1
1
Trends in analytical chemistry
Application of membrane-based pre-separation
techniques in analysis of environmental,
biological and clinical samples
Lecture for students of MU in Brno, 19.10.2017
Pavel Kubáň (kuban@iach.cz)
Department of Electromigration Methods
Institute of Analytical Chemistry of the Czech Academy of Sciences
2
 Complex samples and their pretreatment
 Membrane techniques
 Electrically induced transfer of ions across membranes
 Applications
 Coupling to standard analytical instrumentation
 Conclusions and future perspective
3
Pretreatment of
complex samples
PreconcentrationClean-up
 Matrix effects
 High concentrations of
proteins and salts
 Deteriorated performance
 Analytical system poisoning
 Low concentrations of
analytes
 Analytes not detected
 Poor quantitative
results
4
Human plasma 1:1, essential amino acids
6543210
migration time (min)
3 mV
1
2
3
5
Standard methods for pretreatment of complex
samples
Liquid-liquid extraction (LLE)
Solid phase extraction (SPE)


Automation (SPE)
High consumption of organic solvents and complex samples
Time consuming
Costly
Additional instrumental equipment
6
Membrane techniques for pretreatment of
complex samples
Dialysis (MWCO membranes, hollow fibers)
2
7
Ultrafiltration (flat sheet
membranes, hollow fibers)
8
Supported liquid membrane (SLM)
Support – porous PP, PTFE (thickness 25 – 300 µm)
9
Liquid phase microextraction
membranedonor acceptor
A
M
A+
M
membranedonor acceptor
A+
M+
A+
M+
Diffusion Electric potential
Phase interfaces – membranes
10
Electric potential in sample treatment
 Short extraction times
 High extraction efficiencies
 High selectivity
 Simple instrumentation
 Membrane selection
 Electrode reactions
 High electric current collapse of system
?
?
!
11
Electromembrane extraction – EME
 LLE large volumes of organic solvents
 1996 – LPME (µL volumes of organics)
Liu and Dasgupta, Anal. Chem. 68 (1996) 1817-1821
Jeannot and Cantwell, Anal. Chem. 68 (1996) 2236-2240
 Stability of organic phase
 1999 – HF-LPME (inert porous polypropylene hollow fibre)
Pedersen-Bjergaard and Rasmussen, Anal. Chem. 71 (1999) 2650-2656
 Long extraction times
 2006 – EME (short extraction times due to use of DC voltage)
Pedersen-Bjergaard and Rasmussen, J. Chromatogr. A 1109 (2006) 183-190
!
!
!
12
+
+ analytes
matrix
 Hollow fiber impregnated with organic solvent (~ 10 L) – SLM
 Cheap disposable extraction units (< 10 h/cm), no carry-over
 DC voltage source (0 – 400 V)
 Donor (~ mL) and acceptor (~ 20 L) are aqueous
++
acceptor
sample
1 – 2 mm
200 – 300 m
Hollow fiber:
total length: 2 – 5 cm
Pedersen-Bjergaard and Rasmussen, J. Chromatogr. A 1109 (2006) 183
Electromembrane extraction – EME
3
13
Parameters affecting EME
 Composition of liquid membrane
 pH a composition of acceptor and donor
 Electric potential / current
 Extraction time
 Stirring/agitation
14
EME of basic drugs – model example
 SLM – NPOE
 Acceptor – 10 mM HCl
 Donor – drugs in 10 mM HCl
 Extraction time – 5 min at 300 V
1 µg/mL
Pedersen-Bjergaard and Rasmussen J. Chromatogr. A 1109 (2006) 183
15 16
1 µg/mL
17
Comparison of LPME and EME of basic drugs
Gjelstad et al. J.Chromatogr. A 1157 (2007) 38
18
EME in portable format – use of 9 V battery
 SLM – ENB
 Acceptor – 10 mM HCl
 Donor – plasma, blood, urine in 10 mM
HCl
 Extraction time – 5 min at 9 V
Eibak et al. J. Chromatogr. A 1217 (2010) 5050
4
19
Drop-to-drop EME
 SLM – NPOE
 Acceptor – 10 mM HCl
 Donor – plasma, urine in 10 mM HCl
 Extraction time – 5 min at 15 V
Petersen et al. J. Chromatogr. A 1216 (2009) 1496
A – spiked urine no EME
B – spiked urine after EME
20
Microchip EME
Petersen et al. Microfluid Nanofluid 9 (2010) 881
a) PMMA – top cover
b) PP membrane
c) 50 µm channel
d) PMMA – base plate
21
Microchip EME
 SLM – NPOE
 Acceptor – 10 mM HCl
 Donor – urine in 10 mM HCl
 Extraction time – 10 min at 15 V
 FR donor = 3 µL/min
A – raw urine
B – urine spiked with drugs
C – urine spiked with drugs + EME
22
EME at constant electric current
1. Faraday’s law m =
A – electrochemical equivalent
2. Faraday’s law A =
zF
Mm

tIA 
F = 96485 C mol-1
z – charge number
n =
zF
tI


R
U
I =Ohm’s law  Poor repeatability of EME
(RSD up to 30%)
23
EME of basic drugs
Constant U (5 V)
Constant i (4 µA)
 SLM – ENB
 Acceptor – 10 mM HCl
 Donor – STD, urine in 10 mM HCl
 Extraction time – 5 min
CE-UV in 15 mM phosphate buffer (pH 2.9)
4.03.53.02.52.01.5
migration time (min)
10 mAU
RSD = 3 – 8%
0
1
2
3
4
5
6
0 1 2 3 4 5
extraction time (min)
current(A)
1
2
3
4
5
4.03.53.02.52.0
migration time (min)
10 mAU
RSD = 7 – 15%nortriptyline
haloperidol
loperamide
24
RSD = 6 – 12%
RSD = 3 – 7%
Constant i (4 µA)
Constant U (5 V)
EME of spiked urine at constant electric current
 Improved repeatability
 Similar performance for other analytical parameters
Linearity
LOD
 Supplies operating at constant electric current are not cheap
4.03.53.02.52.01.5
migration time (min)
10 mAU
nortriptyline
haloperidol
loperamide
Šlampová et al. J. Chromatogr. A 1234 (2012) 32
!
5
25
Pulsed EME
Rezazadeh et al. J. Chromatogr. A 1262 (2012) 214
26
phenolphthalein, colorless  pink at pH 8 – 10
0 s – 0 mC
60 s – 0.19 mC
120 s – 0.59 mC
180 s – 0.87 mC
240 s – 1.00 mC
300 s – 1.05 mC
0 s – 0 mC
15 s – 0.07 mC
30 s – 0.18 mC
45 s – 0.39 mC
60 s – 0.50 mC
90 s – 0.54 mC
120 s – 0.56 mC
180 s – 0.57 mC
0 s – 0 mC
30 s – 0.04 mC
60 s – 0.07 mC
120 s – 0.12 mC
180 s – 0.17 mC
300 s – 0.30 mC
360 s – 0.38 mC
420 s – 0.48 mC
in DI water in 1 mM HCl in 50 mM HAc
Q, F, Vd/a ~ 7 mM (H+/OH–)
Q, F, Vd/a ~ 4 mM (H+/OH–)
Q, F, Vd/a ~ 3 mM (H+/OH–)
EME and electrolysis
Kubáň J. Chromatogr. A 1398 (2015) 11
27
12
10
8
6
4
2
0
pHvalue
4035302520151050
extraction time (min)
donor solution
acceptor solution
80
60
40
20
0
extractionrecovery(%)
4035302520151050
extraction time (min)
procaine
nortriptyline
papaverine
4
3
2
1
0
pHvalue
4035302520151050
extraction time (min)
donor solution
acceptor solution
Šlampová et al. Anal. Chim. Acta 887 (2015) 92
Non-optimized acceptor
1 mM HCl
Optimized acceptor
500 mM formic acid
pKa ~ 9
pKa ~ 6
80
60
40
20
0
extractionrecovery(%)
4035302520151050
extraction time (min)
procaine
nortriptyline
papaverine
28
Selected applications of EME in analysis of
biological, environmental and other complex
samples
29
EME of drugs used for treatment of alcohol
and opiates abuse in biological fluids
Rezazadeh et al. J.Chromatogr. B 879 (2011) 1143
30
 SLM –NPOE/DEHP
 Acceptor – 100 mM HCl
 Donor – plasma and urine in 10 mM HCl
 Extraction time – 20 min at 100 V
6
31
EME of peptides in biological fluids
 SLM – 1-octanol/DEHP
 Acceptor – 0.1 M HCl
 Donor – plasma in 0.1 M HCl
 Extraction time – 5 min at 50 V
Balchen et al. J.Chromatogr. A 1194 (2008) 143
A – STD at 300 V
B – STD at 0 V
32
EME of amphetamines in biological fluids
 SLM – NPOE/TEHP
 Acceptor – 100 mM HCl
 Donor – urine in 1 mM HCl
 Extraction time – 7 min at 250 V
Seidi et al. J. Chromatogr. A 1218 (2011) 3958
A – urine spiked with amphetamines
B – urine of amphetamine user
33
EME of decomposition products of nerve
agents in environmental samples
Xu et al. J. Chromatogr. A 1214 (2008) 17
 SLM – 1-octanol
 Acceptor – DI water
 Donor – samples in DI water
 Extraction time – 30 min at 300 V
 Real samples – environmental waters
(a) – STD
(b) – river water spiked with
phosphonic acids
34
EME of heavy metals in water samples and
food supplements
5.04.54.03.53.0
migration time (min)
2 mV
Mg2+ Mn2+ Cd2+ Zn2+ Pb2+
Co2+
Cu2+ Ni2+
1 M HMs after EME
Kubáň et al. Electrophoresis 32 (2011) 1025
 SLM – 1-octanol/DEHP
 Acceptor – 100 mM HAc
 Donor – samples in DI
 Extraction time – 5 min at 75 V
 Real samples – tap water, infant
food supplements
35
EME and CE-C4D of zinc
3.53.02.52.01.5
migration time (min)
5 mV
a
b
c
Ca2+/Na+/Mg2+
Zn2+
3.53.02.52.01.5
migration time (min)
2 mV
Ca2+/Na+/Mg2+
K+
Zn2+
b
a
+ 2 M Zn2+
MP 1:50
+ 10 M Zn2+
+ 5 M Zn2+
TW
Tap water
Milk powder
36
 Endogenous concentrations ~ 100 M
 Metabolic disorders concentration (MSUD ~ 500 M)
(PKU ~ 350 – 1500 M)
EME of amino acids in biological fluids
 SLM – ENB/DEHP
 Acceptor – 2.5 M HAc
 Donor – serum, plasma, blood, urine in 2.5 M HAc
 Extraction time – 10 min at 50 V
7
37
1210864
migration time (min)
0.3 mV
12 34 5 6 7 8 9 10 11 12serum
MSUD – branched amino acids
(Val, Leu, Ile)
1210864
migration time (min)
0.5 mV
12 34 5 6 7 8 9 10 11 12blood
PKU – phenylalanine (Phe)
CE-C4D in 2.5 M HAc
Strieglerová et al. J. Chromatogr. A 1218 (2011) 6248
EME of amino acids in biological fluids
38
EME summary
• Clean-up and preconcentration in one step
• ~ 10 L of organic solvent/analysis
• Disposable extraction units
• Short extraction times
• High selectivity of SLM
• Suitable for biological samples
• SLM selection
• EME parameters
!
!
39
Coupling of membrane extraction techniques
with state-of-the-art analytical instrumentation
Extraction and subsequent analysis off-line
40
1. On-line coupling of SLM to HPLC
Lindegard et al. Anal. Chem. 66 (1994) 4490
Acceptor channel volume ~ 10 µL
41
Determination of basic drugs in plasma
I – Amperozide
II –Amperozide metabolite
III – unknown compound
42
2. On-line coupling of SLM to GC
Shen et al. Anal. Chem. 70 (1998) 946
8
43
Palmarsdottir et al. Anal. Chem. 69 (1997) 1732
(a) – STD
(b) – plasma
A – IS
B – bambuterol
3. On-line coupling of SLM to CE
44
4. On-line coupling of ED to CE
Buscher et al. J. Chromatogr. A 788 (1997) 165
45
On-line coupling of ED to CE – inositol triphosphates analysis
A – without ED
B – with ED
46
5. On-line microchip EME
 SLM – 0.2 µL NPOE
 Acceptor – 100 mM HCOOH
 Donor – urine in 10 mM HCl
 Extraction time – 10 min at 15 V
 FR donor = 9 µL/min
 FR acceptor = 0 – 3 µL/min
Petersen et al. Anal. Chem. (2011) 44
47
On-line monitoring of amitriptyline metabolism
48
Nozal et al. Electrophoresis 27 (2006) 3075
6. On-line coupling of SLM to commercial CE – Beckman
9
49
Determination of nitroimidazoles in liver
1 – metronidazole
2 – ronidazole
3 – dimetridazole
A – sample
B – spiked sample
50
4.44.24.03.83.63.4
migration time (min)
0.2 mV
Chol
Crea
Orn
Lys
Arg
His
serum
plasma
C4D
C
Pt Pt
h
HV
6.05.04.03.0
migration time (min)
3 mV
serum
breast milk
SCN-
ClO4
Kubáň
et al. Electrophoresis 33 (2012) 2695
Applications
Extraction ~ 0 – 10 min
7. In-line coupling of disposable SLM to lab-made CE
Kubáň and Boček J. Chromatogr. A 1234 (2012) 2
51
Electrode Capillary
Sample vial
Compression spring
Donor
SLM
Acceptor
 SLM ~ 5 µL of organic solvent
 Sample volume – 10 – 40 µL
 Disposable extraction devices !!!
Acceptor
Donor
SLM
Extraction Injection
8. In-line coupling of disposable
SLM devices to commercial CE
52
30
20
10
0
mAU
1.61.41.21.00.80.60.4
migration time (min)
10 mM
1 mM
blank
Undiluted serum – healthy individual
formate
LOD: 30 µM undiluted serum,
35 µM undiluted whole blood
Endogenous concentrations: 0 – 0.4 mM
Methanol poisoning: 1 – 40 mM
 10 mM serious health consequences
Formate in human serum and whole blood
chloride
20 mM
80
60
40
20
0
mAU
1.61.41.21.00.80.60.4
migration time (min)
formate
chloride
Undiluted serum– patient after methanol intoxication
10.4 ± 0.4 mM
Pantůčková et al. J. Chromatogr. A 1299 (2013) 33
Total analysis time: ~ 4 min!!!
 Support: PP foil (100 µm)
 SLM: 10 µL MeOH
 Donor: undiluted serum, whole blood
 Acceptor: DI water
 Volume: 10 µL
 Extraction time: 60 s !!!
53
Polymer inclusion membranes (PIMs)
Base polymer – Cellulose triacetate (CTA)
Plasticizer – 2-nitrophenyloctyl ether
Ion carrier – Aliquat 336
PIM – dry, homogenous, non-porous
500 nm
Resulting PIM:
60% (w/w) CTA
40% (w/w) Aliquat 336
Dissolve in dichloromethane and evaporate
Schow et al. J. Membr. Sci. 111 (1996)
54
Formate in whole blood after methanol poisoning
PIM extraction Coupling to CE CE-C4D analysis of raw blood
2.52.01.51.00.5
migration time (min)
13 mV
fresh PIM
30 days old PIM
90 days old PIM
formate
chloride
Long-term
performance of
PIM
Pre-assembled disposable units
for clinical applications?
Pantůčková et al. Anal. Chim. Acta 887 (2015) 111
Total time ~ 7 min
10
55
10. Free liquid membranes (FLMs)
OD1.6mm
ID1.0mm
donor FLM acceptor
Total length 20 mm
donor FLM acceptor
PFA tubing
 Extraction units – PFA, PTFE, PP tubing (ID 0.5 – 1.0 mm)
 Minimum consumption of solvents/samples (350 nL – 1.5 µL/extraction)
 Cheap, disposable extraction units (~ 1 kč/cm), no sample carry-over
 Stable and precisely defined phase interfaces
56
µ-EME across FLM
0 s
60 s
120 s
180 s
240 s
300 s
0 s
5 s
15 s
30 s
60 s
120 s
300 s
Anions Cations
 FLM: 1.5 µL 1-pentanol
 Donor: SPADNS or
crystal violet
 Acceptor: DI water
 Volume: 1.5 µL
 Extraction voltage: 100 V
 Extraction time: 5 min
+
GND GND
-
57
µ-EME across FLM
µ-EME principle
EME fundamentals
Donor: SPADNS3- in DI water
Acceptor: DI water
FLM: 1-pentanol
Voltage: 100 V
Time: 3 min
Volumes: 1.5 µL
Donor: phenolphthalein in 1 mM HCl
Acceptor: DI water
FLM: 1-pentanol
Voltage: 100 V
Time: 3 min
Volumes: 1.5 µL
58
12
10
8
6
4
2
0
electriccurrent(A)
300250200150100500
extraction time (s)
µ
1 mM SPADNS
100 µM SPADNS
10 µM SPADNS
DI water
12
10
8
6
4
2
0
electriccurrent(A)
300250200150100500
extraction time (s)
µ
1 mM crystal violet
100 µM crystal violet
10 µM crystal violet
DI water
µ-EME process – electric currents monitoring
59
20
15
10
5
0
UVabsorbance(mAU)
3.02.52.01.51.00.50.0
migration time (min)
Standard
Urine
Serum
ceatinine
nortriptyline
haloperidol
loperamide
µ-EME of basic drugs across FLM
 FLM: 1.5 µL ENB
 Donor: urine and serum
+ 20 μg/mL drugs
 Acceptor: 10 mM HCl
 Volumes: 1.5 µL
 Voltage: 100 V
 Time: 5 min
60
CONCLUSIONS AND PERSPECTIVES
 Membrane extraction techniques are green, cheap, fast
and efficient
 Electric potential is suitable for pretreatment of complex
samples
 Resulting acceptors are compatible with standard
analytical instruments
 On-line and in-line coupling to analytical instrumentation
is very attractive
Ph.D. positions available