Dissociation constants analyte

The pKa is an important physicochemical parameter. The analyte pKa values are especially important in regard to pharmacokinetics (ADME—absorption, distribution, metabolism, excretion) of xenobiotics since the pKa affects the apparent drug lipophilicity [59]. Potentiometric titrations and spectrophome-tric analysis can be used for pKa determination however, if the compound is not pure, is poorly soluble in water, and/or does not have a significant UV chromophore and is in limited quantity, its determination may prove to be challenging. 

Dissociation constants of ionizable components can be determined using various methods such as potentiometric titrations [85] CE, NMR, [86] and UV spectrophotometric methods [87]. Potentiometric methods have been used in aqueous and hydro-organic systems however, these methods usually require a large quantity of pure compound and solubility could be a problem. Potentiometric methods are not selective because if the ionizable impurities in an impure sample of the analyte have a pK similar to that of the analyte, this could interfere with determining the titration endpoint. If the titration endpoint is confounded, then these may lead to erroneous values for the target analyte pKa. 

Liquid chromatography has also been widely used for the determination of dissociation constants [88-92] since it only requires small quantity of compounds, compounds do not need to be pure, and solubility is not a serious concern. However, the effect of an organic eluent modifier on the analyte ionization needs to also be considered. It has been shown that increase of the organic content in hydro-organic mixture leads to suppression of the basic analyte pKa and leads to an increase in the acidic analyte pK compared to their potentiometric pKa values determined in pure water [74]. 

Knowledge of pKa for the target analyte and related impurities is particularly useful for commencement of method development of HPLC methods for key raw materials, reaction monitoring, and active pharmaceutical ingredients. This practice leads to faster method development, rugged methods, and an accurate description of the analyte retention as a function of pH at varying organic compositions. Relationship of the analyte retention as function of mobile-phase pH (spH) is very useful to determine the pK of the particular 

The general procedure for the chromatographic determination of the pKa is to run at least 5 pH experiments isocraticaUy to construct a pH (on the x-axis) versus retention factor (or retention, on the y-axis) plot. The concentration of organic in the mobile phase should be selected to elute the most hydrophilic species (ionized form) with a /c 1. If the compound is acidic, the elution of the fuUy ionized species will be obtained at 2 pH units greater than the analyte pKa. If the compound is basic, the elution of the fully ionized species will be obtained at 2 pH units less than the analyte pKa. The organic composition chosen must also be able to elute the neutral species within a reasonable retention time (i.e., 30min). A short column with narrow internal diameter (i.e., 5.0 X 3.0 mm, using flow rate of 1.5 mL/min) that is stable from pH 2-11 should be used for these studies. The mobile phase could be made from 15 mM potassium phosphate, and the pH can be adjusted with either HCl or NaOH from 2 to 11. 

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A sample contains a weak acid analyte, HA, and a weak acid interferent, HB. The acid dissociation constants and partition coefficients for the weak acids are as follows Ra.HA = 1.0 X 10 Ra HB = 1.0 X f0 , RpjHA D,HB 500. (a) Calculate the extraction efficiency for HA and HB when 50.0 mF of sampk buffered to a pH of 7.0, is extracted with 50.0 mF of the organic solvent, (b) Which phase is enriched in the analyte (c) What are the recoveries for the analyte and interferent in this phase (d) What is the separation factor (e) A quantitative analysis is conducted on the contents of the phase enriched in analyte. What is the expected relative erroi if the selectivity coefficient, Rha.hb> is 0.500 and the initial ratio ofHB/HA was lO.O ... 

Another situation in which an inflection point may be missing or difficult to detect occurs when the analyte is a multiprotic weak acid or base whose successive dissociation constants are similar in magnitude. To see why this is true let s consider the titration of a diprotic weak acid, H2A, with NaOH. During the titration the following two reactions occur. 

In the second limiting situation the analyte is a weaker acid or base than the interferent. In this case the volume of titrant needed to reach the analyte s equivalence point is determined by the concentration of both the analyte and the interferent. To account for the contribution from the interferent, an equivalence point for the interferent must be present. Again, if the acid dissociation constants for the analyte and interferent are significantly different, the analyte s determination is possible. If, however, the acid dissociation constants are similar, only a single equivalence point is found, and the analyte s and interferent s contributions to the equivalence point volume cannot be separated. 

Modern Analytical Chemistry Add Dissociation Constants—continued... 

The ionization eonstant should be a function of the intrinsic heterolytic ability (e.g., intrinsic acidity if the solute is an acid HX) and the ionizing power of the solvents, whereas the dissoeiation constant should be primarily determined by the dissociating power of the solvent. Therefore, Ad is expeeted to be under the eontrol of e, the dieleetrie eonstant. As a consequenee, ion pairs are not deteetable in high-e solvents like water, which is why the terms ionization constant and dissociation constant are often used interchangeably. In low-e solvents, however, dissociation constants are very small and ion pairs (and higher aggregates) become important species. For example, in ethylene chloride (e = 10.23), the dissociation constants of substituted phenyltrimethylammonium perchlorate salts are of the order 10 . Overall dissociation constants, expressed as pArx = — log Arx, for some substanees in aeetie acid (e = 6.19) are perchloric acid, 4.87 sulfuric acid, 7.24 sodium acetate, 6.68 sodium perchlorate, 5.48. Aeid-base equilibria in aeetie acid have been earefully studied beeause of the analytical importance of this solvent in titrimetry. 

The following physico-chemical properties of the analyte(s) are important in method development considerations vapor pressure, ultraviolet (UV) absorption spectrum, solubility in water and in solvents, dissociation constant(s), n-octanol/water partition coefficient, stability vs hydrolysis and possible thermal, photo- or chemical degradation. These valuable data enable the analytical chemist to develop the most promising analytical approach, drawing from the literature and from his or her experience with related analytical problems, as exemplified below. Gas chromatography (GC) methods, for example, require a measurable vapor pressure and a certain thermal stability as the analytes move as vaporized molecules within the mobile phase. On the other hand, compounds that have a high vapor pressure will require careful extract concentration by evaporation of volatile solvents. 

Water solubility, dissociation constant(s) and n-octanol/water partition coefficients allow one to predict how an analyte may behave on normal-phase (NP), reversed-phase (RP), or ion-exchange solid-phase extraction (SPE) for sample enrichment and cleanup. 

Potentiometry is used in the determination of various physicochemical quantities and for quantitative analysis based on measurements of the EMF of galvanic cells. By means of the potentiometric method it is possible to determine activity coefficients, pH values, dissociation constants and solubility products, the standard affinities of chemical reactions, in simple cases transport numbers, etc. In analytical chemistry, potentiometry is used for titrations or for direct determination of ion activities. 

Among the possible analytical methods for alkalinity determination, Gran-type potentiometric titration [2] combined with a curve-fitting algorithm is considered a suitable method in seawaters because it does not require a priori knowledge of thermodynamic parameters such as activity coefficients and dissociation constants, which must be known when other analytical methods for alkalinity determination are applied [3-6],... 

In cases where fluorescence sensing is accompanied by binding of the analyte (classes 2 and 3), the dissociation constant of the complex should match the ex-... 

Figure 10.6. Relations between analyte concentration, dissociation constant, and relative concentration of each form of the probe.

Equation (10.23) describes the relations of the preexponential factors to the analyte concentration in the same way as relative concentration of free and bound forms given by Eqs. (10.12) and (10.13). The preexponential factor analyte response function may be shifted toward lower or higher analyte concentrations compared to those obtained from the absorbance or/and intensity measurements (Figure 10.6) because of the apparent dissociation constant (Kd) given by Eq. (10.24). 

If the probe display shifts in absorption and/or emission spectra, the apparent dissociation constant will depend not only on the measured parameter but also on the excitation and emission wavelengths. For probes that display a shift in the absorption spectrum on analyte binding, the value of f/ bdepends on excitation wavelength, which affects the value of Koa (see Eq. (10.24)). For probes which display shift in emission spectrum the value of may depend on observation wavelength. The... 

The agreement between the observed and predicted k values of aromatic acids was within 10%. The correlation coefficient was 0.954 (n = 32). An error of greater than 10% for 3-hydroxy-2-naphthoic acid and 2-hydroxybenzoic acid was attributed mainly to an error in their K.A values.25 The partition coefficient, logP, and dissociation constant, pKA, of analytes can be obtained by simple calculations and by computational chemical calculations, and thus the retention time can be predicted in reversed-phase liquid chromatography. 

The dissociation constant of an analyte can be calculated mathematically from Hammet s equation.27 The organic solvent effect on the pA a has also been examined 26... 

It is perfectly possible for some substrate-modulator combinations to result in an increase in substrate affinity, an increase in the rate of product formation, or both. The same analytical approaches may be used to study such compounds as have been described earlier to assess inhibitory mechanisms and potencies. However, with an allosteric activator, the dissociation constant might better be termed and values for a and p are more likely to be less than one, and greater than one, respectively. As is the case for inhibition, allosteric enzyme activation would be expected to exhibit substrate dependence (Holt et al., 2004). 

Other evidence for the contribution of electrostatic interactions to retention on SOIL phases may be found in a study by Sun and Stalcup [67]. In this work, it was reported that while the LSFER approach successfully accounted for intermolecular interactions responsible for retention of nonpolar solutes, inclusion of ionizable solutes such as pyridine or nitrophenol isomers seriously degraded the correlation between experimental and predicted retention. Successful global application of an LSFER approach for a training set which includes ionizable analytes required incorporation of an additional descriptor to account for the degree of ionization [68] of the analytes as well as to account for the impact of electrostatic interactions. The additional descriptor incorporated the mobile-phase pH as well as the acid dissociation constant of the analyte. 

Appendixes Tables of solubility products, acid dissociation constants (updated to 2001 values), redox potentials, and formation constants appear at the back of the book. You will also find discussions of logarithms and exponents, equations of a straight line, propagation of error, balancing redox equations, normality, and analytical standards. 

Fraction of easily accessible cavities Fluorescence intensity in the presence of analyte Initial fluorescence intensity in the analyte absence Current of anodic peak in LSV or CV Retention factor Acid dissociation constant Complex dissociation constant... 

Dissociation constant Kd is the most frequently used parameter for determination binding affinity of analyte to the aptamer. The lower Kd corresponds to higher affinity. This constant can be expressed as follows ... 

By combining equations (2.15), (2.16), and (2.18), a distribution diagram (Figure 2.10) for acetic acid can be prepared given that the acid dissociation constant is 1.8 x 10 5 with an assumed concentration of 0.01 M. The vertical line in Figure 2.10, positioned at x 4.74, is a reminder that when the pH of the solution is equal to the pKa of the analyte, the a value is 0.5, which signifies that the concentration of HA is equal to the concentration of A. The distribution diagram can be used to determine the fraction of ionized or nonionized acetic acid at any selected pH. 

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