IEF 1
Isoelectric focusing
K. Šlais
10/3/2017 IEF 2/7910/3/2017 IEF 2/79
Isoelectric focusing - IEF
 Electromigration separation analytical method based on existence of
isoelectric state of ampholytes, where the effective charge is zero.
 pH = pI
 Analytes - proteins
 Separation - DpI < 0.01
 Focusing – concentratrion
 Characterization - pI
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Protein as ampholyte
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Simul 5
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Kinds of IEF
 Gel IEF
– With carier ampholytes
 With immobilised gradient (IPG)
 Two dimmensional electrophoresis 2D = IEF+SDS PAGE
 Capillary IEF
 Preparative IEF
 Free flow IEF
 Chamber IEF
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Typical result of gel IEF
IEF 2
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pI standards - proteins
? stability
? purity
? price
? color
? solubility at pI
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Low molecular mass colored pI markers
 Demands on pI markers
- Scale of pI from ~ 2 up to 11, step ~ 0.5 pI
- good ampholytes, -dz/dpH ~0.5 > 0.05, DpK ~2 < 4
- water solubility at pH = pI, > 1 mg/ml
- different colours lmax > 400 A1% > 100
- Purity > 99 % ,
 - Availability, price of pI marker
- Stability - hydrolysis, oxidation, photodegradation, microorganisms
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Aminomethylated nitrophenols
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Yellow pI markers
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Example of colored
pI marker
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pK1 = 3.50
± 0.02
pK2 = 5.92
± 0.02
pI = 4.71
± 0.02
Spectrophotometric determination of pI
N N N N
Me
Me
HO2
C
IEF 3
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Determination of pI by interpolation in gel IEF
Gradient pH
Mixture of 30 simple buffers
Biolyt 3 – 10
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Dynamic of focusing in gel IEF
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Dynamic of pH gradient Biolyt 3-10
Linear gradient
pH 4 - 10
After ½ hour small
changes in pH gradient
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Dynamic of focusing in gel IEF determination
of focusing end
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Development of fluorescent pI markers
Vis
fluorescence
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pI markers
10.1
9.3
8.4
7.5
6.4
5.3
3.9
Cytochrome C
Myoglobin
Albumin
β – amylase
2μl 4μl
Mass spectrometric characterization of low-molecular-mass
color pI markers and their use for direct determination of pI
value of proteins
Mazanec, K, Slais, K., Chmelik, J.
J. Mass Spectrom. 41 2006 1570-1577
0
50
100
%Int.
230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380
Mass/Charge
1[c].17
2[c].17
3[c].11
4[c].11
36 mV 8.4 mV 7.6 mV 42 mV
45B_0002, 22A_0002, 03A_0001, Smeska2_0002
Kratos PCKompact SEQ V2.3.0
247.4
296.0
237.5
239.5
280.0
267.5 358.6295.4 345.6251.4 373.6230.9 333.7
266.0
264.0
277.5
317.9
357.5265.5
246.8
297.2278.0246.9
251.2
296.5
301.9
237.0
343.7
318.9 331.8232.9
358.0345.1
302.9
372.9360.2
376.0
346.3
327.9
306.9 333.0
313.6
Mass spectra of nitro-substituted pI markers
Pardubice 2005
yellow markers
IEF 4
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2D Gel electrophoresis
SDS
PAGE
IEF
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0 2 4 6 8 10 12 14 16 18 20
0
1000
2000
3000
4000
5000
6000
7000
8000
Voltage[V]
0 2 4 6 8 10 12 14 16 18 20
0
5
10
15
20
25
30
35
40
Time [h]
Current[µA]
Voltage practical
Voltage theoretical
Current practical
Credit:
Deng, X., Hahne, T., Schröder, S., Redweik, S., Nebija, D., Schmidt, H., Janssen, O., Lachmann,
B., Wätzig, H., (2012). The challenge to quantify proteins with charge trains due to isoforms or
conformers. Electrophoresis, 33(2), 263–9. doi:10.1002/elps.201100321
Voltage and current record in IEF on IPG strip
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Strips rehydrated 2 hours under Kerosene
run native 7 hours with Nitrogen
Amersham Multiphor.
Test of color pI markers - LM ladder
Hanspeter Schickle,
ETC Elektrophorese-Technik GmbH, Kirchentellinsfurt, Germany
Courtesy of Dr. Hanspeter Schickle,
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in Clinical Proteomics. From Diagnosis to Therapy. J. Van Eyk and M.J. Dunn (Eds.),
Chapter 2, Protein Separation by Two-Dimensional Electrophoresis
Pamela M. Donoghue, Miroslava Stastna, Michael J. Dunn, p 13,
2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Immobiline Dry Strip (Amersham Biosciences) pH 3–10, 18 cm.
Apparatus: Protean IEF Cell (BioRad).
Sample: 10 ml of pI markers mixture diluted with 340ml of IEF buffer (8M urea, 2M thiourea, 4%
CHAPS, 1% DTT, 0.01% bromophenol blue, 1.5% (v/v) hydroxyethyl disulfide, 0.2% (v/v) IPG
buffer pH 3–10).
The acidic end is on the left and the basic end on the right side of the strip.
The pI values of individual pI markers are marked in the picture
IEF of mixture of chosen pI markers in the first dimension strip of 2D gel
electrophoresis
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2D - typical result – silver staining
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2D Gel electrophoresis - Software
IEF 5
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Protein identification by 2D gel electrophoresis -MS
Use of coloured pI - markers to
determine the slope of the pH
gradient and the position where
to ‚cut‘ and remove the
individual Sephadex fractions in
order to fit to the corresponding
narrow pH range IPGs
Courtesy of Carsten Lück
Methods in Molecular Biology, vol. 424: Volume 1: Sample Preparation and PreFractionation,
Edited by: A. Posch ,
Chapter 22, Sample Prefractionation in Granulated Sephadex IEF Gels
Angelika Görg, Carsten Lück, and Walter Weiss, p 277,
Humana Press Inc., 2007, Totowa, NJ
IEF in Granulated Sephadex Gels
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pI markers - LM ladder
Home made strip,
linear gradient pH 4-10,
11cm,
1 min 30V,
50 min 30V -> 3500V,
2 hours 3500V.
IEF in Sephadex gels and IPG strips
Hodný Z., Přidalová J.,
Institute of Experimental Medicine AV ČR, v.v.i., Prague
Courtesy of Z. Hodný
Courtesy of J. Přidalová
10/3/2017 IEF 28/79
Micropreparative sIEF in nonwoven strip
28
10/3/2017 IEF 29/79
sIEF with carrier buffers and colored pI markers
focused mixture:
0,15 ml stock of 12 carreir buffers
0,05 ml stock pf colored pI markers
0,15 ml ethylen glycol; 0,05 ml butanolu 0,1 ml water29 10/3/2017 IEF 30/79
Animation of separation course
• Time of separation cca 12 hours
– Evenimg - Sampling and power switch on
– Next morning – fraction harvest
30
IEF 6
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Harvest and extraction of fractions
31 10/3/2017 IEF 32/79
Whey fractionation
• Aim – pure caseinomacropeptide
– sample – 1% (m/V) solution of raw dry whey
– Additional spacers – IMAC (imidazol-1-yl-acetic acid) and Tris
(tris(hydroxymethyl)aminometane)
program of power source:
100 V – 200 V 4 h.
200 V – 1000 V 4 h.
1000 V – 3000 V 4 h.
3000 V  harvest
vzorek:
0,375 ml 1% (w/v) whey;
0,05 ml stock colored pI
markers;
0,1 ml ethylen glycol;
0,05 ml butanol;
0,025 ml 0,1 mol·l-1 IMAC;
0,1 ml of 0,1 mol·l-1 Tris;
extraction: 6mm strip segment
in to 100 µl water
32
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HPLC analysis of sIEF fractions
Kolona Microbore Poroshell 300SB-C18 (5 μm částice, 1×75 mm) + C18 předkolonka, při 70 °C
Průtok 20 μl·min-1
Voda/ACN s 0.1% (v/v) TFA lineární gradient od 5 do 80 % (v/v) ACN (30 min)
dávkovaný objem: 4 µl
33 10/3/2017 IEF 34/79
Content of proteins in fractions
Výtěžek:
glykosylovaný CMP 44 % neglykosylovaný CMP 80 %
α-laktalbumin 77 % β-laktoglobulin 101 %
34
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Capillary IEF
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Capillary IEF of standards
proteins pI markers
IEF 7
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CIEF of microorganisms
Sample: E. coli, C. albicans, S. epidermidis, E. faecalis in
physiological saline solution, 4 x 108 cell ml-1.
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CIEF viruses with UV detection
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Patogenes of different sources Tapered capillary in cIEF
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Effect of treatment of 100 μm i.d. fused silica capillary with supercritical
water in semi-dynamic mode. Experimental conditions: 400 °C, 32 MPa, 20
replacements of supercritical water.
Supercritical water in preparation of
tapered fused silica capillaries
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Dependence the local internal diameter of etched fused silica
capillary on the capillary length.
The cutout of the segment used as the tapered capillary in cIEF
and the detection window are indicated.
IEF 8
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Resolution of several Dickeya bacterium species with
similar isoelectric points by capillary isoelectric focusing
employing
cylindrical (left) and tapered (right) capillary.
Combination of Capillary Isoelectric Focusing in a Tapered Capillary with MALDI-TOF MS for
Rapid and Reliable Identification of Dickeya Species from Plant Samples
Horka, M; Salplachta, J; Karasek, P; Kubesova, A; Horky, J; Matouskova, H; Slais, K ; Roth, M
ANALYTICAL CHEMISTRY Volume: 85 Pages: 6806-6812 , JUL 16 2013
10/3/2017 IEF 45/7915. dubna 2013 IEF, 45/79
Preparative Liquid phase IEF
Total 2.5 ml sample
Ten 0.25 ml fractions
2 hours run time
Rotofor
MicroRotofor
pI = 5.3pI = 3.9
Preparative autofocusing
of peptides + pI markers
Tomas, R; Yan, LS; Krenkova, J; Foret, F.
ELECTROPHORESIS, 28 (13): 2283-2290
2007
10/3/2017 IEF 46/7946
ZOOM® IEF Fractionator
(Invitrogen)
• Focusing chambers are separated
by polyacrylamide discs with
immobilized pH
• Proteins have to pass through
discs toward their pI
• Chamber volume 650 µl
• Sample preparation: dissolve,
denaturation, alkylation
Use of pI-dye markers as online
trackers for the focusing
of peptides during
electrophoresis on the
OFFGEL fractionator device
(OGE). 22 hours runtime
Courtesy of M. Heller DKF,
University of Bern, Switzerland
P.E. Michel, F. Reymond, I. L.
Arnaud, J. Josser, H. H. Girault, J.
S. Rossier,
Electrophoresis 2003, 24, 3–11
Agilent 3100 OFFGEL Fractionator
pI-based fractionation of proteins and
peptides with liquid-phase recovery.
introduced May 30, 2006, Codeveloped
with DiagnoSwiss S.A.
OFF - GEL electrophoresis Use of pI-dye markers as on-line trackers for the focusing of peptides
during electrophoresis on the OFFGEL fractionator device (OGE).
•pI-marker dyes were added at 10 ug (dark orange, pI 3.9; violet, pI 5.2; red, pI 6.2; bright
orange, pI 8.0) or 30 ug (yellow, pI 10.1), respectively.
•Peptide/dye solution was distributed into the 13 wells of the OGE.
•IPG strips pH 3-10 from BioRad re-hydrated in OGE buffer were used.
•Focusing was done by setting a maximal potential (1250 or 1500 V) and a current limit of 50 uA.
Courtesy of M. Heller, DKF, University of Bern, Switzerland
IEF 9
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Free-Flow Electrophoresis
The miniaturization of FFE implies several advantages especially considering sample volume and
separation speed. In contrast to the tens of milliliters of sample consumed by conventional large scale FFE
devices, microfluidic FFE systems require only tens of nanoliters up to hundreds of microliters of sample.
This is especially interesting in clinical analysis where often only low sample volumes are available.
Furthermore, instead of residence times of up to tens of minutes, microfluidic FFE (µ-FFE) devices
separate within several seconds.
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FFIEF of seven fluorescent IEF markers
Voltage = 150 V, current = 50 mA
Markers (pI 4, 5.1, 6.2, 7.2, 8.1, 9, and 10.3) are fully separated
within less than 2 s.
The sample flow rate was 0.4 mL/min (v = 2 mm/s).
The apparent kinks in the fluorescent tracer paths are caused by
merging multiple photographs.
Copyright American Chemical Society. © 2008
colorless fluorescent markers
Microfluidic high-resolution free-flow isoelectric focusing
Kohlheyer, D., Eijkel, J. C. T., Schlautmann, S., van den Berg, A., Schasfoort, R. B. M.,
Anal. Chem. 2007, 79, 8190–8198.
10/3/2017 IEF 51/79
WEBER, Gerhard; Margeritenweg 23 85551 Kirchheim (DE).
WO/2002/050524, 07.12.2001,
Preparative Free Flow Electrophoresis
FFE Service GmbH
Dr. Gerhard Weber
D-85551 Kirchheim
Germany
3.10.2017 24th Nov. 08 5th CECE 52
Basic ideas
 Fluidics - continuous widening of the flat channel while
the liquid flows from channel inputs toward the outputs
which generates a divergent flow
and, at the same time
 IEF - small transversal voltage drop at the channel input and
high transversal voltage drop at the channel output.
Divergent Flow IEF (DF IEF)
Šlais K. Electrophoresis 29 2008 2451-2457
27th Nov. 07 soutez 07 53
Carriers & analytes
anolyte catholyte
High flow velocity Short separation path
Fluidics – divergent flow
Divergent flow IEF
Membranes
27th Nov. 07 soutez 07 54
+
-
high
low
k
Separated fractions
Carriers & analytes
anolyte catholyte
High flow velocity
Low voltage drop
Short separation path
Fast separation
Fluidics – divergent flow
IEF – electricity control
by electrolyte cnductivity k
Divergent flow IEF
High voltage drop Efficient focusing
Membranes
IEF 10
27th Nov. 07 soutez 07 55
+
-
high
low
k
Separated fractions
Carriers & analytes
anolyte catholyte
High flow velocity
Low voltage drop
Short separation path
Fast separation
Fluidics – divergent flow
IEF – electricity control
by electrolyte cnductivity k
Simple device :
Membranes eliminated
by porous layer bed
Divergent flow IEF
High voltage drop Efficient focusing
27th Nov. 07 soutez 07 56
+
-
high
low
k
Separated fractions
Carriers & analytes
anolyte catholyte
High flow velocity
Low voltage drop
Short separation path
Fast separation
Fluidics – divergent flow
IEF – electricity control
by electrolyte cnductivity k
Simple device :
Membranes eliminated
by porous layer bed
Separation area,
flow inputs and outputs
made from
non-woven fabric
Divergent flow IEF
High voltage drop Efficient focusing
27th Nov. 07 soutez 07 57
+
-
high
low
k
Separated fractions
Carriers & analytes
anolyte catholyte
High flow velocity
Low voltage drop
Short separation path
Fast separation
Fluidics – divergent flow
IEF – electricity control
by electrolyte cnductivity k
Simple device:
Membranes eliminated
by porous layer bed
Separation area,
flow inputs and outputs
made from
non-woven fabric
Electrode contacts made from
non-woven fabric
Flow generated by
hydrostatics
Divergent flow IEF
High voltage drop Efficient focusing
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Divergent flow IEF
The polypropylene nonwoven web 0.1 mm thick lies on white
polyvinylchloride flexible sheet
input strips dipped in Petri dishes containing:
above left – anolyte
above middle - solution of carriers and pI markers
above right – catholyte
middle left - carbon rod anode
middle right – carbon rod cathode
output strips - bottom - microplate
Streamlines of red pI markers from left pI
= 3.3, 4.7, 6.2, 7.6, 11.0
Flow due to hydrostatics and capillary elevation
Constant power load 1 W
No cooling
Šlais K. Electrophoresis 29 2008 2451-2457
10/3/2017 IEF 59/79 10/3/2017 IEF 60/7910/3/2017 IEF 60/73
Dynamics of divergent flow IEF
1 W constant power load
switched off at 11 hod 30 min
switched on at 11 hod 40 min
Flow inputs:
Anolyte: 0.05 M H3PO4 , 5.2 mS/cm,1 mL/h
Catholyte: 0.05 M NaOH, 11mS/cm, 1 mL/h
Carriers and pI markers: 0.75 mS/cm, 4 mL/h,
Holdup volume: 1 ml
Separation area: 71 cm2
Streamlines of red pI markers from left
pI = 3.3, 4.7, 6.2, 7.6, 11.0
Šlais K. Electrophoresis 29 2008 2451-2457
IEF 11
24th Nov. 08 5th CECE 61
time stability
0
50
100
150
200
250
300
0 2 4 6 8 10 12 14 16
time [hours]
position[pixels]
MO
pI 5.2
hemoglobin
cytochrom C
Streamlines fluctuation 3.96 %, 3.94 %,
1.26 % and 1.88 %, respectively.
Šťastná M., Šlais K., Electrophoresis, accepted -00293
Streamlines from left :
orange – marker pI 2.6; lavender - marker pI
5.2; brown – hemoglobin, 0.5 mg/ml; brick –
cytochrom C, 0.5 mg/ml; flow 0.18 mL/min
Performance stability of SI DF IEF
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Preparative DF IEF of bear
Mazanec K., Bobalova J., Slais K. Anal Bioanal Chem 2009, 393, 1769-1778
10/3/2017 IEF 63/79
DF IEF of barley extract, malt and bear
Continuously sampled pI markers:
orange – marker pI 2.5
pink - marker pI 11
Input- Raw barley extract + buffers + markers pI 2.5 and 11
flow rate - 0.23 ml/ min,
conductivity- 1.0 mS/cm
Input electrodes 4 mA, 20 V
Output electrodes: 6 mA, 800 V
1 drop of pI markers mixture
Mazanec K., Bobalova J., Slais K.
Anal Bioanal Chem 2009, 393, 1769-1778
3.10.2017 63/79 10/3/2017 IEF 64/79
Combined scan of IEF
gel fractions from DF IEF
of bear
Colored pI markers scanned immediately after gel IEF
Proteins scanned after Commassie staining
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
3
pI
11
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
3
pI
11
MALDI-MS spectra of 20 DF
IEF fraction of proteins from
raw malt extract.Mazanec K., Bobalova J., Slais K.
Anal Bioanal Chem 2009, 393, 1769-1778
3.10.2017 64/79
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Preparative divergent flow IEF without carrier ampholytes
for separation of complex biological samples
DF IEF without carrier ampholytes with
yeast lysate sample and colored pI
markers.1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
3.7
5.3
6.3
fractions
8.5
11.0
pIs
+ +
-
input
output
2.6
M S 8 9 10 11 12 13 14 15 16 17 18 19 M
Separation of proteins in individual
yeast lysate DF IEF fractions by
polyacrylamide gel IEF.
M. Stastna, K. Slais, Electrophoresis,31, 2010,433-439
Desalting, preconcentration, preseparation
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Electrolyte system for fast preparative focusing in wide pH
range based on bidirectional isotachophoresis (BITP)
,
L- leading anion of strong
acid
L+ - leading cation of strong
base,
C- - anionic counter ions,
C+ - cationic counter ions,
S- - anionic spacers,
S+ - cationic spacers,
T- - the fastest C- in LB in
anionic ITP part and
T+ - the fastest C+ in LA in
cationic ITP part.
IEF 12
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The composition of LB, LA and spacer electrolytes used
for simulation and in the experiment verification
catholyte pK
u x10-9
[m2V-1s-1] Mw
simulated
conc [mM]
conc [mgL-
1]
lysine 10.79 -26.4 146.19 7 1023.3
ACA 10.80 -28.8 131.20 10 1312.0
GABA 10.56 -29.0 103.10 10 1031.0
β-alanine 10.24 -30.8 89.10 10 891.0
glycine 9.78 -37.4 75.00 7 525.0
asparagine 9.02 -31.6 130.00 5 650.0
TAPS 8.30 -25.0 243.28 5 1216.4
TAPSO 7.70 -26.0 259.28 5 1296.4
NaOH 13.70 +51.9 40.00 60 2400.0
anolyte
glutamic acid 2.16, 4.32 +27.4, +27.6 147.13 5 735.7
β-alanine 3.55 +38.5 89.10 10 891.0
GABA 4.03 +28.8 103.10 10 1031.0
ACA 4.37 +29.8 131.20 10 1312.0
creatinine 4.83, 9.20 +37.0, 37.2 113.10 5 565.5
IDPN 5.29 +30.0 123.20 5 616.0
BisTris 6.40 +26.0 209.20 5 1046.0
HEMorf 6.80 +30.2 131.20 5 656.0
H2SO4 -82.9 98.00 25 2450.0
pK
u x10-9
[m2V-1s-1]
Mw
simulated
conc [mM]
conc [mgL-
1]
spacers
MOPS 7.20 -26.9 209.30 3 627.9
ACES 6.70 -31.3 182.20 3 546.6
MES 6.09 -28.0 213.25 3 639.8
picolinic acid 5.30 -29.6 123.11 3 369.3
ammonium acetate 4.76 -42.7,-39.4 77.09 3 231.3
glycolic acid 3.89 -42.4 118.10 3 354.3
phosphoric acid 2.16,7.21,12.67 34.6,61.4,71.5 192.12 3 576.4
imidazol 7.15 +52.0 68.10 3 204.3
EtMorf 7.70 +30.0 115.20 3 345.6
Tris 8.08 +29.5 121.10 3 363.3
morpholine 8.35 +41.3 87.00 3 261.0
ammediol 8.70 +33.5 105.10 3 315.3
aminomethylpropano
l 9.60 +30.0 89.14 3 267.4
etylaminoethanol 10.00 +30.0 89.14 3 267.4
piperidin 11.10 +39.8 85.10 3 255.3
10/3/2017 IEF 68/79
Computer simulation of dynamics in newly suggested
electrolyte system based on bidirectional ITP
10/3/2017 IEF 69/79
The animation of the experiment with colored
indicators subjected to BITP electrofocusing in newly
suggested electrolyte system and carried out on
nonwoven strip in V-shape trough during 30 min.
10/3/2017 IEF 70/79
0 min
12 min
30 min
cathodeanode
[cm]
loading area
XIXVIIIVIIIISPADNSI
The examples of representative images displaying
bidirectional ITP electrofocusing process in nonwoven
strip in V-shape trough with colored pH indicators taken
at 0, 12 and 30 minutes.
10/3/2017 IEF 71/79
simulation
experiment carried out
on linear nonwoven
strip in the V-shape
trough
The electrofocusing dynamics shown as dependence of
zone position on analysis time
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210 µL/min
LC
S
LA
cathode
anode
X
IX
VIII
VI
III
SPADNS
210 µL/min
LC
LA
S
cathode
anode
X
III
SPADNS
a b
The images of bidirectional ITP electrofocusing with
continuous flow in rectangular (a) and trapezoidal (b)
separation beds under the same experimental conditions
IEF 13
10/3/2017 IEF 73/79
210 µL/min cathode
anode
LC
S
LA
X
SPADNS
cytC
myo
a
b LC
LA
S
c
The example of bidirectional ITP separation and
electrofocusing in continuous flow of cytochrome C
(cytC) and myoglobin (myo) B
C
A
anode
cathodecontact strips
LA
LB
S
input
output
plastic foil holders
anolyte/catholyte drain
Šťastná M., Šlais K. Electrophoresis 36 2015 2579-2586
Continuous fast focusing in trapezoidal void channel based on
bidirectional isotachophoresis in wide pH range.
The separation of colored indicators in instrumentation with a larger void closed channel.
anode
cathode
S
LA LB
The details of the instrumentation output with collected fractions in twelve well plate.
A B
C 1 2 3 4 5 6 7 8 9 10 11 12
The separation of two colored indicators and cytochrome C in smaller void channel.
anode cathode
S
LA LB
A
anode cathode
LA LB
S
B
The separation of two colored indicators and myoglobin in smaller void channel.
anode cathode
LA LB
S
A
cathode
LA LB
S
anode
B
IEF 14
24th Nov. 08 5th CECE 79
Conclusions
Divergent flow isoelectric focusing (DF IEF)
 combines
• speed and low demand of electricity typical for micro fluidic
channels
• sample loadability and separation efficiency of preparative
devices
 has a potential
• for further shape and material optimization
• for scaling up and down
 Single input design (SI DF IEF) simplifies miniature devices
 Carriers based on mixtures of simple buffers are cheap and
advantageous for further processing of collected fractions
 Stability of streamlines is promising for the designs with more
collected fractions
 Thickness of separation layer can conveniently be adjusted by
sandwiching of non woven fabric or glas plate distance
10/3/2017 IEF 80/7980/79