Derivatization preferences for compounds containing active hydrogens.
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\\n\\nLaunching 2021
\\n\\nArtificial Intelligence, ISSN 2633-1403
\\n\\nVeterinary Medicine and Science, ISSN 2632-0517
\\n\\nBiochemistry, ISSN 2632-0983
\\n\\nBiomedical Engineering, ISSN 2631-5343
\\n\\nInfectious Diseases, ISSN 2631-6188
\\n\\nPhysiology (Coming Soon)
\\n\\nDentistry (Coming Soon)
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\\n\\nNote: Edited in October 2021
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\n\nDesigned to cover fast-moving research fields in rapidly expanding areas, our Book Series feature a Topic structure allowing us to present the most relevant sub-disciplines. Book Series are headed by Series Editors, and a team of Topic Editors supported by international Editorial Board members. Topics are always open for submissions, with an Annual Volume published each calendar year.
\n\nAfter a robust peer-review process, accepted works are published quickly, thanks to Online First, ensuring research is made available to the scientific community without delay.
\n\nOur innovative Book Series format brings you:
\n\nIntechOpen Book Series will also publish a program of research-driven Thematic Edited Volumes that focus on specific areas and allow for a more in-depth overview of a particular subject.
\n\nIntechOpen Book Series will be launching regularly to offer our authors and editors exciting opportunities to publish their research Open Access. We will begin by relaunching some of our existing Book Series in this innovative book format, and will expand in 2022 into rapidly growing research fields that are driving and advancing society.
\n\nLaunching 2021
\n\nArtificial Intelligence, ISSN 2633-1403
\n\nVeterinary Medicine and Science, ISSN 2632-0517
\n\nBiochemistry, ISSN 2632-0983
\n\nBiomedical Engineering, ISSN 2631-5343
\n\nInfectious Diseases, ISSN 2631-6188
\n\nPhysiology (Coming Soon)
\n\nDentistry (Coming Soon)
\n\nWe invite you to explore our IntechOpen Book Series, find the right publishing program for you and reach your desired audience in record time.
\n\nNote: Edited in October 2021
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He is currently a professor in the Department of Mechanical and Intelligent Engineering, Utsunomiya University, Japan.",institutionString:"Utsunomiya University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"0",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"Utsunomiya University",institutionURL:null,country:{name:"Japan"}}},coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"124",title:"Vehicle Engineering",slug:"vehicle-engineering"}],chapters:[{id:"79180",title:"Introductory Chapter: Propulsion and Movement",slug:"introductory-chapter-propulsion-and-movement",totalDownloads:102,totalCrossrefCites:0,authors:[{id:"42387",title:"Prof.",name:"Kazuo",surname:"Matsuuchi",slug:"kazuo-matsuuchi",fullName:"Kazuo Matsuuchi"}]},{id:"76789",title:"Hybrid Propulsion System: Novel Propellant Design for Mars Ascent Vehicles",slug:"hybrid-propulsion-system-novel-propellant-design-for-mars-ascent-vehicles",totalDownloads:260,totalCrossrefCites:0,authors:[{id:"332637",title:"Dr.",name:"Ozan",surname:"Kara",slug:"ozan-kara",fullName:"Ozan Kara"}]},{id:"74791",title:"Keeping the Dream Alive: Is Propellant-less Propulsion Possible?",slug:"keeping-the-dream-alive-is-propellant-less-propulsion-possible-",totalDownloads:436,totalCrossrefCites:1,authors:[{id:"335130",title:"Emeritus Prof.",name:"James F.",surname:"Woodward",slug:"james-f.-woodward",fullName:"James F. 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One is the continuous demand for more sensitive and accurate analytical methods. The other is the desire for simpler methods that require as little as possible human intervention. One of the various procedures to make the analytical methods more sensitive and accurate is the use of specific chemical changes (e.g., derivatization) applied on the analytes or even on the whole sample. However, these changes frequently involve more human intervention than the direct use of advanced instrumentation. For this reason, the methods involving chemical changes such as derivatizations are not necessarily the first choice when selecting an analytical method. Nevertheless, in many cases, the benefits of derivatization are more important than the disadvantage of requiring human intervention, and for this reason, derivatization is still frequently used in the analytical practice. Also, modern GC, GC/MS (or GC/MS/MS) instrumentation may offer autosampling with the capability of adding reagents to the sample, as well as stirring, heating, and injecting the sample at specific time intervals in the GC system. This type of instrumentation may reduce significantly the human handling involved in derivatization.
\nVarious chemical changes can be performed on an analyte in order to make it suitable for a specific method of analysis. The most common is derivatization, but other chemical changes can be utilized, for example, pyrolytic decomposition and, in the case of polymers, polymer fragmentation using reagents. The choice depends on the nature of the analyte, the sample matrix, the intended changes in the analyte properties, and the analytical method to be used.
\nThe addition of a reagent on a sample may produce a chemical reaction only with the analytes without affecting the matrix. However, it is also possible that some matrix components are derivatized unintentionally. Usually, it is preferable to have only the analytes derivatized since in this way a better separation from the matrix is expected. Some derivatizations are used in the sample cleanup or concentration process. Also, the derivatization process may be combined with simultaneous extraction and concentration of the sample or may be followed by a second preparation step before the chromatographic analysis. More frequently, the derivatization is done to change the analyte properties for the core analytical procedure (GC, GC/MS, etc.).
\nDerivatization can be applied before the core chromatographic process or after it. Precolumn derivatization takes place before the separation and postcolumn derivatization after it. In GC precolumn derivatization is much more common and most derivatizations are performed “offline.” There are however derivatizations that can be done “online,” for example, in the injection port of the GC such as some methylations using tetramethyl ammonium hydroxide (TMAH). Postcolumn derivatizations are performed only for enhancing the detectability of the analytes. Typically, they must be done “online” and should be completed in the specific time frame needed by the analyte to reach the detector.
\nA wide variety of derivatization reagents and procedures are described in the literature, with the reagents carrying specific moieties that provide a desired property to the analytes, as well as with specific reactive groups that permit the reaction with the analyte. Multiple step derivatizations as well as derivatizations followed by a second one are known.
\nDerivatization is not always the first step in sample preparation. Sample preparation typically includes other operations, besides derivatization. Some of these steps are more complex such as sample cleanup or concentration and others more simple such as pH adjustments, addition of proton acceptors or donors, change of the medium (from one solvent to another), and addition of catalysts to enhance the derivatization, and these may be necessary for a successful derivatization.
\nAlthough derivatization is performed in order to make possible or to improve the results of a chemical analysis, there are also some disadvantages of using derivatization. Besides the potential need of more manpower for the analysis, the addition of more operations applied on the sample (including the analytes) can be a source of additional errors. In particular the involvement of a chemical reaction that may not be perfectly controlled can bring significant errors in the analytical results. To minimize the potential errors when using derivatization, specific aspects of the derivatization must be considered in its choice, such as the efficiency of the chemical reaction used in the derivatization, the stability of the derivatized analytes, the availability of reagents and necessary equipment, and the time necessary for performing the analysis. For a given analyte or group of analytes, the reaction with the derivatization reagent must be complete or at least close to complete, must take place in a length of time that is not prohibitive, and must have very little loss of the analyte with formation of artifacts or decomposition products. Only when such criteria are satisfied can a specific chosen derivatization be applied successfully.
\nThe application of derivatization in chromatography is the subject of many studies. Numerous derivatizations have been reported in journals (e.g.,
For GC analysis, the effect of derivatization can be beneficial in a variety of circumstances. Some of the most common uses of derivatization for improving the GC separation are the following:
(a) Derivatization that replaces active (polar) hydrogen atoms in the analyte to decrease its boiling point. The active hydrogens in a chemical compound typically enhance the capability to form hydrogen bonds and increase the compound polarity. For this reason, many compounds containing active (polar) hydrogens are not volatile, the volatility being necessary for using GC or GC/MS as a core analytical method. Derivatization can be used to replace active hydrogens from an analyte Y-H (or Y
In reaction (1), the reagent R-X contains an “active” group X and a group R that carries a desired property (e.g., lack of polarity for GC). Group R in the reagent can be a low molecular mass fragment such as CH3 or C2H5, a short-chain fluorinated alkyl in alkylation reactions, Si(CH3)3 or other silyl groups in silylations, COCH3 or short-chain fluorinated acyl groups in acylations, etc. An example of a chromatogram resulting from the GC/MS analysis of a silylated tobacco sample is given in Figure 1. Tobacco contains many hydroxy acids such as malic, trihydroxybutanoic, citric, quinic, glucuronic, and chlorogenic. Also, it contains monosaccharides (e.g., glucose, fructose), disaccharides (e.g., sucrose), and even trisaccharides. None of these compounds are volatile, having numerous active hydrogens. The replacement of these hydrogens with Si(CH3)3 by silylation renders these compounds volatile, and they can be analyzed by GC/MS as seen in Figure 1.
(b) Derivatization for enhancing the separation. Specific moieties added to an analyte may be necessary for enhancing the separation. This is frequently practiced for general GC separations and is also very useful for the separation of chiral molecules (see Section 4). The derivatized analytes may have significantly different properties from each other, for example, regarding polarity and implicitly in their boiling point, allowing separations that are difficult to achieve otherwise. Also, derivatization may generate more significant differences between the analytes and the matrix components.
(c) Derivatization that replaces active hydrogens in the analyte to improve the behavior of the analyte in the chromatographic separation. The chromatographic column (e.g., a capillary column coated with a bonded stationary phase) may display additional capability to interact with polar molecules, besides the intended interactions due to its bonded phase. This may come, for example, from the silica wall of the column. Secondary interactions taking place with only a portion of the molecules of the analyte generate peak tailing. This is exemplified in Figure 2 which shows a hypothetical case of two different types of interaction between the column and a specific molecular species.
(d) Derivatization for the improvement of stability of a compound. This stability may refer to thermal stability, a property which overlaps to a certain extent to what was described at point (a). However, even some volatile compounds may be further thermally stabilized by derivatization. Also, chemical stability can be enhanced by protecting specific groups in the analyte using derivatization. For example, thiols can be protected using derivatization against oxidation by the traces of oxygen in the heated injection port of the GC.
GC/MS chromatogram of a silylated tobacco sample, with separation on a DB-5 MS column from Agilent (Agilent Technologies Inc., Wilmington, DE, USA) (Note: an internal standard I.S. was added to the sample).
Peak tailing due to multiple retention mechanisms.
The choice of the appropriate derivatization is not always a simple task. The replacement of a hydrogen atom with a group of atoms may increase the molecular weight of the derivatized analyte. In such cases, it must be verified that the increase in the molecular weight by derivatization brings no or only a small increase in the boiling point of the analyte. Most of the time, low molecular weight substituents such as CH3 or Si(CH3)3 are preferable for GC analysis to the active hydrogens for achieving the previously described goals. Large substituents may increase the boiling point too much and make the compound not acceptable for GC analysis.
\nBesides replacement of active hydrogens, other derivatization reactions can be utilized. For example, condensation reactions may decrease the boiling point and improve the thermal stability of an analyte. However, the generation of new active hydrogens must be avoided in condensation reactions or must be followed by a second derivatization.
\nThe compounds with structures that are mirror images to each other are indicated as enantiomers, and their molecules are not superimposable, having the property called chirality. Chirality is commonly caused by the existence in the molecule of at least one tetrahedral carbon atom substituted with groups that are different. However, chiral molecules may be generated with a phosphorus or a sulfur chiral atom. Not only chiral centers (such as an asymmetric carbon) generate enantiomers, but a chiral axis or a chiral plane can lead to enantiomers. The chirality in an enantiomer is specified using the symbols R and S based on specific rules. For the assignment of a symbol R or S to a chiral carbon, the substituents are arranged in a sequence a > b > c > d. For the four atoms directly attached to the asymmetric carbon, a higher atomic number outranks the lower, and a higher atomic mass outranks the lower mass. For the same atoms directly attached to the asymmetric carbon, the priorities are assigned at the first point of difference. After the sequence is established, the molecule is oriented in space with the group “d” of the lowest priority behind the asymmetric carbon. When viewed along the C─d bond (from C) and the three substituents a, b, and c are oriented clockwise, the compound contains an R asymmetric carbon, and it contains an S asymmetric carbon for counterclockwise arrangement.
\nMore than one asymmetric carbon can be present in a molecule, as in the case of carbohydrates. The stereoisomers generated by more than one asymmetric carbon can be mirror image one to the other (enantiomers) or may have different steric arrangements being diastereoisomers. These types of molecules are schematically shown in Figure 3.
\nCompounds with two chiral centers.
The (S,S)- and the (R,R)-compounds from Figure 3 are enantiomers, while the (S,R)-compound is a diastereoisomer to both (S,S)- and to (R,R)-compounds (it is an enantiomer to the (R,S)-compound). The gas chromatographic separation of enantiomers can be done only using chromatographic columns having chiral stationary phases. The derivatization of enantiomers with non-chiral reagents generates molecules that remain enantiomers. This type of derivatization may improve the chromatographic separation from other molecules, but the derivatized compounds of remaining enantiomers cannot be separated except on chiral stationary phases. Sometimes, better separation can be obtained even between the enantiomers (on chiral chromatographic columns) after derivatization. One such example is the separation of (R)- and (S)-nornicotine derivatized with isobutyl chloroformate on a chiral Rt-BDEXsm column with separation improved compared to that of underivatized enantiomers [6]. The derivatization reaction is indicated below:
\nDiastereoisomers can be separated on chromatographic columns with non-chiral stationary phases which offer a much wider possibility to select the column. For this reason, an alternative procedure toward the separation of enantiomers is using derivatization with chiral reagents. This type of derivatization generates diastereoisomers which can be separated on non-chiral stationary phases.
\nA discussion on the separation of enantiomers on chiral phases without derivatization is beyond the purpose of this chapter. Numerous publications are dedicated to this subject, including papers published in general chromatography journals or in dedicated journals (e.g.,
The separation after derivatization with a pure enantiomer reagent is based on formation of diastereoisomers that can be separated on regular stationary phases. Depending on the nature of the analyte and of the derivatization, different separation techniques can be applied. A variety of common columns are used for such GC separations. The choice of the column is again dependent on the analyte and the derivatization procedure. For example, α-substituted organic acids such as α-chloropropionic, α-bromocaproic, etc. can be derivatized with a specific enantiomer of an amino acid ester (e.g., ethyl 2-aminopropanoate) in the presence of a peptide coupling reagent (benzotriazol-1-yl-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate or BOP) in a reaction of the type:
The derivatized acids that are now diastereoisomers (R,S) and (S,S) can be separated on a common capillary column (e.g., a DB-1701 column from Agilent). Another example of derivatization with a chiral reagent is that of methamphetamines with (R)-menthyl chloroformate. This derivatization allows the separation of over-the-counter (R)-methamphetamine from the illicit (S)-methamphetamine. The reaction of the (R)-enantiomer is indicated below [8]:
The separation of the (R,R) and (S,R) derivatives was possible on a non-chiral column for a GC/MS analysis.
\nGas chromatography (not coupled with mass spectrometry, GC/MS being separately presented) used as an analytical technique can involve various detectors. The variety of such detectors is rather large, and several types include the following: thermal conductivity detector (TCD), flame ionization detector (FID), nitrogen-phosphorus detector (NPD), electron capture detector (ECD), flame photometric detector (FPD), photoionization detector (PID), electrolytic conductivity (Hall), sulfur chemiluminescence, nitrogen chemiluminescence, aroyl luminescence detector (ALD), atomic emission detector (AED), helium ionization detector (HID), vacuum ultraviolet (VUV) absorbance, infrared Doppler (IRD) absorption, FID with catalytic conversion of all analytes in CH4 (e.g., Polyarc system [9]), etc. The derivatization with the purpose of improving detectability in GC is determined by the type of detector utilized. Most derivatizations are performed precolumn, even if they are applied only with the purpose of improving detection. However, it is important that the derivatization for improving detection does not deteriorate the separation. Preferably, both the detection and the chromatographic separation are improved by the same derivatization. Some specific postcolumn reactions applied to the analytes are part of certain types of detectors such as chemiluminescence detectors, atomic emission detectors (AED), and FID with catalytic conversion into CH4. Some of these chemical changes in the analytes are not necessarily classified as derivatization reactions.
\nNo specific derivatization is usually recommended to improve sensitivity when using nonselective detectors such as TCD and FID. However, in some cases when the detector is not sensitive to a specific analyte, such as formaldehyde or heavily halogenated compounds, derivatization can be used to enhance detection.
\nIn case of NPD detector, derivatization with nitrogenous compounds can be done, which should give a higher sensitivity. However, this type of derivatization is not very common. An adverse result occurs for the NPD detectors when silylation is performed on the sample. Besides a possible reduction in the NPD response on silylated compounds containing nitrogen, a drastic decrease in the lifetime of the detector may occur, probably due to the excess of silylating reagent that commonly is injected with a derivatized sample and affects the alkali active element of the NPD.
\nThe response of the photoionization detector (PID) depends on the ionization potential of the analyte, and compounds with higher ionization potential are not sensitive in PID, while those with lower ionization potential may have excellent sensitivity, as low as 10−12 mg of sample. A derivatization resulting in lowering the ionization potential of the analyte may be beneficial for PID detection. However, derivatization for enhancing PID response is not frequently used.
\nSome detectors such as electron capture detectors (ECD) may benefit very much from certain derivatization types. ECD (as well as negative chemical ionization mass spectrometry or NCI-MS) can be extremely sensitive, but they are selective to compounds that are able to form more stable negative ions. ECD, for example, can have sensitivity as low as 10−13 mg of analyte in the detector compared to the best sensitivity of FID that can be 10−8 to 10−11 mg of analyte. The efficiency of the process seems to be related to the ease of attaching an electron on the molecule. In ECD this process can be written as follows:
With some exceptions, ECD response can be correlated with the electron affinity of the analyte [4]. In general, the halogen substituents increase the sensitivity in ECD in the order I > Br > Cl > F. Multiple substitutions seem to have a cumulative effect. Besides halogens, nitro groups seem to have an effect similar to chlorine groups. For aromatic compounds, the substituents affect the sensitivity of the ECD according to their electron withdrawing capability. Strong electron withdrawing groups such as NO2 increase the sensitivity of the detection, while electron donating groups reduce it.
\nA variety of substitution groups containing electronegative elements (halogens) or nitro groups can be attached to an analyte. The procedure to attach these groups is in most cases the typical substitution of an active hydrogen in the analyte Y-H with a group R from a reagent R-X that has the appropriate active X group. Some groups used for enhancing ECD (as well as NCI-MS) sensitivity following an alkylation or aryl derivatization reaction are shown in Figure 4, and several substitution groups introduced by acylation, chloroformylation, or sulfonation used for the same purpose are shown in Figure 5. Besides alkylation or aryl derivatization, other derivatization techniques used to replace an active hydrogen are applied to introduce into a molecule as a substituent containing halogens or nitro groups enhancing significantly the detectability of the derivatized analytes by ECD (as well as NCI-MS). Silylation, for example, can be used for this purpose when silyl groups used for derivatization contain halogens. Several silyl groups containing halogens that can be attached to an analyte by silylation with special reagents are given in Figure 6 [4].
\nSubstitution groups used in alkylation and aryl derivatization for enhancing ECD (and NCI-MS) detectability (the masses are considered only for the most abundant isotope.).
Substitution groups used in acylation chloroformation and sulfonation for enhancing ECD (and NCI-MS) detectability.
Substitution groups used in silylation for enhancing ECD (and NCI-MS) detectability.
The most powerful tool used for compound identification purposes is very likely mass spectrometry (spectroscopy). This technique is capable to provide information from very low amounts of material such as that eluting from a chromatographic column and can be easily coupled with a gas chromatograph. Most analyses performed with MS detection (GC/MS or GC/MS/MS) are using EI+ ionization mode with electron impact at 70 eV. The electrons interact with the molecule A to eject an additional electron leaving a positively charged species (with an odd number of electrons) of the type A▪+. The ions also receive energy during electron impact and the excess of energy induces fragmentation. For most molecules, this process can be written as follows:
The fragments Bi+ are commonly but not always with an even number of electrons. The formation of molecular ions takes place with a range of internal energies, and more than one fragmentation path is possible for a given molecule. Also, the fragments can suffer further fragmentations. In general, the most abundant fragment ion results from the fragmentations that form the most stable products (ion and neutral radical). The abundance of a fragment ion is affected by its stability. For this reason, the intensity of the response of a mass spectrometric detector can be very different for different molecular species, and the prediction of this intensity is difficult. As a result, the improvements in the sensitivity in EI + −type mass spectrometry (in GC/MS using EI+ ionization) are not usually sought (but not impossible) through derivatization.
\nDerivatization for enhancing sensitivity is, however, frequently applied in NCI-MS. In this technique, the electrons interact with the molecules of the CI gas which is lowering their energy but without forming ions. The ionization of analyte molecules takes place by interaction with the low-energy electrons or with already formed negative ions by electron capture, dissociative electron capture, ion pair formation, or ion molecule reaction. The ionization process with the formation of negative ions is efficient only for molecules with positive electron affinities. For this reason, the sensitivity in NCI-MS is highly dependent on the electron affinity of the analyte, similarly to the sensitivity in ECD. For enhancing the electron affinity, the derivatization with reagents containing, for example, fluorinated moieties (indicated in Figures 4, 5, 6) is practiced. The sensitivity of the analytical methods where such derivatization is applicable can have very good sensitivity. For example, derivatization with heptafluorobutyric anhydride of aromatic amines that are present at low trace level in cigarette smoke leads to limit of detection (LOD) values as low as 0.05 ng/cig. for compounds such as 4-aminobiphenyl [10, 11].
\nThe fragmentation pattern generated by EI+ ionization mode that generates a specific mass spectrum of a molecule is very likely the most utilized technique for the identification of the molecular species. For this identification, large libraries of mass spectra are available, and computer algorithms are used for automatic searches. The identification of compounds using mass spectroscopy is not a simple process even with the capabilities offered by the electronic searches in the mass spectral libraries. This is particularly true for analysis of complex mixtures or when the analyzed compound is present in traces. Some compounds do not have a very characteristic mass spectrum, or during the chromatographic process, the separation is not achieved, and it is difficult to make an identification due to the spectra overlapping. Also, numerous compounds may have a mass spectrum that matches more than one compound (with a good quality fit). In such cases, a derivatization with the purpose of obtaining a compound that forms more informative fragments in the mass spectrum can be very useful.
\nThe fragments from derivatized compounds can be used for the identification of unknown compounds using library searches and even when the mass spectrum is not available in the libraries. As an example, the derivatization by silylation allowed the identification of a new pentacyclic triterpenoid present in several bioactive botanicals [12]. An unidentified compound with MW = 456.7 was detected by LC/MS/MS in a rosemary extract. The structure of the compound was elucidated after silylation of the plant material based on the comparison of mass spectrum of the unidentified compound with that of silylated betulinic acid. The new compound was identified as (3β)-3-hydroxy-lupa-18,20(29)-dien-28-oic acid (or betul-18-en-oic acid). The mass spectra of the two acids are shown in Figure 7.
\nMass spectrum of silylated betulinic acid and that of silylated betul-18-en-oic acid.
The two mass units difference between different fragments from the mass spectra of the two compounds allowed the identification of the new compound structure. Neither free betulinic acid nor betul-18-en-oic acid are volatile, such that the use of GC/MS for identification was possible only after derivatization.
\nAnother special procedure that may be utilized for compound identification based on mass spectra is the use of two parallel derivatizations, one of them being done with an isotope-labeled reagent. Common labeling isotopes are 2H (deuterium, d), 13C, 15N, etc. One such isotopic labeling can be done, for example, using silylation with d18-N,O-bis(trimethylsilyl)-trifluoroacetamide (d18-BSTFA). Derivatization of an aliquot of sample with regular BSTFA and another with d18-BSTFA provides a pairing chromatogram with peaks at retention times that have only small differences from the first but with spectra differing by a number of units. The comparison of the spectra for corresponding peaks (based on retention time) of a given compound allows the calculation of the number of silyl groups attached to that compound. In addition, the fragmentation in the spectra can be better interpreted allowing easier compound identification.
\nDerivatization in GC/MS analysis may have multiple other utilizations and benefits. For example, quantitative analysis frequently utilizes isotopically labeled internal standards. In an analysis with multiple analytes, addition of an isotopically labeled internal standard for each analyte may become a complex process. When a derivatization is involved in the analysis, this can be done with a non-labeled reagent for the analytes in the sample, while the internal standards are obtained by derivatization of standards with the same reagent but isotopically labeled. Such technique has been proven to be very successful, for example, in the analysis of multiple amino acids (but using an LC/MS/MS procedure [13]).
\nDerivatizations as chemical reactions can be classified as follows: (1) reactions with formation of alkyl or aryl derivatives, (2) silylation reactions, (3) reactions with formation of acyl derivatives, (4) reactions of addition to carbon-hetero multiple bonds, (5) reactions with formation of cyclic compounds, and (6) other reactions specific to a certain analysis. The selection of the derivatization reaction is typically done based on the desired property to be brought to the analyte and its possible reactivity. For this reason, the reagent is selected to have moieties that add the desired property to the analyte and also to have the capability to react with the specific functional group of the analyte. The matrix of the sample also has a role in the choice of a specific derivatization procedure. Initial matrix of the sample is not always suitable for derivatization, and in some cases preliminary sample preparation is necessary to change this matrix. The change can be as simple as drying the initial sample but can also be rather complex [14]. Table 1 gives a simplified view of preferences for the choice of a derivatization reagent for compounds containing active hydrogens [14].
\nDerivatization preferences for compounds containing active hydrogens.
Besides functionalities with active hydrogens, other functionalities can also be derivatized. Compounds containing carbonyls can be derivatized, for example, using condensation reactions. Some analytes may contain multiple functional groups such as the amino acids. Specific derivatization reactions can be selected for such cases.
\nThe formation of alkyl or aryl derivatives is applied to replace the active hydrogens from an analyte with an alkyl (R) or aryl (Ar) group. The replacement can be done in functionalities such as OH, COOH, SH, NH, or CONH. For example, the derivatization with short-chain alkyl bromides or iodides has numerous analytical applications for compounds such as steroids, amino acids, catecholamines, sulfonamides, phenols, barbiturates, organic acids, and mono- and oligosaccharides. A large number of reagents R-X are known, and in a simplified approach, it can be considered that R is carrying a specific property and X a specific reactivity, although the reactivity of a reagent is influenced by both R and X components of the molecule. The type of moiety R and that of reactive group X are guiding the selection process of selecting a reagent for a specific derivatization.
\nIn most alkylation reactions, the analyte acts as a nucleophile (Y
Various reagents and conditions were utilized in the derivatizations for analytical purposes. As reagents R-X for alkylations, one of the most commonly used are the alkyl halides, especially alkyl iodides and alkyl bromides. Because some of the derivatizations can be slow and inefficient depending on the analyte and on the reagent, the reaction rate becomes an important parameter for the analytical applicability. The reaction with an alkyl halide for the preparation of methyl or ethyl substituents, for example, is frequently performed either with a specific methylation reagent, in the presence of a catalyst, or in some instances using a particular solvent. The enhancement of the alkylation efficiency can be achieved using several other procedures. For example, for the analytical alkylation of carboxylic acids, specific cryptands such as crown ethers can be used to solvate the alkali metal portion of an organic acid salts, allowing the anion to be freer and increasing the rate of nucleophilic substitution. One other approach for enhancing the alkylation efficiency is the use of phase transfer alkylation. This approach is based on the formation of a compound easily extractable in an organic phase and on the displacement of the equilibrium in the direction of the formation of the desired product.
\nOne different way of enhancing the alkylation efficiency is the use of different alkylating reagents besides short-chain alkyl bromides or iodides. One example of a halide that is particularly reactive is pentafluorobenzyl bromide. This reagent can be used for the derivatization of a variety of compounds containing active hydrogens. Another reactive halide is 2-bromoacetophenone (phenacyl bromide). This reagent is used mainly for the alkylation of compounds containing more acidic hydrogens such as carboxylic acids. Another example of methylation using a special reagent R-X is applied on carbohydrates [15]. This methylation uses methylsulfinylmethanide anion. The reagent is prepared from dry DMSO and NaH or KH in a reaction as follows:
A polyol or a monosaccharide dissolved in DMSO is easily methylated with methylsulfinyl-methanide anion.
\nOther alkylating reagents are known (different X in R-X), also reacting in a nucleophilic substitution. For example, dimethyl sulfate can be used for alkylations. Alkylfluoromethyl-sulfonates are even more reactive than sulfates, and the reaction may take place with the active hydrogen even from alcohols or amines as follows:
Even tertiary amines, such as pyridine, also react with this type of reagent forming quaternary ammonium salts. The alkylation with alkylfluorosulfonates can be catalyzed as other alkylation reactions for increasing the reaction rate. A catalyst that can be used in this reaction is Hg(CN)2.
\nDiazomethane is another common alkylating (methylating) reagent. The alkylation using diazomethane is assumed to take place as follows:
Diazomethane is a gaseous unstable substance, which cannot be stored for long periods of time. It is usually prepared in small quantities and used immediately with or without an intermediate step of dissolution in ether. The preparation can be done from different N-nitroso-N-alkyl compounds in a reaction with a base. A common preparation uses N-nitroso-N-alkyl-p-toluenesulfonamide (Diazald). Methylation with diazomethane may require addition of a Lewis acid catalyst such as BF3. The methylation of partly acetylated sugars and amino sugars using diazomethane and BF3 in ether leads to the methylation of the free OH groups without the migration or substitution of the existent acyl groups.
\nA common alkylation of acidic analytes such as carboxylic acids, phenols, and thiols is performed using another type of alkylating reagent, namely, N,N-dimethylformamide dialkyl acetals. N,N-Dimethylformamide dimethyl acetal (Methyl-8®) is commonly used for methylations. For a compound containing a COOH group, the reaction with this reagent takes place as follows:
The compounds with acidic hydrogens can also be alkylated (methylated) using trimethyl orthoacetate, alkyl-p-tolyltriazenes (R─NH─N═N─C6H4─CH3), and O-alkyl isoureas are also used for the formation of analytes containing acidic hydrogens, imino esters, etc.
\nAlcohols can also act as alkylating reagents in particular when the analyte contains a more acidic hydrogen. Catalyst such as HCl, BF3, CF3 COOH or a cation exchange resin in H+ form is also frequently added to facilitate the reaction. The addition of HCl can be made as a water solution or as gaseous HCl that does not bring additional water to the reaction medium. The formation of alkyl or aryl derivatives of acids is a particularly important reaction known as esterification. Derivatization by esterification has been used with acids as the analyte and the alcohol as the reagent and also with the alcohol as the analyte and the acid the reagent. The esterification can be viewed either as the acid alkylation or as the acylation of the alcohol (see also the esterification mechanism). This reaction is typically catalyzed by strong acids and can be written as follows:
The mechanism of ester formation can be summarized by the following series of reactions:
The esterification efficiency can be improved by removing the water formed in this reaction. This can be done using a chemical reagent or distillation when the compounds of interest boil above 100°C. Among the materials able to eliminate water are desiccants such as anhydrous MgSO4, molecular sieves, or substances that react with water such as CaC2, (CH3)2C(OCH3)2 (2,2-dimethoxypropane), and even an appropriately chosen acid anhydride that reacts faster with water than with the reacting alcohol. The derivatization also may be performed in the presence of SOCl2 (thionyl chloride), which reacts with the water assisting in its removal, and when present in excess, may react with the alcohols forming alkyl chlorides or with the acids forming acyl chlorides. Chloride is a better leaving group in a nucleophilic alkylation reaction, and the efficiency of alkylation increases. Acids also can be esterified using a mixture of an alcohol and an acyl halide.
\nOne procedure for the formation of esters with less active organic acids applies the addition of dicyclohexylcarbodiimide (DCCI) in the derivatization process, to facilitate esterification. The reaction can be performed by adding to the acids that need to be analyzed the appropriate alcohol and DCCI usually in a solvent such as pyridine. Dicyclohexylurea, which is formed in the reaction, is not soluble in pyridine and can be separated. Besides DCCI, other carbodiimides can be used in the reaction of acids and alcohols. Among these are carbonyldiimidazole (CDI), 6-chloro-1-p-chlorobenzensulfonyloxybenzotriazole (CCBBT), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDAC), etc. Also, 2-chloro-1-methylpyridinium iodide, 2,4,6-triisopropylbenzenesulfonyl chloride, trialkyloxonium fluoroborate, etc. can be used to facilitate esterification.
\nTransesterification is another technique applicable for obtaining certain alkyl derivatives of acids (or acyl derivatives of alcohols). The reaction can be written as follows:
Transesterification can be catalyzed by acids (or Lewis acids) such as HCl, BF3, and H2SO4 or by bases such as CH3OK, CH3ONa, or C4H9ONa. The basic catalysts are commonly used for the methanolysis of triglycerides, followed by the analysis of the fatty acid methyl esters using GC or GC/MS [16].
\nA special alkylation can be achieved online during the heating in the injection port of a gas chromatograph using tertraalkylammonium hydroxides or alkylarylammonium hydroxides. Tetramethylammonium hydroxide (TMAH) is the most common reagents of this type. The reaction takes place as follows (Δ indicates heating):
Numerous other reactive compounds may be used for replacing active hydrogens in specific compounds. For example, epoxides, aziridines, and episulfides react easily with compounds with active hydrogens. Formation of a second group containing an active hydrogen may preclude the use of such reagents for analytical purposes.
\nBesides the desired derivatives, certain unexpected compounds that can be considered artifacts for the particular analysis can also be formed in alkylation reactions. The artifacts may be formed from unexpected interactions of the reagent with the analyte or may be a result of undesired effects of the catalysts or medium used for derivatization. In some cases, the control of the alkylation process may be difficult. Longer or shorter reaction times or intervals between derivatization and analysis may lead to errors, even when an internal standard is used for quantitation.
\nOne common case of artifact formation occurs during the reaction with compounds containing O-acyl or N-acyl groups, such as previously acylated carbohydrates, glycolipids, or glycoproteins, in particular when the reaction is done with short-chain alkyl bromides or iodides. When the OH groups of different sugars or NH2 groups of amino sugars were already protected with acyl groups, it was noted that, depending on the catalyst and the chosen medium, these acyl groups can be replaced by alkyl groups, or they may migrate from one position (such as C1) to other positions.
\nOxidation is another common side reaction when using Ag2O as a catalyst. The oxidation effect of Ag2O can be seen on free sugars as well as when attempting to permethylate peptides. Sulfhydryl groups are particularly sensitive to oxidation with Ag2O as a catalyst. The use of methylsulfinyl carbanion as a methylating reagent may also produce undesired side reactions with certain esters generating methylsulfinylketones. Also, strong alkylating reagents may produce undesired artifacts by unexpected alkylations.
\nThe derivatization with the purpose of obtaining aryl derivatives is similar in many respects to the alkylation reaction. The reaction takes place with compounds containing active hydrogens. Simple aryl halides are generally resistant to be attacked by nucleophiles and do not react similar to alkyl halides. This low reactivity can be significantly increased by changes in the structure of aryl halide or in the reaction conditions. The nucleophilic displacement can become very rapid when the aryl halide is substituted with electron attracting groups such as NO2.
\nSilylation is the chemical reaction of replacing a reactive hydrogen atom in OH, COOH, SH, NH, CONH, POH, SOH, or enolisable carbonyl with a silyl group, most frequently with trimethylsilyl (TMS). A large number of analytical methods involve silylation applied to alcohols including carbohydrates [17], phenols [18], amines, sterols [19], etc. The purpose of silylation in chromatography is mainly to reduce the polarity of the analyte, increase its stability, and improve the GC behavior. The differences in the mass spectra of the silylated compounds as compared to the initial analyte may also be an advantage for detectability. However, the mass spectra of many silylated compounds may not be available in common mass spectral libraries. Also, the silylated compounds plus the commonly present excess of silylating reagent may deteriorate some types of stationary phases such as that of Carbowax (polyethylene glycol)-type columns, and for this reason, their separation cannot be done on such columns.
\nSilylation can be performed on specific analytes or directly on complex samples such as a plant material (see, e.g., [12]). The silylating agent and the solvent can play the double role of extractant and silylating reagent. Many publications describe the use of silylation reactions for analytical purposes (e.g., [1, 5, 20]). The reaction of an analyte Y
The molecular weight for TMS is 73.047 calculated considering in the elemental composition of only the masses of the most abundant isotope. Numerous reagents have been synthesized to be used in silylations. Various aprotic solvents can be used as medium for silylation. The analysis can be focused on one analyte or on a mixture of analytes. The main factors contributing to the increase of the efficiency and the rate of the silylation reaction are the silyl donor ability of the reagent and the ease of silylation of different functional groups in the analyte. The solvent (or mixture of solvents) used as a medium and the compounds present or added in the silylation medium may also play a role for silylation efficiency. The reagent excess is sometimes important for displacing the equilibrium in the desired direction, and usually an excess up to ten times larger than stoichiometrically needed is used for silylation. Temperature also increases reaction rate, as expected, and heating of the sample with the reagents at temperatures around 70°C for 15 to 30 min is common. Some reagents used for trimethylsilylation are shown in Figure 8 [14].
\nSome reagents used for trimethylsilylation.
The approximate order of the increasing silyl donor ability for the reagents shown in Figure 8 is HMDS < TMCS < MSA < TMSA < TMSDEA < TMSDMA < MSTFA < BSA < BSTFA < TMSI. This order may be different on particular substrates where other reagents or reagent mixtures may be more reactive.
\nSilylation reagents can be used pure or in mixtures of two or even three reagents. The reagent mixtures may provide a more efficient silylation for specific compounds. For example, silylation of 3,4-dimethoxyphenylethylamine with BSA leads to the substitution of only one active hydrogen in the NH2 group, while the silylation with BSA in the presence of 5% TMCS produces silylation of both hydrogens in the NH2 [21]. A common silylating mixture is BSTFA with 1% TMCS.
\nOne of the determining factors regarding the silylation efficiency is the nature of the molecule Y
Several functional groups that can be silylated (listed in the approximate order of decreasing ease of silylation).
In general, the silylation of OH and COOH groups takes place with better results than that of NH2, CONH, or NH groups. Excellent results are obtained, for example, for the analysis of phenols after silylation [19]. A chromatogram of a solution containing a mixture of phenols at concentrations between 2.0 and 2.5 μg/mL in DMF, derivatized with BSTFA, separated on a BPX-5 chromatographic column (SGE Anal. Sci.), followed by MS analysis in single-ion monitoring (SIM) mode is shown in Figure 9. Details regarding the analyzed phenols are given in Table 3.
\nChromatogram of a set of phenol standards in DMF with the concentrations between 2.0 and 2.5 μg/mL derivatized with BSTFA, separated on a BPX-5 chromatographic column followed by MS analysis.
No. | \nCompound | \nRet. time | \nm/z | \nAbrrev. | \nNo. | \nCompound | \nRet. time | \nm/z | \nAbrrev. | \n
---|---|---|---|---|---|---|---|---|---|
(1) | \nPhenol | \n6.88 | \n166 | \nPh | \n(14) | \n3,4-Dimethylphenol | \n12.32 | \n194 | \n3,4-diMePh | \n
(2) | \no-Cresol | \n8.57 | \n180 | \no-Cr | \n(15) | \n3-Methoxyphenol | \n13.17 | \n196 | \n3-MeOPh | \n
(3) | \nm-Cresol | \n8.76 | \n180 | \nm-Cr | \n(16) | \n4-Methoxyphenol | \n13.47 | \n196 | \n4-MeOPh | \n
(4) | \np-Cresol | \n9.08 | \n180 | \np-Cr | \n(17) | \nCatechol | \n13.88 | \n254 | \nCa | \n
(5) | \n2-Ethylphenol | \n10.28 | \n194 | \n2-EtPh | \n(18) | \nResorcinol | \n16.05 | \n254 | \nRe | \n
(6) | \n2,5-Dimethylphenol | \n10.70 | \n194 | \n2,5-diMePh | \n(19) | \n4-Methylcatechol | \n16.27 | \n268 | \n4-MeCa | \n
(7) | \n3,5-Dimethylphenol | \n11.07 | \n194 | \n3,5-diMePh | \n(20) | \nHydroquinone | \n16.73 | \n254 | \nHy | \n
(8) | \n2,4-Dimethylphenol | \n11.20 | \n194 | \n2,4 diMePh | \n(21) | \n3-Methylcatechol | \n16.71 | \n268 | \n3-MeCa | \n
(9) | \n2-Methoxyphenol | \n11.28 | \n196 | \n2-MeOPh | \n(22) | \n5-Methylresorcinol | \n18.19 | \n268 | \n5-MeCa | \n
(10) | \n4-Ethylphenol | \n11.59 | \n194 | \n4-EtPh | \n(23) | \n2-Methylresorcinol | \n18.66 | \n268 | \n2-MeRe | \n
(11) | \n4-Chlorophenol | \n11.71 | \n185 | \n4-ClPh | \n(24) | \n4-Ethylresorcinol | \n19.90 | \n282 | \n4-EtRe | \n
(12) | \n2,6-Dimethylphenol | \n11.79 | \n194 | \n2,6-diMePh | \n(25) | \n2,5-Dimethylresorcinol | \n20.18 | \n282 | \n2,5-diMeRe | \n
(13) | \n2,3-Dimethylphenol | \n12.02 | \n194 | \n2,3-dimePh | \n\n | \n | \n | \n | \n |
Details regarding the analyzed phenols with the chromatogram shown in Figure 9.
Besides organic active hydrogens, several inorganic compounds with active hydrogens can also react with silylating reagents. Among these are H2O, H2O2, and strong inorganic acids. Also, some salts of the acids may be silylated. The reaction of silylating reagents with water imposes that water should be at the low level in the matrix or the solution of the analytes. The reaction with water takes place as follows:
In many solvents used as medium for derivatization, the trimethylsilanol formed in the reaction with water is separated as a distinct layer of solvent. The formation of two layers impedes a proper sampling of the derivatized material in the GC/MS instrument. In addition to that, the presence of an excess of water suppresses the derivatization of other compounds. The silylation is not recommended on samples with a water content higher than about 10%.
\nThe silylation reaction is commonly performed in a solvent that does not have active hydrogens. The most commonly used solvents as a medium for silylation are dimethylformamide (DMF), pyridine, and acetonitrile. The main role of the solvent is to dissolve the analyte and the reagents. The by-product HX of silylation shown in reaction (17) can be an acid, a base, or a neutral compound. As examples, for TMCS the by-product is HCl, for HMDS the by-product is NH3, for BSTFA the by-product is N-TMS-trifluoroacetamide, and for TMSI the by-product is imidazole. When the silylation reagent generates an acid as a by-product of the reaction, this may interfere with the silylation. For this reason, silylation can be promoted by any acid acceptor used as solvent or present in the solvent. Among such solvents are pyridine, triethylamine, and to a lower extent DMF. They can be used as both solvents and acid acceptors. Mixtures of solvents are commonly used for both enhancing solubility and promoting silylation. For example, formamide in the presence of pyridine may react with an acidic by-product generating CO and an ammonium salt. The addition of basic compounds to the silylation reaction may also influence the efficiency of the silylation. Also, some compounds may act as catalysts for silylation.
\nAlthough the TMS derivatives are by far the most commonly used in the derivatization for analytical purposes, other substituents in the silyl group can be used as reagents. Several such groups are indicated in Figure 10. The groups can be present in a variety of reagents connected to leaving groups “X-” such as Cl-, imidazolyl, F3C-(CO)-N(CH3)-, etc. For example, a common reagent containing
Examples of silyl groups different from TMS used in silylation reagents.
The use of different groups than TMS may serve different purposes. For example, a fluorinated or brominated group may enhance significantly the detection sensitivity when using ECD or NCI-MS. Also, the stability toward hydrolysis of compounds silylated with different groups than TMS may be higher, and such silylation can be advantageous. This is, for example, the case of
As an example, silylation of amino acids with MTBSTFA is commonly used [22, 23], and it is preferred to the silylation generating TMS derivatives. The chromatogram of a set of amino acid standards with the concentration of 0.05 μmol/mL derivatized with MTBSTFA and separated on a DB-5MS chromatographic column (from Agilent) followed by MS analysis is shown in Figure 11. Details regarding the analyzed amino acids are given in Table 4.
\nChromatogram of a set of amino acid standards with the concentration of 0.05 μmol/mL derivatized with MTBSTFA separated on a DB-5MS chromatographic column.
Peak No. | \nAmino acid | \nAbbrev. | \nMW | \nFormula + x TBDMS | \nMW + x TBDMS | \nCharact. ion | \nRet. time | \n
---|---|---|---|---|---|---|---|
(1) | \nα-Alanine | \nα-Ala | \n89.09 | \nC15H35NO2Si2 | \n317 | \n260 | \n31.69 | \n
(2) | \nGlycine | \nGly | \n75.07 | \nC14H33NO2Si2 | \n303 | \n246 | \n32.63 | \n
(3) | \nSarcosine | \nSar | \n89.09 | \nC15H35NO2Si2 | \n317 | \n260 | \n33.85 | \n
(4) | \nα-Amino-n-butyric acid | \nα-ABu | \n103.10 | \nC16H37NO2Si2 | \n331 | \n274 | \n34.36 | \n
(5) | \nβ-Alanine | \nβ-Ala | \n89.09 | \nC15H35NO2Si2 | \n317 | \n260 | \n35.58 | \n
(6) | \nUrea | \n\n | 60.06 | \nC13H32N2OSi2 | \n288 | \n231 | \n36.01 | \n
(7) | \nβ-Aminoisobutyric acid | \nβ-ABu | \n103.10 | \nC16H37NO2Si2 | \n331 | \n274 | \n36.11 | \n
(8) | \nValine | \nVal | \n117.15 | \nC17H39NO2Si2 | \n345 | \n186 | \n36.15 | \n
(9) | \nLeucine | \nLeu | \n131.17 | \nC18H41NO2Si2 | \n359 | \n200 | \n37.71 | \n
(10) | \nNorleucine | \n\n | 131.17 | \nC18H41NO2Si2 | \n359 | \n200 | \n38.8 | \n
(11) | \nIsoleucine | \niLeu | \n131.17 | \nC18H41NO2Si2 | \n359 | \n200 | \n38.8 | \n
(12) | \nγ-Aminobutyric acid | \nγ-ABu | \n103.10 | \nC16H37NO2Si2 | \n331 | \n274 | \n39.79 | \n
(13) | \nProline | \nPro | \n115.13 | \nC17H37NO2Si2 | \n343 | \n184 | \n39.87 | \n
(14) | \n2-Phenylglycine | \nPhGly | \n151.17 | \nC20H37NO2Si2 | \n379 | \n220 | \n46.16 | \n
(15) | \n5-Oxoproline | \noPro | \n129.13 | \nC17H35NO3Si2 | \n357 | \n300 | \n46.18 | \n
(16) | \nMethionine | \nMet | \n149.20 | \nC17H39NO2SSi2 | \n377 | \n320 | \n46.68 | \n
(17) | \nSerine | \nSer | \n105.09 | \nC21H49NO3Si3 | \n447 | \n390 | \n47.52 | \n
(18) | \nThreonine | \nThr | \n119.12 | \nC22H51NO3Si3 | \n461 | \n404 | \n48.43 | \n
(19) | \nPhenylalanine | \nPhe | \n165.19 | \nC21H39NO2Si2 | \n393 | \n336 | \n50.35 | \n
(20) | \nAspartic acid | \nAsp | \n133.10 | \nC22H49NO4Si3 | \n475 | \n418 | \n52.47 | \n
(21) | \nHydroxyproline | \nHyPro | \n131.13 | \nC23H51NO3Si3 | \n473 | \n314 | \n53.23 | \n
(22) | \n3-Methyl-L-histidine | \n3MeHys | \n169.20 | \nC19H39N3O2Si2 | \n397 | \n340 | \n55.15 | \n
(23) | \nGlutamic acid | \nGlu | \n147.13 | \nC23H51NO4Si3 | \n489 | \n432 | \n55.53 | \n
(24) | \nOrnithine | \nOrn | \n132.20 | \nC23H54N2O2Si3 | \n474 | \n286 | \n55.64 | \n
(25) | \n1-Methyl-L-histidine | \n1MeHys | \n169.20 | \nC19H39N3O2Si2 | \n397 | \n302 | \n57.03 | \n
(26) | \nLysine | \nLys | \n146.19 | \nC24H56N2O2Si3 | \n488 | \n300 | \n58.02 | \n
(27) | \nα-Aminoadipic acid | \n\n | 161.20 | \nC24H53NO4Si3 | \n503 | \n446 | \n58.06 | \n
(28) | \nHistidine | \nHys | \n155.16 | \nC24H51N3O2Si3 | \n497 | \n440 | \n62.29 | \n
(29) | \nTyrosine | \nTyr | \n181.19 | \nC27H53NO3Si3 | \n523 | \n302 | \n63.29 | \n
(30) | \nArginine | \nArg | \n174.20 | \nC24H56N4O2Si3 | \n516 | \n144 | \n64.26 | \n
(31) | \nTryptophan | \nTrp | \n204.22 | \nC29H54N2O2Si3 | \n546 | \n244 | \n67.98 | \n
(32) | \nCystine | \nCys | \n240.30 | \nC28H64N2O4S2Si4 | \n668 | \n348 | \n72.65 | \n
(33) | \nHomocystine | \nhCys | \n268.30 | \nC32H72N2O4S2Si4 | \n724 | \n362 | \n76.59 | \n
Details regarding the analyzed amino acids with the chromatogram shown in Figure 11.
In most situations, silylation generates only the desired derivatives. However, there are cases when the expected silylated compound is not formed, and either the silylation is not complete, or some compounds such as aldehydes, ketones, or esters with no obvious active hydrogen generate silylated compounds. Incomplete silylation is usually the result of inappropriate reaction conditions. However, when compounds with multiple functionalities are silylated, it is possible to generate a variety of derivatized compounds, regardless of the intention to obtain fully silylated or partly silylated compounds.
\nIn some cases, artifacts are formed due to the modification of the analyte under the influence of the reagents during derivatization. For example, when the silylation is done in basic or acidic conditions, the analytes that are sensitive to acidic or basic media may suffer unexpected transformations. The most frequent artifacts with compounds not containing obvious active hydrogens occur with aldehydes. Some aldehydes are able to undergo two types of chemical reactions with formation of OH groups, namely, enolization and acetal formation in the presence of water. The OH groups formed in this manner react with different silylating reagents and give the corresponding silylated products. Although the enolization or the acetal formation is negligible for the initial aldehyde, the reactions may be significantly displaced toward the formation of the silylated compounds of the enol or of the acetal. Artifacts can also be generated when the reaction is allowed to continue for an extended period of time. Other uncommon reactions with a specific silylation reagent and analyte may occur. An example of an uncommon reaction is the ring opening of flavanones.
\nThe formation of acyl derivatives is applied for replacing the active hydrogens from an analyte in functionalities such as OH, SH, NH [11, 24], CONH, etc. The acylation is also used for reducing polarity and improving the behavior of the analytes in the chromatographic column. Acylation may confer a better volatility of the analytes, although not as marked as for silylation or methylation. Only the derivatization with acetyl groups or with fluorinated acyl groups (not heavier than heptafluorobutyryl) improves volatility, while other heavier acyl groups are not suitable for this purpose. Acetylation, for example, can be used for compounds such as monosaccharides and amino acids to allow GC analysis. The detectability improvement on the other hand is a very common purpose for acylation. Acylation with fluorinated compounds is frequently used for enhancing detectability in GC with ECD or NCI-MS detection. Other uses of acylation include the enhancement of separation of chiral compounds, etc.
\nMost acylation reactions are nucleophilic substitutions where the analyte is a nucleophile (Y
Some common acyl groups present in acylation reagents are indicated in Table 5.
\nSome common groups present in acylating reagents used in derivatizations for GC analysis [14].
As shown in Table 5, the acyl groups in the reagent can be attached to various “X” groups. One such group is OH and among the acylating reagents are some free acids. When nucleophile is an alcohol, the reaction is known as esterification and has been discussed in Section 7. The acylation with acids can be applied besides alcohols to certain thiols, phenols, amines, etc. and can be written as follows:
The reaction can be displaced toward the formation of the acyl derivatives by eliminating the water using compounds such as anhydrous MgSO4, molecular sieve, or substances that react with water such as CaC2, or (CH3)2C(OCH3)2. Dicyclohexylcarbodiimide (DCCI) also is used for modifying the yield of the desired product. The reaction with reagents containing a carboxylic acid reactive group also can be done in the presence of 2,4,6-trichlorobenzoyl chloride or with various sulfonyl chlorides such as 2,4,6-triisopropyl-benzenesulfonyl chloride or 2,4,6-trimethyl-benzenesulfonyl chloride. The reaction of amines with acids can be displaced toward the formation of the amides using a peptide coupling reagent such as benzotriazol-1-yl-oxy-tris(dimethyl-amino)-phosphonium hexafluorophosphate (BOP), diethyl cyanophosphonate, O-benzotriazol-1-yl-N,N,N′,N′-bis(tetramethylene)uronium hexafluorophosphate, 2,2′-dipyridyl disulfide + triphenylphosphine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDAC), etc.
\nCommon acylating reagents are acyl halides such as chlorides or bromides, which are reactive compounds suitable for acylation. The reaction of an acyl chloride with an amine, for example, takes place as follows:
Since the reactivity of amides is lower than that of amines, the second hydrogen in the amine is more difficult to replace. Also, steric hindrance may negatively influence the reaction. The generation of a strong acid such as HCl is a disadvantage in the reaction with acyl halides, and usually the acid should be removed either by adding basic compounds such as Na2CO3 or MgCO3 or using pyridine as the reaction medium. The high reactivity of acyl halides is used for the acylation of compounds with less reactive hydrogens. Certain carbonyl cyanides react similarly to acyl chlorides.
\nThe disadvantage of generating a strong inorganic acid in the acylation with acyl halides also can be avoided by having, instead of the acyl halide, an anhydride. The reaction of Y
The acid resulting together with the acylated compound is not a strong acid such as HCl. The anhydrides of trifluoroacetic acid (TFA), pentafluoropropionic anhydride (PFPA), and heptafluorobutyric (HFBA) acids are commonly used for derivatization of alcohols, phenols, amines, etc., with the purpose of enhancing detectability (by ECD or NCI-MS) and also for improving the chromatographic behavior (higher volatility, better thermal stability, better separation). The volatility of fluorinated compounds allows the GC applications. The reactivity of the perfluorinated anhydrides increases in the order HFBA < PFPA < TFA. However, the differences are not significant. Once formed, the heptafluorobutyrates are more stable to hydrolysis than the trifluoroacetates. An inert solvent such as CH2Cl2, ether, ethyl acetate, acetone, tetrahydrofuran or in CH3CN, etc. can be used as a medium for the reaction with perfluoroanhydrides. For the neutralization of the acids formed during derivatization, the basic compounds such as triethylamine, pyridine, or even solid NaHCO3 can be utilized.
\nIn order to avoid the formation of water or of a strong acid in the acylation reaction, certain amides such as N-methyl-bis(trifluoroacetamide), bis(trifluoroacetamide), or 2,2,2-trifluoro-N-methyl-N-(2,2,2-trifluoroacetyl)acetamide (MBTFA) can be used as reagents. Acylation of amines takes place at room temperature. Solvents such as CH3CN, pyridine, DMSO, or THF can be used as a reaction medium:
One other procedure successfully applied to obtain acyl derivatives is the use of acyl imidazoles as reagents. This class of compounds reacts with analytes containing alcohol, primary and secondary amino groups, or thiols. The reaction generates as a by-product imidazole:
Succinimidyl esters also can be used for acylation purposes. Amines and the amino group in amino acids also can be acylated using urethane-protected α-amino acid-N-carboxyanhydrides or oxycarbonyl-amino acid-N-carboxyanhydrides. Alkylketenes and their dimers may be used for acylation.
\nA special type of acylation is that using chloroformates. Carbonic acid, O═C(OH)2, can form amides, esters, halides, etc., due to the presence of two OH groups bonded to the CO group. Carbonic acid ester halides, also called chloroformates or chloroformate esters, with the formula R─O─C(═O)─X, where R is an alkyl or aryl group and X is F, Cl, Br, or I, can react with various compounds containing active hydrogens, such as acids [25], amines, alcohols, thiols, and amino acids. Amino acids, for example, in the presence of an alcohol in water form carbamate esters (urethanes) reacting as follows [26]:
The formation in reaction (25) of the alcohol Ra–OH may lead to traces of a resulting compound with both substituted radicals being Ra. For this reason it is typically recommended to perform the reaction in the presence of an alcohol having the same radical as the chloroformate reagent (Ra = Rb). Chloroformates containing in the alkyl or aryl group halogen substituents are particularly reactive. Even tertiary amines can react with specific chloroformates, such as pentafluorobenzoyl chloroformate or with trichloroethyl chloroformate, by displacing an alkyl group connected to the nitrogen atom and forming the carbamate ester.
\nSimilar in many respects to that of acyl derivatives R–CO–X are the reactions of sulfonyl derivatives R–SO2–X. Sulfonyl halides are in general less reactive than halides of carboxylic acids. The reaction of a sulfonyl derivative may take place with alcohols, phenols, amines, etc. The reactivity toward the sulfonyl sulfur is RNH2 > CH3COOR > H2O > ROH.
\nHigh reactivity toward active hydrogens in alcohols, amines, etc. can also be achieved using reagents with other functionalities. These functionalities include isocyanates, isothiocyanates, carbonyl azides, etc. These reactions can be seen as a replacement of an active hydrogen with a CO-R group or CS-R group as it occurs in other acylations.
\nA variety of other derivatization reactions are reported in the literature (see, e.g., [1]) and used for GC and GC/MS analyses. Among these are the addition to hetero multiple bonds in functional groups such as C═O, C═S, C═N, or C☰N. Many such reactions are additions to multiple bonds. Such reactions are, for example, the additions to the C═O groups in aldehydes and ketones. Reagents containing active hydrogens in groups such as NH2, OH, H2N-NH-, etc. can react, for example, with aldehydes and ketones. Alcohols, for example, form hemiacetals or acetals with aldehydes and ketals with ketones, and although most of such compounds are not stable enough to be suitable for derivatization, cyclic acetals and ketals may be stable and used for analytical purposes. A common reaction of carbonyl compounds is with amines. The initial addition reaction usually continues with water elimination forming a substituted imine or a Schiff base. Similar to the reaction of amines is the reaction with substituted hydroxylamines or hydrazines. A typical reaction of derivatization of carbonyl compounds is that using dinitrophenylhydrazine (DNPH). The derivatized compound can be analyzed either by LC [27] or by GC/MS [28]. The reaction takes place as follows:
The groups Ra and Rb can be H or alkyl or various other substituents.
\nAnother reagent that can be used for ketone derivatization is N-aminopiperidine in the presence of catalytic amounts of acetic acid. The resulting substituted hydrazone can be used in GC analysis:
β-Diketones may react differently with hydrazines generating pyrazole derivatives as shown below:
Several other classes of compounds similar to hydrazines react with the carbonyl compounds. Among these are hydrazones (NH2─N═CR2), hydrazides (NH2NH-COR), and semicarbazide (NH2NH-CONH2). Hydroxylamines also react with carbonyl compounds forming oximes. Hydroxylamine itself, hydroxylamine hydrochloride (STOX® reagent), or derivatives such as H2N-OSO3H in a solvent like pyridine can be used in this reaction:
When the reaction is performed with hydroxylamine, the generated oxime contains an active hydrogen. This can be further derivatized, for example, by silylation in a reaction with a common silylation reagent.
\nFor derivatization purposes other reagents can be used, such as substituted hydroxylamines like methoxyamine hydrochloride NH2OCH3•HCl (MOX® reagent) and O-(pentafluorobenzyl)-hydroxylamine hydrochloride (FLOROX® reagent). The reaction of a ketone or aldehyde with FLOROX is shown below:
The oximes existing in
The transformation of the oximes into nitriles generates one single compound from the two (syn- and anti-) isomers and can be used to simplify the chromatograms of sugars derivatized as oximes.
\nAlcohols, amines, and thiols also can react at other hetero multiple bonds leading to analytical applications. This addition may occur at the isocyanates (─N═C═O), ─C═O group in an amide, at a nitrile, at CS2, or at other groups. One example is the addition under special conditions of alcohols to dimethylformamide. The resulting acetals are very reactive and are used themselves as reagents, as shown previously for N,N-dimethylformamide dimethyl acetal (see reaction 12). Another example is the reaction of CS2 with alcohols in the presence of a base, leading to the formation of xanthates. Amines also react with CS2, and the formed isothiocyanate can be analyzed using GC analysis. The reaction takes place as follows:
Formation of new cycles from noncyclic compounds or replacement of old cycles with new ones that are more stable or have a desired property is also exploited in sample processing using derivatization. Epoxides, for example, can be formed in the reaction of a compound with a carbon–carbon double bond and a peroxy acid. Among the peroxy acids more frequently used for the formation of epoxides are peracetic, performic, perbenzoic, trifluoroperacetic, and 3,5-dinitroperoxybenzoic acids. However, in this reaction a mixture of enantiomers is formed, as shown below for a
The separation of the epoxides may be easier to achieve than that of olefins, and this type of derivatization has been utilized, for example, for better separation of various
Another reaction with formation of new cycles is that of amino acids with phenyl isothiocyanate leading to a thiohydantoin derivative:
This reaction has been successfully used for the analysis of amino acids in proteins [29, 30].
A variety of aromatic cycles can be formed in reactions involving bifunctional compounds. Addition reactions to hetero multiple bonds in bifunctional molecules frequently lead to cyclic compounds. For example, formaldehyde can react with tryptophan or tryptamine generating a β-carboline derivative as follows:
The new compound can be analyzed by GC, usually after further derivatization by silylation of the carboxyl group.
\nA typical reaction leading to pyrazoles is the reaction of hydrazines with diketones such as 2,4-pentandione (acetylacetone). For example, the reaction between hydrazine or methylhydrazine and acetylacetone takes place as follows:
Activated carbonyl groups such as those in hexafluoroacetone are known to react with difunctional compounds. The reaction may take place with an amino acid as follows:
Amino acids can react with an activated anhydride such as trifluoroacetic anhydride (TFAA):
The reaction takes place by heating the amino acids with an excess of TFAA. The reaction mixture is then dissolved in ethyl acetate and analyzed by GC.
\nNumerous other types of derivatization reactions were used for making the analytes suitable for GC and GC/MS analyses. These include formation of various cyclic types of compounds such as azines, siliconides, boronates, etc., that are thermally stable and do not have polar hydrogens such that GC or GC/MS analysis is possible. In addition to reagents that add specific moieties to the analytes, oxidation and reduction were sometimes used for the analyte modification (see, e.g., [4]).
\nSolid-phase reagents are polymeric materials with specific groups that are reactive and can be transferred to the analyte molecule producing derivatization. For an analyte of the form Y
Solid-phase reagents must work analogously to the corresponding small-molecule reagents containing the group R (a tag). Reagents that are insoluble in certain solvents at high concentrations can often provide a high ratio of analyte/substrate in a polymeric microenvironment that yields a high kinetic rate for the heterogeneous reaction.
\nA variety of materials can be used as solid support, such as specifically bound reagents on a silica support (used, e.g., for online derivatization in HPLC analysis), ion exchange resins, as well as other supports [31]. One example of solid-phase support that can produce derivatization is trifluoroacetyl nylon 6,6. This solid-phase reagent can be obtained from poly(hexamethylene adipamide) (nylon 6,6) and trifluoroacetyl anhydride. This solid-phase reagent can be used in amine derivatization in a reaction as follows:
This derivatization of the amine is done by mixing the solid-phase reagent with a solution of amine solution in CH3CN. Following derivatization, the solid-phase reagent is separated by centrifugation, and the solution is concentrated by evaporating part of the solvent and analyzed by GC (an amine internal standard must be used in this procedure). However, some such derivatizations require a long time of interaction between the solid-phase reagent and the analytes and found only limited applications.
\n(Another) alternative of derivatization of specific analytes is using the reaction between the reagent and the analyte both adsorbed on a solid support. This type of derivatization has been used, for example, in connection with a solid-phase microextraction (SPME) technique [32]. In this technique a reagent is initially adsorbed in the SPME fiber, followed by exposure to the analytes. The derivatized analytes are further desorbed in the injection port of the GC and analyzed using a detector such as MS. For example, formaldehyde from air can be analyzed using a polydimethylsiloxane (PDMS) fiber containing
Human beings in the global ecosystem are no longer consumers, but wasters. To achieve “Transforming our World: the 2030 Agenda for Sustainable Development (2030 Agenda)” and set the Sustainable Development Goals (SDGs) consisting of 17 global goals in 2015 [1], human beings need to become former consumers and stay within the energy and matter cycles of the global ecosystems. One of the examples is the sixth goal of “Clean Water and Sanitation” in the 17 SDGs. The cause of anthropogenic eutrophication and subsequent organic pollution is the imbalance in the ecological matter cycle that occurs in human social life.
In Japan, the anthropogenic eutrophication and the organic pollution in 1960’ had simultaneously been caused by the direct influx of wastewaters from industries and households (Figure 1a) [2]. On the other hand, most of such eutrophication in recent years is induced by the anthropogenic influx of nutrient salts, such as phosphate and nitrate salts, which are the essential nutrients of living organisms (Figure 1b) [3]. It is known that orthophosphate ion, that is, phosphate ion (Pi) is the most causative nutrient. These nutrient salts are contained in the effluent of sewage treatment plants or industrial wastewater treatment plants, or in leaching water from farms or live stocks [3, 4]. In the cases of these plants, only an ecological decomposer is employed for the biodegradation of organic matters contained in the wastewater (Figure 1b). This is the root cause of current eutrophication and subsequent organic pollution. Therefore, such anthropogenic influx of nutrient salts causes the water bloom by phytoplankton as an ecological producer, and the remains of phytoplankton cause organic pollution. To investigate the ecological phenomenon of such anthropogenic eutrophication and subsequent organic pollution, we observed the water ecosystem and water qualities of a eutrophied pond as a model water body [5]. The results led to a study on hydroponics [6] and water chemical remediation (WCR) that was developed for simultaneous removal of Pi and phytoplankton from the anthropogenically eutrophied pond [7]. A series of the studies will be described elsewhere.
(a) Schematic diagram in comparison on the causes of organic pollution in the 1960s and the recent past [
In our studies, we have also developed measurement methods for these water pollutions. As one of the ways to measure the degrees of water pollution, several biosensors have been developed. The biosensor consists of a molecular recognition element and a transducer and has the features of a simple and rapid measurement device (Figure 2a). The first biosensor was studied for medical use by Updike and Hicks in 1967 and developed to determine glucose concentration employing an enzyme-catalyzed reaction (enzymatic reaction) by glucose oxidase (GOD) [12]. This enzymatic glucose biosensor used a Clark-type electrode [13] and measured dissolved oxygen (DO) consumption caused by the GOD reaction. Since then, biosensors as measurement devices have been developed not only for medical uses [14] but also for food or environmental uses [10]. Thus, many biosensors and their associated techniques have been studied and developed [10]. In particular, the biosensor for environmental use requires highly sensitive and wide-determination range (dynamic range) measurement of the analyte. In addition, depending on what is being analyzed, the environmental biosensors require the feature to perform either specific or nonspecific measurements. Furthermore, for practical use, it is also required the application style of the biosensor, such as on-site use, continuous use, or laboratory use.
Biosensor. (a) Principles and (b) automatic Pi biosensor instruments of a desktop-type [
To satisfy these requirements, many kinds of biosensors for environmental water pollution have been developed worldwide [3, 10, 15]. In the second section of this chapter, the development of enzymatic Pi biosensors for eutrophication and in the third section, the development of microbial biochemical oxygen demand (BOD) biosensors for eutrophication are described. In the final fourth section, the contents of this chapter are concluded.
In the early times of biosensor development for environmental use, there were many technological problems for practical use. To solve the problems, a wide variety of challenges have been carried out. In this section, the main breakthrough technics for the practical application in the field of biosensor development for eutrophication are described based on the previous literature [3, 10, 14, 15].
As mentioned above, the most causative nutrients of the eutrophication are phosphates, which are classified into Pi, condensed phosphates (pyro-,
In Japan, a spectrophotometric molybdenum blue method for Pi determination is employed as the standard method [16], which is based on the method by Lowry and Lopez in 1946 [17]. By this method, Pi concentration can be determined between 0.1 and 3.0 mg/L Pi (3.2–96 μM) at a relative standard deviation (RSD) of 2–10%. The detection limit in this method is 0.03 mg/L Pi (0.32 μM Pi).
These values obtained by the standard method are barely applicable as an indication of the eutrophication (0.64 μM Pi), however, they are insufficient for the estimation of the eutrophication, which is classified into the five categories in lakes (between around 0.032–3.2 μM Pi). In addition, the maximal permissible concentration in Japanese lakes is 0.32 μM Pi for drinking and 3.2 μM Pi for environmental protection [18]. Thus, the influences of Pi on eutrophication are caused at extremely low concentrations. Therefore, it was found that the estimation of the eutrophication requires highly sensitive and wide-range Pi determination techniques.
For ideal estimation of the eutrophication, the standard method has difficulties in both accurate and high-precision measurements. In accurate measurement, dilution of sample solution needs due to that the dynamic range is narrow and causes error. In high-precision measurement, the minimum limit of determination (3.2 μM Pi) is insufficient to estimate the water quality in all categories of eutrophication.
To make ideal estimations for all categories of the eutrophication, a measurement method that was more sensitive and had a wider dynamic range (if it is possible; 0.032–3.2 μM Pi) than the standard methods was needed. However, considering the biosensor performance at that time, such high sensitivity was not realistic. Even if such highly sensitive Pi measurements cannot be made, we should be able to determine 0.32 μM Pi with a biosensor at a concentration that can confirm signs of eutrophication and monitor the quality of the lake water for drinking. In addition, we considered that the maximum limit of Pi determination obtained by the biosensor should be kept at least 3.2 μM or more as the practical dynamic range.
Like the other problems, the standard method is complicated and time-consuming to operate. Further, it requires the use of strong acid and heavy metal ions. The use of such chemicals is subject to limitations when applied to on-site monitoring and continuous monitoring because leakage of the chemicals into the environment has to be prevented. In addition, the standard method affects the influences of co-existing matters in a sample, such as Ca2+, Fe3+, NH4+, NO2−, NO3−, and AsO43−.
As is clear here, the standard method is not sufficient to evaluate eutrophication. For this reason, the development of practical Pi biosensors has been performed.
It turns out that there were many issues that need to be resolved to actually estimate the eutrophication. Thus, we have set five requirements for the practical application of the Pi biosensors [19].
For practical use, our Pi biosensors are able to;
Have a practical Pi determination range (0.32–3.2 μM, no sample dilution required) required for environmental water control,
Maintain a practical minimum limit of Pi determination (0.32 μM Pi or less in the calibration curve) required for environmental water control for at least 2 weeks,
Establish a pretreatment method for removing interfering matters,
Measure a real sample of environmental water, and
Automate a biosensor system that meets the above conditions.
We tried to develop practical Pi biosensors that meet these requirements (Table 1). As a reference, the history of the studies on the Pi biosensor development has been reviewed in several articles [3, 10, 15].
Instrument type | Submersible buoy1 | Desktop | |||
---|---|---|---|---|---|
Enzymatic system | PO | PO | MP | MP2 | IP (PPi) |
Ref. No. | [9] | [8] | [20] | [11] | [21] |
Requirement | |||||
(1) Calibration: | Satisfied | Satisfied | Satisfied | Satisfied | Satisfied at PPi |
Range (at least 0.32–3.2 μM) | 0.16–32 μM | 96 nM–32 μM | 10 nM–32 μM | 0.1–30 μM | 0.1–100 μM (PPi) |
Coefficient | |||||
(2) Sability test: | Satisfied | Satisfied | Not performed | Satisfied? | Not performed |
as the term to maintain the practical lower limit of Pi determination in the calibration curve (0_32 μM Pi or less | Calibration curves of 0.32–32 μM for 48 days | Calibration curves of 0.16–32 μM for at least 2 weeks | 1.0 μM for at least 2 weeks | 0.1 μM for at least 2 months | (30 μM PPi for at least 2 weeks) |
(3) Sample pretreatment: | |||||
as countermeasures against interfering matters | Not performed | Performed | Performed | Performed and established the method | Not performed |
(4) Real sample application: | Performed | Performed | Performed | Performed | Not performed |
(5) Automation: | Semi-automated | Automated | Not automated | Not automated | Automated |
Looking back on the five requirements that were set for the practical use of our automated CL-FIA systems as Pi biosensors (performed by Nakamura et al.).
Tests with the Pi biosensor on a submersible buoy have not been conducted.
Pre-treatment method was investigated in detail for real sample application.
A biosensor has characteristics that it can measure analyte using biological reactions by applying the molecular recognizing function in living organisms (Figure 2a). A Pi biosensor can measure directly the concentration of the bioavailable Pi existing in the environmental water. This means that the Pi biosensors have the possibility to be able to monitor the status of the water ecosystem. This section briefly explains the history of the development of Pi biosensors with such potential, which is above described [3].
The first Pi biosensor was studied by Guilbalt and Nanjo in 1975. Using the Clark-type DO electrode, they studied an enzymatic Pi biosensor that was based on the inhibition of alkaline phosphatase activity by Pi [22]. By employing the inhibitory reaction, this biosensor lacked sensitivity with the detection limit of 0.1 mM Pi. In addition, the inhibitory reaction is low selectivity in general. In 1990, d’Urso and Coulet studied a two-enzyme Pi biosensor using nucleoside phosphatase and xanthine oxidase [23]. Enzymatically generated hydrogen peroxide (H2O2) by the existence of Pi was electrochemically measured. In this study, the Pi biosensor could have a dynamic range between 0.1 and 10 μM Pi. In 1992, Wollenberger et al. improved the two-enzyme system to a multiple-enzyme system for Pi recycling [24]. The Pi biosensor was fabricated by incorporating a Clark-type DO electrode into a flow injection analysis (FIA) system. As the result, this Pi biosensor realized an excellent detection limit at 25 nM Pi. However, this biosensor was not suitable for practical use due to its short lifetimes by using an unstable enzyme. As another reason, it was reported that inosine used as another substrate was also unstable [25].
Karube et al. have studied several kinds of enzymatic Pi biosensors using pyruvate oxidase (PO) from
The Pi biosensor using PO is superior to other Pi biosensors because it requires only one step of a catalytic reaction for selective Pi detection. Using PO
In 1996, Ikebukuro et al. examined the combination of a luminol chemiluminescence (CL) reaction and a FIA system for enzymatic Pi biosensor (CL-FIA system) [27]. At that time, the CL reaction was known as a highly sensitive reaction, and the FIA system was also known as a highly repeatable measurement system [30, 31]. The reason why the latter repeatability contributes to high-precision analysis was that the closer the multiple measurement results obtained from the same standard solution were to their average values, the smaller the standard deviation value. Along with this, by increasing the significant difference between the standard measurement value and the blank value, the standard measurement value closer to the blank value could be set as the detection limit. As a result, by combining CL and FIA technics, it was possible to realize a high-precision analytical method due to the synergistic effect of both. This meant that the CL-FIA system can turn the enzymatic Pi biosensors into highly sensitive analytical instruments. Thus, the CL-FIA biosensor systems have been widely studied [32].
In the PO
In 1997, Nakamura et al. used highly sensitive luminol catalyzing peroxidase from
In 1999, Nakamura et al. reexamined the development of the CL-FIA system for an enzymatic Pi biosensor using a new enzyme, PO
On the other hand, in 1995, Conrath et al. studied a new electrochemical Pi biosensor using an analyte recycling system consisting of four enzymes, maltose phosphorylase (MP), acid phosphatase (AcP), mutarotase (MUT), and GOD [33]. The four enzymatic system enabled the successful detection of 10 nM Pi. However, the system was too complicated, and we thought it would be difficult to reproduce in manufacturing as well as other multiple-enzyme systems [24]. In 1999, Nakamura et al. modified the MP system and applied it to our CL-FIA biosensor system using a tri-enzymatic reaction of MP-MUT-GOD without the analyte recycling by AcP [20]. Then, we could obtain the same results with Conrath et al. at a detection limit of 10 nM Pi. In addition, an excellent calibration between 10 nM and 30 μM Pi was obtained and stability to detect 1.0 μM Pi was observed for at least 2 weeks.
In 2003, Nakamura et al. improved the MP-MUT-GOD system for freshwater measurements [11]. Our previous studies revealed that the Pi biosensors employing the CL-FIA system were affected by the cations contained in the real sample solutions. Then, we have tried to examine several pretreatment methods to remove the cations from the sample [8, 11, 20]. As the result, we could find and establish a pretreatment method using a cation-exchanging resin. A total of 31 samples of freshwaters were taken from the river and pond. These real samples were pretreated by our method and measured for Pi determination by both this Pi biosensor and the conventional molybdenum-blue method. The results showed that the value from the conventional method was 2.78 times higher than that from the Pi biosensor. One reason for this outcome was considered that there were large differences in the reacting conditions between the Pi biosensor (natural pH) and the conventional method (under strong acid conditions). Therefore, an enzymatic Pi biosensor may determine the more accurate and realistic Pi concentration as free and bioavailable Pi, which is needed to understand the water ecosystem.
Furthermore, Nakamura et al. studied a pyrophosphate ion (PPi) biosensor in 2004 [21]. As well as the Pi concentration, the PPi concentration is also an indicator of the eutrophication and the organic pollution. For the enzymatic PPi biosensor, inorganic pyrophosphatase (IP) was added to the PO
Here, an author looks back on the five requirements that were set for the practical use of our Pi biosensors [8, 9, 11, 20, 21]. In the first requirement, the CL-FIA systems were successfully applied to the Pi biosensors and made it possible to highly sensitive and practical Pi measurements [8, 9, 11, 20, 21]. In the second requirement, our Pi biosensor was able to continue to make practical calibration curves as the stability tests and demonstrate the practicability [8, 9]. In the third requirement, the sample pretreatment method was finally established by the countermeasures against interfering matters [11]. In the fourth requirement, the establishment of the sample pretreatment method was enabled to perform real sample applications [11]. In the fifth requirement, the automation of the Pi and PPi biosensor systems was realized by employing the CL-FIA systems [8, 9, 21].
As described above, we studied several types of Pi biosensors for the estimation of eutrophication, and finally, two trial Pi biosensor instruments of the desktop and the submersible buoy were developed. Although thorough examinations using the real samples were necessarily employing the automatic Pi biosensor instrument for practical use and commercialization, various factors made it impossible to conduct the study (laboratory relocation, running costs, stop production of enzymes, etc.). In conclusion of our study for practical use of the enzymatic Pi biosensors, the highest risk was the stop production of the enzymes. As the general concerns, the development and subsequent practical use of the enzymatic biosensor tend to be expensive running costs for the enzyme. In particular, the FIA system consumes a lot of reagents and our CL-FIA system needs to contain the enzyme (HRP or ARP) in the CL reagent.
Subsequent development of the Pi biosensors by other groups was introduced here. In 2005, Kwan et al. studied a screen-printed electrochemical Pi biosensor using PO. The enzymatic Pi biosensor had a linear calibration range from 75 to 625 μM Pi [35]. The PO reaction needs TPP, which releases Pi. For practical use, an investigation of the storage condition of TPP would be needed. In 2001, Mousty et al. studied an electrochemical tri-enzymatic Pi biosensor employing an MP-MUT-GOD reaction system [36]. This system had a linear calibration range from 1 to 50 μM and was stable for at least 2 weeks. In 1998, Fernandez et al. studied another type of electrochemical tri-enzymatic Pi biosensor [37]. The three enzymes, a substrate, a cofactor, and a mediator, were incorporated into hydrogels. The enzymatic Pi biosensor had a detection limit of 2 mM Pi. Quite complicated principles and unstable enzymes would be a problem for practical use. These enzymatic Pi biosensors had insufficient sensitivity for actual eutrophication. For practical use, the influences of reducing matters in a sample solution on the biosensor response cannot be ignored. In 2013, Lawal and Adeloju studied measures against reducing matters in electrochemical Pi biosensors [38]. For this aim, they used conductive polymer of polypyrrole to the electrochemical bi-enzymatic Pi biosensors and could not observe the obvious influences of uric and ascorbic acids on both amperometry and potentiometric methods. However, the sensitivity of the Pi biosensor was insufficient for natural waters, therefore, the effects of the polypyrrole on the Pi measurement might be unclear. In 2020, Korkut et al. also used the polypyrrole to a PO-Pi biosensor and successfully performed accurate Pi determination of eutrophied water at 91% [39].
The enzymatic Pi biosensors have been developed not only for environmental use but also for medical use (urine Pi) [40, 41] and food use [42]. In the case of food use, in 2020, He and Liu successfully developed a highly sensitive PO-Pi biosensor employing “Nano-Enabled Biosensing” techniques that combined with gold nanorods (AuNRs) as nanomaterials and conductive materials. The PO-Pi biosensor was able to detect 0.4 nM Pi, which is sufficient to estimate eutrophication. On the other hand, these conductive materials have the potential to perform well in both food and medical applications, where samples are high in reducing matters [38, 39, 42].
On the other hand, studies using molecular recognition elements other than enzymes have also been carried out. The molecular recognition elements can be categorized into two types. One is bacterial phosphate-binding proteins (PBPs) and another is an ionophore that is made of lipid or polymer. The PBP is a component of a phosphate transport system and a highly selective recognition element of Pi (
In 2002, Kubo et al. studied an electrochemical PBP Pi biosensor. They extracted the PBP from
In the cases applying the ionophores to the Pi biosensors, Carey and Riggan in 1994 applied cyclic polyamine to an ion-selective electrode (ISE) for Pi [49]. This Pi sensor specifically detected dibasic phosphate (HPO42−) and obtained a linear response between 1.0 μM and 0.1 M Pi. For the real sample application, the influences of sample pH have to be concerned. The ionophores from microbes were used for the Pi biosensors [50, 51]. As an example, the Pi biosensor studied by Wygladacz et al. obtained a linear calibration range from 1.0 μM to 2.5 mM Pi. However, the lifetime was the order of days [51].
Other principles for the Pi biosensors were also studied. In 2001, Schreiter et al. studied a Pi bioavailability assay employing a luminescent cyanobacterial reporter strain for the replacement of the conventional AGP test. The method enabled the highly sensitive detection of Pi from 0.3 to 8 μM, although it took 8 hours of incubation [52]. In 2003, Dollard and Billard studied a
In this section, the author explained the representative studies on the development of Pi biosensors and Pi bioavailability assays for eutrophication. The biomaterials, such as enzymes, PBPs, and ionophores, were used for the Pi biosensors. These biomaterials need purification from living organisms. Therefore, the issue of cost and risk of production outage will be a challenge to the practical application of the Pi biosensors in this field. Further, in 2021, Becker et al. calculated the complete environmental factor (E-factor) of the enzyme [54]. The complete E-factor, including required waste and water, was calculated as 37,835 g-waste/g-enzyme. Therefore, the use of such biomaterials will require environmental consideration and not be neglected for the sustainability assessment of bioprocesses in the future. Furthermore, new technologies will be introduced one after another in this field, such as smartphones, 3D printers, nanomaterials, miniaturization, and automation.
To estimate the degree of organic pollution, several indicators of total organic carbon (TOC), COD, BOD, and DO have been employed. In general, COD is employed for closed water bodies of both natural water and seawater, and BOD is employed for flowing water, such as rivers. The differences in the usage of COD and BOD estimation methods are simply determined by the presence or absence of the flux, which depends on the decomposition rate of organic matters dissolved in the water body by aerobes, although they are often employed as the references in each field.
In the estimation methods for organic pollution, only BOD involves the results obtained by a biological reaction. The conventional standard method of the BOD estimation is referred to as the 5-day BOD (BOD5) method [55, 56]. It requires 5 days of incubation to obtain the results. The BOD value (mg O2/L) is calculated from the amount of DO consumed by the aerobic decomposition of organic matters during incubation (primary fermentation). From this principle, BOD is also called biochemical oxygen consumption.
At the peak of organic pollution in advanced countries in the 1960s, the standard method of BOD was limited to the BOD5 method [55, 56]. The BOD5 method has several problems that prevent it from satisfying the needs for practical use in wastewater control, that is, this method is time-consuming and requires tedious operations. Therefore, a method that can be used to monitor the BOD value in real-time or continuous was urgently needed.
In 1960’, there were many issues that need to be resolved to actually estimate the BOD. To solve these problems, many kinds of BOD biosensors have been studied and developed for practical use [57].
As described above, the BOD5 method is time-consuming and requires tedious operations. For example, it is not possible to detect the abnormality of wastewater before and after the treatment because it takes 5 days to obtain the measurement result. In other words, even if abnormal wastewater flows into the treatment facility or is not sufficiently treated at the treatment facility, it can be detected only after the wastewater has flowed out to the environmental water. In addition, the tedious operations of the BOD5 method also make it difficult to make accurate and accurate measurements.
In 1977, Dr. Karube studied a practical microbial biosensor for BOD [58]. The key technique was the immobilization of microbes to a thin collagen membrane. The microbial membrane was put onto the surface of a DO electrode. By the addition of a sample solution into a batch system, microbial respiration was activated by the decomposition of organic matters, and the degree of DO consumption by the microbes was determined by the DO electrode. The microbial BOD biosensor indicating DO consumption (BODDO) could successfully determine the BOD value at drastically shortened incubation and measurement times (ca. 30 minutes). By the study, the possibilities for solving the problems of wastewater control were enhanced.
In 1979, Hikuma and Karube et al. developed a flow system of the BODDO biosensor [59]. In the study, omnivorous yeast
Since the first microbial biosensor was reported, many kinds of microbial biosensors have been studied for not only environmental applications but also food applications, including fermentation. These studies on both environments and foods by microbial biosensing methods were reviewed [3, 10, 15, 62].
Dr. I. Karube was widely studied all-fields of biosensor development as one of the leading scientists in the world. His notable study on biosensor development was summarized in the review [10] and the detailed history of his study on microbial biosensor development was described in one chapter of Encyclopedia [57].
The notable studies on the microbial BOD biosensor development performed by Karube et al. are briefly described as follows. As the study on the BOD biosensors, a microbial fuel cell (MFC) type biosensor has also been developed. However, the MFC biosensor at that time used expensive materials, easily deteriorated electrodes, anaerobes, etc., and had low cathode reaction efficiency due to low electron transfer from the anaerobes to the cathode. In addition, a flow-type cathode chamber of the MFC biosensor has a low exchange efficiency of sample solution, making it difficult to repeat and rapid BODMFC measurements.
After two types of BODDO-
Another practical study was the development of portable type instruments for on-site monitoring. To realize the on-site monitoring, it was required to stop using air-supply equipment, to reduce the size of measurement devices, to miniaturize and single-use biosensors, to employ omnivore and vital microbes, etc. Dr. Hiroaki Suzuki has been studying the miniaturization of the biosensors to be used as disposable sensor chips [64]. In 1996, Suzuki and Yang et al. studied BODDO-
To solve the problem, in 2000, Yoshida et al. studied two types of BOD biosensor principles for on-site monitoring. One was a single mediator (SM) type of an electrochemical BOD biosensor [67]. In the study, omnivorous bacteria
On the other hand, in 1999, Chee et al. studied highly sensitive BODDO biosensors for low BOD measurements [71]. The background of this study was that the spread of sewage treatment facilities improved the water quality of rivers at that time, and it became necessary to measure low BOD including persistent organic matters in the sample solution. To properly measure the river water quality at that time, isolation of the microbe that biodegrades persistent organic matters was needed, and they isolated
The studies on the practical BOD biosensor were successfully performed by Dr. Karube et al. and the practical instruments including the potable instruments for on-site monitoring were developed.
After these excellent studies on the practical BOD biosensors by Dr. Karube et al., the author explored other possibilities of BOD biosensors with better functionality, for example, improvements of (1) detection limit, (2) signal repeatability of the microbial biosensor, and (3) suitability of microbe used.
The detection limit can be improved to enhance the reaction efficiency of microbial degradation of organic matters. However, most of the microbial biosensors developed to date immobilized the microbes. To enhance the reaction efficiency of microbial degradation of organic matters, microbes should be dispersed to uniform the suspension.
The signal repeatability of the microbial biosensor can be enhanced by a fully unified operation of repeating measurements under the same conditions. It is important to keep constant at least both temperature and reaction time. This enhancement also leads to highly sensitive measurements. This is because by increasing the significant difference between the standard measurement value and the blank value, the standard measurement value closer to the blank value can be set as the detection limit (it is known that the reproducibility of the signals obtained by the microbial biosensor is around 10% (as RSD value) [70]).
On the other hand, the improvement of microbial suitability can also be achieved by employing easily available, omnivorous, and vital microbe.
Then, the author tried to satisfy these requirements by employing the absorptiometric BODRCI measurement method and a temperature-controlled three-cuvette-stir system [73, 74]. As the usable (easily available, omnivorous, and vital) microbe, Baker’s dry yeast
A key principle for the simple absorptiometric measurement method using redox color indicator [
In general, the suspension is not suitable for absorptiometry due to the occurrence of light scattering. However, by a combination of the cuvette-stir system and the time difference method, the influences of the light scattering were efficiently canceled and only absorbance change of DCIP was accurately determined. Further, by repeating the exact same measurement operation three times, the fluctuation of the measured value became small, and the reproducibility was improved, so that highly precise measurement became possible. As a result, the significant difference from the blank value became large, and highly sensitive measurement became possible.
BODRCI-
BODDM-
BODCL-
BODDM:Trinder-
In the studies that the author principally conducted, excellent functions in the BOD biosensor were achieved in (1) the practical detection limit, (2) the signal reproducibility, and (3) the suitability of the microbe used. In addition, some principles of both microbial BOD biosensors and measurement methods have been studied for practical use, but none of the studies the author have principally conducted has reached practical use. The most practical BOD measurement method in the studies might be the BODRCI-
Practically suitable microbe (easily available, omnivorous, and vital yeast)
Very simple operation (just added three solutions into a cuvette; a
Rapid measurement time (only 10 minutes incubation)
Highly sensitive measurements (highly repeatable results were obtained by employing a temperature-controlled three-cuvette-stir system and provided highly precise results, enabling highly sensitive measurements; and it was also important to use RCI of high absorption coefficient)
Practical dynamic range (same as available BOD biosensor instrument)
The patent registered for practical use (the patent right did not continue [74])
For future study, automatic instrumentation of the BOD biosensor would be required having the features that we obtained. Then, further suitability of the microbe might be needed to be considered, for example, use of thermally killed microbes [84] or cell crushed microbes, or direct use of available dry yeast [85].
Even after our studies on the BOD biosensors have been reported, the studies on next-generation BOD biosensors are ongoing. In this section, the progress of our studies on the BOD biosensors performed by other groups is introduced.
In our studies that the author principally conducted, the most practical BOD biosensor or measurement method was the BODRCI-
As recent progress of the DM-type microbial biosensors, several groups were reported. For example, in 2017, Gao et al. reported a DM-type
As recent progress of the BODCL-
As the other progress, MFC biosensors have many potentials not only as BOD biosensors but also as self-powered devices for biosensors [92]. As a new insight into the future perspectives of the BOD biosensor fields, the author have one idea that is our past application study on a damped glycolytic oscillation induced in living yeast cells for toxicity assays (Figure 4) [93, 94]. We extracted six indexes from the wave shape and observed that these indexes were changed depending on both toxicity and the concentration of each toxic matter. By applying this principle, wave changes depending on both bioavailability and the concentration of the organic matters dissolved in a sample solution might be determined. In fact, excellent correlation was obtained between one of the indexes and the concentration of glucose (
(a) Typical damped glycolytic oscillation induced in living yeast cells. (b) Six indexes in an oscillation wave shape. (c) Response to glucose. These were permitted from Springer Nature [
In this chapter, the author described the study on biosensor development for both eutrophication and organic pollution as one of my studying fields. By focusing on the two keywords of eutrophication and organic pollution, the author was able to summarize for the first time a series of our developmental flows and their aims. In conclusion, our biosensors introduced here could not be put into practical use. Nonetheless, the author hope that the practical use of both Pi and BOD biosensors as a replacement of the conventional standard methods will be realized based on the knowledge obtained by our developmental study.
In the relevant studies of the present chapter, the author acknowledges Professor Dr. Isao Karube (February 2020 passed away), his laboratory members, the relevant parties, for their mentoring, collaborations, and supports, and also acknowledge my laboratory members for the contributions to their experiments.
The authors declare no conflict of interest.
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\\n\\nAdrian Assad De Marco joined the company as a Director in 2017. With his extensive experience in management, acquired while working for regional and global leaders, he took over direction and control of all the company's publishing processes. Adrian holds a degree in Economy and Management from the University of Zagreb, School of Economics, Croatia. A former sportsman, he continually strives to develop his skills through professional courses and specializations such as NLP (Neuro-linguistic programming).
\\n\\nDr Alex Lazinica
\\n\\nAlex Lazinica is co-founder and Board member of IntechOpen. After obtaining a Master's degree in Mechanical Engineering, he continued his Ph.D. in Robotics at the Vienna University of Technology. There, he worked as a robotics researcher with the university's Intelligent Manufacturing Systems Group, as well as a guest researcher at various European universities, including the Swiss Federal Institute of Technology Lausanne (EPFL). During this time he published more than 20 scientific papers, gave presentations, served as a reviewer for major robotic journals and conferences and, most importantly, co-founded and built the International Journal of Advanced Robotic Systems, the world's first Open Access journal in the field of robotics. Starting this journal was a pivotal point in his career since it proved to be the pathway to the foundation of IntechOpen with its focus on addressing academic researchers’ needs. Alex personifies many of IntechOpen´s key values, including the commitment to developing mutual trust, openness, and a spirit of entrepreneurialism. Today, his focus is on defining the growth and development strategy for the company.
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\n\nCo-founded by Alex Lazinica and Vedran Kordic: “We are passionate about the advancement of science. As Ph.D. researchers in Vienna, we found it difficult to access the scholarly research we needed. We created IntechOpen with the specific aim of putting the academic needs of the global research community before the business interests of publishers. Our Team is now a global one and includes highly-renowned scientists and publishers, as well as experts in disseminating your research.”
\n\nBut, one thing we have in common is -- we are all scientists at heart!
\n\nSara Uhac, COO
\n\nSara Uhac was appointed Managing Director of IntechOpen at the beginning of 2014. She directs and controls the company’s operations. Sara joined IntechOpen in 2010 as Head of Journal Publishing, a new strategically underdeveloped department at that time. After obtaining a Master's degree in Media Management, she completed her Ph.D. at the University of Lugano, Switzerland. She holds a BA in Financial Market Management from the Bocconi University in Milan, Italy, where she started her career in the American publishing house Condé Nast and further collaborated with the UK-based publishing company Time Out. Sara was awarded a professional degree in Publishing from Yale University (2012). She is a member of the professional branch association of "Publishers, Designers and Graphic Artists" at the Croatian Chamber of Commerce.
\n\nAdrian Assad De Marco
\n\nAdrian Assad De Marco joined the company as a Director in 2017. With his extensive experience in management, acquired while working for regional and global leaders, he took over direction and control of all the company's publishing processes. Adrian holds a degree in Economy and Management from the University of Zagreb, School of Economics, Croatia. A former sportsman, he continually strives to develop his skills through professional courses and specializations such as NLP (Neuro-linguistic programming).
\n\nDr Alex Lazinica
\n\nAlex Lazinica is co-founder and Board member of IntechOpen. After obtaining a Master's degree in Mechanical Engineering, he continued his Ph.D. in Robotics at the Vienna University of Technology. There, he worked as a robotics researcher with the university's Intelligent Manufacturing Systems Group, as well as a guest researcher at various European universities, including the Swiss Federal Institute of Technology Lausanne (EPFL). During this time he published more than 20 scientific papers, gave presentations, served as a reviewer for major robotic journals and conferences and, most importantly, co-founded and built the International Journal of Advanced Robotic Systems, the world's first Open Access journal in the field of robotics. Starting this journal was a pivotal point in his career since it proved to be the pathway to the foundation of IntechOpen with its focus on addressing academic researchers’ needs. Alex personifies many of IntechOpen´s key values, including the commitment to developing mutual trust, openness, and a spirit of entrepreneurialism. Today, his focus is on defining the growth and development strategy for the company.
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Through comparative analysis, the chapter investigates sustainability potential of vernacular architecture in the region to derive core concepts as guidelines of reproducing the characteristics of society and reveal identity of contemporary architecture in the Arab World.",book:{id:"8260",slug:"urban-and-architectural-heritage-conservation-within-sustainability",title:"Urban and Architectural Heritage Conservation within Sustainability",fullTitle:"Urban and Architectural Heritage Conservation within Sustainability"},signatures:"Maha Salman",authors:[{id:"258226",title:"Dr.",name:"Maha",middleName:null,surname:"Salman",slug:"maha-salman",fullName:"Maha Salman"}]},{id:"51000",doi:"10.5772/63726",title:"Towards Sustainable Sanitation in an Urbanising World",slug:"towards-sustainable-sanitation-in-an-urbanising-world",totalDownloads:3202,totalCrossrefCites:11,totalDimensionsCites:17,abstract:"Urban sanitation in low‐ and middle‐income countries is at an inflection point. It is increasingly acknowledged that conventional sewer‐based sanitation cannot be the only solution for expanding urban areas. There are other objective reasons apart from the lack of capital. The lack of stable energy supplies, of spare parts and of human resources for reliable operation, and the increasing water scarcity are factors that seriously limit the expansion of centralised systems. This chapter argues that a new paradigm for urban sanitation is possible, if the heterogeneity within developing cities is reflected in the implementation of different sanitation systems, adapted to each urban context and integrated under one institutional roof. This new paradigm entails: (1) innovative management arrangements; (2) increased participation and the integration of individual, community and private sector initiatives; (3) thinking at scale to open new opportunities; (4) improved analysis of the situation and awareness raising. Moving beyond conventional approaches towards sustainable urbanisation needs to follow both a top‐down and a bottom‐up approach, with proper incentives and a variety of sanitation systems which, in a future perspective, will become part of the ‘urban ecosystem’.",book:{id:"5235",slug:"sustainable-urbanization",title:"Sustainable Urbanization",fullTitle:"Sustainable Urbanization"},signatures:"Philippe Reymond, Samuel Renggli and Christoph Lüthi",authors:[{id:"181079",title:"Dr.",name:"Christoph",middleName:null,surname:"Lüthi",slug:"christoph-luthi",fullName:"Christoph Lüthi"},{id:"182136",title:"Mr.",name:"Philippe",middleName:null,surname:"Reymond",slug:"philippe-reymond",fullName:"Philippe Reymond"},{id:"182137",title:"Mr.",name:"Samuel",middleName:null,surname:"Renggli",slug:"samuel-renggli",fullName:"Samuel Renggli"}]},{id:"42926",doi:"10.5772/55736",title:"Disaster Risk Management and Social Impact Assessment: Understanding Preparedness, Response and Recovery in Community Projects",slug:"disaster-risk-management-and-social-impact-assessment-understanding-preparedness-response-and-recove",totalDownloads:10044,totalCrossrefCites:3,totalDimensionsCites:11,abstract:null,book:{id:"3364",slug:"environmental-change-and-sustainability",title:"Environmental Change and Sustainability",fullTitle:"Environmental Change and Sustainability"},signatures:"Raheem A. Usman, F.B. Olorunfemi, G.P. Awotayo, A.M. Tunde and\nB.A. Usman",authors:[{id:"156875",title:"Dr.",name:"Usman A",middleName:null,surname:"Raheem",slug:"usman-a-raheem",fullName:"Usman A Raheem"},{id:"166449",title:"Dr.",name:"A.M",middleName:null,surname:"Tunde",slug:"a.m-tunde",fullName:"A.M Tunde"},{id:"167886",title:"Dr.",name:"F.B.",middleName:null,surname:"Olorunfemi",slug:"f.b.-olorunfemi",fullName:"F.B. Olorunfemi"},{id:"167887",title:"Dr.",name:"G.P.",middleName:null,surname:"Awotayo",slug:"g.p.-awotayo",fullName:"G.P. Awotayo"}]},{id:"44263",doi:"10.5772/54339",title:"Conservation and Sustainability of Mexican Caribbean Coral Reefs and the Threats of a Human-Induced Phase-Shift",slug:"conservation-and-sustainability-of-mexican-caribbean-coral-reefs-and-the-threats-of-a-human-induced-",totalDownloads:2352,totalCrossrefCites:4,totalDimensionsCites:11,abstract:null,book:{id:"3364",slug:"environmental-change-and-sustainability",title:"Environmental Change and Sustainability",fullTitle:"Environmental Change and Sustainability"},signatures:"José D. Carriquiry, Linda M. Barranco-Servin, Julio A. Villaescusa,\nVictor F. Camacho-Ibar, Hector Reyes-Bonilla and Amílcar L. Cupul-\nMagaña",authors:[{id:"158136",title:"Prof.",name:"Jose D.",middleName:"D.",surname:"Carriquiry",slug:"jose-d.-carriquiry",fullName:"Jose D. Carriquiry"},{id:"160078",title:"Dr.",name:"Julio A.",middleName:null,surname:"Villaescusa",slug:"julio-a.-villaescusa",fullName:"Julio A. Villaescusa"},{id:"160079",title:"MSc.",name:"Linda M.",middleName:null,surname:"Barranco-Servin",slug:"linda-m.-barranco-servin",fullName:"Linda M. Barranco-Servin"},{id:"160082",title:"Prof.",name:"Victor F.",middleName:null,surname:"Camacho-Ibar",slug:"victor-f.-camacho-ibar",fullName:"Victor F. Camacho-Ibar"},{id:"167394",title:"Dr.",name:"Hector",middleName:null,surname:"Reyes-Bonilla",slug:"hector-reyes-bonilla",fullName:"Hector Reyes-Bonilla"},{id:"167395",title:"Dr.",name:"Amilcar L.",middleName:null,surname:"Cupul-Magaña",slug:"amilcar-l.-cupul-magana",fullName:"Amilcar L. Cupul-Magaña"}]}],mostDownloadedChaptersLast30Days:[{id:"64381",title:"Sustainability and Vernacular Architecture: Rethinking What Identity Is",slug:"sustainability-and-vernacular-architecture-rethinking-what-identity-is",totalDownloads:4441,totalCrossrefCites:8,totalDimensionsCites:22,abstract:"Sustainability has often been a fundamental part of the composition of both tangible and intangible cultural resources; sustainability and preservation of cultural identity are complementary. Elements of sustainable design are integral to vernacular architecture that have evolved over time using local materials and technology emerging from ambient natural and cultural environment creating optimum relationships between people and their place. This chapter aims to redefine what identity is as a concept and the impact of globalization on contemporary architecture especially on regions with rich heritage and unique culture as the Arab World. To accomplish this, the chapter examines the emergence of “local identity” as a reaction to the globalization of cultural values, uniform architectural styles, and stereotype patterns through discussing sustainability as a motivation for identity in culture and architecture. The research methodology is based on conducting a qualitative analysis of literature review to the main concepts discussed in this chapter such as: identity, culture, vernacular architecture, and sustainability. Through comparative analysis, the chapter investigates sustainability potential of vernacular architecture in the region to derive core concepts as guidelines of reproducing the characteristics of society and reveal identity of contemporary architecture in the Arab World.",book:{id:"8260",slug:"urban-and-architectural-heritage-conservation-within-sustainability",title:"Urban and Architectural Heritage Conservation within Sustainability",fullTitle:"Urban and Architectural Heritage Conservation within Sustainability"},signatures:"Maha Salman",authors:[{id:"258226",title:"Dr.",name:"Maha",middleName:null,surname:"Salman",slug:"maha-salman",fullName:"Maha Salman"}]},{id:"67342",title:"Introductory Chapter: Heritage Conservation - Rehabilitation of Architectural and Urban Heritage",slug:"introductory-chapter-heritage-conservation-rehabilitation-of-architectural-and-urban-heritage",totalDownloads:2616,totalCrossrefCites:3,totalDimensionsCites:6,abstract:null,book:{id:"8260",slug:"urban-and-architectural-heritage-conservation-within-sustainability",title:"Urban and Architectural Heritage Conservation within Sustainability",fullTitle:"Urban and Architectural Heritage Conservation within Sustainability"},signatures:"Kabila Faris Hmood",authors:[{id:"214741",title:"Prof.",name:"Dr. Kabila",middleName:"Faris",surname:"Hmood",slug:"dr.-kabila-hmood",fullName:"Dr. Kabila Hmood"}]},{id:"76898",title:"The Relationship between Land Use and Climate Change: A Case Study of Nepal",slug:"the-relationship-between-land-use-and-climate-change-a-case-study-of-nepal",totalDownloads:700,totalCrossrefCites:1,totalDimensionsCites:2,abstract:"Land Use and Climate change are interrelated to each other. This change influences one another at various temporal and spatial scales; however, improper land uses are the primary causal factor on climate change. It studies relevant literature and Nepal’s case to assess the relationship between land use and climate change. Similarly focuses on how land-use impacts climate change and vice versa. In recent centuries land-use change significant effects on ecological variables and climate change. Likewise, understanding the research on both topics will help decision-makers and conservation planners manage land and climate.",book:{id:"10754",slug:"the-nature-causes-effects-and-mitigation-of-climate-change-on-the-environment",title:"The Nature, Causes, Effects and Mitigation of Climate Change on the Environment",fullTitle:"The Nature, Causes, Effects and Mitigation of Climate Change on the Environment"},signatures:"Pawan Thapa",authors:[{id:"349566",title:"M.Sc.",name:"Pawan",middleName:null,surname:"Thapa",slug:"pawan-thapa",fullName:"Pawan Thapa"}]},{id:"50282",title:"Relation Between Land Use and Transportation Planning in the Scope of Smart Growth Strategies: Case Study of Denizli, Turkey",slug:"relation-between-land-use-and-transportation-planning-in-the-scope-of-smart-growth-strategies-case-s",totalDownloads:4667,totalCrossrefCites:0,totalDimensionsCites:1,abstract:"In the decision-making process of planning residential areas in developing countries, importance of the commercial areas and need for a sustainable urban transportation infrastructure have generally been ignored based on several sociopolitical reasons. Meanwhile, decision-making periods of location choice and determining areal densities are conducted without quantitative spatial/technical analyses. Those urban matters bring along new planning paradigms like smart growth (SG) and new urbanism. SG is a land use planning paradigm which indicates that traffic problems should be minimized by transit alternatives, effective demand management and providing a balance between land use and transportation planning. This study aims to apply SG strategies to the land use planning process and evaluate the accuracy of land use planning decisions in the perspective of sustainable transportation. In order to reveal the effects of land use planning decisions on the available transportation infrastructure, two scenarios are investigated for 2030. In the first scenario “do nothing” option is considered, while the residential area densities and trip generation rates are regulated based on SG strategies in the second scenario. The results showed that the land use and traffic impact analyses should simultaneously be conducted before land use configuration process.",book:{id:"5235",slug:"sustainable-urbanization",title:"Sustainable Urbanization",fullTitle:"Sustainable Urbanization"},signatures:"Gorkem Gulhan and Huseyin Ceylan",authors:[{id:"182126",title:"Dr.",name:"Gorkem",middleName:null,surname:"Gulhan",slug:"gorkem-gulhan",fullName:"Gorkem Gulhan"},{id:"185555",title:"Dr.",name:"Huseyin",middleName:null,surname:"Ceylan",slug:"huseyin-ceylan",fullName:"Huseyin Ceylan"}]},{id:"42926",title:"Disaster Risk Management and Social Impact Assessment: Understanding Preparedness, Response and Recovery in Community Projects",slug:"disaster-risk-management-and-social-impact-assessment-understanding-preparedness-response-and-recove",totalDownloads:10045,totalCrossrefCites:3,totalDimensionsCites:11,abstract:null,book:{id:"3364",slug:"environmental-change-and-sustainability",title:"Environmental Change and Sustainability",fullTitle:"Environmental Change and Sustainability"},signatures:"Raheem A. Usman, F.B. Olorunfemi, G.P. Awotayo, A.M. Tunde and\nB.A. Usman",authors:[{id:"156875",title:"Dr.",name:"Usman A",middleName:null,surname:"Raheem",slug:"usman-a-raheem",fullName:"Usman A Raheem"},{id:"166449",title:"Dr.",name:"A.M",middleName:null,surname:"Tunde",slug:"a.m-tunde",fullName:"A.M Tunde"},{id:"167886",title:"Dr.",name:"F.B.",middleName:null,surname:"Olorunfemi",slug:"f.b.-olorunfemi",fullName:"F.B. Olorunfemi"},{id:"167887",title:"Dr.",name:"G.P.",middleName:null,surname:"Awotayo",slug:"g.p.-awotayo",fullName:"G.P. Awotayo"}]}],onlineFirstChaptersFilter:{topicId:"136",limit:6,offset:0},onlineFirstChaptersCollection:[{id:"82644",title:"Climate-Driven Temporary Displacement of Women and Children in Anambra State, Nigeria: The Causes and Consequences",slug:"climate-driven-temporary-displacement-of-women-and-children-in-anambra-state-nigeria-the-causes-and-",totalDownloads:24,totalDimensionsCites:0,doi:"10.5772/intechopen.104817",abstract:"With increasing periods of extreme wet seasons, low lying geographic position, with socioeconomic, and political factors; some communities in Anambra State, Nigeria experience heightened floods annually resulting in loss of shelter, displacement of people with breakdown of livelihoods, particularly in rural communities worsening their risks and vulnerabilities. In 2012, a major flood event in the state temporarily displaced about 2 million people. In this chapter, we used a community-based adaptation approach to investigate the causes and consequences of climate-related temporary displacement on community members in Ogbaru LGA, Anambra State following flood events. We used global positioning system to obtain the community’s ground control points and gathered our data via field observation, transects walks, focus group discussions, photography, and in-depth interviews. Our findings reveal a heightened magnitude of flood related disasters with decreased socio-economic activities, affecting their health and well-being. Also, the community members have a practice of returning to their land, after flood events, as a local mitigating risk management strategy. For multilevel humanitarian responses at the temporary shelter camps, it becomes imperative to meaningfully engage the community members on the challenging risks and vulnerabilities they experience following climate-driven temporary displacement to inform adaptation and resilience research, policy change and advocacy.",book:{id:"7724",title:"Climate Change in Asia and Africa - Examining the Biophysical and Social Consequences, and Society's Responses",coverURL:"https://cdn.intechopen.com/books/images_new/7724.jpg"},signatures:"Akanwa Angela Oyilieze, Ngozi N. Joe-Ikechebelu, Ijeoma N. Okedo-Alex, Kenebechukwu J. Okafor, Fred A. Omoruyi, Jennifer Okeke, Sophia N. Amobi, Angela C. Enweruzor, Chinonye E. Obioma, Princess I. Izunobi, Theresa O. Nwakacha, Chinenye B. Oranu, Nora I. Anazodo, Chiamaka A. Okeke, Uwa-Abasi E. Ugwuoke, Uche M. Umeh, Emmanuel O. Ogbuefi and Sylvia T. Echendu"},{id:"79637",title:"Evaluation of the Spatial Distribution of the Annual Extreme Precipitation Using Kriging and Co-Kriging Methods in Algeria Country",slug:"evaluation-of-the-spatial-distribution-of-the-annual-extreme-precipitation-using-kriging-and-co-krig",totalDownloads:53,totalDimensionsCites:0,doi:"10.5772/intechopen.101563",abstract:"In this chapter, we have conducted a statistical study of the annual extreme precipitation (AMP) for 856 grid cells and during the period of 1979–2012 in Algeria. In the first step, we compared graphically the forecasts of the three parameters of the generalized extreme value (GEV) distribution (location, scale and shape) which are estimated by the Spherical model. We used the Cross validation method to compare the two methods kriging and Co-kriging, based on the based on some statistical indicators such as Mean Errors (ME), Root Mean Square Errors (RMSE) and Squared Deviation Ratio (MSDR). The Kriging forecast error map shows low errors expected near the stations, while co-Kriging gives the lowest errors on average at the national level, which means that the method of co-Kriging is the best. From the results of the return periods, we calculate that after 50 years the estimated of the annual extreme precipitation will exceed the maximum AMP is observed in the 33-year.",book:{id:"7724",title:"Climate Change in Asia and Africa - Examining the Biophysical and Social Consequences, and Society's Responses",coverURL:"https://cdn.intechopen.com/books/images_new/7724.jpg"},signatures:"Hicham Salhi"},{id:"77854",title:"Flooding and Flood Modeling in a Typhoon Belt Environment: The Case of the Philippines",slug:"flooding-and-flood-modeling-in-a-typhoon-belt-environment-the-case-of-the-philippines",totalDownloads:162,totalDimensionsCites:0,doi:"10.5772/intechopen.98738",abstract:"Flooding is a perennial world-wide problem and is a serious hazard in areas where the amount of precipitable water has potential to dump excessive amount of water. The warming of the Earth’s climate due to the increase in greenhouse gases (GHGs) increases the availability of water vapor and hence, of extreme precipitation as observed and forecasted by researchers. With rainfall intensity too high, the torrential rains coupled with weather systems that enhances its effects, flooding not only submerges anything low-lying, it also washes away living and non-living things along the course of the river and the floodplain. The flooding is even worsened by the increase in velocity of flow caused by unsustainable urbanization and denudation of the watershed at the headwaters. Nature’s strength is an order of a magnitude that is way beyond that of the strength of men but human ingenuity enables us to transform our living environment into models that could help us better understand it. Flood modeling provides us decision support tools to deal better with nature. It also enables us to simulate the future especially nowadays that changes in our climate is imminent and even happening already in many parts of the world. Therefore, strategies on how to cope with our ever changing environment is very important particularly to countries that are at more risk to climate change such as the archipelagic Philippines.",book:{id:"7724",title:"Climate Change in Asia and Africa - Examining the Biophysical and Social Consequences, and Society's Responses",coverURL:"https://cdn.intechopen.com/books/images_new/7724.jpg"},signatures:"Fibor J. Tan"},{id:"77797",title:"Adapting to Climatic Extremes through Climate Resilient Industrial Landscapes: Building Capacities in the Southern Indian States of Telangana and Andhra Pradesh",slug:"adapting-to-climatic-extremes-through-climate-resilient-industrial-landscapes-building-capacities-in",totalDownloads:98,totalDimensionsCites:0,doi:"10.5772/intechopen.98732",abstract:"There is now greater confidence and understanding of the consequences of anthropogenic caused climate change. One of the many impacts of climate change, has been the occurrence of extreme climatic events, recent studies indicate that the magnitude, frequency, and intensity of hydro-meteorological events such as heat waves, cyclones, droughts, wildfires, and floods are expected to increase several fold in the coming decades. These climatic extremes are likely to have social, economic, and environmental costs to nations across the globe. There is an urgent need to prepare various stakeholders to these disasters through capacity building and training measures. Here, we present an analysis of the capacity needs assessment of various stakeholders to climate change adaptation in industrial parks in two southern states of India. Adaptation to climate change in industrial areas is an understudied yet highly urgent requirement to build resilience among stakeholders in the Indian subcontinent. The capacity needs assessment was conducted in two stages, participatory rural appraisal (PRA) and focus group discussion (FGD) were conducted among various stakeholders to determine the current capacities for climate change adaptation (CCA) for both, stakeholders and functional groups. Our analysis indicates that in the states of Telangana and Andhra Pradesh, all stakeholder groups require low to high levels of retraining in infrastructure and engineering, planning, and financial aspects related to CCA. Our study broadly supports the need for capacity building and retraining of functionaries at local and state levels in various climate change adaptation measures; likewise industry managers need support to alleviate the impacts of climate change. Specific knowledge, skills, and abilities, with regard to land zoning, storm water management, developing building codes, green financing for CCA, early warning systems for climatic extremes, to name a few are required to enhance and build resilience to climate change in the industrial landscapes of the two states.",book:{id:"7724",title:"Climate Change in Asia and Africa - Examining the Biophysical and Social Consequences, and Society's Responses",coverURL:"https://cdn.intechopen.com/books/images_new/7724.jpg"},signatures:"Narendran Kodandapani"},{id:"77460",title:"Changing Climatic Hazards in the Coast: Risks and Impacts on Satkhira, One of the Most Vulnerable Districts in Bangladesh",slug:"changing-climatic-hazards-in-the-coast-risks-and-impacts-on-satkhira-one-of-the-most-vulnerable-dist",totalDownloads:210,totalDimensionsCites:0,doi:"10.5772/intechopen.98623",abstract:"Changes in the climate due to anthropogenic and natural variation are indicated by parameters including temperature and rainfall. Climate change variability with changing trends of the two have been unpredictable and unprecedented globally leading to changing weather patterns, natural disasters, leading to sectoral impacts on food and water security, livelihood, human health among others. This research analyses the changing patterns of these parameters over the last 35/37 years of Satkhira district of Bangladesh to assess the state and trend across spatial and temporal dimensions. Such, the study validates to rationalize the observed seasonal changes that persist in Satkhira of Bangladesh. Both in terms of intensity and frequency of the occurrences of natural disasters, the series of natural events have been triangulated, with impacts and vulnerability being assessed from temperature variations, erratic rainfall, cyclone, flood and water logging etc. The study’s prime contribution remains in attribution of climate change in relation contextual circumstances in the region including sea level rise, salinity intrusion. Therefore, the risk and climatic hazards and its resulting impacts over time has been assessed to draw deeper connection between theoretical and practical values. The series of analyses also draw conclusion that assets are at risk from changing climatic condition.",book:{id:"7724",title:"Climate Change in Asia and Africa - Examining the Biophysical and Social Consequences, and Society's Responses",coverURL:"https://cdn.intechopen.com/books/images_new/7724.jpg"},signatures:"Md. Golam Rabbani, Md. Nasir Uddin and Sirazoom Munira"},{id:"76915",title:"The Impacts of Climate Change in Lwengo, Uganda",slug:"the-impacts-of-climate-change-in-lwengo-uganda",totalDownloads:101,totalDimensionsCites:0,doi:"10.5772/intechopen.97279",abstract:"Climate Change has become a threat worldwide. Vulnerable communities are at foremost risk of repercussions of climate change. The present study aimed at highlighting a case study of climate change impacts on Lwengo District of Uganda. Out of the total geographical area of the district, 85% hectares are under cultivation and most of its population depends majorly on the rain- fed agriculture sector to meet the food requirement and as a major income source. With the changing climatic conditions, agriculture is the major sector which is being impacted. The region has experienced disasters from some time, usually the second seasons rains used to result in such disasters but since 2016 both seasons have occurred disasters, which majorly include hailstorm, strong wind, long dry spells, pests and diseases. The situation became more severe due to shortage of availability of skilled human resources, quality equipment for disaster management, limited financial resources and weak institutional capacity, which resulted in increasing vulnerability of small farm holders. Some of the adaptation strategies are being taken up by the government but there is a need to understand prospects of decision-making that are site specific and more sustainable for smallholder communities. Climatic changes possess many obstacles to farming communities which require sustainable adaptation to enhance the adaptive capacities of the communities through continued production systems, which are more resilient to the vagaries of weather. Farmers are practising such options which are location specific, governed by policy framework and dependent on dynamism of farmers. 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