Most Complex Formation Reactions Biology Essay


Most complex formation reactions are usually measured in an aqueous media. However, ligands that are organic soluble are employed too, and their stability constants are often determined in mixed solvent systems such as dioxane - water. For completely organic systems such as acetonitrile, metal complex can be formed quite readily but their stability constants are generally not known. Approximate values have been used for such systems as there is no mathematical expressions that can be related the equilibrium constants in such systems to the formation constant or stability constant in water or water/organic mixture (Martell and Hancock, 1996).

The dramatic increase in publication in this discipline without any goals of developing the concepts of coordinating chemistry, or of providing essential information to other fields, has led to a major decline in the prestige of this research area, and work in stability constants came to be considered as a routine, this problem has been further aggravated by the publication of a large number of poor papers, whereby a minor change in ligand structure was taken as a sole justification for yet another publication, and in which controls and conditions were neglected, resulting in the reporting of poor data (Martell and Motekaitis, 1992, p.1).

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Two major developments have risen providing a remarkable role for Equilibrium constants determination. Firstly, the development of the new discipline of bioorganic chemistry, which in turn requires knowledge of the complexes formed in multicomponent systems containing many ligands and metal ions. Secondly, the availability of computers which has remarkably expedited changed and facilitates the methods of determining stability constants. Computers have been used for deferent systems because of their ability to remarkably reduce errors of resulting stability constants comparing to those obtained graphically. However, there are other advantages of stability constants computerisation, not the least liberation of researchers from the diligence of repetitive arithmetic. Currently, there is software that are able to perform a systemic search through all possible species, eliminating preconceived ideas on the nature of the complex of interest. It should be noticed that All computer software require careful consideration of the parameter determining the best available set of constants, the relative importance of data from various concentration ranges, and the effects of possible experimental errors.

Stability constant, is a constant describing the equilibrium that exists between a metal ion surrounded by aqueous molecule ligands and the same metal ion surrounded by ligands of another kind in a ligand displacement reaction:

aA + bB ↔ cC + dD

The logarithm of the equilibrium constant is directly related to the Gibbs free energy of the products of the reaction minus the Gibbs free energy of the reactants in their standard state and it is therefore a measure of the difference in reactivities of the reactants and their products, Gibbs free energy is a thermodynamic potential that measures the "useful" or process-initiating work obtainable from a thermodynamic system at a constant temperature and pressure (Kotrly and Sucha, 1985, p 34).

Measuring the activities of the ion-ligand complexes under real conditions is a complex and time consuming process which is beyond the capability of most researchers. However, because concentration closely parallel activities under carefully controlled conditions involving both temperature and ionic strength, it is practical to determine equilibrium concentration constant instead of activities constant (Martell and Hancock, 1996).

Researchers would like to determine the activities of all the reactants, but it is not possible to measure this activity for a single specie/ion, in the best scenario it would be possible to determine the mean ionic activities occasionally. However, it is currently possible to do that for at least some of the species of interest through the use of modern experimental techniques (Kotrly and Sucha, 1985).

If and are the total concentrations of metal and ligand in a solution respectively, considering [ML], [M], and [L] as the concentrations of individual free species, then we can write (from now and on we shall consider the symbols [X], {X}, and 𝛾X refer to the concentration, the activity, and the activity coefficient, respectively) (Hutton and Linder, 2011).

= [M] + [ML] = +

= +

Factors affecting Stability of metal complexes

Several factors contributing to stability constants have been defined, and these factors can be categorized into metal ion depending factors and ligand depending factors.

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Metal ion depending factors:

Size and charge: in complexation reactions, a positively charged metal ion and anionic or polar neutral ligands carrying high electron density in their lone pair, a purely electrostatic contribution will definitely take place.

Metal class and ligand preference: electropositive metals (i.e. lighter and/or more highly charged ones such as ) prefer lighter p-block donor from such as N and F donor. While less electropositive metals (i.e heavier and/or lower charged ones such as) prefer heavier p=block donors from the same families such as P, and I donors to produce higher significantly M - L covalent bond, the second case should be applied. Beside, a grey area of metals and ligand who do sit in either set at, at least not easily, is present.

Natural order of stability for transition metal ions: These ions with incomplete sets of d electron, present a contribution to stability from the crystal field stabilization energy (CFSE), where is ions such as (metal ion with full set of electron) there is no stabilization energy. CFSE of metal ions in metal - ligand complexes has a demonstrated influence on the value of stability constant K of transition metals and this fact can be noticed in the experimentally determined stabilities for the series of metal ions from to (Lawrance, 2010).

Ligand depending factors:

Base strength: since the earlier attempts to determine K, it was obvious the relationship between Brønsted base strength of a ligand and its ability to form a stable complex, which can be explained that base strength is a measure of the capacity to bind a proton. So, substitution of by is reasonable, allowing basicity to define complex stability.

Chelate effect: thermodynamically, the equilibrium constant reports the enthalpy change (∆in the reaction and the entropy change (∆) resulted from a reaction. The greater the amount of energy evolved, the more stable the complex is. Furthermore, the greater amount of entropy change will result in greater stability of the products. Generally, chelating is beneficial for complex stability, and ligands from stronger complexes than comparable monodentate ligand sets.

Chelate ring size: the size of the chelate ring directly influences the size of the stability constants. Since we are restraining the metal ions by binding donor atoms, it is not surprising that there is a correlation between the formed chelate ring size and stability.

Steric strain: Ligands vary so much more in size and shape than metal ions, other consequences arise, including simply size effect in terms of fitting around the central atom. These effects of ligand bulk, resulting from molecules being necessarily requires binding different regions of space, and thus required to avoid "bumping" against each other when confined around central metal ions, which is known as steric effect. As a general rule, the bulkier a molecule the weaker the complex formed when there is a set of ligands involved (Lawrance, 2010).

Sophisticated effects:

There are some other factors that result from molecular shape and may add complications. For example, macrocycle effect, which is a large cyclic ligand that carry at least three donor atoms has a hole (i.e. central cavity), so the fit of the metal ions into this hole is an important considerations. As a matter of fact, metal ions fitting or missing into predefined shaped ligands is an important aspect of metal coordination chemistry, as it becomes familiar meeting more sophisticated ligand systems (Lawrance, 2010).

Over all stability constants

Since most metal ions usually provide more than one coordination site, they can bind more than only one donor group, or in most cases more than one donor (ligand), the stability constants equation must be extended to take into account the attachment of a set of n ligands. This process occurs in a sequential manner, this is because donor replacement is a result of molecular encounters, within the complex required to make contact with an incoming donor with sufficient velocity and with unit required direction of approach so as to permit a donor exchange to occur. The sequential substitution steps for formation of M by a series of equilibria can be presented in the following equation:

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The overall reaction occurring through combination of the previous steps can be expressed as:

And the overall stability constant is:

This constant defines the formation of the overall complex, where all of the donor can be considered replaced by another: it does not infer anything about the mechanism of the process (Lawrance, 2010).


Validated values of stability constants are remarkably useful and necessary in many aspects of technology, such as developing analytical methods involving ion-exchange processes, chromatography, separations of metals, complexometric titrations, as well as many aspects of academic, medical, environmental, and industrial research.

Quantitative data of the stability and extent of complex formation is essential in developing methods for separation of specific elements such as metals from mixtures and for their determination by electrochemical techniques (Martell and Motekaitis, 1992, p.195).

Accurate stability constants data are required to develop better understanding of metals biological activity. Many medical applications are based on complex formation to remove toxic metals or to insert metals such as radioactive indium for diagnostic purposes (Martell and Motekaitis, 1992, p.195).

In the environment, the toxic effect of some metals becomes increased when derived complexes can be leached by underground water from a mine, waste dump, and land-fill site or waste dumps. On the other hand, highly stable complexes can act as very effective detoxificants and it is important to be able to calculate the effect of defined concentrations of a particular ligand or mixture of ligands on the final composition of elements and compounds in the environment (Anderegg et al., 2005).

In agriculture, accurate values of stability constants are used to determine the extent of complex formation of metals in soils, which is greatly affecting their availability as well as toxicity to plants. Synthetic chelating agents are now employed in plant nutrient to form soluble metal complex which are available to plants. Stability constants are then employed to determine the effectiveness of these artificial carriers (Martell and Motekaitis, 1992, p.195).

In another discipline, using of complexing agents in detergents requires vast knowledge of metal complexes stabilities, for the both detergent action effect of these complexing agents on waste water treatment when detergent solutions are employed (Martell and Motekaitis, 1992, p.196).

Developing synthetic membranes is an important field of research in both university and industry. Many of the membrane being designed are based on models that utilize our current knowledge of metal ligand affinities. Thus a membrane channels may contain functional groups which may react with metals ions in such a way as to promote selective migration though the membrane pores (Martell and Motekaitis, 1992, p.197).

In metallurgy, the actions of complexing agents aqueous solution on ores is frequently involved. Secondary separation, including solvent extraction and ion exchange also use complexing agents which selectively react with metal ions. Stability constants are required for a better understyaning of the chemical reactions involved (Martell and Motekaitis, 1992, p.197).

Determination of the distribution of all the species in a multicomponent system such as biological fluids (e.g. blood plasma), is often referred to as speciation. Arguably, the best way of carrying out speciation on a particular system is by making use of reliable stability constant values introduced into appropriate forms of mass action and mass balance equations. Several databases have been constructed and several suitable computer programs have been developed for this purpose. For example, a recent version of a blood plasma database incorporates 10 metal ion species, over 100 types of ligand and 10000 complexation reactions between them. Critically evaluated stability constants are to be found in several modern databases (Hutton and Linder, 2011).

Techniques used for stability constant determination:

Any method which can be employed to determine with accuracy the concentration of at least one of the species in equilibrium provides the information needed, together with the known analytical composition of the experimental solution, to calculate the concentration of all the remaining species present, and hence the stability constant. If a sufficient number of such measurements are performed over a wide range of conditions, accurate values of stability constants that apply within the range of reactions conditions employed can be obtained (Martell and Motekaitis, 1992, p.13).


It is an electrochemical technique which depends on measuring the potential of a galvanic cell, usually without the drawing appreciable currency. In this method, cell potential is governed by the potential of an indicator electrode which in turn responds to changes in the activity of the species of interest. It is the most widely used electrochemically technique, it can be used for quantitative determination of many species in a solution over a wide range of concentrations (, with relative precision of 0.1 - 5 %. Potentiometric titrations are especially useful for coloured or turbid samples or for mixtures. On the other hand, this kind of titrations is slow and time consuming unless automated (Fifield and Kaeley, 2000, p 232-234) (Bakker and Pretsch, 2007).

Determining stability constants involves solving of mass-balance equations written for the total metal-ion, ligand [] and [] respectively. Refinement of stability constants that involves the simultaneous solution of these mass-balance equations is only possible if either the free-metal-ion or free-ligand [M] or [L] respectively, which can vary over a wide concentration range, can be monitored with high accuracy throughout an experiment. Thus, it is not surprising that potentiometry is currently the most powerful and most widely used analytical technique in the study of metal/ligand equilibria (Cukrowski et al., 2004). If an electrode reversible to ions of M is introduced into a solution of known and in a medium of constant ionic strength and the solution is combined with a reference electrode through a suitable conducting bridge, the measured emf at a temperature T K is given by the Nernest equation:

E = + ( In [M]

must be determined at the particular ionic strength of the experiment and incorporates the intrinsic electrode parameter as well as relative activity coefficient and liquid junction potentials (Shultz et al., 2002). Suitable half-cells and various amalgam electrode have been used to measure [M] and hence to obtain the stability constant of metal complexes of Cu+, Cu2+, Ag+, Au+, Zn2+, Cd2+, Hg2+, Hg22+, Co2+, Ni2+, Sn2+, Pb2+, Bi3+, Fe2+, Pd2+ (Hutton and Linder, 2011).

Polarograpghy and Stripping voltametry:

Another electrochemical analysis technique based on the measurement of the diffusion - controlled current flowing in an electrolysis cell in which a single electrode is polarized. The current is directly proportional to the concentration of electroreactive species.

Usually used for both quantitative and qualtitative determination of metal traces and organics especially in the range of ( with relative precision of 2 - 3%. The main limitation is that the measurement is too sensitive to solution composition (i.e. dissolved oxygen and/or impurities may remarkably effect sensitivity) (Fifield and Kaeley, 2000, p 247-250). The simplicity of the polarograghy instrumentation allows this technique to be widely used in equilibrium chemistry. If the reversible electrode is assured, half-wave electrode potential is given by Heyrorky-Ilkovic equation:

E = ()s =

Where z is the change in electron number

is the diffusion current

i is the current strength at half - wave potential

A major advantage of this polarograghy is its usefulness as a complement to potentiometry for determining stability constants (Hutton and Linder, 2011).

Stripping voltametry can be useful in the determination of the complexation as well. The techniques can be used to determine metal concentration as low as mol/l. Stability constant can be measured in a way close to that used in polarograpghy, and the principle involves deposition of a metal ion in reduced form on a mercury electrode followed by reoxidation through reversing polarity so the reoxidation current become directly related to metal ions concentrations in the media (Hutton and Linder, 2011).

Cation exchange resins:

It is an insoluble matrix in the form of small beads. The separation here takes place through the developed structures of pores of the surface of which are sites with easily retained and released ions. Here the trapping of ions takes occur only with simultaneous release of other ions, where ions migrate at different rate due to differences in adsorption, solubility, charge or size. The main disadvantage in this techniques is gravity flow separation slow; separated components accompanied by a large excess of eluting electrolyte (Fifield and Kaeley, 2000, p 160-162). .

This technique can be used in determining stability constant by partitioning a cationic species Mbetween and aqueous phase and the sodium containing exchange media (ion exchanger) according to the equation:

z(Na+)R + (MLnz+) −−−−−−−− (MLnz+)R + z(Na+)

The stoichiometric partition coefficient for each metal species is given by:

And will be constant as long as k and the ratio of sodium ion concentration are constant (Hutton and Linder, 2011).

Nuclear Magnetic Resonance Spectroscopy (NMR):

This developed technique depends on absorption of electromagnetic radiation in the radio frequency region of the spectrum resulting in alteration of the orientation of spinning media in a magnetic field. Used for identification and structural analysis of organic substance. It is remarkably useful for quantitative analysis. The limitations are that it's expensive and involves complex instrumentation, moderate to poor sensitivity with continuous wave instrument, finally limited range of solvents for analysing proton spectra unless deuterated (Fifield and Kaeley, 2000).

Using NMR to study metal-ligand equilibrium becomes a greater interest and use. Beside its ability to determine stability constant, this technique can also provide sufficient information about the complex structure as well as binding location (Hutton and Linder, 2011).

Spectrophotometry methods:

Spectrophotometry is an analytical technique for quantitative measurements of the reflection or transmission properties of the substance as a function of wavelength. It involves the use of spectrophotometer, which can measure intensity as a function of a light source wavelength. It is widely used for measuring reflectance or transmittance of solutions, transparent or opaque solids, and gases (Fifield and Kaeley, 2000, p426-428). Spetcrophtotometer is currently successful in determining equilibrium constant. As well known, a lot of the chemical reactions may take place in forward and reverse directions where reactants from products and products break down to reactant. At a certain point, the reaction reaches the equilibrium point at which reactants and products respective concentrations can be determined through testing light transmittance by a spectraphotometer. The amount of light transmitted is a direct indication of the concentration of certain substance.

Drawbacks of using spectrophotometer are the complexity of the instrumentations and process, also high cost instrument, and difficult maintenance

When using spectrophotometer for stability constant determination, the absorbance of specie, M, of concentration c in a cell length d is:

A = εcd

Where: ε is the molar absorption coefficient at a specific wave length.

So: [MLn] = A/εd

One of the terms needed to determine a series of stability constants can be computed provided its spectrum is sufficiently distinguishable.

In the following equilibrium

pM+ qL ↔MpLq

A series of solution can be prepared for which (Mt + Lt) is constant and the ratio x = varies from 0 to 1.

Absorbance in visible spectrometer to E is proportional to [MpLq] and can be measured and plotted against x to give two straight lines intersecting at X = p/q c(Hutton and Linder, 2011).

Other methods:

Other physical techniques have been used for determining stability constants including polarimetry, Raman spectroscopy, infrared spectroscopy, conductivity, along with depression of the freezing points, and solubility.

Production Artificial Saliva

Natural saliva is a watery, visco-elastic substance with a distinct surface activity produced in the mouth of mammalians. It's secreted from the three pairs of major salivary glands; the parotid, sublingual and submandibular glands. In addition, there are hundreds in minor salivary glands contributing to the production of saliva.

Generally the amount of saliva secreted by a healthy person is estimated to be in the range of 0.75-1.5 litre/day, while it is accepted that the amount drops to almost zero during sleep (Humphrey and Williamson, 2001)

Produced in salivary glands, human saliva is 98% water, and the remaining 2% compose of many important substances, including mucus, electrolytes, various enzymes, antibacterial compounds (Walters, 2003).



2-21 mmol/L sodium.

10-36 mmol/L potassium.

1.2-2.8 mmol/L calcium.

0.08-0.5 mmol/L magnesium

5-40 mmol/L chloride.

25 mmol/L bicarbonate.

1.4-39 mmol/L phosphate.



mainly consists of mucopolysaccharides and glycoproteins

Antibacterial compounds

thiocyanate, hydrogen peroxide, and secretory immunoglobulin A.


α-amylase (EC3.2.1.1). Amylase starts the digestion of starch and lipase fat before the food is even swallowed. It has a pH optima of 7.4.

lingual lipase. Lingual lipase has a pH optimum ~4.0 so it is not activated until entering the acidic environment of the stomach.

Antimicrobial enzymes that kill bacteria.


Salivary lactoperoxidase

Minor enzymes such as salivary acid phosphatases A+B, N-acetylmuramoyl-L-alanine amidase, NAD(P)H dehydrogenase (quinone), superoxide dismutase, glutathione transferase, class 3 aldehyde dehydrogenase, glucose-6-phosphate isomerase.

Epidermal growth factor or EGF

Proline rich proteins

Contribute to in enamel formation, Ca2+-binding, microbe killing and lubrication.


as 8 million human and 500 million bacterial cells per ml.


researched pain-killing substance found in human saliva.

Artificial Saliva:

These are commercial preparations used in salivary glands disorder (e.g. dry mouth), and they should resemble normal saliva particularly in the biophysical properties.

The existing artificial substitutes fall short of required biophysical criteria and modifications are necessary in order to improve them. Several essential components are required for the production of near ideal artificial saliva such as surface active, shear thinning and muco-adhesive polymer. (preetha and Banerjee, 2005).

Although no ideal artificial saliva have been developed to date. These preparations have successfully replaced the components and hence functions of natural saliva.

Artificial saliva is supposed to react with the test material in a similar manner to that of natural saliva, which is a basic requirement of any artificial substitutes in order to be used in in-vitro studies of oral therapy and health care products. It is important to notice that the exact duplication of the properties of human natural saliva is impossible due to the inconsistency and instability of natural saliva (Leung and Darvell, 1997).

Studies about the saliva components over the last two decades using advanced technologies (e.g. molecular biology, protein chemistry and biophysics) have demonstrated the complexity of salivary functional and structural requirements. Nevertheless, several principles control the salivary function has been evolved. First, the majority of salivary molecules are multifunctional. Secondly, the conformation of a salivary molecule is an important factor in its biological activity. Third, a lot of the molecules have overlapping functions (e.g. both amylase and mucin interact with streptococcus viridians). Fourth, salivary molecules may be amphifunctional, which mean that different functions of a single molecule could be either protective or potentially harmful depending on the intraoral site of action (e.g. amylase). Last, but not least, functional interactions may exist between different molecules (Lavine, 1993).

In general, the salivary molecules should be used to fight microbial induced diseases as well as xerostomia. Artificial saliva should be long lasting, biocompatible, biodegradable, and provide high protection to the oral environment (Lavine, 1993).

In order to construct a near real oral cavity, particularly in respect to ion/molecular composition, temperature, pH and ionic strength, we looked for several recipes for artificial saliva focusing mainly on comparing their composition with natural human saliva. All of the available preparation, including commercial ones used for managing dry mouth disorder ( such as Saliva Orthane, Biotene Oralbalance, Salivix) ( BNF 53, 2007),provide almost all the electrolyte naturally present in the natural saliva. On the other hand, they lack major components, mainly enzymes and proteins, which make them far from being a good media to simulate natural saliva in in-vitro studies such as ours (Tang et al., 2003).

In 1997, van Ruth and colleagues published their vision to the best artificial saliva, and by comparison to the human natural saliva composition, it was found to contain most of the components with major functions in the natural saliva (i.e. electrolyte, protein and enzymes). The components were (Deibler and van Ruth, 2003)


Weight - units


5.208 g

1.369 g


0.877 g


0.477 g


0.441 g


0.5 g

Mucin ( porcine stomach mucin)

2.160 g

α-Amylase (hog pancreas amylase)

200,000 units

Brought to one litre with distilled water and adjusted to pH 7