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Nanoscale Analytical Chemistry: Separation Methods
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16/04/2020 Outline • Introduction. – Interests and specificities of nanometric scale for separation methods. • Last insights in nanometric scale for chromatography, Nanoscale in Analytical Chemistry – from micro to nano flow rate. Evolution of new stationary phases, i.e. monoliths. Miniatirization of separation methods • Another alternative to chromatography: electrophoretic separation. – From 2D Gel to capillary electrophoresis. • Lab on chip, the future of miniaturization for separation methods. - Microsystem production - Microsystems for sample preparation Contact : Yannis FRANCOIS, Lab. de Spectrométrie de Masse des Interactions et des Systèmes, Institut Lebel, 4 rue Blaise Pascal, 67000 Strasbourg email: [email protected] Key points of miniaturization history 60’: What the need in separation chemistry? Gaz chromatography (GC) using packed columns Characteristic of the method First generation of mass spectrometer (elemental analysis) Speed, high throughput Reduction of analysis time 70’: Introduction of GC with capillary columns Low cost Decreased consumption of solvents and samples, releases Introduction of HPLC « in situ » analysis Introduction of capillary electrophoresis Characteristic of samples Miniaturiation of the device Integrated systems 90’: Mass spectrometry: API, MALDI, ICP,… Separation methods coupled to MS: >50% of instrumental market Growing role of biology in analytical developments Small volume Diminution of sample consumption Complexity of the mixture Highly resolutive methods Low concentration level 2000 -…: Miniaturization is the key word in every analytical domains. coupling with sensible detector Bioanalytical chemistry requests high throughput and rapid diagnostic 1 16/04/2020 HPLC column: diminution of diameter Reduction of solvent consuption Description Dimensions HPLC 4 mm i.d. 5 mm narrow-bore column 2 mm i.d. 4 mm Tipical flow rate 0,3 – 3 mL/min micro-bore column 1 mm i.d. 2 mm 50 – 1000 µL/min Capillary column 100 µm i.d. 1 mm 0,4 – 200 µL/min Nano-column 25 µm i.d. 100µm 25 – 4000 nL/min 1 – 5 mL/min Inner diameter(mm) Section (mm2) Flow rate (µL/min) Solvent consuption 4,6 16,6 1200 100% 2 3,1 225 19% 1 0,8 56 5% 0,5 0,2 15 1,2% 0,25 0,05 3,5 0,3% nanoLC HPLC Improvement of sensitivy Sensitivity Detection of concentration: Response = Peak area A = Response.dt h = Response max Inner diameter(mm) Section (mm2) Flow rate (µL/min) 4,6 16,6 1200 1 2 3,1 225 5,3 1 0,8 56 21 Gain in sensitivity * 0,5 0,2 15 80 0,25 0,05 3,5 335 * for identical optical paths 2 16/04/2020 Theoretical aspect The plate theory The plate theory is probably the best theory for explaining chromatographic separation phenomena. Gaussian peak Calculation of theoretical plate number Theoretical aspect The kinetic theory The kinetic theory considers the chromatographic peak as representative of the statistical distribution of the retention times of the molecules of a given substance on the column. Limitations : Absence of consideration of diffusion phenomena Absence of kinetic consideration (speed of exchanges between the two phases Impossibility of introducing the entire sample in an infinitely small volume The kinetic theory considers the diffusion phenomena and mass transfer Theoretical aspect The kinetic theory Diffusion phenomena Longitudinal molecular diffusion Theoretical aspect The kinetic theory Mass transfert a b t0, molecules a and b of the same substance are on the same line ti, a will stay in the grain pore of the stationary phase and b in the mobile phase Turbulent diffusion tf, b will go faster than the molecule a 3 16/04/2020 Theoretical aspect The kinetic theory Theoretical aspect The kinetic theory Solution to minimize diffusion phenomena Improvement of the homogeneity of the stationnary phase: Absence of heterogeneities Absence de bubbles Homogenize the flow rate of the mobile phase Réduction of the diameter of particules dp Van Deemter equation H = A + B/ū + C.ū Decrease the size of particule pores ū = mean linear velocity of flow of the mobile phase in the column Theoretical aspect Increase in efficacity (N) The kinetic theory Van Deemter equation Summary For particules Hopt = f(dp) Reduction of the diameter of particules uopt = f(1/dp) Small size Low porosity dp1 dp2<dp1 Hopt Chromatography Rapid separation With miniaturized stationnary phases uopt Experimental condition Limitation: Increased pressure at the top or the column At low temperature By reducing dead volumes Darcy’s low: P = Lu / k0dp2 Limited particule diamenter between 1,5 - 3µm permeability 4 16/04/2020 Non-porous particles of particles with a porous surface layer Monoliths Inorganic or organic Improvement of mass transfert phenomena Continuous structure containing interconnected pores Bimodal structure: macroporous and mesoporous network High permeability Faster separation Improvement in mass transfert HEPT (µm) 35 P (bars) 300 Column: Poroshell 300SB-C18 75 mm x 2.1mm i.d. Mobile phase: (A) 0.1%TFA, (B) 0.07%TFA in ACN 30 colonne particulaire 3µm 250 Gradient: 5-100%B in 1 min 20 colonne particulaire 5µm 150 T=70°C colonne particulaire 3µm 15 100 UV à 215nm colonne particulaire 5µm 25 200 Flow rate: 3mL/min - P=260bar 10 50 colonne monolithique 5 colonne monolithique 0 0 0 2 4 6 8 10 0 2 4 6 8 10 débit (mL/min) débit (mL/min) Characteristics of the porous network Inorganic monolith synthesis Tetramethoxysilane (TMOS) Bimodal distribution of macropores and mesopores + Acetic acid (catalyst) + PEG : porogene Independent control of both pore sizes Modling Freezing - Aging Structure of the monolith Si(OR)4 + H2O Si-OH + Si-OH Si-OH + Si-OR x Si-O-Si Si(OH)(OR)3 + ROH Si-O-Si + H2O Si-O-Si + ROH (Si-O-Si)x Dissolution-Reprecipitation Generation of mesoporous Macroporosity : generated by PEG Mesoporosity: generated by NH4OH treatment (or urea) Hydrolysys Condensation Polycondensation Calcination Drying PEG removal Skeletal strenghening 5 16/04/2020 Interest of organic monolith Preparation of organic monolith acrylamide-based Synthesys in aqueous medium: low solubility of "hydrophobic" monomers in water, difficulty in controlling pore size Simple synthesis Synthesys in organic medium Adaptable to microdevice Methacrylic ester-based Major work on monolith Stability in a high range of pH : 1 -13 Simple synthesis Polystyrene-based (LC packing, Dionex) Physical aspect of silicium monolith Initiation By heating: T=55-70°C, t20h Easy to make By UV irradiation: =365nm, t<1h Fast reaction Polymerization zone easy to delimit Slow Possibility of cracks Low penetration depth Difficulty to delimit polymerization zone More complex implementation Monolith made from a mold: L=10cm, d=1cm Micro-column Micro-system Thesis work J. Chamieh 6 16/04/2020 Monoliths Column 4,6 mm i.d. Inorganic or organic Continuous structure containing interconnected pores Bimodal structure: macroporous and mesoporous network High permeability Faster separation Improvement in mass transfert HEPT (µm) 35 P (bars) 300 30 colonne particulaire 3µm 250 colonne particulaire 5µm 25 200 20 colonne particulaire 5µm 150 colonne particulaire 3µm 15 100 10 50 colonne monolithique 5 colonne monolithique 0 0 0 2 4 6 8 10 débit (mL/min) 0 2 4 6 8 10 7mL/min 91bars débit (mL/min) Why the nano-flow rate? Column 100 µm i.d. When the sample concentration is very low need of miniaturization in classical columns, pressure problem 7 16/04/2020 Issues concerning nano-flow rate Technological constraints: Pumping process The ability to generate gradients Measurement and control of nano-flow rate Leak detection,… Capillary electrophoresis: Fundamental notions Options: The "classic" pumping system can be used with “split " Electrokinetic pumping Contact : Yannis FRANCOIS, Lab. de Dynamique et Structure Moléculaire par Spectrométrie de Masse, institut de Chimie, 1 rue Blaise Pascal, 67000 Strasbourg email: [email protected] History History 1937 1939 Separation of protein By electrophoresis on paper 1954 1967 Separation of protein S. Hjerten : 300 µm i.d. capillaries In the Human serum 1981 From J.L. Veuthey, Univ. de Genève J. Jorgenson : 75 µm i.d. capillaries From J.L. Veuthey, Univ. de Genève 8 16/04/2020 Electrophoresis: a big familly Outline 1. Migration phenomenon in CE Electrophoresis Isoelectric focalisation Isotachophoreris 1.1 Electrophoretic mobility 1.2 Electroosmotic mobility 2. The separation in CE Zone electrophoresis 2.1 Efficacity 2.2 Resolution 3. Improvement of selectivity Paper Gel Capillary 4. Quantitative analysis 4.1 Injection 4.2 Detection CZE CGE CEC MEKC 5. Capillary isoelectric focalisation (CIEF) Introduction capillary electrophoresis CE as a separation method HPLC Principle CE analyte E soluté Mobile phase Stationary phase No Stationary phase instrumentation: No pressure device, no injection device + - +Capillary Injection Detector MINIATURISATION Low sample consumption Inexpensive method High EFFICACITY of separation High voltage generator Low SENSIBILITY of detection 9 16/04/2020 Principle of separation under electric fields Experimental device Capillary + Detector Important question to ask - o So.. How it works? Electrodes o How to see neutrals and anions ? o How to calculate electroosmotic and electrophoretic mobilities ? Electrolyte vials o How to detect all anions and cations during the separtation ? High voltage generator o What are the parameters to optimize the separation ? Classical capillaries: bare fused silica lenght : 20 - 100 cm inner diameter : 20 - 100 µm Voltage: 5 - 30 kV Principle of separation under electric fields Principle of separation under electric fields Voltage 30 kV Voltage 30 kV Electric Field Detector Electric Field Detector Cathode Background electrolyte = Salted solution Anode Cathode Cathode Background electrolyte = Salted solution Anode + Conductivity – ++ Conductivity – 10 16/04/2020 Principle of separation under electric fields Principle of separation under electric fields Voltage 30 kV Voltage 30 kV Electric Field Cathode Cathode ++ Detector Electric Force Friction Force + Electric Field Anode Cathode Cathode – ++ Principle of separation under electric fields + + FF = 6 r vep : viscosity r : hydrodynamic radius vep : ion velocity Detector ++ + Electrophoretic Mobility µep – ANODE FF: friction force + FE: electric force Cathode Cathode Anode Electrophoretic Mobility µep Electrophoresis Voltage 30 kV Electric Field Detector Anode – - CATHODE qE Vep = 6r FE = q E q: ion charge E: electric field q µep = 6r 11 16/04/2020 + + ++++ + ++ ++ + - + - + - ++ ++ + ++++ + - 12 16/04/2020 + - ++ ++ + + ++ - ++ + Principle of separation under electric fields Voltage 30 kV Detector + ++ ++ + + Cathode Cathode ELECTROPHORESIS can separate: Molecules bearing DIFFERENT CHARGES, Molecules with the SAME CHARGE but with a DIFFERENCE of SIZE. Anode ++ – – 13 16/04/2020 Principle of separation under electric fields Principle of separation under electric fields Voltage 30 kV Voltage 30 kV Detector + Detector µep + Cathode Cathode Anode Cathode Cathode ++ – ++ µep Anode – µep – µep – Electropherogram + Time Principle of separation under electric fields Principle of separation under electric fields Important question to ask Voltage 30 kV o So.. How it works? Detector o How to see neutrals and anions ? + µep o How to calculate electroosmotic and electrophoretic mobilities ? Cathode Cathode Anode ++ o How to detect all anions and cations during the separtation ? – µep Electropherogram – + How to separate neutrals and anions o What are the parameters to optimize the separation ? Time 14 16/04/2020 Electroosmotic mobility Electroosmotic mobility Bare Fused Silica Capillary Voltage 30 kV Detector Detector Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH Cathode Background Electrolyte = Salted solution Anode Cathode Anode + Conductivity – + – OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Electroosmotic mobility Electroosmose Bare Fused Silica Capillary Acidic condition Protonation of silanols Si-OH Surface net charge = 0 Basic condition Deprotonation of silanols Si-O Surface net charge < 0 Si Si Si Si Si Si OH OH OH OH OH OH Si Si Si Si Si Si Charge capillary surface + Neutralization of opposite charge of electrolyte DOUBLE LAYER O- O- O- O- O- Odisplacement of the solvent that occurs under the effect of the electric field Surface net charge depending the pH value of Background Electrolyte 15 16/04/2020 Electroosmotic mobility Bare Fused Silica Capillary At basic conditions Electroosmotic mobility Bare Fused Silica Capillary At basic conditions Detector Detector ----------------------------- Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- Cathode Anode Cathode Background Electrolyte = Salted solution Anode + – + Conductivity – -O -O -O -O -O -O -O -O -O -O -O -O -O -O -O -O -O -O -O -O -O -O -O -O -O -O -O -O -O Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Si Electroosmotic mobility Bare Fused Silica Capillary At basic conditions Electroosmotic mobility Bare Fused Silica Capillary At basic conditions Detector ----------------------------Cathode + Background Electrolyte = Salted solution Anode Cathode Conductivity – + Detector ----------------------+ – + – + – + – + – + – – – – – – – + + + + + + + + + + + + ---+ – – + – + – – – – – – – – – – – – – + + + + + + + + + + + + + – + – – – – – – – – – – – – – -+ – + – Anode – + – Voltage 16 16/04/2020 DOUBLE LAYER: STERN Model Electroosmotic mobility Creation of an ion movement due to the formation of double ionic layer = electroosmotic mobility Bare Fused Silica Capillary At basic conditions + ----------------------------+ + + + + + + + + + + + + + + – + – + – + – + – + – + – + + – + – + – – + – – – + – – – – – – – – – – – – – – – – – – – + + – – + – + – + – + – + – + – + + – + – – -- + -- + + + + + + + -+ + + -- + + --- + electroosmose --- + + -- Anode silanols – + distance – 1/x Voltage potential The decrease of the double layer potential determines the velocity of solvent. E 0 s Influence of pH on silica group of the capillary Electroosmotic flow 4 In solution Diffuse layer -- + - + + + + + Electroosmotic mobility µeo Veo = - + - - - - - - - - - - - - - - - - - - - - - - - Cathode + Bare fused silica pI 2 µeo = 4 : Zeta potentiel zéta : dielectric constant of electrolyte : viscosity Increase of (Charge density) 17 16/04/2020 Principle of separation under electric fields Bare Fused Silica Capillary At basic conditions Principle of separation under electric fields Bare Fused Silica Capillary At basic conditions Voltage 30 kV Voltage 30 kV Detector Detector ----------------------------- ----------------------------- + + Cathode Cathode Anode Cathode Cathode ++ – ++ Anode – µep – Principle of separation under electric fields Bare Fused Silica Capillary At basic conditions – Principle of separation under electric fields Bare Fused Silica Capillary At basic conditions Tension 30 kV µeo µep Detector - - - - - - - -µep- - - - - - - - - - - - - - - - - - - - + Cathode Cathode ++ µep – What compounds will I be able to detect ? µeo µeo µeo Apparent mobility µapp = µep + µeo + Anode µep and µeo same direction µapp > 0 Detection µep = 0 then µapp = µeo Detection µep et µeo opposite direction µep < µeo Detection µep < µeo No detection – Electropherogram + – – Time 18 16/04/2020 Principle of separation under electric fields Apparent mobility µapp = µeo + µep Important question to ask Bare fused silica capillary: o So.. How it works? + o How to see neutrals and anions ? ep - - - - - - - - - - - - - - - - - - eo - eo + eo ep eo o How to calculate electroosmotic and electrophoretic mobilities ? o How to detect all anions and cations during the separtation ? + + o What are the parameters to optimize the separation ? - time Measurement of mobilities Ld Principle of separation under electric fields + Lt détecteur Important question to ask - + + - o So.. How it works? o How to see neutrals and anions ? tapp Electroosmotic mobility Apparent mobility t0 Electrophoretic mobility µapp=µep + µeo veo/E vapp/E o How to calculate electroosmotic and electrophoretic mobilities ? o How to detect all anions and cations during the separtation ? o What are the parameters to optimize the separation ? µep=µapp - µeo 19 16/04/2020 The modification of the capillary surface, Why? To modulate the electroosmotic flow whatever the pH value To cancel or reverse the electroosmotic flow To avoid adsorption phenomenon in the capillary wall The modification of the capillary surface, Why? Anion separation: + ep - - - - - - - - - - - - - - - - - eo ep eo app app Dynamic coating Presence of additives in the electrolyte détecteur + To realize short time analyses The modification of the capillary surface, How? - - eo - Amino « quenchers » : polycationic polymers CH3 N+ CH3 2Br (CH2)6 CH3 N+ (CH2)3 CH3 polybrene Permanent coating Chemical linkage 1- Silica activation by a reaction of silylation 2- Coating by functional group + - - - +- - - - +- - - - +- - - - +- - + + + + + Thermal immobilization 20 16/04/2020 Separation on coated capillary Other additives Positive coated capillary Neutral polymers Voltage - 30 kV HPMC PEO µeo PVA Detector + + + + + + + +µep+ + + + + + + + + + + + + + + + + + + + + Neutral surfactants Triton X-100 Brij-35 Tween 20 µeo – Anode Cathode µeo –+ Zwitterionic surfactants n=9,11,15 CH 3(CH2)n N+ SO3- µep + µeo Cathode + Electropherogram – + SB-n+1 Apparent mobility µapp = µep + µeo Separation on coated capillary Time The modification of the capillary surface, How? Positive coated capillary What compounds will I be able to detect ? – + µep et µeo same direction µapp > 0 Detection µep = 0 then µapp = µeo Detection µep et µeo opposite direction µep < µeo Detection µep < µeo No detection Rapid Commun. in Mass Spectrom., 1997, 11, 307 21 16/04/2020 Efficacity of the separation Flow profiles HPLC Peak distortion following Van Deemter model In terms of height of theoretical plate (H) : x x Hydrodynamic flow: parabolic profile POMPE H = A + B/u + C.u Hopt Turbulence (A): CE uopt Electroosmotic flow: flat profile - + Molecular diffusion (B) : Mass transfert (C) : INCREASE OF EFFICACITY Voltage issue Separation factors 1 Limitation: Joule effect 2 P L t HPLC CE Retention or migration k’ µep = k’1/k’2 = µep,1/µep,2 = C r2 V2 L2 L : capillary length r : capillary radius C : electrolyte concentration Selectivity Resolution Rs = 1 -1 k’ 4 k ’+1 N Rs = 1 µep N 4 µep,moy + µeo : Molecular conductance Low inner diameter capillary 22 16/04/2020 Principle of separation under electric fields Outline 1. Migration phenomenon in CE Important question to ask 1.1 Electrophoretic mobility 1.2 Electroosmotic mobility o So.. How it works? 2. The separation in CE o How to see neutrals and anions ? 2.1 Efficacity 2.2 Resolution o How to calculate electroosmotic and electrophoretic mobilities ? 3. Improvement of selectivity o How to detect all anions and cations during the separtation ? 4. Quantitative analysis o What are the parameters to optimize the separation ? 4.1 Injection 4.2 Detection 5. Capillary isoelectric focalisation (CIEF) IMPROVMENT OF SELECTIVITY CZE: Capillary Zone Electrophresis Factors which can modifiy electroosmotic mobility MEKC µeo = Non-aqueous CE SELECTIVITE 4 : charge density of capillary surface EKC: cyclodextrines,… : viscosity : double layer thickness CGE Electrochromatography (CEC) Background electrolyte : nature and concentration of ions, pH, organic solvents Nature of capillary Temperature 23 16/04/2020 Influence of pH on silica group of the capillary Factors which can modify electrophoretic mobilities q µep = Bare fused silica pI 2 6r pH : modification of analyte apparent charges Nature of the electrolyte Addition of organic solvent Temperature Increase of (Charge density) Acidic or basic properties: pKa Usual buffer used in CE LH ↔ H+ + L- [L-] pKA = pH - log 1 [HL] charge 0,8 0,6 0,4 Buffer pKA Phosphate Citrate Formate Succinate Acetate Borate MES HEPES TRIS 2.12 - 7.21 -12.32 3.06 - 4.74 - 5.40 3.75 4.19 - 5.57 4.74 9 6.15 Weak 7.55 conductivity 8.30 0,2 pK -6 A-6 pK-4A-4 pK -2A-2 0 pK 0A pKA2+2 pKA+4 4 pKA6+6 24 16/04/2020 Influence of ionic strength Influence of ionic strength = K.(T/Cizi2)1/2 µeo = 4 Increase of concentration decrease of Phosphate buffer : charge density of capillary surface : viscosity : double layer thickness = K.(T/Cizi2)1/2 Tech. Prot. Chem. II, 3-19 (1991). Influence of organic modifier Influence of organic modifier q Polaires ( >30) Protiques Eau MeOH Apolaires ( <30) Aprotiques ACN DMF DMSO Protiques Aprotiques EtOH THF PrOH Dioxane Influence on mobility and/or constant of dissociation (pKA, ion pairing,…) µep = Influence on viscosity 6r Influence on pH Influence on solvation effect Solvent Cations Anions Water ++ ++ Methanol +/- ++ Ethanol - ++ Acetonitrile -- -- 25 16/04/2020 General rules: Influence of organic modifier veo (10-3 m.s -1) 2 1,8 1,6 µeo = 1,4 1,2 4 To DECREASE the electroosmotic mobility: 1 0,8 When the pH decreases decrease of 0,6 0,4 0,2 0 0 20 40 60 80 % v/v Influence on viscosity Influence on zeta potential (10-4kg.m-40 1 -1 s ) Polar Solvents (ex : water): zeta potential which can be up to 100mV. 35 30 Non-polar Solvents (ex : heptane): No zeta potential , except in the presence of additives. 25 20 15 When the concentration of electrolyte increases decrease of When the percentage of organic modifier increase decrease of ACN < acetone < MeOH < EtOH, PrOH < DMSO 10 5 •Increased percentage of organic modifier 0 0 20 40 60 80 decrease of 100 % v/v DMSO acetone ACN ACN < acetone < MeOH < EtOH, PrOH < DMSO IMPROVEMENT OF SELECTIVITY CZE: Capillary Zone Electrophresis Non-aqueous capillary electrophoresis NACE weak electric currents MEKC increase of inner diameter of capillaries semi-preparative increase of efficacity (N/t /2) Non-aqueous CE SELECTIVITE EKC: cyclodextrines,… Water methanol CGE Electrochromatography (CEC) / /2 88,2 6924 22,7 552 NMF 110,3 20075 acetonitrile 110,3 4136 Changes on selectivity Better compatibility with MS detection Increase of solubility (ex: cyclodextrins) 26 16/04/2020 Non-aqueous capillary electrophoresis NACE Non-aqueous capillary electrophoresis NACE 3 1 10 11 9 5 1 A 8 67 3 24 12 9 5 24 10 8 6 B 11 12 7 Separation of a 12 compounds mixture Bare fused capillary 58;5cm x 50µm i.d. - 30kV electrolyte : (A) ethanol/acetonitrile/acetic acid (50:49:1) in 20mM CH 3COO-, NH4+ (B) methanol/acetonitrile/acetic acid (50:49:1) in 20mM CH 3COO-, NH4+ 1 amphétamine, 2 éphédrine, 3 levorphanol, 4 dextromoramide, 5 morphine, 6 hydrochlorothiazide, 7 acide benzoïque, 8 acide meso-2,3-diphénylsuccinique, 9 probenecid, 10 chlorothiazide, 11 acide phénylènediacétique, 12 acide éthacrynique. Chromatographia, 2000, 52, 403-407 J. Chromatogr. A, 1997, 792, 13-35. IMPROVMENT OF SELECTIVITY CZE: Capillary Zone Electrophresis Non-aqueous CE SELECTIVITE CGE Micellar eletrokinetic chromatography MEKC EKC: cyclodextrines,… Electrochromatography (CEC) = separation technique that combines type of phenomena : electroosmose electrophoresis chromatography Partition between mobile phase and pseudo stationary phase No instrumental development Separation of neutral molecules 27 16/04/2020 Separation of neutral molecules Micellar eletrokinetic chromatography sufficiently soluble electrolyte to form micelles No electrophoresis mobility under electrical field UV transparent Co-elution of all de toutes ces molécules avec le flux électroosmotique Homogeneity of micelles Low viscosity Strategies: 1. Formation of charged complexes CMC(10 -3 M) à 25°C dans l ’eau Surfactant ex : S + THA+ S(THA)+ + THA+ Sodium dodecylsulfate (SDS) Sodium tetradecylsulfate (STS) Sodium N-lauroyl-N-methyltaurate (LMT) Sodium cholate Cetyltrimethylammonium bromide (CTAB) S(THA)+ S(THA)22+ 2. Ionic micelles 8.1 2.1 (50°C) 8.7 13-15 0.92 The most commonly used Micellar eletrokinetic chromatography Micellar eletrokinetic chromatography + - - - - - - - - - - - - - - - - - - vep,mc - - - - - - - - veo - vep,mc - - vep,mc - - - - - - - - - = Neutral compound - - - - - - - = micelle - - 28 16/04/2020 injection detection Free micelle Analyt e Micellar eletrokinetic chromatography water analyte Free micelle eau Migration time t0 tR tmc t0< tR < tmc Window of detection Different type of Pseudo-phases used in MEKC IMPROVMENT OF SELECTIVITY CZE: Capillary Zone Electrophresis Non-aqueous CE SELECTIVITE CGE MEKC EKC: cyclodextrines,… Electrochromatography (CEC) 29 16/04/2020 Chiral separation (identical charge density) Chiral separation (identical charge density) = separation technique that combines type of phenomena : electroosmose electrophoresis On-line separation Off-line separation - - - - - - - - - - - - A- A+ AA- A- A+ A+ A+ A- A+ - - - - - - - - - - - - Principle based on chiral recognition by addition of chiral selector Formation of diastereoisomers = chiral selector A- A- A+ A+ No instrumental development Chiral recognition Electrophoretic separation Separation of enantiomers cyclodextrins Chiral selectors Complexation by inclution , , -cyclodextrins éthers-crowns Complexation by chelation -hydroxy ou -aminoacids et metals (Cu) Association with chiral polymers maltodextrins heparin, sulphated dextran Micellar separation -hydroxy ou -aminoacides with alkyl chain Formation of ion pairing camphrosulfonates quinine and derivatives -CD Interaction by affinity proteins 30 16/04/2020 Characteristics of cyclodextrins -CD -CD Cyclodextrine Number of glucose Molecular weight Inner diameter of cavity/nm Diameter/nm 6 972.9 0.47–0.52 1.46 Height of cavity/nm Solubility in water at 25°C -CD -CD 7 1135.0 8 1297.2 Large number of modified cyclodextrins: 0.62–0.64 0.75–0.83 1.54 1.75 Reaction with OH in position 2,3 et 6 neutral 0.79–0.80 0.79–0.80 0.79–0.80 140mM 16mM 140mM Positively charged Negatively charded OH O O COO- H N Hydroxy-CD in 4M urea : 89mM Amino-CD NH2 OO Carboxy-CD in 8M urea : 226mM Sulfonato-CD -O 3S OO SO3- -O3S SO3- Allow separation of netraul compounds 4,3Å Chiral selector IMPROVMENT OF SELECTIVITY 5,0Å -CD 8,0Å -CD HS--CD HS- -CD CZE: Capillary Zone Electrophresis 6,2Å -CD Non-aqueous CE Rs = 2.2 Rs = 2.9 SELECTIVITE 10.0 temps (min) alprenolol 2.0 4.0 temps (min) isoproterenol EKC: cyclodextrines,… Rs = 1.8 CGE 5.0 MEKC HS--CD 5.0 Electrochromatography (CEC) 10.0 temps (min) atenolol Capillary 50 µm i. d. x 31.5 cm - separation under -15 kV, T = 22°C 5%HS--CD in 25mM tetraethylammonium phosphate, pH 2.5 31 16/04/2020 Influence of cyclodextrins in MEKC IMPROVMENT OF SELECTIVITY CZE: Capillary Zone Electrophresis Non-aqueous CE MEKC CGE Electrochromatography EKC: cyclodextrines,… SELECTIVITE Electrochromatography (CEC) Electrochromatography Advantages = separation technique that combines type of phenomena : electroosmose electrophoresis chromatography Partition between mobile phase and stationary phase o o o o o o o Selectivity Injection capacity Low dispersion in mobile phase and stationary phase Low diffusion effect Allow the use of very low particle size High efficiency Hyphenation with MS detection o o o o o o Manufacture of columns Fragility of columns Control of mobile phase flow rate Nature of the stationary phases Difficult to perform gradient Increase of the analysis time Drawbacks No instrumental development, but modification of capillary Separation of neutral molecules 32 16/04/2020 Comparison LC/CEC Comparison LC/CEC HPLC CE Classical column volumes 4 mL 2 µL Classical injection volumes 1-10 µL 1-10 nL Decrease of efficacity Limits of detection 10-7-10-8 M 10-5-10-6 M (UV detection) Electrochromatography IMPROVMENT OF SELECTIVITY CZE: Capillary Zone Electrophresis Non-aqueous CE SELECTIVITE CGE MEKC EKC: cyclodextrines,… Electrochromatography (CEC) 33 16/04/2020 Capillary Gel Electrophoresis (CGE of CE-SDS) Capillary Gel Electrophoresis (CGE of CE-SDS) = separation technique that combines type of phenomena : electroosmose electrophoresis Separation due to the presence of polymer in the electrolyte: Sieving gel No instrumental development Addition of polymer in the electrolyte to create a sieve with a control grid Separation of compounds which have a uniforme distribution of electric charge. Separation in function of mass gain in selectivity Separation by size exclusion Capillary Gel Electrophoresis (CGE of CE-SDS) Capillary Gel Electrophoresis (CGE of CE-SDS) 34 16/04/2020 Outline INJECTION 1. Migration phenomenon in CE 1.1 Electrophoretic mobility 1.2 Electroosmotic mobility Most classical injection modes 2. The separation in CE Electrokinetic injection Hydrodynamic injection 2.1 Efficacity 2.2 Resolution 3. Improvement of selectivity Injected sample quantity Q is defined by: 4. Quantitative analysis Q = l.r2.C 4.1 Injection 4.2 Detection avec 5. Capillary isoelectric focalisation (CIEF) l, lenght of the sample zone r, capillary radius C, sample concentration Hydrodynamic Injection By difference of pressure, Electrokinetic injection Realized by the application of an electrical field in the sample vial. The injected volume Vinj is proportional to: l = tinj (veo+ vep) Time of injection tinj, Difference of pressure ΔP0 Vinj = Qinj = r4.Po.tinj 8.L (eo+ ep)V.r2.C.tinj L Due to electrophoretic mobility of analytes, injected quantity is diferent in funtion of sample nature. Equation is not valid if sample conductivity is different to electrolyte conductivity. Electrokinetic mode is always used in CGE. 35 16/04/2020 Outline DETECTION 1. Migration phenomenon in CE Most often used: 1.1 Electrophoretic mobility 1.2 Electroosmotic mobility UV detection 2. The separation in CE Mass spectrometry detection Fluorescence detection 2.1 Efficacity 2.2 Resolution OFF-COLUMN Detection ON-COLUMN Detection 3. Improvement of selectivity 4. Quantitative analysis 4.1 Injection 4.2 Detection + - + - 5. Capillary isoelectric focalisation (CIEF) ON-COLUMN: DIRECT MODE ON-COLUMN: DIRECT MODE Detection directly on the capillary, near the capillary tip Detection directly on the capillary, near the capillary tip operated through a window formed by removing the polyimide protection of the capillary. operated through a window formed by removing the polyimide protection of the capillary. UV Detection Fluorescence detection Generaly well adapted to bare fused capillary which present a weak luminescence require the use of transparent capillaries up to 170 nm Generaly need a derivation step of the sample: Equiped all commercial apparatus limited sensitivity because of the low capillary loading capacity and low diameter : 10-5 mol.L-1 dansyl/fluorescein-thiocarbamyl for amino acids Fluorescamine for amino acids or peptides ex : phenol, LOD = 67 fmol ex : -chymotrypsinogen, LOD = 2 fmol Develpment of capillary with a bubble cell of a Z cell to increase the optilcal lenght Linearity: 10-3 - 10-7 M CE-LIF commercialized with an argon laser at 488 nm 36 16/04/2020 OFF-COLUMN Mass spectrometry detection Need an adapted interface Méthode LDD (mol) LDD (M) UV- Vis 10-13 - 10-16 10-5 - 10-8 Universel Possibilité d’information spectrale Fluorescence 10-15 - 10-17 10-7 - 10-9 Sensible Requiert souvent une dérivatisation Fluorescence induite par laser 10-18 - 10-20 10-14 - 10-16 Extrêmement sensible Requiert souvent une dérivatisation Cher Ampérométrie 10-18 - 10-19 10-10 - 10-11 Sensible Sélective mais seulement pour analytes electroactifs Requiert une électronique spéciale et des modifications du capillaire Conductivité 10-15 - 10-16 10-7 - 10-8 Universel Requiert une électronique spéciale et des modifications du capillaire Spectrométrie de masse 10-16 - 10-17 10-8 - 10-9 Sensible Informations structurales Maintain the electrical field No succion effect Increase of sensitivity due to ultra-low flow separation Détection indirecte 10 - 100 moins qu’en direct (UV, fluorescence, ampérométrie) Optimization of sensitivity 1. On-line preconcentration before separation 2. Isotachophoresis (ITP) Avantages/ inconvénients Universel Plus faible sensibilité qu’en direct On-line preconcentration: amplification of the electric field Conductivity of the sample zone (к0) Conductivity of the electrolyte (к) Electric field Limitations • axial dispersion • Joule effect Optimal conditions (hydrodynamic injection) • weak voltage • 8 < к/к0 <10 • Injected volume: x 8 to 10 37 16/04/2020 On-line preconcentration: amplification of the electric field On-line preconcentration: amplification of the electric field (large volume) Hydrodynamic injection Elimination of low conductivity zone by reversing the polarity Separation in classical condition On-line preconcentration: transient Isotachophoresis On-line preconcentration: transient Isotachophoresis Principe: sample preconcentration in a gradient of conductivity BGE Leader Sample Background electrolyte : (HCOOH) Sample contain a leader ion (NH4+) + 1 2 3 4 - 5 Electrical field Conductivity Injection of large sample zone 38 16/04/2020 On-line preconcentration: transient Isotachophoresis On-line preconcentration: transient Isotachophoresis A B Capillaire silice fondue 60 cm x 75 µm d.i. (10 cm au détecteur) avec greffage dynamique HPC; Voltage : - 25 kV ; Température : 25°C ; Détection UV à 200 nm ; Electrolyte : acide formique 50 mM, pH 2,7 ; Echantillon : digeste de BSA 20 pmol/µL A : Sample with leader ions in the sample, Vinj = 10% of the capillary B : Sample without leader ions in the sample, Vinj = 10% of the capillary Outline 1. Migration phenomenon in CE 1.1 Electrophoretic mobility 1.2 Electroosmotic mobility 2. The separation in CE CIEF Characteristics: • Separation in function of isoelectric point. • Migration in a pH graditn (presence of ampholytes) • Two step: Focusing and Mobilization 2.1 Efficacity 2.2 Resolution 3. Improvement of selectivity 4. Quantitative analysis 4.1 Injection 4.2 Detection 5. Capillary isoelectric focalisation (CIEF) 39 16/04/2020 CIEF : Principe CIEF Filling the capillary 2D gel electrophoresis Acidic pH Basic pH Detector CIEF : Principe CIEF : Principe Filling the capillary Acidic pH Second step: Mobililsation pH Gradient Basic pH Acidic pH - Basic pH - + Pressure + pH Gradient Detector Power supply Detector Power supply 40 16/04/2020 CIEF Separation of proteins By CIEF Lab-on-chip Production and applications Contact : Yannis FRANCOIS, Lab. de Dynamique et Structure Moléculaire par Spectrométrie de Masse, institut de Chimie, 1 rue Blaise Pascal, 67000 Strasbourg email: [email protected] TOWARDS THE MICROFLUIDIC SCALE Microfluidics: it typically regulates flows in labs on a chip. Channels with diameter smaller than 100 microns or less than μL. 1930 1980 1990 2000 Chip µ-TAS IMPORTANT SURFACE FORCES AND OFTEN PREDOMINANT Van der Waals interaction (associated with charged surfaces in the presence of ionic solutions) Surface tension (liquid/liquid or liquid/gas interactions) Electrophoresis Capillary zone electrophoresis MEKC, CEC,.. Detection: LIF, MS If you return a microchip, water stays on the surface! Surface treatments have more influence than gravity! In these flows: no TURBULENCE and mixtures are made by DIFFUSION. The drops keep their integrity and the bubbles behave like obstacles in the channels. 41 16/04/2020 MICRO-CE « HARD » TECHNOLOGY: SILICIUM electrolyte RESIN DEPOSIT sample waste DEVELOPMENT electrolyte COLLAGE ENGRAVING Cross device ENGRAVING PROCESS ENGRAVING PROCESS HUMID ENGRAVING: Chemical attack in liquid phase Ex: Spherical cavity SF6, CF4 SiF4 DRIED ENGRAVING: ISOTROPIC ENGRAVING: it develops indifferently in 3 directions HF/HNO3/CH3COOH for silicium HF for glass Spray Chemical + Physico-chemical + ANISOTROPIC ENGRAVING: it develops preferentially according to certain crystalline planes Température°C KOH for silicium Structure cristalline du silicium Taux de gravure µm/h Glass = amorphous no anisotropic engraving ANISOTROPOUS 100Å/min Etching by physical action of the incident ion flux Etching by chemical action of reactive species Target placed on the cathode Diffusion of reactive species towards the target and adsorption Ions accelerated by an electric field Ex: faceted cavities ISOTROPOUS ANISOTROPOUS 1000Å/min Engraving by chemical action of reactive species assisted by ion bombardment = RIE (Reactive Ion Etching) Reaction with the target material and formation of a volatile compound 42 16/04/2020 PALSTIC TECHNOLOGY: PDMS (polydimethylsiloxane) REPLICATION MOLDING (ex : PDMS) Polyméthylméthacrylate (PMMA) Polycarbonate Crosslinking agent Polydimethylsiloxane (PDMS) + T (70°C) Polytetrafluoroethylene Pression + T (>T° of glassy transition) Pression + T (170°C) MATRICAGE (ex : PMMA) MICRO-INJECTION Possibility of multi-dimentional separation PDMS : hydrophobic material O2 Plasma Surface treatment (polyvinylpyrrolidone, poly-L-lysine,…) Without surface treatment With surface treatment 43 16/04/2020 Possibility of multi-dimentional separation MICRO-Chip: Injection Fixed volume = volume of the intersection « Pinched » injection Sample electrolyte E Esample Purification Concentration Derivation/Reaction Separation (s) Detection electrolyte E buffer buffer Ewaste sample waste Esample Ewaste sample microCE CE waste µ1 µ2 µ3 Eanalysis electrolyte Eanalysis Good injection Bad injection electrolyte Kirchhoff’s law (law of the nodes): At an intersection, the sum of the currents is zero Loading Injection + Analyse Esample + Ebuffer + Eanalysis= Ewaste Esample + Ewaste + Eanalysis = Ebuffer Sample preparation MICRO-Chip: Injection Volumes between 10 and 400 pL Sample treatment « Gated » injection sample sample Esample waste electrolyte Eanalysis Esample Ewaste Ebuffer Ebuffer Eanalysis electrolyte waste electrolyte waste electrolyte Ewaste Ebuffer sample Esample Eanalysis electrolyte On-chip Off-chip Ewaste electrolyte E1 + E2 = E3 + E4 Loading Injection Analyse Esample Ewaste ou Ebuffer Eanalysis Esample Ewaste ou Ebuffer Eanalysis Esample + Ewaste + Eanalysis = Ebuffer Grinding Linked to the separation Dissolution Purification Derivation Lié à la détection Elimination of large particles Lyse des cellules Biochemical treatment (amplification, enzymatic digestion, …) Preconcentration 44 16/04/2020 Sample derivation Current limits: Detection Chip design Chip voltages Optical detection: A = lc 2cell=V2cell Reactor Pre-column derivation Laser induced fluoresence detection (« gated » injection) Injection Detection Separation Allows to focus the detection on a width of 5-20μm or less than linj Injection Ar+: exc = 488nm Post-column derivation (« gated » injection) He-Cd: exc= 350/ 442nm Separation Detection Reactor Requires optically transparent substrates: glass, quartz, some plastics Nitroaromatic and nitrosamines separation in MEKC Current limits: Detection Mass spectrometry ESI-MS Agilent µ-Chip L=65mm Capillary L=50cm 7 3 4 10 8 9 Electrolyte:10mM borate de sodium, 50mM SDS Detection: Fluorescence inverse 6 5 1=TNB, 2=DNB, 3=NB, 4=TNT, 5=tetryl, 6=2,4-DNT, 7=2,6-DNT, 8=2-,3-,5-NT, 9=2-Am-4,6-DNT, 10=4-Am-2,6-DNT 2 MALDI-MS Less popular Development of the ROACHE « Rapid Open-Access Channel Electrophoresis » Analytes are separated in open channels. 1 La matrice est ajoutée à l’électrolyte avant la séparation. Wallenborg S. R. et al. Anal.Chem. 2000, 72, 1872-1878. Wallenborg S. R. et al., Electrophoresis 2000, 21, 3257-3263. At the end of the separation, a laser pulse leads to the ionization of the analytes which are then directly directed to the MS 45 16/04/2020 Current limits: To mix LOW NUMBER OF REYNOLDS: LAMINARY DISCHARGE Molecular diffusion mixing Hydrodynamic focalising Chaotic advection trésidence~d tdiffusion Flow separation Creating a chaotic flow ~d2 Division and recombination of flows 46
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