters, (v) the use of two unique SP for quantification of one mAb to gauge possible degradation/modification in biological system and thus enhancing data reliability (e.g., rejection of data if the deviation between the two SP is greater than 25%), and (vi) the utilization of purified target protein as the calibrator to eliminate the risk of severe negative biases that could occur when a synthesized peptide is used as calibrator [30].
To show a proof of concept, this strategy is applied in the quantification of cT84.66, a chimeric, anti-CEA antibody, in preclinical mouse models. The strategy employed in this study can be easily adapted to the sensitive and accurate analysis of other mAb and therapeutic proteins [30]. Some applications of nano-LC on biomedical analysis including the proteome and its nano-LC conditions are explained in Table 2.
Recent proteomic and peptide analyses using nano-LC techniques coupled to MS detection.
3.3 Environmental analysis
The use of nano-LC for the analysis of compounds of environmental interest has not been so widely extended up to now, although HPLC is one of the major techniques for the analysis of pollutants and their metabolites. In fact, very few works deal with the application of nano-LC in environmental analysis [33].
MS detection was coupled through a nanospray interface, obtaining good intra- and interday precision and complete separation of zeralanol (ZAL) and E2 isomers in less than 20 minutes. The nano-LC-MS method was applied for the detection of the target compounds in mineral waters, extracting the analytes by SPE with commercial zearalenone (ZEN)-molecular imprinted polymer (MIP) cartridges, and obtaining LODs. The method was compared with CEC-MS, but less satisfactory limits of detection (LOD) and limit of quantification (LOQ) results were achieved due to the lower injection with respect to nano-LC and the MS signal suppression caused by the presence of the buffer in the MP [33].
Also, regarding monolithic columns, Rahayu and coworkers in situ prepared a 300 μm i.d. monolithic column attaching polyethylene glycol (PEG) groups into a glycidyl methacrylate monolith polymer for the separation of inorganic anions (IO3-, BrO3-, NO3-, Br-, and NO3-). It constituted a very simple and fast method to analyze seawater and public drinking water, and a simple filtration was needed before injection [34].
3.4 Food analysis
The applications of miniaturized LC techniques to the analysis of foodstuffs are scarce with reference to other fields. The analysis of anthocyanins in commercial red fruit juices was assessed by nano-LC, and therefore the method was compared with conventional HPLC by Fanali and coworkers. A C18 capillary column of 100 μm i.d. and MS was employed within the first case, while a 2.1 mm i.d. narrow-bore C18 column and diode array detector (DAD) was utilized in the second. Both methods were fully validated to achieve higher sensitivity with HPLC. However, nano-LC offered good quantitative leads to a shorter analysis time. The column, full of fully porous particles, allowed the separation of all studied compounds in but 7 minutes and gave better results with respect to the column containing partially porous particles. On the opposite hand, the nano-LC system provided higher separation efficiency and worse peak symmetry and determination than conventional C18 columns by HPLC. The developed nano-LC method was applied to wine pomace samples and represented a suitable system for the analysis of anthocyanin dyes [15].
Besides the control of food quality and nutraceutical analysis, food safety has experienced an excellent rising concern. Food products reach the buyer through human handling and action, which generally results in the introduction of exogenous molecules that could modify and accelerate food deterioration or even endanger human health. In recent years, nano-LC and CLC have been applied for the determination of harmful compounds in food matrices such as antibiotics/drugs, pesticides, mycotoxins, and endocrine-disrupting compounds (EDCs) like phthalates [35].
3.5 Future prospects
Nano-LC techniques are emerging as an accessible, robust, and easy-to-use platform. The hyphenation techniques of nano-LC with Mass spectrometry are offering the sensitivity, selectivity, and high resolution demanded in the analyses of highly regulated biopharmaceuticals. Thus, future analytical works in complex biological samples will rely heavily on nano-LC techniques.
As this chapter has shown, nano-LC is a cutting-edge separation method with a lot of promise, particularly in the analytical scale arena. It has been used in a variety of industries, including biomedical interest, agro-chemical, food, pharmaceutical, and environmental analyses techniques and samples. Pharmaceutical and biological applications require methodologies that are sensitive enough to identify and measure. Chemicals with biological significance are present in trace amounts. Hence, the used procedures, as demonstrated by nano-LC–MS and nano-LC–MS-MS hyphenations, must have outstanding detectability and undeniable identification, particularly for these low-concentration compounds. As an adjunct to immunoassays and electrophoresis in the analysis of biological molecules, nano-LC may soon establish a dominant position. In the near future, however, nano-LC has the potential to reach a consolidated position in the analysis of biological molecules as a complement to electrophoresis and immunoassays.
References
1.Tsuda T, Novotny M. Packed microcapillary columns in high performance liquid chromatography. Analytical Chemistry. 1978;50:271-275
2.Tsuda T, Novotny M. Band-broadening phenomena in microcapillary tubes under the conditions of liquid chromatography. Analytical Chemistry. 1978;50:632-634
3.Knox JH, Gilbert MT. Kinetic optimization of straight open-tubular liquid chromatography. Journal of Chromatography. 1979;186:405-418
4.Tsuda T, Hibi K, Nakanlshl T, Takeuchi T, Ishii D. Studies of open-tubular micro capillary liquid chromatography: II. Chemically bonded octadecylsilane stationary phase. Journal of Chromatography. 1978;158:227-232
5.Fanali S. Identification of chiral drug isomers by capillary electrophoresis. Journal of Chromatography. A. 1996;735:77-121
6.Chankvetadze B. Enantioseparations by using capillary electrophoretic techniques: The story of 20 and a few more years. Journal of Chromatography. A. 2007;1168:45-70
7.Jáč P, Scriba GKE. Recent advances in electrodriven enantioseparations. Journal of Separation Science. 2013;36:52-74
8.Calcerrada M, González-Herráez M, García-Ruiz C. Recent advances in capillary electrophoresis instrumentation for the analysis of explosives. TrAC Trends in Analytical Chemistry. 2016;75:75-85
9.Sánchez-López E, Marina ML, Crego AL. Improving the sensitivity in chiral capillary electrophoresis. Electrophoresis. 2016;37:19-34
10.Iadarola P, Fumagalli M, Bardoni AM, Salvini R, Viglio S. Recent applications of CE and HPLC-MS in the analysis of human fluids. Electrophoresis. 2016;37:212-230
11.Scriba GKE. Recent advances in peptide and peptidomimetic stereoisomer separations by capillary electromigration techniques. Electrophoresis. 2006;27:222-230
12.Szumski M, Buszewski B. State of the art in miniaturized separation techniques. Critical Reviews in Analytical Chemistry. 2002;32:1-46
13.Rieux L, Sneekes E-J, Swart R. Nano LC: Principles, evolution, and state-of- the art of the technique. LC-GC North America. 2011;29:926-934
14.Zotou A. An overview of recent advances in HPLC instrumentation. Central European Journal of Chemistry. 2012;10:554-569
15.Fanali C, Dugo P, Dugo L, Mondello L. Capillary-liquid chromatography (CLC) and nano-LC in food analysis. Trends in Analytical Chemistry. 2013:3-4
16.Ahmed M, Yajadda MM, Han ZJ, Su D, Wang G, Ostrikov KK, et al. Single-walled carbon nanotube-based polymer monoliths for the enantioselective nano-liquid chromatographic separation of racemic pharmaceuticals. Journal of Chromatography. A. 2014;1360:100-109
17.Ciogli A, Pierri G, Kotoni D, Cavazzini A, Botta L, Villani C, et al. Toward enantioselective nano ultrahigh-performance liquid chromatography with whelk-O1 chiral stationary phase. Electrophoresis. 2014;35:2819-2823
18.Fanali S, D'Orazio G, Farkas T, Chankvetadze B. Comparative performance of capillary columns made with totally porous and core-shell particles coated with a polysaccharidebased chiral selector in nano-liquid chromatography and capillary electrochromatography. Journal of Chromatography. A. 2012;1269:136-142
19.Aturki Z, D'Orazio G, Rocco A, Si-Ahmed K, Fanali S. Investigation of polar stationary phases for the separation of sympathomimetic drugs with nano-liquid chromatography in hydrophilic interaction liquid chromatography mode. Analytica Chimica Acta. 2011;685:103-110
20.Auditore R, Santagati NA, Aturki Z, Fanali S. Enantiomeric separation of amlodipine and its two chiral impurities by nanoliquid chromatography and capillary electrochromatography using a chiral stationary phase based on cellulose tris(4-chloro- 3-methylphenylcarbamate). Electrophoresis. 2013;34:2593-2600
21.D'Orazio G, Fanali S. Enantiomeric separation by using nano-liquid chromatography with on-column focusing. Journal of Separation Science. 2008;31:2567-2571
22.Heus F, Giera M, de Kloe GE, van Iperen D, Buijs J, Nahar TT, et al. Development of a microfluidic confocal fluorescence detection system for the hyphenation of nano-LC to on line biochemical assays. Analytical and Bioanalytical Chemistry. 2010;398:3023-3032
23.D'Orazio G, Rocco A, Fanali S. Fast-liquid chromatography using columns of different internal diameters packed with sub-2μm silica particles. Journal of Chromatography. A. 2012;1228:213-220
24.Vega ED, Crego AL, Lomsadze K, Chankvetadze B, Marina ML. Enantiomeric separation of FMOC-amino acids by nano-LC and CEC using a new chiral stationary phase, cellulose tris(3-chloro-4-methylphenylcarbamate). Electrophoresis. 2011;32:2700-2707
25.Zheng M, Wu Y, Lu L, Ding K, Tang F, Lin Z, et al. Simultaneous analysis of acetaminophen, p-aminophenol and aspirin metabolites by hydrophilic interaction and strong anion exchange capillary liquid chromatography coupled to amperometric detection. Journal of Separation Science. 2011;34:2072-2078
26.Pröfrock D. Progress and possible applications of miniaturized separation techniques and elemental mass spectrometry for quantitative, heteroatom-tagged proteomics. Analytical and Bioanalytical Chemistry. 2010;398:2383-2401
27.She YF, Smith RD. Advanced nanoscale separations and mass spectrometry for sensitive high-throughput proteomics. Expert Review of Proteomics. 2005;2:431-447
28.Mateos J, Lourido L, Fernández-Puente P, Calamia V, Fernández-López C, Oreiro N, et al. Differential protein profiling of synovial fluid from rheumatoid arthritis and osteoarthritis patients using LC–MALDI TOF/TOF. Journal of Proteomics. 2012;75:2869-2878
29.Joseph R, Srivastava OP, Pfister RR. Differential epithelial and stromal protein profiles in keratoconus and normal human corneas. Experimental Eye Research. 2011;92:282-298
30.Duan X, Dai L, Chen S-C, Balthasar JP, Qu J. Nano-scale liquid chromatography/mass spectrometry and on-the-fly orthogonal array optimization for quantification of therapeutic monoclonal antibodies and the application in preclinical analysis. Journal of Chromatography A. 2012;1251:63-73
31.Hernáez ML, Ximénez-Embún P, Martínez-Gomariz M, Gutiérrez-Blázquez MD, Nombela C, Gil C. Identification of Candida albicans exposed surface proteins in vivo by a rapid proteomic approach. Journal of Proteomics. 2010;73:1404-1409
32.Marie B, Zanella-Cléon I, Guichard N, Becchi M, Marin F. Novel proteins from the calcifying shell matrix of the pacific oyster Crassostrea gigas. Marine Biotechnology. 2011;13:1159-1168
33.Javier S, Barbara R, Javier B, Maria A, Miguel D. Evaluation of two molecularly imprinted polymers for the solid-phase extraction of natural, synthetic, and mycoestrogens from environmental water samples before liquid chromatography with mass spectrometry. Journal of Separation Science. 2015;35:2692-2699
34.Rahayu A, Wah Lim L, Takeuchi T. Polymer monolithic methacrylate base modified with tosylated-polyethylene glycol monomethyl ether as a stationary phase for capillary liquid chromatography. Talanta. 2015;134:232-238
35.Upadhyay R, Roy S, Dalwadi M. Umesh Upadhyay a review of advance study on nano liquid chromatography and its application: The future of the field. International Journal of Creative Research Thoughts. 2022;10:392-395
Written By
Esayas Tesfaye, Ayenew Ashenef, Ariaya Hymete and Tadios Niguss
Submitted: 11 June 2024Reviewed: 17 June 2024Published: 03 October 2024
Open access peer-reviewed chapter - ONLINE FIRST
