Constant Wavelength Synchronous Fluorescence Spectroscopy Research Articles

Synchronous fluorescence techniques for the detection and monitoring of selected fluorescent dyes in binary and multifluorophoric mixtures

The present study deals with non-destructive conventional emission, constant-wavelength synchronous fluorescence spectroscopy (CWSFS) and first derivative of CWSFS for selective estimation of two fluorescent xanthene dyes viz. rhodamine B (RhB) and fluorescein (Fls) in their binary and multifluorophoric mixtures via spike method in an aqueous environment. Various parameters (Δλ, pH, ionic strength, etc.) affecting these methods were optimized. Based on the results, measurement of RhB and Fls were performed at Δλ, 20.0 nm, pH 7.0, 25.0 ⁰C temperature and scan rate of 240.0 nm/min without their prior separation in mixtures. CWSFS and first derivative CWSFS measurements were carried out at 556.0 nm and 547.0 nm for RhB (R2 = 0.988 and 0.983) and 493.0 nm and 484.0 nm for Fls (R2 = 0.999 and 0.999) over the concentration range of both dyes at 0.10–0.70 mg/L. The mean percentage recoveries from aqueous binary mixtures of RhB and Fls were found to be 98.95, 99.97 (RhB) and 100.68, 100.73 (Fls) for CWSFS and first derivative CWSFS respectively. Results were statistically analyzed in terms of accuracy and precision.

Rapid detection of four polycyclic aromatic hydrocarbons in drinking water by constant-wavelength synchronous fluorescence spectrometry.

Based on the advantages of the good selectivity and high sensitivity of the synchronous fluorescence method, an efficient method using constant-wavelength synchronous fluorescence spectrometry (CWSFS) for simultaneous and rapid determination of four polycyclic aromatic hydrocarbons (PAHs) (acenaphthene, phenanthrene, benzo[a]anthracene and fluoranthene) in drinking water was established in this study. When the difference in wavelength (Δλ) at 100nm was chosen for CWSFS scanning, the synchronous fluorescence spectra of the four PAHs could be well separated with only one single scan. Different from conventional fluorescence analysis, the established method can avoid the interference among the four PAHs each other and the interference of the drinking water sample matrix, so the four PAHs in drinking water could be well distinguished and determined. The concentrations of four PAHs in the range of 0.05-100μg/L, 0.1-400μg/L, 0.05-100μg/L and 0.5-2000μg/L showed a good linear relationship with fluorescence intensity. The limits of detection were 0.0058μg/L, 0.021μg/L, 0.0061μg/L and 0.056μg/L, respectively. The recoveries were in the range of 86.55-98.74%. Overall, the established CWSFS had the characteristics of simple, rapid, sensitive and accuracy, and had been applied to the determination of the four PAHs in various drinking water with satisfactory results.

Using constant-wavelength synchronous fluorescence spectroscopy in nanoparticle-based sensors: a minireview.

This study focuses on optical nanosensors based on constant-wavelength synchronous fluorescence spectroscopy (SFS) and reviews their applications for analysis purposes in the last few decades. In comparison to conventional fluorescence, SFS shows a higher selectivity owing to the narrowing of spectral bands and the simplification of spectra. The reported SFS-based nanosensors are classified based on their mechanism for analyte detection into two types including quenching based methods and enhancement based methods. Herein, almost all studies performed in this field are reviewed and the details of each study are carefully explained. Moreover, the analytical properties of the reported nanosensors are tabulated in relevant tables. It is hoped that this study will stimulate further investigations in this field with similar nanosensors.

Constant-wavelength synchronous fluorescence spectrometry for simultaneous and rapid determination of five polycyclic aromatic hydrocarbon residues in dairy products.

A new method for simultaneously determining five polycyclic aromatic hydrocarbons (PAHs) (fluorene, benzofluorene, pyrene, benzo(a)pyrene, and perylene) in dairy products using constant-wavelength synchronous fluorescence spectrometry (CWSFS) was established in this study. Acetonitrile was chosen as the extraction solvent to extract the five PAHs from the dairy products, and an ultrasound extraction method was adopted. The supernatants were filtered using a 0.45-μm microporous filter membrane and concentrated to dryness with a nitrogen dryer. The extracts were then re-dissolved in cyclohexane for analysis. To overcome interference from the background matrix and between PAHs, the difference in wavelength (Δλ) at 40 nm was chosen for CWSFS scanning. With only one single scan, the five PAHs in dairy products could be distinguished and determined using the standard curve method without the need for previous chromatographic separation of the analyte solution. Detection limits of fluorene, benzofluorene, pyrene, benzo(a)pyrene, and perylene were 0.0051 μg·L-1 , 0.016 μg·L-1 , 0.021 μg·L-1 , 0.0056 μg·L-1 , and 0.0062 μg·L-1 , respectively. Recoveries were between 85.60% and 98.42%. These five PAHs in dairy products were determined with good results and therefore expected to be a routine detection method for PAHs in dairy products.

Derivative constant wavelength synchronous fluorescence spectrometry for the simultaneous detection of cefadrine and cefadroxil in water samples.

A sensitive accurate spectrofluorimetric technique was developed to detect cefadroxil and cefradine traces in water samples simultaneously, by applying a procedure based on the formation of hydrolysis products corresponding to these compounds by sodium hydroxide (1N NaOH) treatment. The conventional and the synchronous fluorescence spectra of these hydrolyzed products were totally overlapped making resolving of this mixture impossible. The second-derivative constant-wavelength synchronous fluorescence spectra allowed their detection simultaneously in a single scan after experimental conditions optimization, which was measured at 390nm and 379nm for cefadroxil and cefradine, respectively at Δλ=30.0. The calibration curves between derivative synchronous fluorescence intensity and drugs concentration showed suitable linear correlation in the range of 0.1 to 5μg.mL-1 for cefadroxil and 0.5-10μg.mL-1 for cefradine. The proposed fluorimetric method is superior in being simple, environmental friendly and cost effective in comparison to the previously published reported methods.

Dual sensing and synchronous fluorescence spectroscopic monitoring of Cr3+and Al3+ using a luminescent Schiff base: Extraction and DFT studies.

A well designed, new pyrene based small molecule (L) was synthesized from 1:1 condensation reaction of 1-aminopyrene and 6-(1,3-benzodioxal-5-yl)-2-pyridine carboxaldehyde which was characterized by absorption, emission spectrometry, FTIR, NMR and mass studies. Interestingly the UV-vis and fluorescence spectroscopic studies revealed that the ligand (L) works as a dual turn-on luminescent chemosensor for chromium(III) (Cr3+) and aluminium(III) (Al3+) in aqueous environment which were further supported by DFT and TDDFT studies. L shows a significant colour change from pale yellow to reddish yellow with a detection limit of ~10-9M in the presence of Cr3+ and Al3+ whereas there were no noteworthy changes in the presence of other monovalent and divalent metal ions. The molecular signaling in the presence of Cr3+, Al3+, Fe3+ and EDTA was compared with advanced level combinational INHIBIT gate based on 4 input logic gates. Herein, first derivative constant wavelength synchronous fluorescence spectroscopy (1st DCWSFS) was applied for the determination of Cr3+, Al3+ ion concentrations in a mixture via increment of spectral resolution of the respective overlapping peaks. 1st DCWSFS is reported to be used in pharmaceuticals but very few works have been done for determination of metal ion concentration in environmental sample without prior separation. The individual Cr3+and Al3+ ion concentrations in a mixture were determined through liquid-liquid extraction process and the efficiencies were compared with 1st derivative SFS method. It was observed that 1st derivative SFS process is more efficient than conventional liquid-liquid extraction process. Therefore, 1st DCWSFS method using sensor L might be useful as a diagnostic tool for detection of individual metal ion concentrations (Cr3+ and Al3+) from a mixture which will be cost-effective, time saving and more precise.

Steroidal thiazolidinone derivatives: Design, synthesis and their molecular interaction with human serum albumin

A series of steroidal thiazolidinone derivatives have been synthesized through one-pot multicomponent reaction involving steroidal ketone, thiosemicarbazide/methyl-thiosemicarbazide and DMAD in presence of AlCl3 as a Lewis acid catalyst. Among all the synthesized steroidal thiazolidinone derivatives, compound 7–9 (ST 7–9) were investigated for their in vitro molecular interaction with human serum albumin. Intrinsic fluorescence spectroscopy, constant wavelength synchronous fluorescence spectroscopy, circular dichroism and UV–visible absorption techniques have been exploited to characterize the binding phenomena in phosphate buffer solution at pH 7.4. The experimental results indicated that ST 7–9 bind to HSA and the intrinsic fluorescence of HSA was quenched through static quenching mechanism. The binding parameters were calculated and the binding constants obtained were 1.44 × 105 M−1 for ST 7, 0.84 × 105 M−1 for ST 8 and 1.06 × 105 M−1 for ST 9. Circular dichroism analysis confirms that the presence of ST 7–9, altered the secondary structure of HSA due to partial unfolding of the polypeptide chain. Furthermore, hemolytic activity assay demonstrated that the synthesized steroidal thiazolidinone derivatives have good compatibility towards human red blood cells. Finally, molecular docking studies revealed that the steroidal thiazolidinones can bind in the hydrophobic cavity of HSA, by hydrophobic and hydrogen bonding interaction. These results provided valuable information about the binding mechanism of ST 7–9 with HSA and play a pivotal role in the development of steroidal heterocycle inspired compounds.

Interaction studies of lead(II) ion with cyclic β-(1→3),(1→6) glucans extracted from Bradyrhizobium japonicum based on 'chelation enhanced fluorescence' (CHEF) effect.

Cyclic β-(1→3),(1→6) glucans (CBGs) isolated from Bradyrhizobium japonicum bacteria are the periplasmic oligosaccharides having cyclic structures. This paper presents the isolation of CBGs from the bacteria cultured using optimized medium that improved yields to 350-450mg per gram of cellular dry weight along with analytical interaction with lead(II) ions in the range 33.0-2.0ppm with CBG as a binding ligand, using constant wavelength synchronous fluorescence spectroscopy. The binding ability of CBGs towards lead(II) is clearly evident from the scanning electron microscopy (SEM) images. The theoretical calculations using HEX 8.0 gave an insight about the interaction between CBG and lead(II) to be in the ratio of 3:1. The method displayed the sensitivity and selectivity towards lead(II) ions up to 2.0ppm. This observed property of CBGs can potentially hold an application in bioremediation of the soil contaminated with lead.

Simultaneous Determination of 1-Naphthylacetic Acid and Thiabendazole in Strawberry Tree Berries and Citrus Fruits by Fluorescence Spectrometry

An accurate, sensitive spectrofluorimetric method was developed to determine 1-naphthylacetic acid and thiabendazole simultaneously in fruits in a simple, expeditious manner. Broadband overlap between the conventional spectra of the two compounds was resolved by using first-derivative constant-wavelength synchronous fluorescence spectrometry. The proposed method is based on the intrinsic fluorescence of the two agrochemicals in ethanol. The sensitivity and selectivity of the determination were optimized by examining the influence of various factors on the fluorescence intensity. Based on the results, analyses were performed in 30% (v/v) ethanol–water mixtures at a pH 4.7 adjusted with sodium citrate buffer. Using the derivative signals at 304 and 286 nm for 1-naphthylacetic acid and thiabendazole, respectively, provided linear calibration graphs over the range 10–100 and 30–120 ng mL−1, and limits of detection of 1.99 and 0.17 ng mL−1, respectively. The analytical figures of merit of the proposed method were calculated in the light of the error propagation theory. The sensitivity, repeatability, reproducibility and limits of detection obtained are acceptable for determining the two agrochemicals in strawberry tree berries and citrus fruits without the need for a time-consuming prior separation. The proposed method is simple, reliable and cost-efficient, which makes it an attractive choice for fast selective screening purposes.

Identification of nitrogen-polyaromatic compounds in asphaltene from co-processing of coal and petroleum residue using chromatography with mass spectrometry

Asphaltene, from co-processing of coal and petroleum residues is one of the most precious and complex molecular mixtures existing, with tremendous economic relevance. Asphaltene was separated by Soxhlet extraction with methylbenzene and then divided into three parts by distillation. Gas chromatography (GC) and high-performance liquid chromatography (HPLC) were coupled with quadrupole time-of-flight mass spectrometry (Q-TOF MS) to separate and characterize organic nitrogen species in the distillates of asphaltene at molecular level. Molecular mass of compounds was mainly distributed from 150 to 600 μ. Number of rings plus double bonds (rdb) and synchronous fluorescence spectra indicated that most of the organonitrogen compounds (NPAC) contained heterocyclic aromatic rings, including pyridines, anilines, quinolins, pyrroles, carbazoles and indoles plus various alkyl groups. Constant-wavelength synchronous fluorescence spectrometry (CWSFS) indicated NPAC with 2–3 rings were the main structures of organonitrogen compounds and the corresponding structural information was proposed. Some organic nitrogen isomers were separated and identified by atmospheric pressure chemical ionization (APCI) GC-Q-TOF MS and electrospray ionization (ESI) HPLC-Q-TOF MS. The methodology applied here contained chromatographic injection of the diluted sample using conventional columns sets and Data Analysis 4.2 software. Identifying molecular structures provides a foundation to understand all aspects of coal-derived asphaltene, enabling a first-principles approach to optimize resource utilization.

Rapid determination of benzo[a]pyrene by membrane enrichment coupled with solid-phase constant-wavelength synchronous fluorescence spectrometry.

Normal methods for benzo[a]pyrene (BaP) determination, including gas chromatography-mass spectrometry and liquid chromatography with fluorescence detection, involve expensive instruments and complex separation and purification processes. Based on membrane enrichment, coupled with solid-phase constant-wavelength synchronous fluorescence spectrometry, a simple, fast, sensitive method was proposed for the determination of BaP in water samples. A Nylon membrane was used as the solid-phase extraction material for enrichment. After enrichment, a constant-wavelength synchronous fluorescence spectrum was scanned directly on the membrane-concentrated BaP without elution. Spectral measurement and enrichment conditions were optimized. Under optimum conditions, when using 150 mL sample solutions, the relationship between fluorescence intensity and BaP concentrations in the 0.05-10.00 μg L(-1) range could be fitted by binomial function with an R(2) value of 0.9973. Limit of detection (LOD) was calculated to be 0.0137 μg L(-1) . The volume of the sample solution was increased to 1000 mL to test if the method could be applied to determine lower BaP concentrations. A linear relationship still existed in the range 2.0-20.0 ng L(-1) BaP with an R(2) value of 0.9895, and a LOD of 2.4 ng L(-1) . The method was also used to measure the BaP concentration in several natural water samples, and recoveries were in the 90-110% range with relative standard deviations (RSDs) in the 0.58-7.93% range. Copyright © 2016 John Wiley & Sons, Ltd.

Human hemoglobin structural and functional alterations and heme degradation upon interaction with benzene: A spectroscopic study

Here, the effect of benzene on hemoglobin structure, stability and heme prosthetic group integrity was studied by different methods. These included UV–vis absorption spectrophotometry, normal and synchronous fluorescence techniques, and differential scanning calorimetry (DSC). Our results indicated that benzene has high hemolytic potential even at low concentrations. The UV–vis spectroscopic results demonstrated that benzene altered both the globin chain and the heme prosthetic group of hemoglobin increasing met- and deoxy-Hb, while decreasing oxy-Hb. However, with increasing benzene the concentration of all species decreased due to heme destruction. The spectrophotometric results show that benzene has a high potential for penetrating the hydrophobic pocket of hemoglobin. These results were consistent with the molecular docking simulation results of benzene-hHb. Aggregation and thermal denaturation studies show that the increased benzene concentration induced hemoglobin aggregation with a decrease in stability, which is consistent with the DSC results. Conventional fluorescence spectroscopy revealed that the heme degradation species were produced in the presence of benzene. The results of constant wavelength synchronous fluorescence spectroscopy (CWSFS) indicated that at least five heme-degraded species were produced. Together, our results indicated that benzene has adverse effects on hemoglobin structure and function, and heme degradation.

Synchronous spectrofluorimetric study of the supramolecular host–guest interaction of β-cyclodextrin with propranolol: A comparative study

The objective of this work is to assess the use of constant-wavelength synchronous fluorescence spectroscopy (SFS) in comparison to conventional fluorescence spectroscopy (CFS) for the investigation of the supramolecular host–guest interaction of β-CD with propranolol (PPL) in aqueous solutions. Scanning for the optimal Δλ at which the SFS can be performed in the presence of β-CD was examined. The results obtained revealed three distinguishable shapes for PPL using SFS that can be represented by three different Δλ values, namely 10, 40, and 100nm. However, the effect of the β-CD concentration on the fluorescence intensity of PPL was examined using CFS and SFS of PPL at a Δλ of 10 and 100nm. The change in the fluorescence intensity was used to calculate the equilibrium constant (Keq) for the formation of the β-CD:PPL inclusion complex by applying the Benesi–Hildebrand method. Keq values of 108, 112, and 117M−1 were obtained using SFS with a Δλ of 10 and 100nm, and CFS, respectively. Further, the SFS method was successfully employed to examine the iodide quenching effect on the fluorescence intensity of PPL, where the results obtained revealed a Stern–Volmer quenching constant of 42.8M−1, which is in good agreement with results obtained using CFS. All results obtained using the SFS method were compared with the results obtained using the CFS method.

Synchronous fluorescence and multivariate classification analysis as a screening tool for determining Sudan I dye in culinary spices

Spices are a globally traded commodity which has been found to be adulterated with forbidden Sudan dyes. This work proposes a screening method for determining the adulteration of paprika varieties (mild, hot and smoked) with Sudan I dye, based on constant-wavelength synchronous fluorescence spectroscopy with multivariate classification. Different wavelength-intervals (Δλ) were evaluated. Classification models were built with Partial Least Squares-Discriminant Analysis (PLS-DA) at two Sudan I dye concentration levels (1 and 5 mg L−1) and they were tested with samples at a lower level (0.5 mg L−1). Classification results were quite satisfactory when a strategy based on first-derivative spectra was used for improving classification results. Δλ = 60 nm was chosen as the optimum wavelength interval giving a 100% of sensitivity and specificity. These results are promising because the risk of assigning adulterated samples as safe to be consumed is highly minimized. The proposed method is feasible, rapid and simple taking advantage of Sudan I fluorescence phenomena in a direct way.

Investigation of the interactions between indole-3-acetic acid and catalase: A spectroscopic study in combination with second-order calibration and molecular docking methods

The interactions between indole-3-acetic acid (IAA) and the important antioxidant enzyme catalase (CAT) have been studied by Ultraviolet-visible (UV-vis) and fluorescence spectroscopic methods. The decrease of the absorption intensity of CAT at 270 nm and 407 nm in UV-vis spectra with the addition of IAA indicated the interactions between CAT and IAA. The three-way excitation–emission matrix spectra combined with parallel factor analysis (PARAFAC) was used to provide the individual concentration information of IAA, CAT, and IAA–CAT in the reaction system. The PARAFAC analysis showed that a 1:1 IAA–CAT complex formed at the equilibrium point. Constant wavelength synchronous fluorescence spectrometry (SFS) showed that the binding of IAA resulted in the micro-environment changes around tryptophan (Trp) and tyrosine (Tyr) residues. Molecular docking studies provide a good structural basis to explain the experimental results. This work is valuable for providing a deeper insight into the interaction mechanism of IAA with CAT.

Interaction of phenanthrene with dissolved organic matter and its fractions from leachate of different landfill ages

Dissolved organic matter (DOM) in leachate from landfills of three different ages (0, 5, and 10 year) was partitioned into hydrophobic acid, hydrophobic neutral, and hydrophilic matter fractions using the fractionation method employing XAD-8 resin. The spectroscopic characteristics of these fractions were determined through constant-wavelength synchronous fluorescence spectrometry (CWSFS). The interaction characteristic of phenanthrene with these fractions was determined through the fluorescence quenching method and CWSFS. The results revealed that the composition of DOM became complex with the increase in landfill age. The partition coefficients (K doc) for the three DOM samples of different landfill ages are in the order 10 a DOM > 5 a DOM > 0 a DOM. The hydrophobic fraction showed the highest K doc value among the DOM fractions. Lower linearity Stern–Volmer plots were observed in 10 a DOM and its hydrophobic fractions. CWSFS spectra showed that a special site was fully occupied with the addition of the quencher. Desorption was found in some fractions. Therefore, CWSFS is a sensitive tool for the detection of DOM-polycyclic aromatic hydrocarbon interaction.

Determination of critical concentrations by synchronous fluorescence spectrometry

We demonstrate that by using constant wavelength synchronous fluorescence spectrometry (CW-SFS) critical concentrations of some types of aggregation can be quantified. Detection of aggregation associated with hydrogen bonding in Chlorin e6 and Triton X-100 suggest that CW-SFS may be a technique that can explore aggregation at much lower levels (dimer, trimer, oligomer, etc.)

Changes in oxyhemoglobin during emotional oddball task in healthy subject evaluation multi channel NIRS

Here, the effect of benzene on hemoglobin structure, stability and heme prosthetic group integrity was studied by different methods. These included UV–vis absorption spectrophotometry, normal and synchronous fluorescence techniques, and differential scanning calorimetry (DSC). Our results indicated that benzene has high hemolytic potential even at low concentrations. The UV–vis spectroscopic results demonstrated that benzene altered both the globin chain and the heme prosthetic group of hemoglobin increasing met- and deoxy-Hb, while decreasing oxy-Hb. However, with increasing benzene the concentration of all species decreased due to heme destruction. The spectrophotometric results show that benzene has a high potential for penetrating the hydrophobic pocket of hemoglobin. These results were consistent with the molecular docking simulation results of benzene-hHb. Aggregation and thermal denaturation studies show that the increased benzene concentration induced hemoglobin aggregation with a decrease in stability, which is consistent with the DSC results. Conventional fluorescence spectroscopy revealed that the heme degradation species were produced in the presence of benzene. The results of constant wavelength synchronous fluorescence spectroscopy (CWSFS) indicated that at least five heme-degraded species were produced. Together, our results indicated that benzene has adverse effects on hemoglobin structure and function, and heme degradation.

Synchronous fluorescence, UV–visible spectrophotometric, and voltammetric studies of the competitive interaction of bis(1,10-phenanthroline)copper(II) complex and neutral red with DNA

Constant wavelength synchronous fluorescence spectroscopy (CW–SFS), UV–visible absorption spectroscopy, and cyclic and differential pulse voltammetry were applied to investigate the competitive interaction of DNA with the bis(1,10-phenanthroline)copper(II) complex cation ([Cu(phen) 2] 2+) and a fluorescence probe, neutral red dye (NR), in a tris–hydrogen chloride buffer (pH 7.4). The results show that both the [Cu(phen) 2] 2+and the NR molecules can intercalate competitively into the DNA double-helix structure. The cyclic voltammetry method showed that both anodic and cathodic currents of [Cu(phen) 2] 2+ decreased on addition of the DNA and the intercalated [Cu(phen) 2] 2+–DNA complex formed ( β = (4.14 ± 0.24) × 10 3). CW–SFS measurements were facilitated by the use of the three-way resolution of the CW–SFS for NR, [Cu(phen) 2] 2+, and NR–DNA. The important constant wavelength (CW) interval, Δ λ, was shown to vary considerably when optimized (135, 58, and 98 nm for NR, NR–DNA, and [Cu(phen) 2] 2+, respectively). This approach clearly avoided the errors that otherwise would have arisen from the common assumption that Δ λ is constant. Furthermore, a chemometrics approach, parallel factor analysis (PARAFAC), was applied to resolve the measured three-way CW–SFS data, and the results provided simultaneously the concentration information for the three reaction components, NR, [Cu(phen) 2] 2+, and NR–DNA, for the system at each equilibrium point. The PARAFAC analysis indicated that the intercalation of the [Cu(phen) 2] 2+ molecule into the DNA proceeds by exchanging with the NR probe and can be attributed to two parallel reactions. Comprehensive information was readily obtained; the replacement of the intercalated NR commenced immediately on introduction of [Cu(phen) 2] 2+, approximately 50% of NR was replaced by [Cu(phen) 2] 2+ at a concentration of 0.45 × 10 −5 mol L −1, and nearly all of the NR was replaced at a [Cu(phen) 2] 2+ concentration of 2.50 × 10 −5 mol L −1. This work has the potential to improve extraction of information from the fluorescence intercalator displacement (FID) assay.

Synchronous fluorescence and UV–vis spectrometric study of the competitive interaction of chlorpromazine hydrochloride and Neutral Red with DNA using chemometrics approaches

In this study, we have shown with the use of UV–vis spectrophotometry and the constant wavelength synchronous fluorescence spectroscopy (CW-SFS) techniques that the pharmaceutical drug, chlorpromazine hydrochloride (CPZ), intercalates into the deoxyribonucleic acid (DNA) double helix by partial exchange with the Neutral Red (NR) molecular probe. We have also demonstrated that with the use of three-way data plots, it is clear that it is important to have well-defined methodology for the selection of the important CW-SFS method parameter, Δ λ. Ad hoc selection of this parameter, or even that based on experience, can readily lead to serious errors, which subsequently can be transferred to the interpretation of results. The said three-way plots provide a straightforward diagrammatic method, which improves the selection process of a satisfactory value for Δ λ. Finally, we used PARAFAC modeling to resolve the complex three-way CW-SFS data, which provided simultaneously the concentration information for the three reaction components, NR, CPZ and NR-DNA, for the system at equilibrium. This PARAFAC analysis indicated that the intercalation of the CPZ molecule into the DNA proceeds by exchanging with the NR probe, and can be attributed to two parallel reactions.