Metal ion vs. log βMY values.
\\n\\n
Released this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
\\n\\nWe wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
Note: Edited in March 2021
\\n"}]',published:!0,mainMedia:null},components:[{type:"htmlEditorComponent",content:'IntechOpen is proud to announce that 191 of our authors have made the Clarivate™ Highly Cited Researchers List for 2020, ranking them among the top 1% most-cited.
\n\nThroughout the years, the list has named a total of 261 IntechOpen authors as Highly Cited. Of those researchers, 69 have been featured on the list multiple times.
\n\n\n\nReleased this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
\n\nWe wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
Note: Edited in March 2021
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He is a Coordinator of The Center for Studies of Inflammatory and Infectious Diseases and a Professor in the Multi-Centric Post-Graduation Program in Physiological Sciences (PMPGCF-UFVJM), and Post-Graduation Program in Pharmaceutical Sciences (PPGCiFarm-UFVJM). He is a graduate (Licenciate and Bachelor) in Biological Sciences by Herminio Ometto University Center (2002), Master degree in Pharmacology by State University of Campinas - UNICAMP (2007), and PhD in Clinical Medicine by the same university (2011). He has a post-doctoral fellow in Biochemistry and Immunology from the Federal University of Minas Gerais-UFMG and develops studies in clinical pathology, microbiology, virology, immunology and biochemistry, techniques on molecular biology applied to the diagnosis of infectious disease and host-parasite interactions.",institutionString:"Federal University of Vales do Jequitinhonha e Mucuri",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"2",totalChapterViews:"0",totalEditedBooks:"1",institution:{name:"State University of Campinas",institutionURL:null,country:{name:"Brazil"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"1046",title:"Infectious Diseases",slug:"infectious-diseases"}],chapters:[{id:"69359",title:"Introductory Chapter: Human Herpesvirus - A Short Introduction",slug:"introductory-chapter-human-herpesvirus-a-short-introduction",totalDownloads:215,totalCrossrefCites:0,authors:[{id:"81175",title:"PhD.",name:"Ronaldo Luis",surname:"Thomasini",slug:"ronaldo-luis-thomasini",fullName:"Ronaldo Luis Thomasini"}]},{id:"64844",title:"Sunlight and Herpes Virus",slug:"sunlight-and-herpes-virus",totalDownloads:877,totalCrossrefCites:1,authors:[{id:"273350",title:"Prof.",name:"Vittorio",surname:"Mazzarello",slug:"vittorio-mazzarello",fullName:"Vittorio Mazzarello"},{id:"274197",title:"Dr.",name:"Marco",surname:"Ferrari",slug:"marco-ferrari",fullName:"Marco Ferrari"},{id:"283742",title:"Prof.",name:"Maria Alessandra",surname:"Sotgiu",slug:"maria-alessandra-sotgiu",fullName:"Maria Alessandra Sotgiu"},{id:"283744",title:"Dr.",name:"Stefano",surname:"Decandia",slug:"stefano-decandia",fullName:"Stefano Decandia"}]},{id:"64988",title:"Neurologic Complications of Varicella-Zoster Virus Infection",slug:"neurologic-complications-of-varicella-zoster-virus-infection",totalDownloads:1127,totalCrossrefCites:1,authors:[{id:"43262",title:"Dr.",name:"Hideto",surname:"Nakajima",slug:"hideto-nakajima",fullName:"Hideto Nakajima"},{id:"286199",title:"Dr.",name:"Makoto",surname:"Hara",slug:"makoto-hara",fullName:"Makoto Hara"},{id:"286200",title:"Dr.",name:"Akihiko",surname:"Morita",slug:"akihiko-morita",fullName:"Akihiko Morita"},{id:"286201",title:"Prof.",name:"Satoshi",surname:"Kamei",slug:"satoshi-kamei",fullName:"Satoshi Kamei"}]},{id:"71254",title:"Extracranial Herpetic Paresis",slug:"extracranial-herpetic-paresis",totalDownloads:243,totalCrossrefCites:0,authors:[{id:"271733",title:"Prof.",name:"Vesna",surname:"Martic",slug:"vesna-martic",fullName:"Vesna Martic"}]},{id:"67401",title:"Human Cytomegalovirus Infection: Biological Features, Transmission, Symptoms, Diagnosis, and Treatment",slug:"human-cytomegalovirus-infection-biological-features-transmission-symptoms-diagnosis-and-treatment",totalDownloads:410,totalCrossrefCites:0,authors:[{id:"269379",title:"M.D.",name:"Şule",surname:"Gökçe",slug:"sule-gokce",fullName:"Şule Gökçe"}]},{id:"69076",title:"The Role of the Epstein-Barr Virus Lytic Cycle in Tumor Progression: Consequences in Diagnosis and Therapy",slug:"the-role-of-the-epstein-barr-virus-lytic-cycle-in-tumor-progression-consequences-in-diagnosis-and-th",totalDownloads:290,totalCrossrefCites:0,authors:[{id:"188773",title:"Prof.",name:"Emmanuel",surname:"Drouet",slug:"emmanuel-drouet",fullName:"Emmanuel Drouet"}]}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"220812",firstName:"Lada",lastName:"Bozic",middleName:null,title:"Ms.",imageUrl:"https://mts.intechopen.com/storage/users/220812/images/6021_n.jpg",email:"lada@intechopen.com",biography:"As an Author Service Manager my responsibilities include monitoring and facilitating all publishing activities for authors and editors. From chapter submission and review, to approval and revision, copyediting and design, until final publication, I work closely with authors and editors to ensure a simple and easy publishing process. I maintain constant and effective communication with authors, editors and reviewers, which allows for a level of personal support that enables contributors to fully commit and concentrate on the chapters they are writing, editing, or reviewing. I assist authors in the preparation of their full chapter submissions and track important deadlines and ensure they are met. I help to coordinate internal processes such as linguistic review, and monitor the technical aspects of the process. As an ASM I am also involved in the acquisition of editors. 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Silverberg",coverURL:"https://cdn.intechopen.com/books/images_new/544.jpg",editedByType:"Edited by",editors:[{id:"78753",title:"Dr.",name:"Donald",surname:"Silverberg",slug:"donald-silverberg",fullName:"Donald Silverberg"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"72428",title:"Confocal Laser Scanning Microscopy of Living Cells",doi:"10.5772/intechopen.92751",slug:"confocal-laser-scanning-microscopy-of-living-cells",body:'Before cultured cells were available, the main objects of cell physiology were nerve and blood cells. The study of giant axons showed that excitability and irritability are the most important properties of living cells. Russian physiologist E. Bauer used these properties to affirm the principle of “stable nonequilibrium” [1]. This principle explains that in the changing environment, living systems maintain their structural stability through energy supply. Instead of “energy,” however, he uses the term “work” performed by system against equilibrium. Physiological implication of this principle was found upon the study of nonequal redistribution of Na+ and K+ ions in cuttlefish neuron during impulse propagation [2].
Physiological studies of living cells are always done in conjunction with light microscopy methods. Later these methods got resolution sufficient to study cell structures in molecular scale. The maximum resolution that allows observation of hexagonal pattern on diatom algae is reached in basic microscopy by shutting the condenser [3]. Near-field scanning optical microscopy (NSOM) overcomes the diffraction limit by using optical fiber for both illumination and scanning [4]. NSOM was able to visualize small antibody-labeled domains on the plasma membrane of living human skin fibroblasts [5]. The small thickness of these cells uses conventional epi-fluorescence microscopy to study the mitochondrial membrane potential (MMP) [6, 7] using the protocol of automated image analysis of mitochondrial shape originally tested by confocal microscopy [8, 9]. The same functionality but greater performance resides in the method of imaging flow cytometry. The method allows fluorescence quantification using spot masks that are created independently on pixel intensity. This method was applied for the characterization of MMP changes involved in the malignant transformation of cancer stem cells. MMP together with such parameters as glucose uptake, superoxide-anion production, and mitochondria mass served as indicator of tumorigenesis [10].
Besides using CCD camera in conventional fluorescence microscopy, video microscopy suggests built-in image processing algorithms that cause inadvertent effect on pixel intensity, but with additional calibration procedure, absolute intensities are also measurable [11]. Instead of these algorithms, conventional microscopy uses deconvolution to improve the quality of images as in confocal microscopy [12]. For example, deconvolution was applied to wide-field microscopy images to resolve DNA replication units that are studied only by electron and structured illumination microscopy [13]. Unlike these later methods, confocal and classical light microscopy possesses the ability to study these structures in the living cells.
Discrimination of depth is a property of confocal microscopy that relies to confocal diaphragm [14] and can be checked by measuring the point spread function (PSF), which is defined as the image of a single point [15]. To see the changes on the image of real object, which contains convoluted PSFs, is impossible unless deconvolution is applied [12]. Special image processing algorithm is also applied in confocal microscopy for quantitative fluorescence analysis. This algorithm was applied in quantitative studies of mitochondrial [Na+] ([Na+]m) and Ca2+ ([Ca2+]m) and mitochondrial pH (pHm) in MDCK cells stained with one of the ion-sensitive dyes [16, 17, 18]. Obtaining information about cellular organelles such as mitochondria requires creation of mask.
The same analytical approach was applied for the study of MMP changes caused by phytohemagglutinin (PHA) treatment in histiocytic lymphoma U937 cells. The image of TMRE-stained cells was used for both creating the mask and obtaining quantitative data. The value of average intensity obtained from masked image is independent of the wide variation of MMP. Therefore, this image can serve as a measure of MMP. To consider the effect of large mitochondrial depolarization that occurs in cells after PHA treatment, data were presented as integrated density divided by the number of cells on the image.
PHA is a lectin that stimulates growth and division of lymphocytes in the process known as transformation [19]. Previous studies using indirect isotopic method have not found considerable MMP change during mitogenic stimulation [20]. At the same time, lectins are affecting all known metabolic pathways. Early response in lymphocytes to PHA treatment includes stimulation of glycolysis [21], activation of pentose-phosphate cycle [22], and activation of pyruvate dehydrogenase (PDH) [23] that is a key enzyme to connecting glycolysis and citric acid cycle.
The history of CLSM began when Marvin Minsky include in his patent letter the scheme with epi-illumination with two pinholes that play the same role as in “double focusing” scheme which have already been tested with a stage-scanning device [24]. Historical information and many technical details are not covered in the present paper but can be found in an encyclopedia article written by Amos and colleagues [14]. From a biological point of view, the development of CLSM began in 1987 when microscopes with laser beam deflection system were able to scan living kidney epithelium cells [25] and spinal cord neurons [26].
The development of CLSM ranging from 1987 to 1995 is distinctive by exponential rise of citations for the query “microscopy AND confocal” (Figure 1). At this period, the number of studies retrieved by the query “microscopy NOT confocal” shows a rather decelerating trend. In 1995, the number of confocal microscopy studies started to grow linearly, while many microscopic studies remain unpublished. The second period ended with the new millennium when linear trend of CLSM publications was accompanied by extensive growth of other microscopic studies. The third period is therefore noticeable by concomitant development of all microscopic methods. During this period, confocal microscopy constitutes one sixth of microscopic papers. Indeed, this share would be greater if query is restricted only to cytological area. It can be concluded that the first period caused a 10-year delay in the development of other microscopical methods, while the second period caused its rapid growth.
Relation between the studies done by confocal or other microscopical methods from 1972 to 2018 following the records from PubMed. Trends of publications by year were downloaded from PubMed web page [
Ideal confocal microscope has detector pinhole that is small enough to suppress the diffraction in emission light path. This made its resolution equally dependent from excitation and emission light paths [14]. Lateral resolution of real confocal microscope is determined by the illuminating pinhole that participates in the formation of PSF [15]. Therefore, lateral resolution of confocal microscope is given by formula
where FWHM is the distance between the points on distribution where its intensity is half of that of the peak intensity,
In real confocal microscope, the detector pinhole determines its axial resolution. The difference in axial resolution between confocal and conventional microscopy can be demonstrated using thin fluorescent sheet. By switching microscope to epi-fluorescent mode, no changes in fluorescence intensity are observed upon moving the test object out of focus. Back to the confocal microscopy mode, fluorescence intensity will decrease in sigmoid manner, the slope of which will depend on pinhole diameter [14]. Thickness of optical section is then given by the formula
where n is the refractive index of immersion medium (
In the current study, we use a step-function fluorescent object for testing the limits of axial resolution of CLSM. The test object was prepared as described in [14] but with the following modifications. Fluoroshield™ mounting media containing 20 μg/ml rhodamine 6G was placed between a microscopic slide and 24 mm squire coverslip and left under 1 kg weight until drying. Fluorescence intensity profiles were obtained by scanning fluorescent object in X,Z direction at different pinhole sizes. As shown in Figure 2A, an increase of pinhole diameter from 1.05 to 3.0 Airy widens fluorescence intensity profile and fluorescence background. Taking the advantage of normal distribution of fluorescence values, we use Gauss function for obtaining FWHM values of intensity curves. The effect of pinhole diameter ranging from 0.45 to 4 Airy units on profile width is presented in Figure 2B.
Effect of pinhole diameter on the width of fluorescence profile recorded across a layer of Fluoroshield™ media containing 20 μg/ml rhodamine G. (A) Fluorescent intensity profiles of X,Z optical sections are registered by Olympus FV3000 confocal microscope equipped with 60x/1.42 plan apochromat objective. Fluorescence was excited at 488 nm and collected from 500 to 600 nm. Two fluorescent profiles obtained at pinhole diameters of 210 and 600 μm (corresponding to 1.05 and 3 Airy units) are shown. One curve is the mean of seven plots generated using ImageJ program. Fluorescence intensity is kept constant by adjustment of laser power. (B) FWHM is derived from the intensity profiles obtained at pinhole diameters of 0.45, 0.65, 0.8, 1.05, 3, and 4 Airy units.
Changes of profile width are correlated with the changes in the optical thickness upon variation of pinhole diameter up to the 3 Airy units. Namely, changing of the pinhole diameter from 1.05 to 3 Airy units increases the width of intensity profile by 0.6 μm. Changing pinhole in reverse order, from 1.05 to 0.65 Airy units, decreases the width of intensity profile by 0.1 μm (Figure 2B).
The absolute resolution values are usually different from those predicted by Eqs. (1) and (2). Practically it is determined by the measurement of PSF, which is generated by microspheres with a diameter of 170 nm in lateral and axial planes. In one practical study, FWHM was determined as 0.32 and 1.9 μm in X,Y or X,Z directions, respectively [18]. The high value of axial FWHM depth is explained by the necessary use of high pinhole diameter in a study of mitochondrial pH in MDCK cells. Pinhole diameter of about three Airy units was applied in this study to attenuate laser power and minimize its photo-damaging effect on living cells [18, 28].
The resolution of CLSM in large extent depends on the quality of objective lens. Using the correct objective in CLSM is especially important because lens parameters are assumed in the design of particular confocal system. Choosing lens with the same nominal parameters but designed for other microscopes results in more than twice a decrease of resolution [29]. In addition, high-magnification oil-immersion objectives work correctly in media with the refractive index being very close to the refractive index of living cells. Any mismatch of the refractive index decreases the quantity of excited fluorescence and, therefore, resolution of confocal system. Water immersion objectives allow working with deeply lying cells [30].
Earlier confocal systems have difficulties in using multiple dyes. Therefore, studies were done in parallel samples assuming that experimental conditions equally apply for both dyes. SNARF-1 (seminaphtorhodafluor-1, free form) was used for the study of pH of luminal solution along colon crypt. Using CLSM localization SNARF-1 was compared with the localization of dye Lucifer yellow [30]. Pseudo-ratiometric approach utilizes cells stained simultaneously with two dyes localized in the same cellular compartment. MitoTracker Green (MTG) resides in the mitochondria and therefore can be colocalized with the dye of interest [16, 17, 18]. As the latter were used one of the following dyes, Rhod-2 (AM), CoroNa Red, or SNARF-1 (AM), accumulated in the mitochondria and giving information on cytoplasmic concentrations of [Na+]m, [Ca2+]m, and pHm, respectively. By using Fura-2 (AM), it was shown that during metabolic inhibition, the main source for transient increase in cytoplasmic Ca2+ concentration ([Ca2+]c) is the mitochondria [16], and only functionally active mitochondria can have buffering capacity to Ca2+ [31].
A large number of studies are directed to understand mitochondrial heterogeneity, which is manifested as the differences in shape and size of the mitochondria in a single cell [8, 9] or morphological differences between cell types [32]. Some cell mitochondria appear as network but in others as discrete individuals [32]. Fission and fusion that maintain the dynamic structure of mitochondrial network are mechanisms involved in the regulation of cell proliferation and apoptosis [33]. These processes are sensitive to cell metabolic state and MMP. Elongated mitochondria are dominant in mouse embryonic fibroblasts grown in conditions promoting oxidative phosphorylation. The addition of glucose suppresses elongation and causes fragmentation [34]. In the study, reviewed in the next section, the attempt was made to uncover the dynamic nature of MMP changes and its role in maintaining mitochondrial network structure.
In studies of MMP, it is important to consider the thickness of optical section because it determines the affectivity of detection of mitochondrial fluorescence. FWHM of optical section that is calculated by Eq. (2) is almost fit to the dimensions of the mitochondria in U937 cells. Electron microscopic studies follow that the mitochondria in U937 cells are spheroidal in structures with a diameter of 0.6–0.8 μm [35]. Therefore, CLSM is effectively collecting fluorescence from the mitochondria of TMRE-stained U937 cells. The mitochondria in lymphocytes have a diameter of only 0.3–0.4 μm [19, 36, 37], and the affectivity of collection of mitochondrial fluorescent signal is less than U937 cells. The mitochondrial shape is also considered in choosing the right procedure for image processing. Thus, different procedures are required for the study of mitochondria in U937 cells or in skin fibroblasts having threadlike appearance [38].
Using CLSM in the study of MMP, it is possible to select for analysis the whole cell area. This selection includes nonmitochondrial compartment that constitutes about 95–98% of the total cell volume [10, 20]. Its signal can be set as a background and subtracted from total fluorescence. The example of this approach is found in a study of MMP changes in lamprey hepatocytes during prespawning migration. As can be expected, CLSM gave results similar to that of flow cytometry [39]. The reason why this approach is chosen by authors is the absence of parallel control samples that are necessary for analytical procedures involving image processing.
Another simplified approach is based on staining of cells with TMRE and MTG and using MTG as reference. For example, TMRE/MTG fluorescence ratio was decreased in the muscle cells of zebrafish embryo after chronical treatment by rotenone [40]. The utilization of MTG as reference dye however is limited because its uptakes in some types of cells depend on MMP [41]. Therefore, MTG is generally used for the creation of mask [16, 17, 18]. By combination of these approaches, it was shown that both TMRE/MTG ratio and MTG signal increased during malignant transformation of mesenchymal stem cells [10].
Good substitute for MTG is mitochondria-specific green fluorescence proteins (GFP). MitoAcGFP1 located in mitochondrial matrix was used for the study of mitochondrial [Ca2+]m [6]. Photoactivatable GFP was used for tracking individual mitochondria in order to relate their MMP to fission events [42, 43]. Data obtained in later studies lead authors to the conclusion that depolarization triggers fission of the mitochondria and fission serves as the quality control for its functional properties [43]. However, the applicability of GFP is limited by insufficient level of expression in some cell types [32, 42]. Rhodamine ester dyes were also used as reference dye in the study of [Ca2+]m [31]. Evidence was provided that TMRM (tetramethylrhodamine, methyl ester) is not liable to auto-quenching and therefore suitable both for the creation of mask and determination of MMP [6]. This is especially important because data obtained using dye rhodamine 123 do not allow their interpretation in terms of MMP.
CLSM experiments with living cells are preceded by cell staining. During staining, TMRE dye almost completely uptakes the cells; hence, it is important to keep constant the dye-to-cell ratio. This ratio should also be equal in control and treated cells. During image acquisition it is possible to use previously saved settings. The function of “auto-exposure” can be used only once with living cells at the beginning of the study [44]. The reason for this is to keep cells from overexposure from laser irradiation. The optimum settings however allow keeping the dynamic range of detector maximum and preventing most of the pixel on image from saturation. Fluorescence intensity is almost linearly dependent on laser power and nearly exponentially—on voltage of photomultiplier tube (PMT). Therefore, it is generally recommended to set PMT voltage first and then adjust laser excitation intensity [44]. With calibration, PMT voltage also can be varied and used for adjustment [18, 28].
Examples of image processing that can be found in the earliest studies are threshold, gradient filtering, and segmentation. These procedures are applied to images resulted from summing of intensity along the Z-axis [26]. Graphical filters are specific instruments used to eliminate noise or other unwanted information from image. “Rolling ball” algorithm uses rank operators to remove pixels exceeding the local background level and replaces them with pixels of neighborhood intensity. The processed image is then subtracted from the original image [45]. “Top hat” filter is used for processing such complex structures as mitochondrial network in human skin fibroblast [8, 9]. “Rolling ball” filter is suitable for images of U937 cells containing the mitochondria of elliptical shape.
For demonstration of image processing in our study, U937 cells were treated with PHA (30 μg/ml, 2 h) and stained with 25 nM of TMRE dye. Cell suspension was placed in Plexiglas holder and scanned in the middle plane of the most cells. Histogram equalization was used to check the amount of TMRE in nonmitochondrial compartment. This was done by running command “enhance contrast” in ImageJ program. Apart from the mitochondria and cytoplasm, small fluorescence is present in the nucleus (Figure 3A and B). To remove background fluorescence, images were processed by “subtract background” algorithm in ImageJ program that utilizes “rolling ball” operator [46]. On the next step, the processed image is subjected to thresholding, which is used to suppress variations at the background level [45]. ImageJ program allows obtaining quantitative data after setting a threshold and execution of commands “create selection” and “analyze stack.” However, for demonstration of validity of this procedure, binary mask (mask) was applied to histogram-equalized image (HEQ) rather than to original image (RAW). This operation gives possibility to see variations within regions, which appeared uniform in the original image [45]. The procedure of histogram equalization is similar to the linear contrast stretch used in other works [6]. The resulting masked images (MSK) contain nonzero pixels that correspond to mitochondrial compartment (Figure 3B and E). Histogram equalization emphasizes mitochondrial heterogeneity and proves the absence of fluorescence in cytoplasmic compartment. However, HEQ images do not give possibility to see the difference of intensities between control- and PHA-treated cells because changes of intensity caused by this procedure are specific to each image [45].
Demonstration of using image processing for quantitative study of MMP changes in U-937 cell during PHA treatment. Cells were incubated without (A–C) or with p-PHA (30 μg/ml) for 2 h (D–F) in atmosphere of 5% CO2 and stained with 25 nM TMRE for 30 min at 37°C. Excess of media was removed by centrifugation. 20 μl of cell suspension is placed on coverslip and a 1% gelatin solution is attached to Plexiglas holder sealed from another side with coverslip. Fluorescence was excited by 561 nm diode laser and collected at 575–675 nm through pinhole of 178 mm (0.73 Airy units). (A, D), raw images after histogram equalization (EQH); (B, E), masked version of this images (MSK); (C, F), merged images of MSK (red pseudo-color), and EQH (green pseudo-color). Red pixels colocalized with green pixels in an area of high intensity except the blurred regions that are present in some cells. Arrows indicate small aggregates of agglutinated cells.
Using merged images it is possible to see similarity between MSK and nonzero regions of HEQ image. As it can be seen in Figure 3C and F, colocalization is present in all regions except blurred parts that are present in both control- and PHA-treated cells. Good correlation between MSK and HEQ is also seen by “Color inspector 3D” plug-in in ImageJ program. The presence of cell groups that mark the beginning of agglutination process was noticed on the image of PHA-treated cells (Figure 3F, arrows). At this period, however, agglutinated cells are lying in plane of imaging available for study.
For quantitative analysis of MMP changes, two analytical values were used. Average intensity is a measure of absolute MMP, but it underestimates changes of MMP if they occur below the threshold level (Figure 4A). The relative value is obtained by the division of integrated density to cell number considering the presence of cells with completely depolarized MMP. This value is applied more specifically and therefore was named “mean cell fluorescence” (Figure 4B). The decrease of average intensity caused by PHA treatment is lower than the corresponding decrease of mean cell fluorescence by 5%. This dissimilarity shows the presence of cells whose fluorescence changed below the detection limit. The changes in the selection area are calculated as the total selection area on images divided by the number of cells attributing to these fluorescent images (Figure 4C).
Quantification of MMP changes in U937 cells during PHA treatment (30 μg/ml, 2 h). (A–C) quantitative data retrieved from images acquired in one (B, C) and three (A) experiments. Data are expressed as means ±SD. The number of images that is used in the analysis is shown near each bar. (A) Average TMRE fluorescence intensity found in selection area. (B) Mean cell fluorescence calculated as integrated density divided by the number of cells. (C) Mean area of selection calculated as total selection area divided by the number of cells. (D) Protocol of imaging processing and analysis. Most important operations are shown by arrows, while its results are indicated in boxes: RAW, original image; EQH, original image after histogram equalization; BS and BIN, images after running “subtracted background” and “threshold” commands, respectively; mask, mask image; MSK, image obtained by multiplying EQH image by mask image. Dot lines show the operations used for the creation of masked image (MSK).
The relation between these analytical options can be understood from the schematic representation of full analytical process shown in Figure 4D. It started from original image (RAW), the duplicate of which passes three steps until it resulted in the generation of mask or the selection of congruent regions on RAW image with output of numerical results. Histogram equalization, background subtraction, and thresholding were done similarly to those published in earlier works [6, 28]. The method presented in Figure 4D also gives possibility to control mitochondrial selection area and compare it with already known morphological data. From the data presented in Figure 4C and from known value of cell area (120 μm2), it can be concluded that selected area constitutes about 10% of cell area. This is close, for example, to a value of 8% in tumorigenic cell lines obtained by electron-microscopic examination [10].
The decrease of average fluorescence intensity by 34% after 2 h of PHA treatment is attributed to the changes of absolute MMP value. While our data were obtained on the early stage of stimulation, they can be correlated with data on metabolic shift that occurs in lymphoid cells after prolonged treatment. These data suggest that the role of the mitochondria in total energy balance decreases during stimulation. It was shown that glycolytic activity in lymphocytes increases by 36 times but the respiratory activity is only by 43% [21]. In thymocytes that obtain energy mainly by glucose oxidation, lectin treatment leads to the deceleration of this pathway [47]. On the contrary, in mesenchymal stem cell, mitochondrial activity is increased during malignant transformation [10].
The difference in selection area between control- and PHA-treated cells (Figure 4C) suggests that MMP in some cells is completely lost. These changes could be related to the changes of [Ca2+]c because it is known that [Ca2+]c is increased after mitogenic stimulation [48]. The mitochondria play essential role in the regulation of [Ca2+]c [31, 49]. During metabolic inhibition in MDCK cells, [Ca2+]c is transiently increased. Restoration of normal [Ca2+]c occurs by coupled action mitochondrial Na+/Ca2+ and Na+/H+ exchangers. Metabolic inhibition decreases proton gradient on mitochondrial membrane [18] and reduces outward Na+ and Ca2+ movement mediated by these transporters. As a consequence, Na+ and Ca2+ concentration in mitochondrial matrix is steadily increased [16, 17].
Similar situation may exist in stimulated lymphoid cells where the magnitude of proton electrochemical gradient is compromised by the changes of pHc. The Na+ content in lymphocytes is increased rapidly after PHA treatment with maximum of 2 h [50]. Both K+ content and influx are also increased during mitogenic stimulation [50]. Therefore, the increase of cytoplasmic [Na+] ([Na+]c) in response to lectin treatment is mediated by Na+/H+ exchanger. This view agrees with the increase of pHc in both thymocytes and lymphocytes after lectin treatment [51, 52]. To test whether these changes take place in U937 cells during PHA stimulation, we performed investigation of [Na+]c by Na-sensitive dye Asante Natrium Green-2 (ANG). As followed from the flow cytometric data, the fluorescence of ANG was increased after 1 h of PHA treatment (Figure 5). The mean ANG fluorescence increased by PHA treatment from 1.89E+05 to 2.59E+05 relative units. Assuming that changes of fluorescence are proportional to the changes of concentration, [Na+]c will increase from 30 mM in resting U937 cells to 38 mM upon PHA treatment.
Effect of PHA treatment (30 μg/ml, 1 h) on fluorescence of ANG dye. Cells stained with ANG-AM (1 μM) during the last 40 min of PHA treatment (37°C). ANG fluorescence was excited using a 488 nm laser, and emission was detected in the FITC channel with a 525/40 nm bandpass. Data were analyzed using CytExpert 2.0 Beckman Coulter software. The major cell populations were selected for analysis using forward/side scatter plot. Data were obtained using the same samples as data presented in
The demonstrated increase of [Na+]c in PHA-stimulated U937 cells may be considered as part of the regulatory mechanism involved in the increase of [Ca2+]m. The increase of [Ca2+]m in many cell types causes activation of mitochondrial dehydrogenases [49] and accounts for the rapid activation of mitochondrial PDH in response to lectin action [23, 53]. The increase of [Ca2+]m can also lead to mitochondrial injury that was confirmed by serious ultra-structural changes observed in human lymphocytes after 3 days of lectin treatment [36, 54]. The deleterious effect of lectins on mitochondrial morphology, however, was absent in other experiments [19, 37, 54]. Controversy of results obtained in these studies can be explained by the complexity of regulatory mechanisms controlling mitochondrial Ca2+ and Na+ homeostasis. This reason can also be applied in our study in which a large variation in the number of cells with complete mitochondrial depolarization was noticed.
The role of the mitochondria in energy supply is determined by the functional state of the cells. This problem is usually addressed by biochemical, polarographic and optical methods which measure ATP production and oxygen consumption in the living cell. In many studies, it is also corroborated by parallel measurement of mitochondrial membrane potential (MMP). This article describes fluorescent method of measuring MMP in cancer cell using potential-sensitive dye TMRE. This method can distinguish fluorescence of TMRE in mitochondrial and nonmitochondrial compartment by using standard software for image analysis.
I acknowledge TS Goryachaya for maintaining a cell culture and ND Aksenov for assistance with flow cytometry investigations. I am very grateful to JP Battersby for discussions leading to the current study and to GI Shtein for technical consultations. I am also thankful to Prof. VS Saakov for his help in the writing of this manuscript.
Stability constant of the formation of metal complexes is used to measure interaction strength of reagents. From this process, metal ion and ligand interaction formed the two types of metal complexes; one is supramolecular complexes known as host-guest complexes [1] and the other is anion-containing complexes. In the solution it provides and calculates the required information about the concentration of metal complexes.
Solubility, light, absorption conductance, partitioning behavior, conductance, and chemical reactivity are the complex characteristics which are different from their components. It is determined by various numerical and graphical methods which calculate the equilibrium constants. This is based on or related to a quantity, and this is called the complex formation function.
During the displacement process at the time of metal complex formation, some ions disappear and form a bonding between metal ions and ligands. It may be considered due to displacement of a proton from a ligand species or ions or molecules causing a drop in the pH values of the solution [2]. Irving and Rossotti developed a technique for the calculation of stability constant, and it is called potentiometric technique.
To determine the stability constant, Bjerrum has used a very simple method, and that is metal salt solubility method. For the studies of a larger different variety of polycarboxylic acid-, oxime-, phenol-containing metal complexes, Martel and Calvin used the potentiometric technique for calculating the stability constant. Those ligands [3, 4] which are uncharged are also examined, and their stability constant calculations are determined by the limitations inherent in the ligand solubility method. The limitations of the metal salt solubility method and the result of solubility methods are compared with this. M-L, MLM, and (M3) L are some types of examples of metal-ligand bonding. One thing is common, and that is these entire types metal complexes all have one ligand.
The solubility method can only usefully be applied to studies of such complexes, and it is best applied for ML; in such types of system, only ML is formed. Jacqueline Gonzalez and his co-worker propose to explore the coordination chemistry of calcium complexes. Jacqueline and et al. followed this technique for evaluate the as partial model of the manganese-calcium cluster and spectrophotometric studies of metal complexes, i.e., they were carried calcium(II)-1,4-butanediamine in acetonitrile and calcium(II)-1,2-ethylendiamine, calcium(II)-1,3-propanediamine by them.
Spectrophotometric programming of HypSpec and received data allows the determination of the formation of solubility constants. The logarithmic values, log β110 = 5.25 for calcium(II)-1,3-propanediamine, log β110 = 4.072 for calcium(II)-1,4-butanediamine, and log β110 = 4.69 for calcium(II)-1,2-ethylendiamine, are obtained for the formation constants [5]. The structure of Cimetidine and histamine H2-receptor is a chelating agent. Syed Ahmad Tirmizi has examined Ni(II) cimetidine complex spectrophotometrically and found an absorption peak maximum of 622 nm with respect to different temperatures.
Syed Ahmad Tirmizi have been used to taken 1:2 ratio of metal and cimetidine compound for the formation of metal complex and this satisfied by molar ratio data. The data, 1.40–2.4 × 108, was calculated using the continuous variation method and stability constant at room temperature, and by using the mole ratio method, this value at 40°C was 1.24–2.4 × 108. In the formation of lead(II) metal complexes with 1-(aminomethyl) cyclohexene, Thanavelan et al. found the formation of their binary and ternary complexes. Glycine, l-proline, l-alanine, l-isoleucine, l-valine, and l-leucine are α-amino acids, and these are important biologically [6]. These α-amino acids are also investigated by potentiometric technique at 32°C. The mixed ligands were also studied using these methods. 50% (v/v) DMSO-water medium used for the determination of acidity constants and their stability constants these type ligands. In a stepwise manner, the ternary complexes were synthesized.
Using the stability constant method, these ternary complexes were found out, and using the parameters such as Δ log
The above acids (gallic and aliphatic dicarboxylic acid) were taken to determine the acidity constants. For the purpose of determining the stability constant, binary and ternary complexes were carried in the aqueous medium using the experimental conditions as stated above. The potentiometric pH-metric titration curves are inferred for the binary complexes and ternary complexes at different ratios, and formation of ternary metal complex formation was in a stepwise manner that provided an easy way to calculate stability constants for the formation of metal complexes.
The values of Δ log
A study by Kathrina and Pekar suggests that pH plays an important role in the formation of metal complexes. When epigallocatechin gallate and gallic acid combine with copper(II) to form metal complexes, the pH changes its speculation. We have been able to determine its pH in frozen and fluid state with the help of multifrequency EPR spectroscopy [8]. With the help of this spectroscopy, it is able to detect that each polyphenol exhibits the formation of three different mononuclear species. If the pH ranges 4–8 for di- or polymeric complex of Cu(II), then it conjectures such metal complexes. It is only at alkaline pH values.
The line width in fluid solutions by molecular motion exhibits an incomplete average of the parameters of anisotropy spin Hamilton. If the complexes are different, then their rotational correlation times for this also vary. The analysis of the LyCEP anisotropy of the fluid solution spectra is performed using the parameters determined by the simulation of the rigid boundary spectra. Its result suggests that pH increases its value by affecting its molecular mass. It is a polyphenol ligand complex with copper, showing the coordination of an increasing number of its molecules or increasing participation of polyphenol dimers used as ligands in the copper coordination region.
The study by Vishenkova and his co-worker [8] provides the investigation of electrochemical properties of triphenylmethane dyes using a voltammetric method with constant-current potential sweep. Malachite green (MG) and basic fuchsin (BF) have been chosen as representatives of the triphenylmethane dyes [9]. The electrochemical behavior of MG and BF on the surface of a mercury film electrode depending on pH, the nature of background electrolyte, and scan rate of potential sweep has been investigated.
Using a voltammetric method with a constant-current potential sweep examines the electrical properties of triphenylmethane dye. In order to find out the solution of MG and BF, certain registration conditions have been prescribed for it, which have proved to be quite useful. The reduction peak for the currents of MG and BF has demonstrated that it increases linearly with respect to their concentration as 9.0 × 10−5–7.0 × 10−3 mol/dm3 for MG and 6.0 × 10−5–8.0 × 10−3 mol/dm3 for BF and correlation coefficients of these values are 0.9987 for MG and 0.9961 for BF [10].
5.0 × 10−5 and 2.0 × 10−5 mol/dm3 are the values used as the detection limit of MG and BF, respectively. Stability constants are a very useful technique whose size is huge. Due to its usefulness, it has acquired an umbrella right in the fields of chemistry, biology, and medicine. No science subject is untouched by this. Stability constants of metal complexes are widely used in the various areas like pharmaceuticals as well as biological processes, separation techniques, analytical processes, etc. In the presented chapter, we have tried to explain this in detail by focusing our attention on the applications and solutions of stability of metal complexes in solution.
Stability or formation or binding constant is the type of equilibrium constant used for the formation of metal complexes in the solution. Acutely, stability constant is applicable to measure the strength of interactions between the ligands and metal ions that are involved in complex formation in the solution [11]. A generally these 1-4 equations are expressed as the following ways:
Thus
K1, K2, K3, … Kn are the equilibrium constants and these are also called stepwise stability constants. The formation of the metal-ligand-n complex may also be expressed as equilibrium constants by the following steps:
The parameters K and β are related together, and these are expressed in the following example:
Now the numerator and denominator are multiplied together with the use of [metal-ligand] [metal-ligand2], and after the rearranging we get the following equation:
Now we expressed it as the following:
From the above relation, it is clear that the overall stability constant βn is equal to the product of the successive (i.e., stepwise) stability constants, K1, K2, K3,…Kn. This in other words means that the value of stability constants for a given complex is actually made up of a number of stepwise stability constants. The term stability is used without qualification to mean that the complex exists under a suitable condition and that it is possible to store the complex for an appreciable amount of time. The term stability is commonly used because coordination compounds are stable in one reagent but dissociate or dissolve in the presence of another regent. It is also possible that the term stability can be referred as an action of heat or light or compound. The stability of complex [13] is expressed qualitatively in terms of thermodynamic stability and kinetic stability.
In a chemical reaction, chemical equilibrium is a state in which the concentration of reactants and products does not change over time. Often this condition occurs when the speed of forward reaction becomes the same as the speed of reverse reaction. It is worth noting that the velocities of the forward and backward reaction are not zero at this stage but are equal.
If hydrogen and iodine are kept together in molecular proportions in a closed process vessel at high temperature (500°C), the following action begins:
In this activity, hydrogen iodide is formed by combining hydrogen and iodine, and the amount of hydrogen iodide increases with time. In contrast to this action, if the pure hydrogen iodide gas is heated to 500°C in the reaction, the compound is dissolved by reverse action, which causes hydrogen iodide to dissolve into hydrogen and iodine, and the ratio of these products increases over time. This is expressed in the following reaction:
For the formation of metal chelates, the thermodynamic technique provides a very significant information. Thermodynamics is a very useful technique in distinguishing between enthalpic effects and entropic effects. The bond strengths are totally effected by enthalpic effect, and this does not make any difference in the whole solution in order/disorder. Based on thermodynamics the chelate effect below can be best explained. The change of standard Gibbs free energy for equilibrium constant is response:
Where:
R = gas constant
T = absolute temperature
At 25°C,
ΔG = (− 5.708 kJ mol−1) · log β.
The enthalpy term creates free energy, i.e.,
For metal complexes, thermodynamic stability and kinetic stability are two interpretations of the stability constant in the solution. If reaction moves from reactants to products, it refers to a change in its energy as shown in the above equation. But for the reactivity, kinetic stability is responsible for this system, and this refers to ligand species [14].
Stable and unstable are thermodynamic terms, while labile and inert are kinetic terms. As a rule of thumb, those complexes which react completely within about 1 minute at 25°C are considered labile, and those complexes which take longer time than this to react are considered inert. [Ni(CN)4]2− is thermodynamically stable but kinetically inert because it rapidly exchanges ligands.
The metal complexes [Co(NH3)6]3+ and such types of other complexes are kinetically inert, but these are thermodynamically unstable. We may expect the complex to decompose in the presence of acid immediately because the complex is thermodynamically unstable. The rate is of the order of 1025 for the decomposition in acidic solution. Hence, it is thermodynamically unstable. However, nothing happens to the complex when it is kept in acidic solution for several days. While considering the stability of a complex, always the condition must be specified. Under what condition, the complex which is stable or unstable must be specified such as acidic and also basic condition, temperature, reactant, etc.
A complex may be stable with respect to a particular condition but with respect to another. In brief, a stable complex need not be inert and similarly, and an unstable complex need not be labile. It is the measure of extent of formation or transformation of complex under a given set of conditions at equilibrium [15].
Thermodynamic stability has an important role in determining the bond strength between metal ligands. Some complexes are stable, but as soon as they are introduced into aqueous solution, it is seen that these complexes have an effect on stability and fall apart. For an example, we take the [Co (SCN)4]2+ complex. The ion bond of this complex is very weak and breaks down quickly to form other compounds. But when [Fe(CN)6]3− is dissolved in water, it does not test Fe3+ by any sensitive reagent, which shows that this complex is more stable in aqueous solution. So it is indicated that thermodynamic stability deals with metal-ligand bond energy, stability constant, and other thermodynamic parameters.
This example also suggests that thermodynamic stability refers to the stability and instability of complexes. The measurement of the extent to which one type of species is converted to another species can be determined by thermodynamic stability until equilibrium is achieved. For example, tetracyanonickelate is a thermodynamically stable and kinetic labile complex. But the example of hexa-amine cobalt(III) cation is just the opposite:
Thermodynamics is used to express the difference between stability and inertia. For the stable complex, large positive free energies have been obtained from ΔG0 reaction. The ΔH0, standard enthalpy change for this reaction, is related to the equilibrium constant, βn, by the well thermodynamic equation:
For similar complexes of various ions of the same charge of a particular transition series and particular ligand, ΔS0 values would not differ substantially, and hence a change in ΔH0 value would be related to change in βn values. So the order of values of ΔH0 is also the order of the βn value.
Kinetic stability is referred to the rate of reaction between the metal ions and ligand proceeds at equilibrium or used for the formation of metal complexes. To take a decision for kinetic stability of any complexes, time is a factor which plays an important role for this. It deals between the rate of reaction and what is the mechanism of this metal complex reaction.
As we discuss above in thermodynamic stability, kinetic stability is referred for the complexes at which complex is inert or labile. The term “inert” was used by Tube for the thermally stable complex and for reactive complexes the term ‘labile’ used [16]. The naturally occurring chlorophyll is the example of polydentate ligand. This complex is extremely inert due to exchange of Mg2+ ion in the aqueous media.
The nature of central atom of metal complexes, dimension, its degree of oxidation, electronic structure of these complexes, and so many other properties of complexes are affected by the stability constant. Some of the following factors described are as follows.
In the coordination chemistry, metal complexes are formed by the interaction between metal ions and ligands. For these type of compounds, metal ions are the coordination center, and the ligand or complexing agents are oriented surrounding it. These metal ions mostly are the transition elements. For the determination of stability constant, some important characteristics of these metal complexes may be as given below.
Ligands are oriented around the central metal ions in the metal complexes. The sizes of these metal ions determine the number of ligand species that will be attached or ordinated (dative covalent) in the bond formation. If the sizes of these metal ions are increased, the stability of coordination compound defiantly decreased. Zn(II) metal ions are the central atoms in their complexes, and due to their lower size (0.74A°) as compared to Cd(II) size (0.97A°), metal ions are formed more stable.
Hence, Al3+ ion has the greatest nuclear charge, but its size is the smallest, and the ion N3− has the smallest nuclear charge, and its size is the largest [17]. Inert atoms like neon do not participate in the formation of the covalent or ionic compound, and these atoms are not included in isoelectronic series; hence, it is not easy to measure the radius of this type of atoms.
The properties of stability depend on the size of the metal ion used in the complexes and the total charge thereon. If the size of these metal ions is small and the total charge is high, then their complexes will be more stable. That is, their ratio will depend on the charge/radius. This can be demonstrated through the following reaction:
An ionic charge is the electric charge of an ion which is formed by the gain (negative charge) or loss (positive charge) of one or more electrons from an atom or group of atoms. If we talk about the stability of the coordination compounds, we find that the total charge of their central metal ions affects their stability, so when we change their charge, their stability in a range of constant can be determined by propagating of error [18]. If the charge of the central metal ion is high and the size is small, the stability of the compound is high:
In general, the most stable coordination bonds can cause smaller and highly charged rations to form more stable coordination compounds.
When an electron pair attracts a central ion toward itself, a strong stability complex is formed, and this is due to electron donation from ligand → metal ion. This donation process is increasing the bond stability of metal complexes exerted the polarizing effect on certain metal ions. Li+, Na+, Mg2+, Ca2+, Al3+, etc. are such type of metal cation which is not able to attract so strongly from a highly electronegative containing stable complexes, and these atoms are O, N, F, Au, Hg, Ag, Pd, Pt, and Pb. Such type of ligands that contains P, S, As, Br and I atom are formed stable complex because these accepts electron from M → π-bonding. Hg2+, Pb2+, Cd2+, and Bi3+ metal ions are also electronegative ions which form insoluble salts of metal sulfide which are insoluble in aqueous medium.
Volatile ligands may be lost at higher temperature. This is exemplified by the loss of water by hydrates and ammonia:
The transformation of certain coordination compounds from one to another is shown as follows:
A ligand is an ion or small molecule that binds to a metal atom (in chemistry) or to a biomolecule (in biochemistry) to form a complex, such as the iron-cyanide coordination complex Prussian blue or the iron-containing blood-protein hemoglobin. The ligands are arranged in spectrochemical series which are based on the order of their field strength. It is not possible to form the entire series by studying complexes with a single metal ion; the series has been developed by overlapping different sequences obtained from spectroscopic studies [19]. The order of common ligands according to their increasing ligand field strength is
The above spectrochemical series help us to for determination of strength of ligands. The left last ligand is as weaker ligand. These weaker ligand cannot forcible binding the 3d electron and resultant outer octahedral complexes formed. It is as-
Increasing the oxidation number the value of Δ increased.
Δ increases from top to bottom.
However, when we consider the metal ion, the following two useful trends are observed:
Δ increases with increasing oxidation number.
Δ increases down a group. For the determination of stability constant, the nature of the ligand plays an important role.
The following factors described the nature of ligands.
The size and charge are two factors that affect the production of metal complexes. The less charges and small sizes of ligands are more favorable for less stable bond formation with metal and ligand. But if this condition just opposite the product of metal and ligand will be a more stable compound. So, less nuclear charge and more size= less stable complex whereas if more nuclear charge and small in size= less stable complex. We take fluoride as an example because due to their smaller size than other halide and their highest electro negativity than the other halides formed more stable complexes. So, fluoride ion complexes are more stable than the other halides:
As compared to S2− ion, O22− ions formed more stable complexes.
It is suggested by Calvin and Wilson that the metal complexes will be more stable if the basic character or strength of ligands is higher. It means that the donating power of ligands to central metal ions is high [20].
It means that the donating power of ligands to central metal ions is high. In the case of complex formation of aliphatic diamines and aromatic diamines, the stable complex is formed by aliphatic diamines, while an unstable coordination complex is formed with aromatic diamines. So, from the above discussion, we find that the stability will be grater if the e-donation power is greater.
Thus it is clear that greater basic power of electron-donating species will form always a stable complex. NH3, CN−, and F− behaved as ligands and formed stable complexes; on the other hand, these are more basic in nature.
We know that if the concentration of coordination group is higher, these coordination compounds will exist in the water as solution. It is noted that greater coordinating tendency show the water molecules than the coordinating group which is originally present. SCN− (thiocynate) ions are present in higher concentration; with the Co2+ metal ion, it formed a blue-colored complex which is stable in state, but on dilution of water medium, a pink color is generated in place of blue, or blue color complex is destroyed by [Co(H2O)6]2+, and now if we added further SCN−, the pink color will not appear:
Now it is clear that H2O and SCN− are in competition for the formation of Co(II) metal-containing complex compound. In the case of tetra-amine cupric sulfate metal complex, ammonia acts as a donor atom or ligand. If the concentration of NH3 is lower in the reaction, copper hydroxide is formed but at higher concentration formed tetra-amine cupric sulfate as in the following reaction:
For a metal ion, chelating ligand is enhanced and affinity it and this is known as chelate effect and compared it with non-chelating and monodentate ligand or the multidentate ligand is acts as chelating agent. Ethylenediamine is a simple chelating agent (Figure 1).
Structure of ethylenediamine.
Due to the bidentate nature of ethylenediamine, it forms two bonds with metal ion or central atom. Water forms a complex with Ni(II) metal ion, but due to its monodentate nature, it is not a chelating ligand (Figures 2 and 3).
Structure of chelating configuration of ethylenediamine ligand.
Structure of chelate with three ethylenediamine ligands.
The dentate cheater of ligand provides bonding strength to the metal ion or central atom, and as the number of dentate increased, the tightness also increased. This phenomenon is known as chelating effect, whereas the formation of metal complexes with these chelating ligands is called chelation:
or
Some factors are of much importance for chelation as follows.
The sizes of the chelating ring are increased as well as the stability of metal complex decreased. According to Schwarzenbach, connecting bridges form the chelating rings. The elongated ring predominates when long bridges connect to the ligand to form a long ring. It is usually observed that an increased a chelate ring size leads to a decrease in complex stability.
He interpreted this statement. The entropy of complex will be change if the size of chelating ring is increased, i.e., second donor atom is allowed by the chelating ring. As the size of chelating ring increased, the stability should be increased with entropy effect. Four-membered ring compounds are unstable, whereas five-membered are more stable. So the chelating ring increased its size and the stability of the formed metal complexes.
The number of chelating rings also decides the stability of complexes. Non-chelating metal compounds are less stable than chelating compounds. These numbers increase the thermodynamic volume, and this is also known as an entropy term. In recent years ligands capable of occupying as many as six coordination positions on a single metal ion have been described. The studies on the formation constants of coordination compounds with these ligands have been reported. The numbers of ligand or chelating agents are affecting the stability of metal complexes so as these numbers go up and down, the stability will also vary with it.
For the Ni(II) complexes with ethylenediamine as chelating agent, its log K1 value is 7.9 and if chelating agents are trine and penten, then the log K1 values are 7.9 and 19.3, respectively. If the metal ion change Zn is used in place of Ni (II), then the values of log K1 for ethylenediamine, trine, and penten are 6.0, 12.1, and 16.2, respectively. The log βMY values of metal ions are given in Table 1.
Metal ion | log βMY (25°C, I = 0.1 M) |
---|---|
Ca2+ | 11.2 |
Cu2+ | 19.8 |
Fe3+ | 24.9 |
Metal ion vs. log βMY values.
Ni(NH3)62+ is an octahedral metal complex, and at 25 °C its log β6 value is 8.3, but Ni(ethylenediamine)32+ complex is also octahedral in geometry, with 18.4 as the value of log β6. The calculated stability value of Ni(ethylenediamine)32+ 1010 times is more stable because three rings are formed as chelating rings by ethylenediamine as compared to no such ring is formed. Ethylenediaminetetraacetate (EDTA) is a hexadentate ligand that usually formed stable metal complexes due to its chelating power.
A special effect in molecules is when the atoms occupy space. This is called steric effect. Energy is needed to bring these atoms closer to each other. These electrons run away from near atoms. There can be many ways of generating it. We know the repulsion between valence electrons as the steric effect which increases the energy of the current system [21]. Favorable or unfavorable any response is created.
For example, if the static effect is greater than that of a product in a metal complex formation process, then the static increase would favor this reaction. But if the case is opposite, the skepticism will be toward retardation.
This effect will mainly depend on the conformational states, and the minimum steric interaction theory can also be considered. The effect of secondary steric is seen on receptor binding produced by an alternative such as:
Reduced access to a critical group.
Stick barrier.
Electronic resonance substitution bond by repulsion.
Population of a conformer changes due to active shielding effect.
The macrocyclic effect is exactly like the image of the chelate effect. It means the principle of both is the same. But the macrocyclic effect suggests cyclic deformation of the ligand. Macrocyclic ligands are more tainted than chelating agents. Rather, their compounds are more stable due to their cyclically constrained constriction. It requires some entropy in the body to react with the metal ion. For example, for a tetradentate cyclic ligand, we can use heme-B which forms a metal complex using Fe+2 ions in biological systems (Figure 4).
Structure of hemoglobin is the biological complex compound which contains Fe(II) metal ion.
The n-dentate chelating agents play an important role for the formation of more stable metal complexes as compared to n-unidentate ligands. But the n-dentate macrocyclic ligand gives more stable environment in the metal complexes as compared to open-chain ligands. This change is very favorable for entropy (ΔS) and enthalpy (ΔH) change.
There are so many parameters to determination of formation constants or stability constant in solution for all types of chelating agents. These numerous parameters or techniques are refractive index, conductance, temperature, distribution coefficients, refractive index, nuclear magnetic resonance volume changes, and optical activity.
Solubility products are helpful and used for the insoluble salt that metal ions formed and complexes which are also formed by metal ions and are more soluble. The formation constant is observed in presence of donor atoms by measuring increased solubility.
To determine the solubility constant, it involves the distribution of the ligands or any complex species; metal ions are present in two immiscible solvents like water and carbon tetrachloride, benzene, etc.
In this method metal ions or ligands are present in solution and on exchanger. A solid polymers containing with positive and negative ions are ion exchange resins. These are insoluble in nature. This technique is helpful to determine the metal ions in resin phase, liquid phase, or even in radioactive metal. This method is also helpful to determine the polarizing effect of metal ions on the stability of ligands like Cu(II) and Zn(II) with amino acid complex formation.
At the equilibrium free metal and ions are present in the solution, and using the different electrometric techniques as described determines its stability constant.
This method is based upon the titration method or follows its principle. A stranded acid-base solution used as titrate and which is titrated, it may be strong base or strong acid follows as potentiometrically. The concentration of solution using 103− M does not decomposed during the reaction process, and this method is useful for protonated and nonprotonated ligands.
This is the graphic method used to determine the stability constant in producing metal complex formation by plotting a polarograph between the absences of substances and the presence of substances. During the complex formation, the presence of metal ions produced a shift in the half-wave potential in the solution.
If a complex is relatively slow to form and also decomposes at measurable rate, it is possible, in favorable situations, to determine the equilibrium constant.
This involves the study of the equilibrium constant of slow complex formation reactions. The use of tracer technique is extremely useful for determining the concentrations of dissociation products of the coordination compound.
This method is based on the study of the effect of an equilibrium concentration of some ions on the function at a definite organ of a living organism. The equilibrium concentration of the ion studied may be determined by the action of this organ in systems with complex formation.
The solution of 25 ml is adopted by preparing at the 1.0 × 10−5 M ligand or 1.0 × 10−5 M concentration and 1.0 × 10−5 M for the metal ion:
The solutions containing the metal ions were considered both at a pH sufficiently high to give almost complete complexation and at a pH value selected in order to obtain an equilibrium system of ligand and complexes.
In order to avoid modification of the spectral behavior of the ligand due to pH variations, it has been verified that the range of pH considered in all cases does not affect absorbance values. Use the collected pH values adopted for the determinations as well as selected wavelengths. The ionic strengths calculated from the composition of solutions allowed activity coefficient corrections. Absorbance values were determined at wavelengths in the range 430–700 nm, every 2 nm.
For a successive metal complex formation, use this method. If ligand is protonate and the produced complex has maximum number of donate atoms of ligands, a selective light is absorbed by this complex, while for determination of stability constant, it is just known about the composition of formed species.
Bjerrum (1941) used the method stepwise addition of the ligands to coordination sphere for the formation of complex. So, complex metal–ligand-n forms as the following steps [22]. The equilibrium constants, K1, K2, K3, … Kn are called stepwise stability constants. The formation of the complex metal-ligandn may also be expressed by the following steps and equilibrium constants.
Where:
M = central metal cation
L = monodentate ligand
N = maximum coordination number for the metal ion M for the ligand
If a complex ion is slow to reach equilibrium, it is often possible to apply the method of isotopic dilution to determine the equilibrium concentration of one or more of the species. Most often radioactive isotopes are used.
This method was extensively used by Werner and others to study metal complexes. In the case of a series of complexes of Co(III) and Pt(IV), Werner assigned the correct formulae on the basis of their molar conductance values measured in freshly prepared dilute solutions. In some cases, the conductance of the solution increased with time due to a chemical change, e.g.,
It is concluded that the information presented is very important to determine the stability constant of the ligand metal complexes. Some methods like spectrophotometric method, Bjerrum’s method, distribution method, ion exchange method, electrometric techniques, and potentiometric method have a huge contribution in quantitative analysis by easily finding the stability constants of metal complexes in aqueous solutions.
All the authors thank the Library of University of Delhi for reference books, journals, etc. which helped us a lot in reviewing the chapter.
Supporting women in scientific research and encouraging more women to pursue careers in STEM fields has been an issue on the global agenda for many years. But there is still much to be done. And IntechOpen wants to help.
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