Basic CE instrument: High voltage power, capillary, 2 buffer reservoirs, detector → simple, low cost, easily miniaturized. Inside the capillary: 1) Covered deprotonated (SiO-) at pH>2(or adsorption ions from buffer) ; 2)Attracts cations inside capillary. 3) Stern double-layer or diffuse double layer (more tightly held fixed layer; not tightly mobile layer). 4) Stern plane: between fixed and mobile layer 5) Slipping plane: in mobile layer separates mobile fluid from fluid attached to surface.Electroosmosis: Under electric field, excess cations mobile layer pushed towards anode→ Net flow as solvated cations drag along solution (EOF)Zeta potential: ζ = 4πδe/ε - Zeta potential [potential difference at interface between fixed and mobile layers (relative to the bulk liquid)] proportional to EOF. - ζ =Zeta potential ε= dielectric constant , δ= thickness diffuse layer, e= charge per unit surface areaElectroosmotic vs hydrodynamic: 1) EOF flow profile: no pressure drop, flow velocity uniform across capillary. no band broadening → sharp peaks and higher resolution (N) ,better separation efficiencies2) Hydrodynamic flow profile: frictional forces at column wall → pressure drop across column. Electroosmotic mobility: μEOF = ζε / 4πη Mobility depends buffer, independent electric field. μEOF = cm2 V-1S-1, ζ= cm3 V-1S-1 , η= cmElectroosmotic velocity: vEOF = ζεE/4πη Velocity depends electric field(E=V), buffer pH and concentration(higher, higher viscosity) organic solvent , surface of capillary. Higher E, sharper peaks, faster separation.

Joule heating: resistance solution, reduced separation efficiency. Temp not controlled lower viscosity bufferheating dissipation: 1) P/L = χCr2V2/L2 =power/length, r2=capillary area, smaller area better) C= buffer concentration χ= molar conductance solution . 2) smaller capillaries, heat dissipated due large surface area volume ratio (capillary internal surface area = 2πrL) Control EOF by chemical modification of capillary inner surface: 1)Chemicals bonded capillary wall or dissolved in buffer (dynamic coating-temporary) → lower EOF due to shielding charge on capillary,increased viscosity.2) For dynamic coating, surfactants and hydrophilic polymers, e.g (CTAB, cationic surfactant). Measurement EOF: (1) Injection neutral marker (nm):vEOF = Ie / tnm Ie = effective length(cm), Criteria neutral marker: uncharged at pH used, detectable, no interaction capillary wall, soluble in buffer used,(2)Gravimetric method: weighing buffer flushed out of capillary given period time → measure additional mass, accurate balance needed, prevent evaporation. Reverse EOF: anions analyzed, reverse direction EOF, reverse polarity voltage → anions migrate faster and separation time reduced. How to reverse EOF: 1) quanternary amines (e.g. CTAB, CTAC, TTAB, TTAC), 2) proteins (e.g. alpha-lactalbumin) 3) Negative SiO- capillary wall attract positively charged quarternary amines 4) Hydrophobic ends quarternary amines associate other quarternary ends 5) exposed positive charges attract negatively charged anions,migration anodeHysteresis:1) starting acidic pH ,H+ removed pH increase. Starting high pH, H+ added pH decrease.2) Competition between anionic enrichment due adsorption and anionic exclusion due to surface potential repulsion. To minimize effect of hysteresis of EOF: - New capillary prior first use: (1) flush MeOH, water, NaOH, (2) flush buffer. Conditioning between runs 3) try not expose capillary to pH extremes, (4) flush neutrals or basic buffer with NaOH (5) flush acidic buffers with phosphoric acid

Effect EOF on resolution: Increasing EOF decrease time, higher separation efficiencies , too fast EOF , low resolution. Electrophoretic mobility: μep = q/(6πηr) q= charge solute, η =viscosity, r= solute radius greater (q/r), higher μep Electrophoretic velocity: vep = μepE Total velocity analyte (Observed):vobs = vep + veof μ obs = μep + μeof

Vep = Ie/tm – Ie/tnm μep = (Ie/tm – Ie/tnm)(L/V) tm: time migrate from injection end to detector , L=capillary length, E=V/L (Vcm-1)tm = IeL / [(μep + μeof)V]Migration velocity: 1) cations flow cathode +ve μep

2) anions tend to anode -Ve μep but EOF>ep, flow cathode.Resolution: H = A + B/u + Cu. In CE, no A (multipath) as tube open, no C(mass transfer) since no stationary phase, B(longitudinal diffusion) remains, good resolution, high efficiency.N = L/H H = B/v = 2D/v, B,D=m2s-1 v = μE = μV/L N = L / [2D/( μV/L)] = μV/2DEfficiency: number of theoretical plates (N). N = 16 (tm/w)2 N = 5.54 (tm/w1/2)2

Peak variance: σ2 = 2Dt σ2 = 2DIeL / [(μep + μeof)V] N = L2 / σ2 N = (μep + μeof)V / (2D)Selectivity: α = (t2 – tnm) / (t1 – tnm). Resolution: R = ∆t/w = (t2 – t1) / [(w1 + w2) / 2] = 0.25 N1/2 (∆v/vave) = 0.25[(μep+μeof)v/2D]1/2[(μ1-μ2)/(μave+μeof)] = 0.177 (μ1 – μ2) {V/[(μAve + μEOF)D]}1/2 sharper peak and longer migration time, higher efficiency(provided solute zone not diffuse more) Types molecules separated by CE: proteins, peptides, amino acids, nucleic acids, inorganic ions, organic bases, organic acids, whole cells. Applications of CE: simultaneous and fast detection toxic metals and bacteria (advantages: current methods require several days, false positives common, techniques like ELISA, PCR, hybridisation are specific to certain microorganisms, readily miniaturized). Advantages of CE: new selectivity an alternative to HPLC, easy and predictable selectivity, high separation efficiency (105 to 106 theoretical plates), small sample sizes (1-10μl), fast separations (1-45 min), can be readily automated, quantitation (linear), easily coupled to MS, different modes. Disadvantages: cannot preparative scale separations – small capillary, low concentrations , large volumes difficult, sticky compounds, species difficult dissolve in aqueous buffer, reproducibility problems – handling very small amounts.Capillary zone: 1) Separation charged analytes based differences electrophoretic mobility 2) Positive peaks: UV absorbing ions displaced detection zone 3) Negative peaks: UV absorbing ions concentrated detection zone 4) Advantages: faster detection than ion chromatography, able to measure both ions. Capillary gel electrophoresis: 1) Separation differences in solute size as migrate through pores gel-filled capillary → ‘molecular sieving’ 2) Gels anti-convective minimize peak broadening due diffusion, prevent solute adsorption capillary and eliminate EOF (EOF not favorable ,may destroy gel)3) Gels temperature stability and suitable pore sizes 4) Extremely high efficiencies 5) Pore size polyacrylamide gel determined total gel (acrylamide) concentration (%T = [bis+acryl]/V) and concentration cross linking agent bisacrylamide (%C = [bis/(bis+acryl)]) 6)Increase %T decreases pore size → suitable separating smaller proteins and DNA , 5% C smallest pore sizes for all %T7) Polymer solutions forming macromolecules can used as size sieving media in CE,replaceable gels (dilute polymer solutions, if not viscous and band broadening) 8) Slab gel vs CGE:CGE faster (mins vs hours), peaks more quantitative than bands in slab gels 9) Log (MW) vs migration time gives linear plot, normalized (divided by reference tm, neutral marker or first peak) 10) SDS denatures proteins and confers negative charges to polypeptide in proportion to length 11)To overcome limit DNA size: pulsed electric field and voltage gradient (increase voltage separation larger DNA)Micellar electrokinetic chromatography: 1) Vx = Veo + [k*/(1+k*)]/Vmc k* phase capacity ratio = moles in micelle / moles in buffer2) Capacity factor: k’ = (tm – to) / [t0 (1-tm/tmc)] to= time complete insoluble micelles 3) Resolution:

R = 0.25 N1/2 [(s-1)/s] [k2’/(k2’+1)] [(1-(t0/tmc))/(1+(t0/tmc)k1’)] s separation factor = k2’/k1’4) separation partitioning between (organic) micellar phase and (aqueous) solution phase 5) Separation both neutral and charged species6) Micelles form in solution when surfactant is added in concentration above critical micelle concentration (CMC), aggregates surfactant molecules lifetimes less ~10μs 7) Most commonly used SDS (CMC=0.008M) anionic surfactant8) Anionic micelles move slower than neutral and positive charged ions.8) very hydrophobic molecules spend all time inside micelles and migrate slower than neutral.Capillary electrochromatography:1) Separation based distribution equilibria, utilizes a packed or coated capillary 2)Packing materials may enhance EOF due to charges on surfaces 3) Enhanced Veo since particle has electrical double layer 4) interaction with stationary phase(retains strongly, migrates slower), analytes charged or neutral.Capillary isoelectric focusing (CIEF): 1) Separation amphoteric species based differences isoelectric points (for proteins seperation) 2) solution forms pH gradient inside capillary (mixture ampholytes), anodic end in acidic solution (anolyte) cathodic end in basic solution (catholyte) 3) In electric field, charged proteins migrate until reside in region pH electrically neutral and stop migrating, zones focused until steady state. 4) After focusing, zones mobilized from capillary hydrodynamically or adding salt to anolyte (chemical mobilization) 5) Detection without mobilization: by scanning but not preferable since removal polymer coating makes gel fragile. 6) Cathodic mobilization: proteins move towards cathode, Cl- added catholyte, higher pH shift. 7) Anodic mobilization: proteins move towards anode, Na+ added to anolyte, lower pH shift.Capillary isotachophoresis: 1) Performed in discontinuous buffer (different buffers 2 ends) 2) leading electrolyte (low electric field, slower) , termination electrolyte( high electric field, faster) 3) graph: series of steps, each step an analyte zone → zone length proportional amount sample4) On-column preconcentration: inject large plug, concentrate small plug, higher sensitivity.- Injection step: sample injected into column filled leading electrolyte - Focusing step: terminating electrolyte placed into reservoir, voltage applied- Separation step: reservoir replaced leading electrolyte and separation voltage applied- Qualitative: RSH, ratio of the step height of the analyte to that of the terminator 5) iso(same) + tacho(velocity)+ phoresisInclusion complexes with cyclodextrins : 1) Cyclic oligosaccharide molecules built of D-(+)- glucopyranose units bonded via α- (1,4) linkages2) Slightly soluble in water but cavities are non-polar which form inclusion complexes3) Capacity for highly hydrophobic solute, k’ = nmc/nCD = KVmc / VCD where n = total amounts solute, K =distribution coefficient between micelles and CD V=volume mc=micelles 4) Ratio solute incorporated micelle depends on hydrophobicity but inclusion complex formation depends matching solute molecular size with cavity diameter CD5) α,β,γ consists of 5,7,8 glycopyranose units and diameters of 0.47-0.52, 0.60-0.64, 0.75-0.83 nm 6) analyte higher affinity CD faster, follow EOF and analyte higher affinity micelles slower attract anodeChiral separation : 1) Advantage: high resolution, low cost, simple & versatile 2) Disadvantages: Microscale, low UV sensitivity 3) capillary electrophoresis: CD derivatives (CDen, THCMH) , carbohydrates (HS-Cys), ionic liquids (organic salts with mp<1000C, soluble in both polar and non-polar)leucinol and pyrrolidinol derived 4) Capillary electrochromatography: silica monoliths, polymer monoliths, particle-fixedEffect of adsorption: Had=2C2(1-C)[μep+μeo]Etad

1) Had is contribution adsorption to plate height, C fractional concentration free solute, tad mean residence time adsorbed solutes 2) Results peak distortion , irreproducibility (large molecules such as proteins are vulnerable)Effect of mobility difference 1) Fronting: solute anion mobility higher than buffer2) Tailing: solute anion mobility lower than buffer 3) match buffer and analyte mobility for symmetrical peakSample introduction : 1) On-column injection 2) Injection volumes: 1-50nL (should be small to minimize zone spreading (loss efficiency, resolution except on-column pre-concentration dilute sample

Hydrodynamic/hydrostatic injection: 1) performed by gravity, pressure or vacuum suction, 2) advantage no sample biasGravity flow injection (siphoning): 1) Sample vial, capillary raised distance, H, above destination vial, sample solution siphon into capillary. 2) Volume sample injected depends on H, length time vial is raised, sample solution viscosity and capillary dimensions 3) Volume injected: q = ρgπr4∆hti /(8ηL)=B∆hti q = volume (cm3), p= density (gcm-3), B= cm2s-1 ti= injection time 4) Amount injected: w = B∆htiC 5) disadvantage= limited by heightPressure or vacuum injection:1)Amount injected: πr4∆PCtI / (8ηL) = B’∆PtiC 2)Correction capillary travel time between reservoirs: wtot = wi + 2wT = ∆hC(ti+2tT)B ,wi =amount injected during actual injection, wT = amount injected during travel time either before or after injection tT = travel time, ti= actual injection time. 3) disadvantage = viscous sample cannotPressure injection: 1) Sample vial pressurized, forcing sample into capillary. 2) Volume sample injected depends on magnitude, duration pressure applied, sample solution viscosity and capillary dimensions Vacuum injection: 1) vacuum applied to destination vial, pulling sample into capillary. Volume sample injected depends on magnitude, duration vacuum applied, sample viscosity, capillary dimensions 2) vacuum limited compare pressureElectrokinetic / electromigration injection:1) Applying voltage while capillary inserted into sample vial 2) Capillary and anode placed sample vial, voltage applied causes sample migrate into capillary due to μeof and μep 3) Amount sample injected depends on electrophoretic mobility , EOF flow rate, voltage, capillary dimensions ,solute concentrations 4) Length of sample zone: ℓ = (vep + veo) ti = (μeof + μep) Viti / L , ℓ = length zone , ti= injection time5) Amount of sample injected: w = πr2ℓC = πr2C (μeof + μep) Viti / L6) For best resolution and peak shape, concentration injected sample less than concentration buffer, i.e. dilute samples7) Amount sample injected depends migration velocity (sample bias due to effect applied voltage)Sample bias: 1) Due differences in mobility – faster migrating ions injected more 2) Due difference in conductivities between sample solution and buffer3) For same sample, different species:w(1) / w(2) = b C(1) / C(2)

b = (μ(1) + μeo) / (μ(2) + μeo) b = tm,2 / tm,1

tm,i = z / vtot,I = z / [(μi + μeo) E] , z= distance injector to detector, vtot,I = total velocity4) For 2 different samples: w(i;s1) / w(i;s2) = vtot(i;s1) C(i;s1) / vtot(i;s2) C(i;s2) vtot = total volumeField-Amplified sample injection: 1) vep increases at high field and analytes reaches boundary between sample zone and background buffer (stacking → enhance efficiency due preconcentrate samples) 2) plug sample in buffer lower ionic strength injected column filled with buffer higher ionic strength, ions migrate rapidly to boundary between sample and buffers under voltage, resulting stacking sample plug 3) lower ionic strength= lower conductivity= higher field 4) Conventional amount injected = (μeof+μep)ACiEt, A= area , plug length = (μeof + μep)Et 5) FASI amount injected = γ(μeof + μep) ACiEt, plug length = (μeof + μep / γ)Et , γ = ratio concentrations background buffer to sample buffer 6) larger γ, more sample injected and plug length shorter when FASI used.7) samples prepared in low conductivity solution (water) 8) Injecting plug water before sample introduction to enhance field strength 9) To pre-concentrate negative ions: FASI polarity switching

Advantages stacking:1) higher sensitivity 2) sharper peaks, better resolution. Impact of injection size (100nL vs 5nL): LOD improved by factor of 13, some injection mediated band broadening can occurStacking of cations in a low pH buffer:1) When positively charged solutes migrate out of injection zone and encounter BGE, field strength drops and vep slows down 2) Solutes at middle to rear of injection still exposed high field strength and continue move forward full speed 3) Ions in injection band continue narrow until all migrated into BGEStacking of anions in a high-pH buffer: Negatively charged anions migrate towards anode and cross boundary between injection solution and BGE at rear of injection zoneAnti-stacking: 1) When sample with high ionic strength relative BGE injected, electric field over injection zone declines 2)When positive ion electrophoreses into BGE, exposed to high field strength over BGE 3) cation accelerates away from those cations still remaining in injection zone → anti-stacking 4) band broadening occurspH mediated stacking: 1) Upon application voltage, negatively charged peptides(in high pH) migrate toward anode 2) peptides at rear band enter acidic buffer (low pH), charge flips and direction migration reverses 3) Meanwhile, peptides front band still migrating toward anode 4) Band collapses on itself (neutralized) 5) becomes positively charged and migrates toward cathode.Stacking of neutral molecules: 1)application electric field, negatively charged micelle migrates injection zone(high field) 2) Micelles stack and thus enrich neutral 3) surfactant concentration increases, field strength declines until equals BGE, stacking ceases, and separation proceedsSalt mediated stacking of neutral solutes: 1) Because high salt content sample, low field injection plug 2) high pH, EOF directed toward cathode and pushes everything in that direction 3) Counter migrating negatively chargedmicelles enter front of injection plug, exposed low field strength 4) stack up at head of zone 5) Neutral solutes pushed to cathode by EOF, at front of injection zone, encounter high concentration of micelles 6) solute concentration stack at zone.

1) Electrophoregram: Between negative peak and water plug, small peak due polarity switching

UV/Visible detector A = εcℓ ([ε] =cm-1M-1)1) Widely available, suitable chromophore 2) Variable wavelength UV/vis detector: white light all

wavelengths diffracted into individual wavelengths by grating to select wavelength , intensity light passes through capillary measured by photodetector and detector converts intensity to au3) Small capillary, light deviates more from straight path, smaller linear rangeExtend light path: 1) Higher sensitivity if larger pathlength 2) bubble cell –optical slit not wide, good resolution 3) Z cell- longer cellIndirect detection: 1) decrease in background signal detected 2) For species do not absorb UV3) mobile phase contains component that provides actual response, when analytes reach detector displacement results in decrease in background signal (negative peak) 4) Concentration LOD: Clim = Cm / (DR * TR) DR = dynamic reserve (background S/N) and TR = transfer ratio (no background molecules replaced 1 analyte molecule), Cm = concentration background speciesLaser induced fluorescence detector:1) optimum excitation, emission wavelength 2) Highly sensitive and highly specific, less background and interference 3) Derivatization necessary to introduce suitable fluorophore4) Light from laser focused by lens into capillary, emitted light collected at right angles, filters and detected by photomultiplierMass spectrometry detector: 1) Determine structure and identify compound 2) Only volatile buffer be used so that compatible with vacuum 3) sheath liquid line help maintain electrospray –disadvantage= dilution and lower sensitivity.

Contactless Conductivity detection 1) C4D uses transmitter electrode to subject sample region to large amplitude, high frequency electromagnetic signal. 2) A attenuated, AC signal registers at a receiver electrode 3) size of the received signal is affected by conductivity of sample 4) received AC signal de-convoluted to convert amplitude into DC voltage signal appropriate data collection 5) As analyte ions pass detection region, cause small changes to overall sample conductivity 6) Continuous monitoring conductivity signal show series peaks, areas/ heights which are related to analyte concentrations 7) C4D electrodes do not make direct contact sample, electrically isolated from sample and electrode fouling eliminated8) Advantage: Minimal sample preparation, sensitivity similar to UV/Vis detection, universalCoated columns: 1) To reduce adsorption solutes (proteins) 2) Ex: polyethylene glycol,polyacrylamide with SiC bond silica (more pH stable than SiO), polyethyeneimine, LC stationary phases, GC stationary phases, charged reversed coatings 3) Alternatives to polyimide coated fused silica capillary: rectangle tubing to increase pathlength (1-5mm, black coating to block UV), UC transparent outer coating for detection, polymeric tubings (eg. PTFE, not UV transparent, not stable under high T) 3) advantage :more stable focusing 4) disadvantage: need to mobilize

samples after focusing

Packed columns: Capillary electrochromatography1) Packing: 3-5μm 2) Pressure required push liquid through packed particles before separation3) Inlet and outlet frits sintered (partially melted using heat form pores) inside column using packing materials 4) Monolithic (created in one piece) structures may be used ,require lower pressure to push liquid throughGel-filled columns: 1) Bifunctional agent: One end bifunctional reagent carries reactive functional group which bind chemically to silano groups, other end contains second reactive group, form covalent bond with polymeric gel 2) Without bifunctional: for low voltage (<300 V/cm)3) Non- crosslinked: easier empty and refill but lower resolving power 4) Agarose gel: allow UV detection shorter wavelength (232nm) than polyacrylamide but less stable, for restriction fragments, proteins 5) Resolution: R = ∆t / ((4σt) 6) Crosslinked polymers (polyacrylamide): for oligonucleotides, DNA sequencing, native and SDS bound protein 7) Linear polymers (polyacrylamide, polyvinyl alcohol, dextran): for restriction fragments, oligonucleotides, DNA sequencing, proteins 8) Molecularly imprinted polymer:9) Mix monomers with target, crosslinker, initiator – polymerize to form MIP- remove template from cavity of MIP. 10) N = (t / σt)2 R= (Δt /4σt)Dynamic sieving CE: 1) Use size sieving solutions (soluble, linear polymers: methylcellulose and polyethylene glycol) 2) Convenient use since solution removed from column more easily than gel3) Viscosity solution should not high to permit filling and removal 4) Lower efficiencies than gel-filled columns, due to higher diffusivities (N α 1/D)5) Capillary array with sheath flow cuvette– detected simultaneously, increase sequencing speed 6) Gooey matrix: Stretching between yellow tip and vialCE on a chip: 1) Fabricated by photolithography2) Separation channel etched onto a glass plate (insulator) or molded with polymer 3) Microchannels (20-50μm) with dimensions comparable to capillaries 4) Efficiencies limited by voltage breakdown characteristics of device, injection not easy for capillaries 5) Advantages: small volume sample, high speed and high efficiency, integration and automation – synthesis/sample preparation/analysis on a chip, easy construction of parallel systems – high throughput, pumping mechanism is pulseless and generates no back pressure, valveless liquid handling, amenable to miniaturization, higher surface:volume ratio → better heat dissipation, high voltages can be applied, faster separations, potentially higher efficiencies

Micro-fabricated CE

Electrophoresis buffer: 1) phosphate(I) (pH 1.1-3.1), acetate (3.8-5.8), phosphate(II) (6.2-8.2), borate (8.1-10.1), MES (5.5-6.7), Tris (7.3-9.3)2) Choice buffer depends on: stability and solubility of analytes in electrolyte, degree ionization of analytes, influence anions and cations present on electromigration of solutes (due to ion pairing or ionic exchange),effect pH, effect organic modifiers and other additives, dissipation of heatIonization of analytes in weak acid:KHA = (CH+ CA-) / CHA μA’ = μA αA

α = CA- / CA’ = KHA / (KHA + CH+)μA’ = observed mobility of A, μA = intrinsic mobility of AIonization of analytes in weak base:KHB+ = (CH+

CB) / CHB+ μB’ = μHB αB

αB = CBH+ / CB’ = CH+ / (KHB+ + CH+)Ion-exchange: 1) Velocity of analyte, vs = veo+Fvep+(1-F) vep(p)

- F = fraction of analyte ion free from polymer ion, F = [S-]/{[S-] + [S-P+]} = 1/(1+Kip [P+])- Kip = ion-pair formation,Kip=[S-P+]/{[S-][P+]}Crown ethers: Synthetic macrocyclic polyethers forming stable inclusion complexes with various organic and inorganic cations, complex formation based on ion-dipole interactions between host and guest molecules

A B C14-crown-4 0.12 0.1515-crown-5 0.17 0.22 0.1718-crown-6 0.26 0.32 0.2721-crown-7 0.34 0.4324-crown-8 0.4

CE-SELEX procedure: Binding a random nucleic acid library to a target, separating bound and unbound nucleic acids, amplifying the bound nucleic acids by PCR for use in the next round of selection, a smaller pool of nucleic acid sequences binding to the target is retained and the unbound nucleic acids are discarded, these aptamers can then be cloned and sequenced.Non-SELEX: PCR is only done in the last round of selection. Electrophoretic migration of native DNA library and its respective proteins or equilibrium mixture under optimized conditions for estimation of bulk affinity and determination of aptamer-collection window for non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) based partitioning, NECEEM electropherogram of best enriched aptamer pools incubated with the proteins, NECEEM electropherogram of a representative aptamer incubated with the proteins and their structurally similar inactive forms

DNA sequencing by CE

DNA finger-printing

Advantages of photolithography: Conventional photolithography with thin films of organic photoresists as resists against etching is highly developed for silicon processing, 2D and 3D dimensional shapes and patterns can be

reproduced in silicon with high precision using bulk and surface micromachining techniques, silicon devices can be batch fabricated using the technology currently used for fabricating integrated circuits, Si/SiO2 is stable chemically and thermally,Disadvantages: high purity silicon is expensive, brittle and opaque in UV/visible regions, surface chemistry of silicon is complicated to manipulate, photolithography is costly (requires clean room)Advantages of polydimethylsiloxane (PDMS): Elastomeric (can be deformed reversibly and repeatedly without permanent distortion or relaxation of features), moldable at a scale suitable for optical applications (with feature sizes in the range of 0.1-10μm) with high fidelity, optically transparent down to 300nm, durable and chemically inert, non-toxic, commercially available and not too expensive, bond to other materials (glass) quite readilyDisadvantages: require master for molding/fabrication, surface is hydrophilic (not fully compatible with aqueous buffer), very low electroosmotic flow without modification, more costly than other commonly available polymers

Photomask vs etch mask: 1) Etch mass is formed on top of the glass substrate to protect the area that is not intended for etching 2) Photomask is placed on top of the etch mask to transfer the pattern on it by UV exposureIntegrated chip-based microcolumn separation 1) air trapping and air escaping channels

Microreactors:

1) Use of high energy radiation (e.g. X-ray or short wavelength UV) capable of creating higher aspect ratio (e.g. 100:1 depth to width) structures compared with conventional photolithography2) Mold insert in nickel for membrane micropump made by LIGA technique using multiple radiation3) Embossing: to decorate with or as if with a raised design4) Electroforming: electrodeposition in a plating bath over a base form5) Limitations of Si compared with metals: lower mechanical strength, poorer heat transfer6)Two concurrent reactions: acid with sodium acetate (ultrafast), iodide and iodate ions yielding to iodine (fast) 5I-+IO3

-+6H+→3I2+3H2O, if mixing is good, no iodine can be observed (no H+) 7) Example: hydrogenation system using a microreactor with immobilized Pd catalyst

Dielectrochoporesis (DEP):- a non-uniform electric field is needed: by applying AC (1-20MHz) electric field on embedded electrodes within a microchannel or applying a DC electic field only at the inlet and outlet across a channel which consists of an array of insulating posts (results in a spatially non-uniform electric field around the post needed for DEP)- Sterilisation: 70% ethanol, bleach, ethylene oxide, UV radiation or autoclaving