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 doublelayer 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: = 4e/
- Zeta potential [potential difference at interface
between fixed and mobile layers (relative to the
bulk liquid)] proportional to EOF.
- =Zeta potential = dielectric constant , =
thickness diffuse layer, e= charge per unit surface
area
Electroosmotic 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 efficiencies
2) Hydrodynamic flow profile: frictional forces at
column wall pressure drop across column.
Electroosmotic mobility: EOF = / 4
Mobility depends buffer, independent electric field.
EOF = cm2 V-1S-1, = cm3 V-1S-1 , = cm
Electroosmotic velocity: vEOF = E/4
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 buffer

heating dissipation: 1) P/L = Cr2V2/L2

=power/length, r2=capillary area, smaller area
better) C= buffer concentration = molar
conductance solution . 2) smaller capillaries, heat
dissipated due large surface area volume ratio
(capillary internal surface area = 2rL)

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 anode

Hysteresis: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: ep = q/(6r)
q= charge solute, =viscosity, r= solute radius
greater (q/r), higher ep
Electrophoretic velocity: vep = epE
Total velocity analyte (Observed):

vobs = vep + veof obs = ep + eof

Vep = Ie/tm Ie/tnm ep = (Ie/tm Ie/tnm)(L/V)
tm: time migrate from injection end to detector ,
L=capillary length, E=V/L (Vcm-1)

tm = IeL / [(ep + eof)V]

Migration velocity: 1) cations flow cathode +ve ep
2) anions tend to anode -Ve ep 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.
2 -1

N = L/H H = B/v = 2D/v, B,D=m s v =

E = V/L N = L / [2D/( V/L)] = V/2D
Efficiency: number of theoretical plates (N).

N = 16 (tm/w)2 N = 5.54 (tm/w1/2)2

Peak variance: 2 = 2Dt 2 = 2DIeL / [(ep +
eof)V] N = L2 / 2 N = (ep + eof)V / (2D)
Selectivity: = (t2 tnm) / (t1 tnm).
Resolution: R = t/w = (t2 t1) / [(w 1 + w2) /
2] = 0.25 N1/2 (v/vave)
= 0.25[(ep+eof)v/2D]1/2[(1-2)/(ave+eof)]
= 0.177 (1 2) {V/[(Ave + EOF)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 (1-10l), 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 %T
7) 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 t m,
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 buffer
2) 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/k14) separation partitioning between (organic)
micellar phase and (aqueous) solution phase 5)
Separation both neutral and charged species
6) Micelles form in solution when surfactant is
added in concentration above critical micelle
concentration (CMC), aggregates surfactant
molecules lifetimes less ~10s 7) Most commonly
used SDS (CMC=0.008M) anionic surfactant
8) 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 sample
4) 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)+ phoresis
Inclusion complexes with cyclodextrins : 1)
Cyclic oligosaccharide molecules built of D-(+)glucopyranose units bonded via - (1,4) linkages
2) Slightly soluble in water but cavities are nonpolar which form inclusion complexes
3) 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 CD
5) ,, 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 anode
Chiral 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<100 0C, soluble in both polar and nonpolar)leucinol and pyrrolidinol derived 4) Capillary
electrochromatography: silica monoliths, polymer
monoliths, particle-fixed
Effect of adsorption: Had=2C2(1-C)[ep+eo]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 peak
Sample 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 bias
Gravity 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 =
gr4hti /(8L)=Bhti q = volume (cm3), p=
density (gcm-3), B= cm2s-1 ti= injection time
4) Amount injected: w = BhtiC 5)
disadvantage= limited by height
Pressure or vacuum injection:

4) Conventional amount injected =

(eof+ep)ACiEt, A= area , plug length = (eof +
ep)Et 5) FASI amount injected = (eof + ep)
ACiEt, plug length = (eof + ep / )Et , = ratio
concentrations background buffer to sample buffer
6) larger , 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

1) Electrophoregram: Between negative peak and

water plug, small peak due polarity switching

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 eliminated
8) Advantage: Minimal sample preparation,
sensitivity similar to UV/Vis detection, universal
Coated 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

1)Amount injected: r4PCtI / (8L) =

BPtiC 2)Correction capillary travel time
between reservoirs: wtot = wi + 2w T =
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 cannot

UV/Visible detector A = c ([] =cm-1M-1)

1) Widely available, suitable chromophore 2)
Variable wavelength UV/vis detector: white light all
Advantages stacking:1) higher sensitivity 2)
wavelengths diffracted into individual wavelengths
sharper peaks, better resolution.
by grating to select wavelength , intensity light
Impact of injection size (100nL vs 5nL): LOD
passes through capillary measured by
improved by factor of 13, some injection mediated photodetector and detector converts intensity to au
band broadening can occur
3) Small capillary, light deviates more from straight
Stacking of cations in a low pH buffer:1) When path, smaller linear range
positively charged solutes migrate out of injection
Extend light path: 1) Higher sensitivity if larger
Pressure injection: 1) Sample vial pressurized,
zone and encounter BGE, field strength drops and pathlength 2) bubble cell optical slit not wide,
forcing sample into capillary. 2) Volume sample
vep slows down 2) Solutes at middle to rear of
good resolution 3) Z cell- longer cell
injected depends on magnitude, duration pressure injection still exposed high field strength and
Indirect detection: 1) decrease in background
applied, sample solution viscosity and capillary
continue move forward full speed 3) Ions in
signal detected 2) For species do not absorb UV
dimensions
injection band continue narrow until all migrated
3) mobile phase contains component that provides
into BGE
actual response, when analytes reach detector
Stacking of anions in a high-pH buffer:
displacement results in decrease in background
Negatively charged anions migrate towards anode
Vacuum injection: 1) vacuum applied to
signal (negative peak) 4) Concentration LOD: Clim
and cross boundary between injection solution and
destination vial, pulling sample into capillary.
= Cm / (DR * TR) DR = dynamic reserve
BGE at rear of injection zone
Volume sample injected depends on magnitude,
(background S/N) and TR = transfer ratio (no
Anti-stacking:
1)
When
sample
with
high
ionic
duration vacuum applied, sample viscosity,
background molecules replaced 1 analyte
strength relative BGE injected, electric field over
capillary dimensions 2) vacuum limited compare
molecule), Cm = concentration background species
injection
zone
declines
2)When
positive
ion
pressure
Laser induced fluorescence detector:
electrophoreses into BGE, exposed to high field
Electrokinetic / electromigration injection:
1) optimum excitation, emission wavelength 2)
strength over BGE 3) cation accelerates away
1) Applying voltage while capillary inserted into
sample vial 2) Capillary and anode placed sample from those cations still remaining in injection zone Highly sensitive and highly specific, less
background and interference 3) Derivatization
anti-stacking 4) band broadening occurs
vial, voltage applied causes sample migrate into
necessary to introduce suitable fluorophore
pH mediated stacking: 1) Upon application
capillary due to eof and ep 3) Amount sample
4) Light from laser focused by lens into capillary,
injected depends on electrophoretic mobility , EOF voltage, negatively charged peptides(in high pH)
emitted light collected at right angles, filters and
migrate
toward
anode
2)
peptides
at
rear
band
flow rate, voltage, capillary dimensions ,solute
detected by photomultiplier
enter acidic buffer (low pH), charge flips and
concentrations 4) Length of sample zone:
Mass spectrometry detector: 1) Determine
direction
migration
reverses
3)
Meanwhile,
= (vep + veo) ti = (eof + ep) Viti / L , =
structure and identify compound 2) Only volatile
peptides front band still migrating toward anode
length zone , ti= injection time
buffer be used so that compatible with vacuum 3)
4)
Band
collapses
on
itself
(neutralized)
5)
5) Amount of sample injected:
sheath liquid line help maintain electrospray
becomes positively charged and migrates toward
w = r2C = r2C (eof + ep) Viti / L
disadvantage= dilution and lower sensitivity.
cathode.
6) For best resolution and peak shape,
Stacking of neutral molecules: 1)application
concentration injected sample less than
electric field, negatively charged micelle migrates
concentration buffer, i.e. dilute samples
injection zone(high field) 2) Micelles stack and thus
7) Amount sample injected depends migration
enrich neutral 3) surfactant concentration
velocity (sample bias due to effect applied voltage)
increases, field strength declines until equals BGE,
Sample bias: 1) Due differences in mobility
stacking ceases, and separation proceeds
faster migrating ions injected more 2) Due
Salt mediated stacking of neutral solutes: 1)
difference in conductivities between sample
Because high salt content sample, low field
solution and buffer
injection plug 2) high pH, EOF directed toward
3) For same sample, different species:
cathode and pushes everything in that direction 3)
w(1) / w(2) = b C(1) / C(2)
Counter migrating negatively charged
b = ((1) + eo) / ((2) + eo) b = tm,2 / tm,1
micelles enter front of injection plug, exposed low
tm,i = z / vtot,I = z / [(i + eo) E] , z= distance field strength 4) stack up at head of zone 5)
Neutral solutes pushed to cathode by EOF, at front
injector to detector, vtot,I = total velocity
of injection zone, encounter high concentration of
4) For 2 different samples:
w(i;s1) / w(i;s2) = vtot(i;s1) C(i;s1) / vtot(i;s2) C(i;s2) vtot micelles 6) solute concentration stack at zone.
= total volume
Field-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
Contactless Conductivity detection 1) C4D uses
ionic strength= lower conductivity= higher field
transmitter electrode to subject sample region to

Packed columns: Capillary

electrochromatography
1) Packing: 3-5m 2) Pressure required push liquid
through packed particles before separation
3) 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 through
Gel-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 / ((4t)
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 /

t)2 R= (t /4t)
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 gel
3) Viscosity solution should not high to permit filling
and removal 4) Lower efficiencies than gel-filled
columns, due to higher diffusivities (N 1/D)
5) Capillary array with sheath flow cuvette
detected simultaneously, increase sequencing
speed 6) Gooey matrix: Stretching between yellow
tip and vial
CE on a chip: 1) Fabricated by photolithography
2) Separation channel etched onto a glass plate
(insulator) or molded with polymer 3)
Microchannels (20-50m) 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

ionic exchange),effect pH, effect organic modifiers

and other additives, dissipation of heat
Ionization of analytes in weak acid:

KHA = (CH+ CA-) / CHA A = A A

= CA- / CA = KHA / (KHA + CH+)
A = observed mobility of A, A = intrinsic mobility of
A
Ionization of analytes in weak base:

KHB+ = (CH+ CB) / CHB+ B = HB B

B = 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

14-crown-4
15-crown-5
18-crown-6
21-crown-7
24-crown-8

A
0.12
0.17
0.26
0.34
0.4

B
0.15
0.22
0.32
0.43

Microreactors:

C
0.17
0.27

Micro-fabricated CE

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.
DNA sequencing by CE
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 aptamercollection 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 finger-printing

Electrophoresis buffer: 1) phosphate(I) (pH 1.13.1), 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

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 0.1-10m) 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 readily
Disadvantages: 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 exposure
Integrated chip-based microcolumn separation
1) air trapping and air escaping channels

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 photolithography
2) Mold insert in nickel for membrane micropump
made by LIGA technique using multiple radiation
3) Embossing: to decorate with or as if with a
raised design
4) Electroforming: electrodeposition in a plating
bath over a base form
5) Limitations of Si compared with metals: lower
mechanical strength, poorer heat transfer
6)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

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

Dielectrochoporesis (DEP):
- a non-uniform electric field is needed: by applying
AC (1-20MHz) electric field on embedded