3.3.1.1 General principle
Absorption measurements based upon ultraviolet and visible
radiation find widespread application for the identification and determination
of myriad inorganic and organic species. Molecular ultraviolet/visible
absorption methods are perhaps the most widely used of all quantitative
analysis techniques in chemical and clinical laboratories throughout the world.
Important characteristics of spectrophotometric and photometric methods
include: (1) wide applicability to both organic and inorganic systems, (2)
typical sensitivities of 10-4 to 10-6 M, (3) moderate to
high selectivity, (4) good accuracy, (5) ease and convenience of data
acquisition (Skoog et al., 1998).
Enormous numbers of inorganic, organic and biochemical species
absorb ultraviolet or visible radiation and are thus amenable to direct
quantitative determination. Many nonabsorbing species like artemisinin and
derivatives can also be determined spectrophotometrically by causing them to
react with a chromophoric reagent (i.e. hemin) to yield a product that absorbs
in the ultraviolet or visible region.
Absorption spectroscopy is based upon electromagnetic
radiation in the wavelength region of 160 to 780nm. Then, molecular absorption
spectroscopy is based on the measurement of the transmittance
T or the absorbance A of solutions contained
in transparent cells having a path length of d cm. Ordinarily,
the concentration c of an absorbing analyte is linearly
related to absorbance as represented by the equation 3-1.
A = -log T = cd (3-1)
This equation is a mathematical representation of
Beer-Lambert's law, where is the molar extinction coefficient. Absorbance of a
solution is often influenced by such variables as the nature of the solvent,
pH, temperature, electrolyte concentration, reaction time and presence of
interfering substances (Skoog et al., 1998).
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3.3.1.2 Procedural details of
hemin-drugs spectrophotometric titrations.
Spectrophotometry is a valuable tool for elucidating the
composition of complex ions in solution and for determining their formation
constants. In this study, we opted for the mole-ratio method, where a series of
solutions is prepared in which the analytical concentration of one reactant is
held constant while that the other is varied. A plot of absorbance versus mole
ratio of the reactants is then prepared.
In our study, the interactions of hemin with quinoline based
drugs were investigated in water-propylene glycol mixture at pH 9, 8.1, 7.4,
and 6.8 using spectrophotometric method. Temperature was controlled at
25oC. Although quinolines compounds showed some bands of absorption
in UV-Visible domain, the titration was carried out at the hemin characteristic
Soret band at 396 nm by mixing a constant volume (0.3 mL) of hemin solution
with various volumes of drug solutions, and then diluted to 5 mL. Thereby,
hemin remains at constant concentration (19 ) while drugs concentrations are
changed in the range of 0-1300 .
Hemin-artemisinin compounds in water-DMSO or in
water-propylene glycol mixture were studied too, using spectrophotometric
method, at pH 9 and 7.4.
The spectrophotometer was equipped with a thermostatic cell
and temperature was controlled at 37oC. Due to its lack of
chromophores group, artemisinin absorb weakly in the low wavelength region and
made his quantification more difficult. Then, the titration was carried out at
the hemin characteristic Soret band at 398 (in 50 % PREG) and 402nm (in 40 %
DMSO) by mixing a constant volume (0.1 mL) of hemin solution with various
volumes of drug solutions, and then diluted to 10 mL by addition of Tris-PEG or
Tris-DMSO mixtures. Thereby, hemin remains at constant concentration (3 ) while
drugs concentrations are changed in the range of 0-200 . Before each
measurements of absorbance, the work solutions were incubated at
37oC. UV-Visible spectra were carried out after 10 or 24 hours of
incubation.
The spectra have been recorded under the following
instrumental conditions:
Light-path-length cell: 1.0 cm
Mode: absorbance
Slit width: 2nm
Scan speed: fast
Wavelength range: 230-650 nm
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