Gain of fluorescence intensity, obtained with various cuvettes.
\\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 member of secretary of Institution Animal Ethics Committee (IAEC) since the year 2008.",coeditorThreeBiosketch:"Associate Professor, Former Research Development Manager, and investigator involved in the pharmacological evaluation, isolation of bioactive and formulation development. Dr. Irfan has 12 years of experience in the Research & Formulation development of Ayurvedic, Herbal, and Nutraceutical products and successfully developed more than 100 formulations. He has 25-original papers in International journals of repute.",coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"345595",title:"Prof.",name:"Juber",middleName:null,surname:"Akhtar",slug:"juber-akhtar",fullName:"Juber Akhtar",profilePictureURL:"https://mts.intechopen.com/storage/users/345595/images/system/345595.jpg",biography:"Dr. Juber Akhtar completed his B.Pharm in 2005 from Jamia Hamdard University, New Delhi. In 2007 he completed his M.Pharm from Manipal University, Karnataka. 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Dr. Akhtar is actively involved in research activities and his areas of interest include development of nano particulate drug delivery system for targeting to various organs.",institutionString:"Integral University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"0",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"Integral University",institutionURL:null,country:{name:"India"}}}],coeditorOne:{id:"255360",title:"Dr.",name:"Usama",middleName:null,surname:"Ahmad",slug:"usama-ahmad",fullName:"Usama Ahmad",profilePictureURL:"https://mts.intechopen.com/storage/users/255360/images/system/255360.jpg",biography:"Dr. Usama Ahmad holds a specialization in Pharmaceutics from Amity University, Lucknow, India. He received his Ph.D. degree from Integral University on his work titled ‘Development and evaluation of silymarin nanoformulation for hepatic carcinoma’. Currently, he’s working as an Assistant Professor of Pharmaceutics in the Faculty of Pharmacy, Integral University. He has been teaching Pharm.D, B.Pharm, and M.Pharm students and conducting research in the novel drug delivery domain. From 2013 to 2014 he worked on a research project funded by SERB-DST, Government of India. He has a rich publication record with more than 24 original articles published in reputed journals, 2 edited books with IntechOpen, 4 book chapters, and a number of scientific articles published in ‘Ingredients South Asia Magazine’ and ‘QualPharma Magazine’. He is a member of the American Association for Cancer Research, International Association for the Study of Lung Cancer, and the British Society for Nanomedicine. Dr. Ahmad’s research focus is on the development of nanoformulations to facilitate the delivery of drugs that aim to provide practical solutions to current healthcare problems.",institutionString:"Integral University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"2",totalChapterViews:"0",totalEditedBooks:"1",institution:{name:"Integral University",institutionURL:null,country:{name:"India"}}},coeditorTwo:{id:"345932",title:"Dr.",name:"x",middleName:null,surname:"Badruddeen",slug:"x-badruddeen",fullName:"x Badruddeen",profilePictureURL:"https://mts.intechopen.com/storage/users/no_image.jpg",biography:null,institutionString:"Integral University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"0",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"Integral University",institutionURL:null,country:{name:"India"}}},coeditorThree:{id:"345933",title:"Dr.",name:"Mohammad Irfan",middleName:null,surname:"Khan",slug:"mohammad-irfan-khan",fullName:"Mohammad Irfan Khan",profilePictureURL:"https://mts.intechopen.com/storage/users/no_image.jpg",biography:null,institutionString:"Integral University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"0",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"Integral University",institutionURL:null,country:{name:"India"}}},coeditorFour:null,coeditorFive:null,topics:[{id:"19",title:"Pharmacology, Toxicology and Pharmaceutical Science",slug:"pharmacology-toxicology-and-pharmaceutical-science"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"301331",firstName:"Mia",lastName:"Vulovic",middleName:null,title:"Mrs.",imageUrl:"https://mts.intechopen.com/storage/users/301331/images/8498_n.jpg",email:"mia.v@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. Whether that be identifying an exceptional author and proposing an editorship collaboration, or contacting researchers who would like the opportunity to work with IntechOpen, I establish and help manage author and editor acquisition and contact."}},relatedBooks:[{type:"book",id:"1591",title:"Infrared Spectroscopy",subtitle:"Materials Science, Engineering and Technology",isOpenForSubmission:!1,hash:"99b4b7b71a8caeb693ed762b40b017f4",slug:"infrared-spectroscopy-materials-science-engineering-and-technology",bookSignature:"Theophile Theophanides",coverURL:"https://cdn.intechopen.com/books/images_new/1591.jpg",editedByType:"Edited by",editors:[{id:"37194",title:"Dr.",name:"Theophanides",surname:"Theophile",slug:"theophanides-theophile",fullName:"Theophanides Theophile"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3161",title:"Frontiers in Guided Wave Optics and Optoelectronics",subtitle:null,isOpenForSubmission:!1,hash:"deb44e9c99f82bbce1083abea743146c",slug:"frontiers-in-guided-wave-optics-and-optoelectronics",bookSignature:"Bishnu Pal",coverURL:"https://cdn.intechopen.com/books/images_new/3161.jpg",editedByType:"Edited by",editors:[{id:"4782",title:"Prof.",name:"Bishnu",surname:"Pal",slug:"bishnu-pal",fullName:"Bishnu Pal"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3092",title:"Anopheles mosquitoes",subtitle:"New insights into malaria vectors",isOpenForSubmission:!1,hash:"c9e622485316d5e296288bf24d2b0d64",slug:"anopheles-mosquitoes-new-insights-into-malaria-vectors",bookSignature:"Sylvie Manguin",coverURL:"https://cdn.intechopen.com/books/images_new/3092.jpg",editedByType:"Edited by",editors:[{id:"50017",title:"Prof.",name:"Sylvie",surname:"Manguin",slug:"sylvie-manguin",fullName:"Sylvie Manguin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"371",title:"Abiotic Stress in Plants",subtitle:"Mechanisms and Adaptations",isOpenForSubmission:!1,hash:"588466f487e307619849d72389178a74",slug:"abiotic-stress-in-plants-mechanisms-and-adaptations",bookSignature:"Arun Shanker and B. Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"72",title:"Ionic Liquids",subtitle:"Theory, Properties, New Approaches",isOpenForSubmission:!1,hash:"d94ffa3cfa10505e3b1d676d46fcd3f5",slug:"ionic-liquids-theory-properties-new-approaches",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/72.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"314",title:"Regenerative Medicine and Tissue Engineering",subtitle:"Cells and Biomaterials",isOpenForSubmission:!1,hash:"bb67e80e480c86bb8315458012d65686",slug:"regenerative-medicine-and-tissue-engineering-cells-and-biomaterials",bookSignature:"Daniel Eberli",coverURL:"https://cdn.intechopen.com/books/images_new/314.jpg",editedByType:"Edited by",editors:[{id:"6495",title:"Dr.",name:"Daniel",surname:"Eberli",slug:"daniel-eberli",fullName:"Daniel Eberli"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"57",title:"Physics and Applications of Graphene",subtitle:"Experiments",isOpenForSubmission:!1,hash:"0e6622a71cf4f02f45bfdd5691e1189a",slug:"physics-and-applications-of-graphene-experiments",bookSignature:"Sergey Mikhailov",coverURL:"https://cdn.intechopen.com/books/images_new/57.jpg",editedByType:"Edited by",editors:[{id:"16042",title:"Dr.",name:"Sergey",surname:"Mikhailov",slug:"sergey-mikhailov",fullName:"Sergey Mikhailov"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"1373",title:"Ionic Liquids",subtitle:"Applications and Perspectives",isOpenForSubmission:!1,hash:"5e9ae5ae9167cde4b344e499a792c41c",slug:"ionic-liquids-applications-and-perspectives",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/1373.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"2270",title:"Fourier Transform",subtitle:"Materials Analysis",isOpenForSubmission:!1,hash:"5e094b066da527193e878e160b4772af",slug:"fourier-transform-materials-analysis",bookSignature:"Salih Mohammed Salih",coverURL:"https://cdn.intechopen.com/books/images_new/2270.jpg",editedByType:"Edited by",editors:[{id:"111691",title:"Dr.Ing.",name:"Salih",surname:"Salih",slug:"salih-salih",fullName:"Salih Salih"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"872",title:"Organic Pollutants Ten Years After the Stockholm Convention",subtitle:"Environmental and Analytical Update",isOpenForSubmission:!1,hash:"f01dc7077e1d23f3d8f5454985cafa0a",slug:"organic-pollutants-ten-years-after-the-stockholm-convention-environmental-and-analytical-update",bookSignature:"Tomasz Puzyn and Aleksandra Mostrag-Szlichtyng",coverURL:"https://cdn.intechopen.com/books/images_new/872.jpg",editedByType:"Edited by",editors:[{id:"84887",title:"Dr.",name:"Tomasz",surname:"Puzyn",slug:"tomasz-puzyn",fullName:"Tomasz Puzyn"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"72344",title:"Special Cuvettes for Spectrofluorimeters",doi:"10.5772/intechopen.92720",slug:"special-cuvettes-for-spectrofluorimeters",body:'Fluorescence spectroscopy is one of the most high-sensitive methods, which allow to detect very low concentrations of substances (μM, nM, and even pM) and to distinguish one substance from another [1, 2, 3, 4, 5]. Fluorescence spectroscopy is used for studying the functioning of living cells [6, 7, 8]. It is intended also for detecting various dyes and substances in cells and membranes [6, 7, 8, 9].
Fluorescence spectroscopy gives valuable information on spatial organization and behavior of biological structures and about appearance of intermolecular complexes, for example, DNA with proteins, enzymes with substrates, etc. It is applied in studies of conformation properties, mobility, and other properties of macromolecules, biological membranes, and living cells [6, 7, 8, 9].
It is an extremely informative method, since it allows us to receive the data on the distances, orientations, and intermolecular interactions. An advantage of fluorescent spectroscopy is the absence of damage of a sample in the course of research. Also, it is possible to work with native biological preparations.
The measured intensity of fluorescence of a biological sample is determined by its optical density, lifetime of the excited state, fluorescence quantum yield, and effective collection of emission beam.
Fluorescence spectra can be detected by special techniques—spectrofluorimeters. In such devices, a scanning of excitation wavelength by the first monochromator (placed before a cuvette with a testified sample) leads to changes in the intensity of fluorescence according to the form of the absorption spectrum; that is, form and position of the excitation spectrum should coincide with the absorption spectrum. At fixed excitation wavelength, a scanning of the second monochromator (placed after a cuvette with a sample) gives the emission spectrum (named also as spectrum of fluorescence or emission).
Various fluorescence techniques are widely used to increase the sensitivity of the method [1, 2, 3, 4, 5, 6]. One of the approaches to enhance the sensitivity of fluorescence analysis is the increase of the length of optical path of an exciting light in a sample. Usually, it is reached by consecutive reflections between concave mirrors, located in a common spectrofluorimeter near the cuvette [5] filled with a studied solution or suspension. However, only two passes of stimulating light in such systems take place, and also losses of light at media borders exist. Therefore, the gain (amplification factor)
That is why special cuvettes which dramatically increase the fluorescence intensity of diluted solutions and suspensions of isolated biomembranes or living cells were invented [7, 8]. They will be described below.
To overcome the mentioned specified difficulties, the multipass cuvettes (Figure 1), allowing to raise G up to 4–10 times and to remove parasitic reflections, can be used [7, 8]. Such cuvettes allow to lower the concentration of substances and to receive an intensive fluorescence signal.
Mirror cuvette, mirror microcuvette, and TIR cuvette.
Mirror cuvettes are intended for measuring the fluorescence of weakly absorbing solutions and can be used to register the excitation and emission spectra in UV and visible region and lifetimes. The cuvettes provide for many-fold increase of fluorescence intensity due to multiple passage of exciting light through the solution being tested and due to additional fluorescence collected.
The stimulating light in multipass cuvettes is almost wholly absorbed by a solution or suspension, and, thus, there are almost no parasitic reflections at cuvette sides (as against usual cuvettes with external concave mirrors, all reflections here are useful). Penetration of stimulating light into registration channel, therefore, sharply decreases. It is especially essential at low intensities of emission of light-scattered suspensions of cells or membranes. Amplification of fluorescence intensity from a sample in multipass cuvettes allows also to raise the accuracy of measurements of fluorescence polarization degree and lifetime of excited state.
Scheme of Mirror cuvette (1) and TIR cuvette (2); top view. Arrows show the direction of light beams. Mirror layers are shaded.
The gain (
Here
Emission spectra of 10-μM solution of ANS dye in ethanol in standard and mirror cuvettes are given in Figure 3. The intensity of fluorescence in mirror cuvettes is sometimes higher than that in the standard one. The form of the emission spectrum does not change.
Fluorescence spectrum of ANS (in ethanol) in standard cuvette (1) and in mirror cuvette (2). Excitation was 360 nm; slits were 5 nm. Spectrofluorimeter was “Perkin Elmer MPF-44B.”
Table 1 summarizes the calculated and experimental gain G, obtained for various samples (diluted solutions and suspensions) in different cuvettes.
Cuvette | ||
---|---|---|
Standard 1-cm cuvette | 1 | 1 |
Cuvette and two outer concave mirrors | 3.3 | 3 |
Mirror cuvette with aluminum coating | 9 | 6 |
Mirror cuvette with silver coating (excepting UV region) | 17 | 8 |
Total internal reflection cuvette | 20 | 10 |
Gain of fluorescence intensity, obtained with various cuvettes.
If an object is excited by short-time light flare, which has a duration less than the time of emission transition of molecules from
In the presence of only one kind of radiating molecules, decay is described as [1, 2, 3, 4, 5, 6]:
Here
Decay has exponential character. The decrease of decay intensity in
Since the fluorescence intensity of a sample in multipass cuvette strongly increases, the excited-state lifetime measurements become more accurate.
Figure 4 demonstrates the lifetime distributions of tryptophan fluorescence of cod parvalbumin solution measured in a standard cuvette and in a multipass one. Cod parvalbumin was dissolved in a 25 mM of Tris-HCl and 1 mM of CaCl2 with pH of 8.2; the absorbance at 295 nm was less than 0.1; excitation wavelength was 295 nm, and emission was detected at 320 nm; and monochromator slits were 5 nm. The UV excitation was made with the Orsay synchrotron (France).
Lifetime distributions of tryptophan fluorescence of cod parvalbumin in a standard 1-cm quartz cuvette (left figure) and in a multipass one (right figure).
Lifetime distributions were measured through correlation fluorescence spectroscopy. The 0.03 ns band, obtained with standard cuvettes (Figure 4, left), is an artifact, caused by a big noise of small fluorescence signal and also by some penetration of the exciting light into registering channel. It is prevented if multipass cuvettes are used (Figure 4, right).
In the case of subnanosecond lifetimes, the measurements should be made using multipass cuvettes in all channels—for studied sample and for reference blank sample.
Table 2 presents the lifetime components of fluorescence of ribonuclease T1, parvalbumin, and phospholipase A2. Excitation was 295 nm (slits were 5 nm). RNAase T1 was dissolved in the 100 mM acetic buffer (pH 5.5). The calcium form of parvalbumin was used in the 25 mM Tris-HCl buffer with 1 mM CaCl2 (pH 8.2). Pork phospholipase A2 was in the 100 mM acetic buffer (pH 5.8) or in 90% glycerol. Detection of emission at 310 and 320 nm was done in mirror cuvettes to increase the intensity. Emission at other wavelengths was measured in standard 1-cm quartz cuvettes.
Protein | ||||||||
---|---|---|---|---|---|---|---|---|
Ribonuclease | 310 | — | — | 4 | — | — | 1 | 4 |
320 | — | — | 4 | — | — | 1 | 4 | |
340 | — | — | 4 | — | — | 1 | 4 | |
375 | — | — | 4 | — | — | 1 | 4 | |
Parvalbumin | 310 | — | 1.1 | 3.4 | — | 0.15 | 0.85 | 2.7 |
320 | — | 1.3 | 3.3 | — | 0.07 | 0.93 | 3.2 | |
340 | — | 1.7 | 3.3 | — | 0.05 | 0.95 | 3.3 | |
375 | — | — | 3.4 | — | — | 1 | 3.4 | |
Phospholipase | 320 | 0.6 | 2.1 | 5.3 | 0.71 | 0.24 | 0.05 | 1.2 |
350 | 0.7 | 2.7 | 7.2 | 0.63 | 0.32 | 0.05 | 1.7 | |
385 | 0.7 | 2.7 | 6.5 | 0.61 | 0.33 | 0.06 | 1.7 | |
Phospholipase in glycerol | 320 | 0.4 | 2.1 | 5.6 | 0.4 | 0.43 | 0.17 | 2 |
350 | 0.8 | 2.7 | 6.3 | 0.19 | 0.54 | 0.27 | 3.3 | |
385 | — | — | 4 | — | — | 1 | 4 |
Lifetimes and amplitudes of tryptophan emission of ribonuclease T1, parvalbumin, and phospholipase A2.
Multipass cuvettes are especially useful in phase modulation measurements of lifetimes, when, because of narrow slits of entrance monochromator and modulator, the intensity of exciting beam is too small. These cuvettes allow to raise the accuracy of phase modulation measurements. For example, a solution of ANS in ethanol in standard cuvettes gives fluctuations in lifetime > ± 0.5 ns (lifetime was 7.7 ns; it was measured by “SLM-4800” at frequency of 30 MHz; excitation was 370 nm; excitation slits were 1 nm), but in multipass cuvettes (at the same conditions), fluctuations were < ±0.1 ns.
Multipassing does not bring appreciable contribution in measurements of nanosecond lifetimes, since several passes of the beam lead to artifact delay only about of 0.1 ns. In the case of subnanosecond lifetimes, it is necessary to do measurements with differential mode, using multipass cuvettes in all channels and entering an amendment for artifact delay.
Molecules of many organic substances consist of a flat chromophore and various groups attached to it. Their structures are optically anisotropic. Therefore, emission of motionless molecules is polarized (even at the nonpolarized excitation). The greatest contribution to the total emission is brought by those molecules, in which chromophores are located in a perpendicularly position to the excitation light flow. It means that even at a chaotic arrangement of molecules, for example, in suspensions of membranes or cells, the emission is partially polarized. The intensity of fluorescence in some direction can be presented as a sum of two light flows,
If to illuminate an object by linearly polarized light, the fluorescence becomes even more polarized. For example, it is possible to direct the light beam from a lamp on a sample through the polarizer (polarizing prism) and to put the analyzer (the same second element) sideways of the sample, passing through which the emission will get on the photomultiplier. The intensity of emission, measured at identical orientations of polarizer and analyzer, is named as “parallel” component. The intensity, registered at orientations, crossed under 90 degrees between the polarizer and the analyzer, is the “perpendicular” component. The degree of polarization at excitation by linearly polarized light is expressed as [6]:
Fluorescence polarization degree (
Multipass cuvettes may be useful not only for fluorescence analysis but also for many-fold intensification of photo-bleaching or laser flash photolysis of chromophores or dyes in solutions or suspensions.
The energy of UV quantum, absorbed by a cell or biomembrane, is spent mainly for fluctuations, so - on instant strong local heating [7, 8].
If such local heating is too high, it can result to denaturation of proteins and their aggregation. For instance, the influence of UV irradiation on intensity of tryptophan fluorescence of α-crystalline (it is a protein from bovine eye lens) is shown in Figure 5. A solution of the protein, hermetically closed and thermostated at 10°C (here, thermo-aggregation is possible to be neglected) in mirror microcuvette, was continuously irradiated for 120 min with UV light of a 450-W xenon lamp through holographic monochromator, positioned at 280 nm with slits of 16 nm. The intensity of tryptophan fluorescence (excitation was 295 nm; emission was 340 nm; emission slits were 4 nm) was quickly measured during a course of irradiation. The observed decrease in fluorescence of α-crystalline, caused by photoinduced denaturation, is due mainly to the increase of light-scattering during aggregation. Light-scattering can be measured from optical density at 310 nm of the sample in a standard compartment of spectrophotometer or, by the other way, from intensity of scattering light, detected from the sample by the photomultiplier of a spectrofluorimeter under a right angle. In the second case, both monochromators have to be established at an identical wavelength of 310 nm (at slits = 1 nm).
Influence of UV radiation on the intensity of tryptophan fluorescence of α-crystalline (0.7 mg/ml) during photoaggregation of the protein at 10°C. irradiation was at ~280 nm in mirror microcuvette.
Fluorescent kinetics of photo-bleaching of flavin mononucleotide (FMN) in aqueous solution and in diluted suspension of proto-mitochondria [9] after illumination by blue light of the mercury lamp SVD-120A in a mirror cuvette is given in Figure 6.
Dependence of photodestruction of proto-mitochondrial flavins (1) and free FMN (2) at the time of blue irradiation. It was measured by fall in the intensity of flavin fluorescence, appeared in the burning band at 450 nm, and detected as decrease in fluorescence intensity at 525 nm.
Photo-bleaching is induced by photo-destruction of FMN, namely, by oxidation of its triplet state by molecular oxygen. The absorption band of FMN, detected at 450 nm, was “burns out” [7, 9].
Cuvette with transparent diagonal quartz plate can be applied for many-fold increase of fluorescence signal and for many-time using of a single sample, adsorbed on the plate. A schematic diagram of a cuvette with the diagonal transparent quartz plate is shown in Figure 7. The sample in the form of a smear or film is located on the side of the plate facing the exciting light.
Cuvette with the diagonal transparent quartz plate (top view). The sample in the form of a smear or film is located on the side of the plate facing the exciting light.
A sample of cells or isolated organelles is attached on the surface of a transparent quartz plate, which is placed inside a standard cuvette at a right angle to the exciting light. A sample has to be as a smear or film. The cuvette should be filled by water (if cells cannot be desorbed from the plate) or hydrophobic solvent like hexane (if cells are not tightly fixed on the plate).
A transparent quartz plate is used for many-fold increase of the sensitivity of the fluorescence analysis. The sample (matter of inquiry) is placed exactly at the center of the surface of the plate. The size of the attached smear (film of organelles or cells or other samples) should correspond to the light spot, formed by the exciting lens (Figure 7).
To get the best results, the object under study (cells, cellular organelles, or nuclear DNA, etc.) is applied to the surface of the plate in the form of a thin smear (with drying from water within 4–5 min). Fluorescent dyes, dissolved in a solution, are deposited as microdroplets (1–4 μl) to the sample. After that, the sample is dried for 1–2 min. The plate with the dried sample is placed into the cuvette filled by liquid, which weakly interacted with sample, for example, hexane, perfluorodecalin or, in opposite case, for water-insoluble samples, by isotonic aqueous medium or water.
In all experiments, the plate was placed along a diagonal of the cuvette, as shown in Figure 7, to exclude the penetration of the reflected artifact light into the recording emission channel. Virtually, any films of samples (stained cells or organelles, etc.) applied on the surface of the plate produce fluorescence signals that are several times higher than signals yielded by the same quantity of the studied substance in the solution filling the cuvette volume, although the sample on the plate is usually hypochromized (the extinction factor of chromophores is substantially decreased due to the sieve and screening effects).
High intensity of fluorescence of the layer on the plate arises due to several reasons. First, the whole studied substance is concentrated in the small square of layer, but, while being in a solution, it is distributed over the whole volume of the cuvette. Second, the sample is attached on the plate at the same place, where the focus of both lenses is located (the area of the sample is equal to that of the light spot). Third, the excited molecules of the sample in the condensed phase are less subjected to deactivation (quenching) than in solutions.
The cuvette with transparent diagonal quartz plate can be applied for chemical treatments of a sample. For instance, Table 4 presents the data on formation of pyrene excimers (excitation was 335 nm, and emission was 393 nm for monomers and 470 nm for excimers) before and after extraction of mitochondrial lipids by acetone from mitochondrial smear. Also, quenching of tryptophan fluorescence (excitation was 286 nm, and emission was 335 nm) of mitochondrial smear by pyrene (2.4 μM) is shown.
Sample (solution or suspension) | P in standard cuvette | P in mirror cuvette |
---|---|---|
Tryptophan residues of sarcoplasmic reticulum (ex. 295 nm, em. 320 nm) | 0.36 | 0.355 |
Tryptophan residues of mitochondrial suspension | 0.29 | 0.285 |
ANS in bovine albumin | 0.28 | 0.275 |
7-Aminoactinomycin in DNA | 0.31 | 0.305 |
Fluorescence polarization degree of various samples.
Mitochondria | Quenching (%) | |
---|---|---|
Native | 27 | 0.47 |
Without lipids | 23 | 0.1 |
Quenching of tryptophan fluorescence of mitochondrial smear by pyrene and pyrene monomer/excimer ratio before and after extraction of lipids from mitochondrial smear.
During the oxidation of succinate in respiratory chain of mitochondria, a consumption of oxygen, accompanied by increased τm and Fm of pyrene, takes place (Table 5) [8]. At exhaustion of oxygen, luminescence of pyrene leaves on a plateau. Mitochondria were 0.8 mg of protein per ml in the incubation solution: 10 mM Tris-HCl, 10 mM phosphate, 50 mM KCl, 150 mM sucrose, and pH = 7.5. Pyrene was 1 μM. Excitation was 336 nm, and emission was 393 nm (monomers) and 480 nm (excimers); slits were 2 nm.
Oxygen (μM) | |||
---|---|---|---|
130 | 95 | 11.5 | 0.12105 |
~1 | 150 | 23 | 0.15157 |
Fluorescence of pyrene in membranes of mitochondria in aerobic and anaerobic conditions.
In anaerobic conditions, the lifetime of pyrene monomers in mitochondria equals 157 ns (Table 5). In without-oxygen organic solvents, τ of pyrene is much more; for example, τ in cyclohexane is 370 ns.
The highly sensitive sensor was designed to measure the molecular oxygen content in solutions and diluted suspensions, placed in a quartz cuvette, which contains the diagonal plate with a pyrene, protected by a Teflon film (Figure 8). The sensor can be used in any standard spectrofluorimeter. Unlike analogs, this oxygen sensor is not inertial.
Cuvette with the diagonal transparent quartz plate with plastic microcontainer. The sample—suspension of cells or solution of pyrene (oxygen sensor).
An additional multiple increase in the fluorescent signal is attained by the use of special mirror cuvette with diagonal plate. Such cuvette (Figure 9) is very useful when the emission intensity from a sample is too low.
Schematic diagram of mirror cuvette with the diagonal transparent quartz plate (top view). The sample in the form of a smear or film is located on the side of the plate facing the exciting light.
In all experiments, the cuvette with the plate was filled with a solvent, the refractive index of which is close to that of quartz. The filling with a solvent is intended to (i) eliminate spurious light reflections at boundaries, (ii) reliably prevent the fluorescence channel from the incoming exciting light, (iii) prevent the sample from strong overheating by the exciting light, and (iv) allow measurement of the kinetic processes on the sample surface. The matter is that molecules of the studied substance are sufficiently mobile on the sample surface contacting the solution and make it possible to observe the kinetics of chemical and biochemical reactions using fluorescence spectroscopy methods.
The use of the plate ensures another possibility; namely, the film sample on it can be used repeatedly, placing the plate (after rinsing) in the required medium. For example, a thin layer of mitochondria (from rat liver) safely lies on the plate even at a long (1–2 h) period of being in the isotonic water phase (the adhesion and stability of a thick layer are rather worse). In this case, the initial activity of enzymes is not lost. In the inert perfluorodecalin, this layer remains unchanged for many hours.
Figure 10 shows, for example, spectra of the tryptophan fluorescence of proteins of mitochondrial diluted suspension in the standard cuvette (curve
Spectrum of the tryptophan fluorescence of mitochondrial proteins: (1) aqueous solution in the standard cuvette and (2) sample film on the surface of the quartz plate in the mirror cuvette.
Sample | Exciting wavelength (nm) | Maximum of fluorescence spectrum (nm) | Fluorescence intensity of | ||
---|---|---|---|---|---|
Dissolved sample in mirror cuvette | Sample layer on the plate in standard cuvette | Sample layer on the plate in mirror cuvette | |||
Rhodamine B | 540 | 570 | 3.5 | 4.1 | 13.8 |
Pyrene | 335 | 390 | 3.3 | 3.8 | 11.9 |
Tryptophan | 280 | 350 | 3.1 | 3.5 | 10.1 |
7-AAMD | 550 | 660 | 3.6 | 3.8 | 12.9 |
7-AAMD in DNA | 570 | 630 | 3.7 | 4.2 | 14.8 |
Mitochondria | 286 | 340 | 3.4 | 4.7 | 15.2 |
Leucocytes | 450 | 525 | 3.1 | 3.8 | 11.1 |
Fluorescence parameters and intensities (gain, G) of various samples on the plate, diagonally cutting in quartz 1-cm cuvette.
Note: concentrations <1 μM; fluorescence intensity of the dissolved sample in the standard cuvette was assumed to be 1; 7-AAMD is 7-aminoactinomycin D.
Similar results also were observed in experiments with dyes and aromatic hydrocarbons. Table 6 summarizes relative fluorescence values of some substances, when they are placed on the plate’s surface (comparing to the solution of a sample in the cuvette volume).
A number of special cuvettes which dramatically increase the fluorescence intensity of diluted solutions and suspensions are tested. All described cuvettes can be applied for measurements of spectra excitation and emission, excited-state lifetime, and polarization degree. These cuvettes enable a multiple increase in the sensitivity of fluorimetric measurements with common spectrofluorimeters. It is also possible to study, in particular, the kinetics of phenomena on the sample surface, to detect small concentrations of substances, e.g., slight quantities of proteins and DNA in solutions, and this information can be used for biomedical analyses, in crime detection, etc. Also, in principle, the described cuvettes could be applied for photo-bleaching experiments.
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.
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