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Chapter 2 Enantioselective, potentiometric membrane electrodes
Introduction
Enantioselective, potentiometric membrane electrodes (EPMEs) were particularly developed for enantioanalysis. EPMEs based on 2-hydroxy-3-trimethylammoniopropyl-β-cyclodextrin were proposed for the assay of angiotension-converting enzyme inhibitors [1-5] as well as for L-proline [6].
Rapid development of new electrodes materials and more sensitive and stable electronic components in the last two decades has increased the range of analytical applications utilizing potentiometric electrodes. The fast development of this field is a scale of the degree to which potentiometric measurements satisfy the need of the clinical chemist for rapid, low cost and accurate analysis.
The accuracy obtained when EPMEs were used in clinical analysis made their utilization a valuable alternative for chromatographic techniques [7, 8]. The method is rapid, precise, and not expensive. The high reliability of the information obtained using these electrodes made automation of potentiometric technique possible, by the integration of enantioselective electrodes as detectors in FIA [9, 10] and SIA [11, 12] systems. The type of electrode and chiral selector must be selected in concordance with the complexity of the structure of the enantiomer to be determined. The principle of molecular recognition for EPMEs is the selective binding between a molecule with a special chemical.
The log KL is directly proportional to ∆GL and log KD is directly proportional to ∆GD, respectively. This means that a difference in the free energies of the reactions will result in a difference of the stability of the complexes formed between the chiral selector and the L and D enantiomers. Therefore, the stability of the complexes is directly correlated with the response (slope) of the EPMEs [13]. Accordingly, a large difference between the
free energies of the reactions of chiral selector with L- and D-enantiomers will give a large difference between the slopes when L and D enantiomers will be determined. The enantioselectivity of the measurements is given by the difference between the two free energies. Also, the slope is a measure of enantiorecognition. The minimum value tolerable for a 1:n stoichiometry between the enantiomer and chiral selector is 50/n mV/decade of concentration [14].
Design of enantioselective, potentiometric membrane electrodes
The design of enantioselective, potentiometric membrane electrodes (EPMEs) plays a very important role in the reliability of analytical information. The evolution concerning the design of EPMEs made their utilization a very accurate and precise alternative for structural analysis techniques [15]. The reliability of the response characteristics as well as the analytical information obtained using EPMEs is strictly correlated to the reliability of the electrodes design [13]. Only a reliable design of EPME will give reliable response characteristics and reliable analytical information.
One of the designs proposed for sensors is based on the impregnation of a chiral selector on a conducting layer such as PVC; imprinting polymers, and carbon paste matrices. The repartition of chiral selector in the plastic membrane is not homogeneous and not reproducible. The liquid membrane needs a support characterized by certain porosity that assures reliability in construction. Accordingly, the most reliable design is that of EPME based on carbon paste that is preferred due to the simplicity and reliability of the construction of electrode.
Modified paste electrode design
Graphite powder proved to be a very good material for electrode design. Mixing oil (paraffin or nujol oil) with the graphite powder is forming carbon paste. One of the most reproducible designs for EPME based on carbon paste has been proposed by Stefan et al [16-18]. The paraffin oil and graphite powder were mixed in a ratio of 1:4 (w/w) followed by the addition of a solution of chiral selector (ligand) (10-3 mol/L) (100 µL of chiral selector solution is added to 100 mg carbon paste). The plain carbon paste was filled into a plastic pipette peak leaving 3 to 4 mm empty in the top to be filled with the modified carbon paste. The optimum diameter of the designed EPME is 3 mm. Electrical contact is made by inserting a silver wire in the plain carbon paste. The surface of the electrode can be renewed by simply polishing it with alumina paper. Because the electrode response is directly proportional to the complex formed at the membrane-solution interface, different types of chiral selectors were proposed for the design of EPMEs such as crown ether, cyclodextrins and its derivatives, maltodextrins and macrocyclic antibiotics.
Cyclodextrins as chiral selector in the EPMEs design
Cyclodextrins (CDs) are oligosaccharides prepared by enzyme degradation of starch and glycosyltransferases of cyclodextrinases producing a mixture of different CDs [19]. The most frequent used CDs as chiral selectors are those consisting of six (α-CD), seven (β-CD) and eight (γ-CD) glucopyranose units with a truncated cone shape providing a hydrophobic cavity (Figure 2.1). Due to the presence of hydroxyl groups (position 2, 3 and 6 of glucopyranose), the outside ring of CD is hydrophilic [20]. The inner diameter of α-, β- and γ-CDs is increasing as a number of glucose units increases from 0.57 to 0.78 and 0.95 mm, respectively. CDs have a suitable solubility in aqueous medium. β-CD has the lowest solubility (1.85 g/100ml water), caused by the existence of intermolecular hydrogen bonding [21].
Several cyclodextrin derivatives have been developed in order to improve the external enantioselectivity of CDs [23]. EPMEs based cyclodextrin derivatives proved good enantioselectivity and reliable analytical information. Stefan et al. proved the suitability of 2-hydroxy-3-trimethylammoniopropyl-β-cyclodextrin based EPME for the enantioanalysis of the S-enantiomer of angiotension-converting enzyme inhibitors [2, 3].
Maltodextrins as chiral selectors in the EPMEs design
Maltodextrins proved to be suitable chiral selectors for compounds with acidic moieties
- Hydrolysis of starch by means of heat and acid or specific enzymatic treatments or combined acid and enzymatic hydrolysis yields a spectrum of depolimerized oligomers
- These hydrolyzates are described in terms of their dextrose equivalent (DE) value, which is a measure of the total reducing power of all sugar present relative on a dry weight basis. Maltodextrins are hydrolyses products of starch with DE lower than 20. They are produced be enzyme-catalyzed conversion using α-amylase (1,4-α-D-glucan glucanohydrolase, EC 3.2.1.1) from Bacillus subtilis and pullulanase (pullulan 6-glucanohydrolase, EC 3.2.1.41) [26]. In general, linear maltodextrins consists of D-(+)-glucose units connected through Glu-(1-4)-α-D-Glu linkages (Figure 2.2). Up to now, three types of maltodextrins with different DE values (I (4.0-7.0), II (13.0-17.0) and III (16.5-19.5)) were used as chiral selectors in enantioanalysis. Hygroscopicity, solubility, osmolarity, and their effectiveness to reduce the freezing point increase with increasing DE, while viscosity, cohesiveness and coarse-crystal prevention increase as DE decreases. Enantioselective, potentiometric membrane electrodes based on maltodextrins have been applied for the enantioanalysis of several drugs [27].
Macrocyclic antibiotics as chiral selectors for EPMEs design
Macrocyclic antibiotics have been successfully used as chiral selectors for the enantiorecognition of several classes of pharmaceutical enantiomers of drugs and molecules with biological importance. The high selectivity and efficiency in molecule discrimination make antibiotics a typical chiral selector of biological origin. Macrocyclic antibiotic contains several functional groups responsible for multiple stereoselective interactions. All macrocyclic antibiotics exhibit very similar physico-chemical properties, but they show a different stereoselective power [28]. The most used macrocyclic antibiotics in enantioanalysis are vancomycin and teicoplanin [29, 30].
Vancomycin is “basket” shaped (Figure 2.3a) with three fused macrocyclic rings and two side chains, a carbohydrate dimmer and a N-methyl leucine moiety [31]. It has 18 asymmetric centers and several functional groups such as carboxylic, hydroxyl, amino, amido and aromatic rings [28]. Vancomycin is very soluble in water and can dimerize in aqueous solutions depending on vancomycin concentration [32].
Vancomycin solutions are stable at low temperature and in buffered solutions (pH 3.0-6.0) [32, 33]. This antibiotic is very efficient for the enantiorecognition of anionic compounds containing carboxylic groups in their structure, which could be explained by the presence of amine groups [28].
Teicoplanin (Figure 2.3b) has a long hydrophobic tail, which behaves like surfactant properties. The molecular structure of teicoplanin shows a slightly higher solubility in water than vancomycin [30]. The amide and carboxylic groups are the most important functional groups of teicoplanin used for the enantiorecognition of molecules containing carboxylic groups. These groups are ionized over the 3.5-8.0 pH range [34, 35]. Teicoplanin exhibits a very slight basic behavior even at acidic pH. At low pH, teicoplanin favors aggregation and micelle formation [36].The most common teicoplanin glycopeptide (A2-2) has a molecular weight of 1877 [34]. Addition of organic modifiers to teicoplanin, such as acetonitrile, improves the resolution ability. These modifiers enhance the enantioselectivity by alerting and/or inhibiting aggregation of teicoplanin monomer producing more teicoplanin molecules available to interact with solutes [28].
Plastic membrane based electrode design
Nowadays, polyvinylchloride (PVC) and its derivatives are the most used in sensor technology. PVC membranes have some disadvantages such as:
- It is not possible to control the uniformity distribution of the electroactive species in the membrane;
- PVC membranes have low thermal durability and low mechanical strength.
PVC membranes are prepared by dissolving a polymer (PVC), a large amount of plasticizer, and the sensing compound in an organic solvent. Tetrahydrofuran and cyclohexane can be used as organic solvents. The solvent is allowed to evaporate, leaving a dry membrane attached to the body of the electrode. The ratio between plasticizer and PVC is 70:30 (w/w) [37, 38]. The PVC and plasticizer form the medium of electroactive compound formation. The most used plasticizers are dioctyl phthalate (DOP), dioctyl sebacate (DOS), dinonyl phthalate (DNP) and ortho-nitrophenyl octyl ether (o-NPOE). Crown ethers [39-42] and lipophilic cyclodextrins [43] are used as chiral selectors for the design of PVC membrane based EPMEs
Response characteristics of EPME
The functional relation between the potential, E measured at I = 0, and the activity, a, of the enantiomer gives the electrode function (Figure 2.4).
Standard electrode potential, Eo
The standard electrode potential is defined by IUPAC as the value of standard emf of a cell in which molecular hydrogen is oxidized to solvated protons at the left-handed electrode [44].
Eo does not depending on the concentration of the ions in solution and can be determined graphically from the calibration graph of the potentiometric electrode (Figure 2.5).
The value of standard electrode potential is also recommended to be determined using the linear regression method as one of the parameters of the equation of calibration of EPME:
E = E o ± SxpM
where E is the potential of the electrode, Eo is the standard potential, S is the slope, and pM = -log CM.
Response of EPME
The response of EPME is dependant on the slope of the linear part of the calibration graph. It is the main characteristic of the potentiometric electrodes. This value can be computed from the Nernst equation:
E = E o ± S(log a)
where E is the potential of the electrode, Eo is the standard electrode potential, S is the slope, and a is the activity of the ion. The slope of the potentiometric electrode has ideal value given by Nernst (59.16/z mV/decade of concentration) where S = RTzF (R = 8.31 J/K mol, T = 298 K, z is the charge of the ion that has determined, F = 96500 C). The minimum accepted value of the slope of potentiometric electrodes for bioanalysis is 50/z
Nernstian response implies ideal sensitivity, but not necessarily ideal selectivity since interfering ions may also give Nernstian response when present as the sole potential determining species. The response is dependant on the stability of the compound formed at the membrane-solution interface [13]. The value of the slope can be deducted using the equation of dependence of slope on the stability of the compound formed at the membrane-solution interface [13].
Synopsis
Samevatting
Dedications
Acknowledgements
Table of contents
Introduction
Chapter 1 Chirality in clinical analysis
1.1 Introduction
1.2 Chirality and configuration
1.3 Descriptors of chiral molecules (Nomenclature)
1.3.1 The L and D designations
1.3.2 The Cahn-Inglod-Prelog designations (S and R designations)
1.3.3 (-) and (+) designations (l or d)
1.3.4 Helicity (M or P)
1.4 Enantiomeric purity
1.5 Sources of chiral compounds
1.6 Importance of chiral molecules
1.6.1 Chirality and clinical diagnosis
1.6.2 Importance of chirality for pharmaceutical compounds
1.7 Method of chiral recognition
1.7.1 Polarimetry
1.7.2 Chromatographic methods
1.7.3 Capillary electrophoresis
1.7.4 Nuclear magnetic resonance spectroscopy
1.7.5 Circular dichroism
1.7.6 Ferroelectric liquid crystals
1.8 Molecular recognition of enantiomers using electrochemical electrodes
1.8.1 Molecular recognition of enantiomers using enantioselective, potentiometric membrane electrodes (EPMEs)
1.8.2 Molecular recognition of enantiomers using amperometric biosensors
1.8.3 Molecular recognition of enantiomers using amperometric immunosensors
1.9 Electrodes as detectors in flow or sequential injection analysis (FIA or SIA)
1.10 References
Chapter 2 Enantioselective, potentiometric membrane electrodes
2.1 Introduction
2.2 Design of enantioselective, potentiometric membrane electrodes
2.2.1 Modified paste electrode design
2.2.1.1 Cyclodextrins as chiral selectors in the EPMEs design
2.2.1.2 Maltodextrins as chiral selectors in the EPMEs design
2.2.1.3 Macrocyclic antibiotics as chiral selectors in the EPMEs design
2.2.2 Plastic membrane based electrode design
2.3 Response characteristics of EPME
2.3.1 Standard electrode potentials, Eo
2.3.2 Response of EPME
2.3.3 Limit of detection
2.3.4 Linear concentration range
2.3.5 Influence of pH
2.3.6 Influence of the temperature on the response of the electrode
2.3.7 Response time
2.3.8 Ionic strength and activity coefficients
2.4 Selectivity of enantioselective potentiometric membrane electrodes
2.4.1 Mixed solution method
2.4.2 Separate solution method
2.5 Direct potentiometric method
2.6 References
Chapter 3 Amperometric electrodes for enantioanalysis
3.1 Introduction
3.2 Design of amperometric electrodes
3.3 Response characteristics of the amperometric electrodes
3.4 Selectivity of the amperometric electrodes
3.5 Direct amperometric method
3.6 Differential pulse voltammetry
3.7 References
Chapter 4 Amperometric biosensors for enantioanalysis
4.1 Introduction
4.2 Design of amperometric biosensors
4.3 Response characteristics of amperometric biosensors
4.4 Selectivity of the amperometric biosensors and immunosensors
4.5 Chronoamperometry
4.6 Direct amperometry
4.7 References
Chapter 5 Enantioanalysis of L- and D-pipecolic acid in biological samples
5.1 Introduction
5.2 Reagents and chemicals
5.3 Amperometric electrode for enantioselective analysis of pipecolic acid
5.4 Enantioselective, potentiometric membrane electrodes for the determination of L-pipecolic acid in serum
5.5 Amperometric biosensors for the enantioselective analysis of L- and Dpipecolic acids in biological fluids
5.6 Sequential injection analysis utilizing amperometric biosensors as detectors for the simultaneous determination of L- and D-pipecolic acids
5.7 Conclusion
5.8 References
Chapter 6 Diamond paste-based electrodes for the determination of L- and D-fucose using differential pulse voltammetry
6.1 Introduction
6.2 Experimental section
6.3 Results and discussion
6.4 Conclusion
6.5 References
Chapter 7 Enantioselective, potentiometric membrane electrodes for the determination of L- and D-glyceric acids
7.1 Introduction
7.2 Reagents and materials
7.3 Enantioselective, potentiometric membrane electrode based on maltodextrins
7.4 Enantioselective, potentiometric membrane electrode based on cyclodextrins
7.5 Enantioselective, potentiometric membrane electrode based on macrocyclic antibiotics
7.6 Conclusion 194
7.7 References 196
Chapter 8 Diagnosis of L- and D-2-hydroxyglutaric acidurias using enantioselective, potentiometric membrane electrodes
8.1 Introduction
8.2 Reagents and materials
8.3 Enantioselective, potentiometric membrane electrode based on maltodextrins
8.4 Enantioselective, potentiometric membrane electrode based on cyclodextrins for the determination of L- and D-2-hydroxyglutaric acid in urine samples
8.5 Determination of D-2-hydroxyglutaric acid in urine ample using enantioselective, potentiometric membrane electrodes based on antibiotics
8.6 Conclusion
8.7 References
Chapter 9 Enantioanalysis of L-vesamicol in serum sample using enantioselective, potentiometric membrane electrodes
9.1 Introduction
9.2 Reagents and materials
9.3 Enantioselective, potentiometric membrane electrodes based on maltodextrins
9.4 Cyclodextrins based enantioselective, potentiometric membrane electrodes
9.5 Enantioselective, potentiometric membrane electrode based on macrocyclic antibiotics
9.6 Conclusion
9.7 References
Chapter 10 Amperometric biosensor for the enantioanalysis of L-lysine in serum samples
10.1 Introduction
10.2 Reagents and materials
10.3 Diamond paste based amperometric biosensor
10.4 Apparatus
10.5 Recommended procedures
10.6 Determination of L-lysine in serum samples
10.7 Results and discussion
10.8 Conclusion
10.9 References
Chapter 11 Conclusions
Appendix
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