Microchip liquid chromatography
Conventional chromatographic separation (LC) is the main separation method used due to its outstanding separation power and versatility . Untill now it is less used than CE for on chip separation.
The first chromatography miniaturization trials were with gas chromatography and were performed at the end of the 70s [311,338] but the main problem was the bad performance of the miniaturized column due to the difficulty to introduce a homogeneous solid phase inside microchannel .
Liquid chromatography miniaturization presents several advantages over the conventional LC such as superior efficiency per time unit, simple positioning of a detection cell and low cost . Both open (the stationary phase is bound to the wall of the coloumn) and packed columns with particles or monolith layers (the stationary phase is bound to the particles or monolith) are used . Open channel chromatography is very easy to prepare but the loading capacity of stationary phase is limited, therefore only small amount of analytes can be injected to avoid saturation and overloading, and a very sensitive detection method such as fluorescence is needed ( and references therein). The packed columns offer more surface area interaction with solutes but make the fabrication process more complicated (e.g. the frits to keep particles inside channel). The columns can have non-homogeneous particle repartition thus decreasing the separation efficiency. Packed columns necessitate also a high pressure source. In order to solve this problem monolithic columns have been developed. They can be operated with low pressure, but present a low reproducibility. Microfabricated pillar arrays are more reproducible . The high pressure needed for miniaturized packed HPLC necessitates a very tight system. The conventional thermal bonding cannot support high pressure while the solvent bonding procedures that glue the polymer is more resistant . Several stationary phases have been used: open channel material (glass or polymers that can be chemically modified or derivatized), chromatography resin, monolith and nanowires [339,342]. Pumping is achieved either by a pump in the case of packed columns or it can be by EOF in open columns [325,337].
Figure 57: SEM of a polyimide microchannel with trapezoidal cross section packed with 5 μm C18 particles (upper panel); several smaller channels constitute a frit-like structure to contain the packed particles (lower panel). Adapted from  CE has several advantages over chromatographic separation such as high separation efficiency, simplicity, low sample and volume consumption and short analysis time . MCE is a further simplification of CE. In MCE, the injection, separation and detection are done on the same platform which permits portability (Figure 58). The high separation efficiency is due to the homogeneous flow inside the channels due to the absence of packing . Nowadays MCE is used for a wide variety of applications such as biomedical applications ([324,345] and references therein) (pharmaceuticals, genetic components (DNA, RNA, enzymes, aptamers…), proteomics , peptides , amino acids, antibodies, antigens, cells and their lysate ), food analysis , environmental , industrial, biological and life sciences [350-352]. The separation in MCE is similar to that of CE and is based on the difference in migration of analytes based on their effective ionic mobilities and EOF under an electric field. There exist different modes of electrophoresis based on the desired application. zone electrophoresis, electrochromatography (EC), micellar electrokinetic chromatography (MEKC), gel electrophoresis (GE), isoelectric focusing (IEF) and isotachophoresis (ITP).
In zone electrophoresis analytes are separated based on their charge to mass ratio. Positive small particles are the most rapid then big positive molecules. Neutral molecules are carried only by EOF only since they have no charge. Negative ions that have mobility smaller than EOF can migrate with EOF direction but slower. As the charge to mass ration of negative ions increase the ions migrate slower (Figure 58) In MEKC analytes are separated based on their partition coefficient with the micelle (made from surfactant), thus allowing the separation of neutral molecules. Gel electrophoresis is used to separate mixtures of DNA, RNA and proteins based on their molecular mass. IEF is a technique that is used to separate molecules having different isoelectric points. ITP is a technique in analytical chemistry used for selective separation and concentration of ionic analytes. Charged analytes are separated based on their ionic mobility.
Figure 58: a) Schematic representation of microchip electrophoresis, b) migration order of ions ionic
analytes based on their charge and mass. EOF: electrososmotic flow.
Injection techniques in MCE
Injection is one of the key elements in sample handling in microsystem. It can be performed either hydrodynamically or electrokinetically. Electrokinetic injection has some limitations such as electrokinetic bias (when one analyte is preferentially injected into the electrophoresis channel the analytes injected are not proportional to the analytes in the sample due to different mobilities of anaytes) and long injection times. It has however been mostly used as it is technically easier to generate an electrokinetically driven flow than pressure driven flow .
Hydrodynamic injection indeed needs micropumps that render the device more expensive and the operation process more difficult to control .
A good injection is an injection that:
1) Can be controlled in order to introduce enough analyte to be detected while providing a low height equivalent to a theoretical plate (HETP) or high efficiency. Remembering that where ωsample is the length of sample plug (the desirable injected volume of sample in the separation channel) along the separation channel axis, K is the injection profile factor (equal to 12 for a rectangular shaped plug), and did is the length of the separation channel between the injector and the detector .
2) It should have fixed-volume sample plug. Focusing of the sample or limitation of the sample diffusion in the separation channel outside this plug between the time of injection and separation in a way that it provide a precise shape can limit the phenomenon of dispersion (Figure 59a and Figure 60).
3) Limitation of sample leakage into the separation channel during separation, which can lead to band broadening, signal drift and decrease in signal to noise ratio [354,357,358] (Figure 59c).
4) The sample should not be diluted during injections and they should be reproducible. For this, sample pullback by back ground electrolyte (BGE) into the sample loading channel should be limited (Figure 61).
5) Matrix and mobility bias should be avoided in order to have a representative aliquot of the sample .
Figure 59: Simulation of steps of floating injection on a cross injection system. a) injection of sample (in black), b) and c) running of buffer. Adapted from 
Figure 60: Simulation of loading step with different focusing ratios in cross form injection system. Adapted from 
Figure 61: Simulation of steps of pullback floating injection on a cross injection system. A) sample (in black) injection, b) and c) running of buffer. Adapted from 
Scientists have worked on two ways to improve the injection conditions. The first way is the configuration of channels or injection design and the second is the injection mode.
Several channel configurations have been designed such as: T-type injection, cross or double-T injection, Multi-T injection form… The T type injection permits only floating injection where the sample is injected across the separation channel. The control of the volume of injected plug cannot be obtained. To solve this problem, the cross injection configuration has been made. This configuration permits other types of injection such as gated and pinched injections. In the cross channel system the volume of the injected analyte is determined by the geometry of the cross sectional area of the injection channel. In order to increase the injected plug double-T, triple-T and multi-T forms have been made  (Figure 62). In the double T configuration for example, the two branches of the injection channel can be separated by a specific distance where the sample will pass by the separation channel, to control the volume of the plug. A variation of the dimensions of the channels was also used to make injections .
Figure 62: Different injection forms. S: sample reservoir, B: buffer reservoir, BW: buffer waste, SW:
In the second axis, several modes have been applied such as: floating, pinched and gated modes.
In the floating mode the sample is driven from the sample reservoir to the sample waste reservoir (Figure 63: reservoir 1 and 3, respectively) in the cross, double-T, triple-T or multi-T-forms by the simple application of a potential difference leaving the other reservoirs grounded or floating (Figure 63a). Then, in the separation step, the buffer flows under the effect of an electric potential between the buffer and the buffer waste reservoirs (Figure 63: reservoir 2 and 4, respectively), taking with it into the separation channel the sample plug previously injected while the sample and sample waste reservoirs are floating (Figure 63b). The limitations of this process are the diffusion of the sample plug into the separation channel during the injection step and possible leakage during the separation step (Figure 59) . In order to counteract the leakage during the separation step, sample pullback can be performed by applying potentials on reservoirs 1 and 3 in such a way that the buffer also enters into the injection channels (Figure 63c).
Figure 63: Floating injection. Sample introduction loading step (A) and dispensing step with (B) or without (C) sample pullback. Adapted from 
In order to escape from this vicious cycle, a control of the voltage in thedifferent reservoirs at all moments should be done. This control is obtained through pinched and gated injections.
With the pinched injection mode  the sample is focused into the injection channel during the loading step by applying potentials in both the buffer and buffer waste reservoirs (Figure 64B1), thus no sample diffusion occurs outside the desired plug and the sample plug is better controlled. As in the floating mode, a sample pullback methodology is implemented during the dispensing step in order to avoid sample leakage (Figure 64B2). Its limitation is the unsymmetrical distribution along the separation channel which makes dispersion increase in comparison to an ideal sample plug and decreases the number of theoretical plate.
Figure 64: Comparison of floating (A) and pinched injections (B) at 1) injection and 2)separation steps. The white coloration is the sample and the colorless is the back ground electrolyte. No sample diffusion occurs in pinched one. S: sample reservoir, B: buffer reservoir, SW: sample waste reservoir, BW: buffer waste reservoir. Adapted from 
The gated injection  represents, along with pinched injection mode, the most frequently used methodology. With the gated injection mode, buffer and sample circulate continuously in laminar flow. Sample goes to sample waste reservoir and buffer to buffer waste reservoir and sample waste reservoir in such a manner that the sample cannot enters the separation channel during the loading step (Figure 65b). At the injection time, the electric potential is turned off in the buffer and sample waste reservoirs so that a plug of sample enters into the separation channel (Figure 65c). The length of the plug is proportional to the injection time and to the applied potential. The separation step is like the loading step in terms of potential distribution (Figure 65) .In order to avoid leakage of the sample in the separation channel, the potential applied in the sample reservoir should be smaller than the one applied in the buffer reservoir.
Figure 65: Illustration of gated injection. White image of microchip valves (a), and fluorescent image of gated injection loading step (b), injection step (c) and running step (d). adapted from .
In summary every injection type has its advantages and limitations, presented in Table 14.
Several detection methods can be implemented within microsystems (Figure 58). The most widely used are: optical (the most common), electrochemical and mass spectrometric ones.
Optical detection methods
Optical detection can be made by fluorescence, absorbance, refractive index detections, raman spectroscopy, surface plasmon resonance, chemiluminescence and electrochemiluminescence depending on the nature of the analyte ([37,326] and references therein). Laser induced fluorescence is the most widely used detection technique because of its very high sensitivity which is necessary in microfluidic device and for the ease of focusing the laser to the extremity of a very tiny separation channel [34,37]. Its limitation is the absence of fluorescence in most naturally occurring compounds and thus the need for derivatization as well as the commercial availability of only specific excitation wavelengths . Derivatization can be performed before, during, or after on-chip separation ([34,326] and references therein). It also requires sophisticated experimental setup and may be expensive . Despite its simplicity, absorbance detection necessitates large optical pathway in order to have good sensitivity which is not compatible with microchannel dimensions. Microfluidic channels have very small dimensions which limits the use of absorbance detection (LODs obtained for antimicrobial metabolites were in the low mg / L, for 19 basic drugs were ≈ 2 mg / mL and for thiourea 167 µM…) Works in progress done in order to increase the optical path as well as to ameliorate sensor detection .
Mass spectrometric detection is also used with MCE . In comparison to conventional CE-MS, it offers a shorter analysis time and consumption of lower volumes of analytes  but needs sophisticated instrumentation and is not portable . It gives complete analysis information on the mass of the analytes detected and their fragmentation pattern. It is usually employed in proteomics and for the detection of large biomolecules, providing quantitative and qualitative data ( and references therein). Matrix-assisted laser desorption / ionization (MALDI)(off-line) and electro spray ionization (ESI) (on-line) are two ionization methods for interfacing microchip with MS that have been extensively used ([362,327] and references therein).
Electrochemical detection is widely used especially for inorganic ions detection . It offers several advantages to the detection especially because of its remarkable sensitivity, facility of integration in miniaturized systems, miniaturization of detection devices in order to have a complete portable system, independence on the optical path and turbidity of the matrix, low cost and minimal power demand [37,365]. Electrochemical detection used with MCE are amperometry and conductimetry.
Table of contents :
II. Etat de l’art
A. Biologie de NO et des RSNOs
B. Décomposition et quantification des RSNOs
C. Miniaturisation et microfluidique
A. Analyse de la décomposition dans le temps de GSNO par électrophorèse capillaire : cinétique et identification des produits de décomposition
B. Analyse de la décomposition des RSNOs par le Cu+ en milieu réducteur ou en présence de nanoparticules d’or
1) Décomposition des RSNOs par Cu+ en milieu réducteur
2) Décomposition des RSNOs en présence de nanoparticules d’or
1) Détection colorimétrique dans un dispositif microfluidique d’analyse à base de papier
2) Détection électrochimie des RSNOs en microsystème après séparation par électrophorèse de zone.
IV. Conclusion et perspectives
Chapter I: State of Art on Nitric oxide and S-nitrosothiols
I. Chemo and Bio-Properties of Nitric Oxide and Nitrosothiols
A. Nitric Oxide (NO)
1) History of NO discovery
2) NO and NO derivatives characteristics
3) Biological synthesis of NO
i. Enzymatic Synthesis of NO by Nitric Oxide Synthase
ii. Enzymatic synthesis of NO by reduction of nitrite and nitrate 
iii. Non-enzymatic NO synthesis
4) Biological effects of NO
i. Role in cardiovascular system
ii. Role in digestive system
iii. Role in inflammation
iv. Role in cancer
v. Role in central nervous system and neurodegenerative disorders
vi. Role in diabetes
vii. Role in immunity
5) Targets of NO in biological system
i. Reactions of NO with metals / metalloproteins
ii. Reactions of NO with low molecular weight chemicals
iii. Reaction with thiols:
B. NO-donors drugs
1) Organic nitrates (RONO2s)
2) NONOates (Diazeniumdiolates):
3) C-nitroso compounds:
4) Iron nitrosyl complexes:
7) Other NO-hybrid donors
1) Formation of RSNOs in-vivo
i. Auto-oxidation of NO followed by addition to thiolate
ii. Oxidative nitrosylation
iii. Direct nitrosylation
iv. Transition metal ion / protein nitrosation
vi. Decomposition of low molecular weight DNICs with thiolate ligand
vii. Nitrite mediated S-nitrosation
2) RSNOs trans-membrane trafficking
3) Decomposition of RSNOs
i. Enzymatic denitrosylation
ii. Decomposition by metal ions
iii. Decomposition by ascorbate
iv. Decomposition by light
v. Decomposition by heat
4) RSNOs in health and disease
i. RSNOs therapeutic effects
ii. RSNOs as diagnosis indicator
II. Methods of quantification of RSNOs
A. Sample pretreatment
B. Direct vs indirect methods
1) Direct Methods
i. Phosphines-based detection method
2) Indirect methods
i. Colorimetric (Saville reaction)
ii. Fluorescence detection
iv. Biotin Switch Assay (BSA) and derived methods
3) Separation techniques (HPLC, GC, CE) coupled to direct or indirect methods
III. Miniaturization and microfluidics
B. RSNOs detection using microsystems
C. Materials for microfluidic devices
1) Silicon and glass
D. Separation on microfluidic devices
1) Microchip liquid chromatography
2) Microchip capillary electrophoresis (MCE)
i. Injection techniques in MCE
a) Floating injection
b) Pinched injection
c) Gated injection
ii. Detection techniques
a) Optical detection methods
b) Mass spectrometry
c) Electrochemical detection methods
Chapter II: Analysis of GSNO decomposition and reactivity by capillary electrophoresis: kinetics and decomposition products identification
I. EC and MS techniques for the analysis of decomposition products of GSNO at solid state 122
2) Sample synthesis
3) CE apparatus and measurements
4) MS detection
B. Results and discussion
II. EC and C4D for the analysis of the decomposition of GSNO solution under light and heat 132
1) Samples, reagents and solutions
2) Capillary Electrophoresis Instrumentation
3) Decomposition and transnitrosation protocols
B. Results and discussion
1) Characterization of GSNO sample
2) Decomposition of GSNO using light.
3) Decomposition by heat
4) Transnitrosation reaction between GSNO and Cysteine
C. Concluding remarks
Chapter III: Decomposition of S-nitrosoglutathione by Cu2+ / GSH and by gold nanoparticles
I. Quantitation of S-nitrosoglutathione using Saville and electrochemical detection upon its Cu+-catalyzed decomposition
2) Microsensor fabrication and NO detection
3) Colometric assays
B. Results and discussion
II. Quantification of GSNO using gold nanoparticles
A. Experimental section
2) Preparation of gold nanoparticles.
3) S-nitrosoglutathione synthesis.
4) Reconstituted human and mice plasma manipulation.
5) Preparation of the NO selective Pt ultramicroelectrode (UME).
6) Amperometric detection of NO.
B. Results and discussion
1) Effect of AuNPs on the GSNO quantification.
2) Effect of plasma thiols on RSNOs quantification.
3) Detection of total RSNOs in plasma.
C. Conclusion of part II
III. Conclusion of chapter III
Chapter IV: Miniaturization
I. Colorimetric analysis of S-nitrosothiols decomposition on paper-based microfluidic devices
1) Chemicals and materials
2) S-nitrosothiols synthesis
3) Fabrication of μPADs
4) Fabrication of a 3D printed holder
5) Lateral flow procedure and colorimetric analysis
6) Plasma RSNOs detection
B. Results and discussion
II. Electrochemical detection of RSNOs in electrophoretic micro device: preliminary studies
1) Microchip configuration
i. PMMA microchip fabrication
ii. Commercial microchips
2) Operating conditions for the microchip electrophoresis of GSNO
i. PMMA and COC microchip
ii. Commercial glass microchip
3) Chemicals and GSNO synthesis
B. Results and discussions
1) Preliminary study
i. Employement of wireless potentiostat
ii. Optimization of the injection mode
iii. microchip with integrated electrodes
2) Application to the separation and quantitation of RSNOs