These absorbance ratios correspond roughly to the range of CR absorbance ratios (R) encountered in oceanic measurements. The absorbance

Y-27632 cost measurements used to determine the ratios were well within the linear-response characteristics of the Cary 400 spectrophotometer. The temperature and salinity ranges were 278.13 ≤ T ≤ 308.27 K and 20 ≤ S ≤ 40. Initial estimates for the e1 term in Eq.  (2) were obtained by determining the e1 molar absorptivity ratio at a pH where the HI− form of the dye is dominant. Iterative calculations are necessary to account for absorbance contributions at 433 nm and 573 nm from the H2I and I2 − forms of the dye. The overlapping absorbance spectra of H2I, HI− and I2 − are shown in Fig. 1. A speciation model for T = 298.15 K and S = 35 was constructed using the K1 determined as described in Section 2.7 and the K2 reported by Byrne and Breland (1989). At a pH of 4.5, HI− is near

99.91% of the total CR concentration; the fractions of H2I and I2 − are 0.045% and 0.046%. Requisite e1 ABT-199 in vivo absorbance data (573A/433A) were determined with a 0.02 m acetate buffer solution at ionic strength of 0.7 m NaCl. No salinity dependence was observed for the very small e1 term. During preparation of the acetate/acetic acid buffer solution, pHf (free scale) was monitored with a ROSS combination electrode that had been calibrated on the free hydrogen ion scale by titrating a 0.7 m NaCl solution with standard HCl. Because the HI− absorbance signal includes contributions from the H2I and I2 − forms of the dye, the following equation was used to account for these contributions (see also derivation of Liu et al., 2011): equation(6) e1=εHI−573εHI−433=AHI−573/sHI−AHI−433/sHI−=AT573−AH2I573−AI2−573AT433−AH2I433−AI2−433where λεHI is the molar absorptivity at a given wavelength (λ) for the HI− form of the indicator, λAx is the absorbance at wavelength λ of total (T) indicator (all forms) or of individual indicator forms (H2I, HI−, or I2 −), s is the cell pathlength, and [HI−] is the concentration of the HI− form.

Expressing the absorbance terms in Eq. (6) in terms of molar absorptivities and total CR concentrations (IT) via K1 and K2, e1 can be written as follows: isothipendyl equation(7) e1=AT573−εH2I573ITsH+2K1K21+H+K2+H+2K1K2−1−εI2−573ITs1+H+K2+H+2K1K2−1AT433−εH2I433ITsH+2K1K21+H+K2+H+2K1K2−1−εI2−433ITs1+H+K2+H+2K1K2−1 To obtain the K2 value required in this calculation, initial e1 estimates were used to obtain initial K2T estimates by solving Eq.  (2) for − log (K2Te2). The e2 term, required to calculate K2T from − log(K2Te2), was calculated as a function of temperature by using the HI− absorbance at λ = 433 nm in the solution used to determine e1 (i.e., acetate buffer of pH = 4.5 and 0.7 m ionic strength) and the absorbance at λ = 573 nm in the solution used to determine e3/e2 (i.e., modified synthetic seawater of pH = 12 and 0.7 m ionic strength).