Tetrasulphonic Acid Research Articles

Influence of cationic dimeric and trimeric-type surfactants on the aggregation behavior of water-soluble phthalocyanine: A combined experimental and computational study

The aggregation behavior of the water-soluble phthalocyanine, namely zinc 2,9,16,23–tetra(tert–butyl)–29H,31H–phthalocyanine tetrasulphonic acids (ZnPcS4), has been examined in the presence of dimeric and trimeric-type cationic surfactants and their conventional counterpart cetyltrimethylammonium bromide (CTAB). The absorption and fluorescence studies of ZnPcS4 in water showed a different feature than that in methanol suggesting the formation of aggregated ZnPcS4 in an aqueous medium. The fluorescence studies of ZnPcS4 in the presence of examined dimeric-type cationic surfactants showed lower critical aggregation concentration (CAC = 0.62–13.6 µM) compared to that of CTAB (CAC = 16 µM). It was found that the position of the substituents affected the critical micelle concentration (CMC) value, and the ortho-dimeric surfactant had a lower CMC value compared to those surfactants with meta, and para-dimeric surfactant. It was interesting to see that the CMC for the dimeric surfactant with two groups at the para position (13.6 µM) decreased significantly (0.04 µM) by removing the (O-CH2) groups attached to the benzene which can be explained by structural parameters determined by theoretical calculations. By changing the surfactants from dimers to cationic trimers surfactants, a significant decrease in the CAC values was observed (0.04 µM for Trimer-12 with two dodecyl groups and 0.39 µM for Trimer-8 with two octyl groups). This finding indicates that the increase in tail length by four methylene groups of Trimer-12 compared to Trimer-8 results in a decrease in CAC by about one order of magnitude. These new findings suggest that the trimeric-type cationic surfactants are remarkably superior to conventional and dimeric-type surfactants, which could have a profound impact on many applications of ZnPCS4 including, but not limited to, medical, organic electronics, light harvesting, photonics, and optical sensors.

Smartphone-assisted ratiometric fluorescence sensor for sensitive and portable α-glucosidase activity detection and inhibitor screening

α-Glucosidase (α-Glu) is an enzyme that plays an essential role in the digestion of carbohydrates, and its activity detection and inhibitor screening are crucial for the therapy of diseases and development of new drugs. In this study, α-Glu can catalyze the hydrolysis of α-arbutin to produce hydroquinone (HQ), and HQ can regulate the in-situ synthesis of polymer carbon dots (PCDs) with ethylenediamine (EDA). PCDs and tetraphenylporphyrin tetrasulphonic acid (TPPS) are separately designed as green and red fluorescent sensing units. With the increase of α-Glu concentration, the number of PCDs is gradually increased, resulting in enhancement of green fluorescence and quenching of red fluorescence. Conversely, when α-Glu inhibitors (AGIs) are present, the activity of α-Glu is inhibited, leading to weak green fluorescence and strong red fluorescence. Thus a ratiometric fluorescence sensor was designed for the determination of α-Glu activity and screening of AGIs. The developed ratiometric sensor exhibits a linear relationship between fluorescence intensity ratios and α-Glu concentrations (0.01 to 0.04 U/mL) with limit of detection of 0.184 mU/mL, which is much lower than the single fluorescence model. In addition, rapid and portable detection of α-Glu can be achieved using a smartphone with a color picking software installed as a signal detection device, which greatly reduces the cost and time of detection and provides a promising method for qualitative identification and quantitative analysis of α-Glu in the resource-limited areas. Moreover, the method is very promising for high-throughput screening AGIs from natural products.

Organic–inorganic hybrid photocatalyst consisting of a highly conjugated metal complex and graphitic carbon nitride for efficient hydrogen evolution and Cr(vi) reduction

Copper tetraphenyl tetrasulphonic acid grafted g-C3N4is demonstrated as a heterogeneous photocatalyst for superior H2evolution and Cr(vi) reduction.

Antifungal properties of hypericin, hypericin tetrasulphonic acid and fagopyrin on pathogenic fungi and spoilage yeasts

Context: The role of hypericin-mediated photodynamic antimicrobial properties on pathogenic fungi and photodynamic therapy for human cancer cells is known. Antifungal properties of Hypericum perforatum L. (Hypericaceae) and Fagopyrum esculentum Moench. (Polygonaceae) extracts were also studied. The different polarities of solvents can cause complication in the identification of antifungal effects of separate biologically active compounds. In recent experimental work, we compared antifungal properties of purified hypericin, hypericin tetrasulphonic acid (hypericin + S) and fagopyrin, which is analogue of hypericin.Objective: The antifungal properties of aromatic polyketide derivatives such as hypericin, hypericin + S and fagopyrin on the selected pathogenic fungi and spoilage yeasts have been studied.Materials and methods: The antifungal properties of hypericin, hypericin + S and fagopyrin were determined using the broth microdilution method against a set of pathogenic fungi and spoilage yeasts including: Microsporum canis, Trichophyton rubrum, Fusarium oxysporum, Exophiala dermatitidis, Candida albicans, Kluyveromyces marxianus, Pichia fermentans and Saccharomyces cerevisiae. The tested concentrations of hypericin, hypericin + S and fagopyrin ranged from 750 to 0.011 μg/mL and MIC values were evaluated after 48 h incubation at 30 °C.Results: The results confirm different antifungal properties of hypericin, hypericin + S and fagopyrin on the selected pathogenic fungi and spoilage yeasts. For pathogenic fungi, the minimum inhibitory concentrations of hypericin ranged 0.18–46.9 μg/mL, hypericin + S 0.18–750 μg/mL and fagopyrin 11.7–46.9 μg/mL. For spoilage yeasts, the MICs of hypericin and hypericin + S ranged 0.18–46.9 and 0.011–0.73 μg/mL, respectively.Discussion and conclusion: The results obtained herein indicate that various chemical structures of hypericin, hypericin + S and fagopyrin can develop different antifungal properties.

In vivo monitoring of liposomal release in tumours following ultrasound stimulation

Dioeleoylphosphatidylethanolamine (DOPE)-based liposomes were recently reported as a new class of liposomes for ultrasound (US)-mediated drug delivery. The liposomes showed both high stability and in vitro US-mediated drug release (sonosensitivity). In the current study, in vivo proof-of-principle of US triggered release in tumoured mice was demonstrated using optical imaging. Confocal non-thermal US was used to deliver cavitation to tumours in a well-controlled manner. To detect in vivo release, the near infrared fluorochrome Al (III) Phthalocyanine Chloride Tetrasulphonic acid (AlPcS4) was encapsulated into both DOPE-based liposomes and control liposomes based on hydrogenated soy phosphatidylcholine (HSPC). Encapsulation causes concentration dependent quenching of fluorescence that is recovered upon AlPcS4 release from the liposomes. Exposure of tumours to US resulted in a significant increase in fluorescence in mice administered with DOPE-based liposomes, but no change in the mice treated with HSPC-based liposomes. Thus, DOPE-based liposomes showed superior sonosensitivity compared to HSPC-based liposomes in vivo.

Highly efficient in vitro photodynamic inactivation of Mycobacterium smegmatis

Efforts to control tuberculosis (TB) have been hampered by the emergence of multiple-drug resistant strains, necessitating pursuit of alternative approaches to the current antibiotic-based treatments. Herein, we explore the feasibility of photodynamic inactivation (PDI) of mycobacteria. In vitro PDI studies employing Mycobacterium smegmatis as a surrogate for Mycobacterium tuberculosis were performed examining photosensitizer (PS) type, concentration and light dose. M. smegmatis was grown to a concentration of 10(8) colony forming units (cfu) per mL, resuspended in PBS-0.5% Tween-80-containing buffer, incubated with the PS for 5 min and subsequently illuminated with white light (400-700 nm) at a fluence rate of 60 mW/cm(2) for 1, 5, 15 or 30 min (equivalent to 3.4, 18, 54 or 108 J/cm(2)). The percentage survival was determined by the ratio of the colony count from illuminated and non-illuminated control cell suspensions. The PSs examined were 5,10,15,20-tetrakis(1-methyl-4-pyridinyl)porphyrin tetratosylate (TMPyP), 5,10,15,20-tetrakis(4-N,N,N-trimethylanilinium)porphyrin tetrachloride (TNMAP), methylene blue (MB), 5,10,15,20-tetrakis(4-sulphonatophenyl)porphyrin (TSPP), 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin-Pd(II) (TCPP-Pd) and phthalocyanine tetrasulphonic acid (PhCS). Our best results demonstrate that PDI of M. smegmatis can achieve a noteworthy 5-6 log unit reduction in cfu (99.999% + viable cell eradication) when cationic PSs are employed in the nanomolar concentration range. Anionic PSs did not effectively mediate PDI of mycobacteria due to their inability to associate with the negatively charged mycobacterial cell membrane. PDI of M. smegmatis was found to be highly efficient in reducing the number of viable cells in vitro when cationic PSs were employed.

Effects of capillary coating and β-cyclodextrin additive to the background electrolyte on separation of sulphonated azodyes by capillary zone electrophoresis

Fourteen azodyes containing one to five acidic groups were separated by capillary zone electrophoresis (CZE). The effects of the pH and β-cyclodextrin additive to the background electrolyte on the separation of sulphonated azodyes were investigated. The effects of the working conditions significantly differ in non-coated fused silica capillaries and in capillaries coated with polyacrylamide. Splitting of the zones of metal-complex dyes was observed in polyacrylamide coated capillaries and the background electrolyte with 10 mmol/L β-cyclodextrin, due to the separation of stereoisomeric forms of the dyes, which were separated for the first time using CE. Relations between the structure of the sulphonated azodyes and the electrophoretic mobilities are discussed. Naphthalene mono- to tetrasulphonic acids were used as the standards for the calibration of migration scale of the analysed dyes.

Phthalocyanine-mediated photodynamic therapy induces cell death and a G 0/G 1 cell cycle arrest in cervical cancer cells

We have developed a series of novel photosensitizers which have potential for anticancer photodynamic therapy (PDT). Photosensitizers include zinc phthalocyanine tetra-sulphonic acid and a family of derivatives with amino acid substituents of varying alkyl chain length and degree of branching. Subcellular localization of these photosensitizers at the phototoxic IC 50 concentration in human cervical carcinoma cells (SiHa Cells) was similar to that of the lysosomal dye Lucifer Yellow. Subsequent nuclear relocalization was observed following irradiation with 665 nm laser light. The PDT response was characterized using the Sulforhodamine B cytotoxicity assay. Flow cytometry was used for both DNA cell cycle and dual Annexin V-FITC/propidium iodide analysis. Phototoxicity of the derivatives was of the same order of magnitude as for tetrasulphonated phthalocyanine but with an overall trend of increased phototoxicity with increasing amino acid chain length. Our results demonstrate cell death, inhibition of cell growth, and G 0/G 1 cell cycle arrest during the phthalocyanine PDT-mediated response.

Separation of aromatic sulphonic acids by CZE in coated and non‐coated capillaries

AbstractCapillary zone electrophoresis (CZE), besides ion‐pairing mode HPLC and salting‐out HPLC, is well‐suited for the analysis of aromatic sulphonic acids widely used as intermediates in the production of synthetic dyes and optical brighteners. The separation selectivity in CZE of many aromatic acids, including positional isomers, can be controlled by the type and concentration of a cyclodextrin additive. The influence of the concentration of β‐cyclodextrin in the working electrolyte on the separation of the positional isomers of naphthalene (poly‐)sulphonic acids, and their amino‐ and hydroxy derivatives, by CZE was studied, both in non‐coated fused silica capillaries and in capillaries coated with polyacrylamide. The migration time scale was calibrated using 4‐alkylbenzenesulphonic acids as the calibration standards. Limiting mobilities of the free acid anions and of their complexes with β‐cyclodextrin were calculated and the effect of the inclusion guest‐host complex formation on the CZE separation was quantitatively characterized. The migration order in coated capillaries is reversed with respect to CZE in non‐coated fused silica capillaries, the separation selectivity is different and the separation of polysulphonic acids such as naphthalene tri‐ and tetrasulphonic acids is significantly accelerated.

Some observations on the synthesis of polysubstituted zinc phthalocyanine sensitisers for photodynamic therapy

The syntheses and properties of a number of polysubstituted zinc phthalocyanines are described which are suitable as sensitisers for photodynamic therapy. Zinc phthalocyanine tetrasulphonic acid, itself a useful photosensitiser, has been used to prepare tetrasulphonamides by converting it to the tetrasulphonyl chloride and reacting this with various amino compounds. This has afforded lipophilic and water-soluble anionic and neutral tetra-sulphonamido derivatives. Chlormethylation of zinc phthalocyanine under carefully defined conditions followed by reaction with pyridine affords a water-soluble cationic dye with an average of two methylenepyridinium groups per molecule. Hydrolysis of zinc phthalocyanine tetracarboxyamide could not be carried out to completion to afford the tetracarboxylic acid, and the resultant product had an average composition corresponding to a dicarboxylic acid-dicarboxyamide structure. Visible absorption spectroscopic properties and singlet oxygen sensitising efficiencies of the dyes (measured relative to methylene blue) are described. All the dyes are photocytotoxic in vitro and show anti-tumour photoactivity in vivo.

Separation of aromatic sulphonic acid dye intermediates by high-performance liquid chromatography and capillary zone electrophoresis

The possibilities of separating various aromatic sulphonic acids used as intermediates in production of synthetic dyes by reversed-phase high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE) were studied. Isomeric naphthalene mono- to tetrasulphonic acids are best separated by HPLC with aqueous-methanolic mobile phases containing sodium sulphate. A high content of bonded hydrocarbon moieties is essential for successful separation. Components of technological samples of 4,4'-dinitrostilbene-2,2'-disulphonic acid can also be successfully separated using this technique, but the separation is very sensitive to the concentration of methanol in the mobile phase. Various aromatic aminosulphonic acids (letter acids) in technological samples can be separated by ion-pair reversed-phase chromatography with mobile phases containing tetrabutylammonium ions, but a better separation is possible using capillary zone electrophoresis with working electrolytes containing β-cyclodextrin. The opposite applies to the separation of naphthalenesulphonic acids and dinitrostilbenedisulphonic acid isomers, so that the HPLC and CZE are complementary to each other. Both HPLC and CZE techniques are suitable for the quantitative analysis of technical products containing aromatic sulphonic acids.

This article is a serial study's: Dyestuff Aggregation Observed by Small-Angle X-ray Scattering. (Part 1). Evaluation of Size and Shape of Copper Phthalocyanine Tetrasulphonic Acid Tetra Sodium Salt and C.I. Reactive Blue 19 in Aqueous Solution.

The aggregation of two dyes (1) copper phthalocyanine tetrasulfonic acid (CuPc) and (2) C. I. Reactive Blue 19 (RB19) was observed in aqueous solution by small-angle X-ray scattering (SAXS). Two CuPc molecules or six RB19 molecules were found to form the aggregate of a disk shape or an ellipsoidal shape in aqueous solution without salt, respectively. By adding salt, they aggregate further into an inclined cylinder composed of five CuPc molecules or an ellipsoid composed of 18 RB19 molecules. The detail of molecular arrangements in the aggregates was considered by comparing the scattering profiles calculated from the molecular models with the observed SAXS profiles.

In vivo action spectra, absorption and fluorescence excitation spectra of photosensitizers for photodynamic therapy

Recently the importance of measuring in vivo action spectra for photodynamic therapy (PDT) with various photosensitizers was pointed out in this journal [1]. It was shown that the effect of PDT with zinc phthalocyanine tetrasulphonic acid (ZnPcS4) increases considerably when the wavelength of the activating light is increased from 680 to 692 nm. In this regard a further comparison with haematoporphyrin derivative/photofrin and other recently presented information about phthalocyanines may be useful. In particular we wish to point out in this note the possible relevance of the fluorescence excitation spectrum and the possibility to measure the in vivo absorption spectrum of a photosensitizer by reflection spectroscopy. Preclinical and clinical studies on PDT with hae

On the photodynamic therapy action spectrum of zinc phthalocyanine tetrasulphonic acid in vivo

The photodynamic therapy (PDT) activity of zinc phthalocyanine tetrasulphonic acid in a rodent tumour model was shown to be critically dependent on the wavelength of the excitation laser light over a relatively small wavelength range. Thus the sensitizer showed a doubling of the PDT activity with fibrosarcoma LSBD 1 in BDIX rats when the wavelength of the illuminant was displaced from 680 to 692 nm. Under these conditions, the sensitizer is approximately three times more effective than polyhaematoporphyrin, whereas previously it has been considered to be of low PDT activity. This wavelength effect is attributed to a red shift of the absorption spectrum of the sensitizer in cells compared with that in solution. Fluorescence excitation studies with sensitizer absorbed in mouse 3T3 fibroblast cells are consistent with such a red shift.

Liquid chromatography and capillary electrophoresis analysis of polyanionic quinobene

Quinobene is the tetrasodium salt of an organic tetrasulphonic acid. Its unusual solubility characteristics makes the development of LC analysis difficult. However, a specific, precise and accurate LC assay was eventually achieved for quinobene. The assay required gradient elution and was not efficient for quinobene with respect to plate number. As an alternative, a capillary electrophoresis (CE) assay was also developed for quinobene. The CE assay was comparable to the LC assay in precision and accuracy. It was unaffected by the unusual solubility characteristics of quinobene and was more specific, efficient and rugged than the LC assay.

Modulating effects in human diets of dietary fibre and beef, and of time and dose on the reactive microcapsule trapping of benzo[a]pyrene matabolites in the rat gastrointestinal tract

Trapping by magnetic polyethyleneimine (PEI) microcapsules was utilized to investigate the influence in male rats of dose, human dietary composition and time-dependence on reactive metabolites of benzo[a]pyrene (B[a]P) in the gastrointestinal (GI) tract; also, PEI microcapsules modified with copper phthalocyanine tetrasulphonic acid (CPTS) were tested in vivo for trapping of endogenous mutagens having planar molecular structure. In a preliminary experiment the PEI microcapsules were administered by gavage at 0, 24 and 48 h, with [14C]B[a]P at 2 h to chow-fed BDVI rats; microcapsules were recovered from faeces collected at 24, 48 and 72 h, and then subjected to an extraction sequence showing that the trapped B[a]P metabolites were inconsistent with B[a]P diol epoxide trapping (as previously found) and unaltered by elapsed time or 5-fold dose alteration of B[a]P. Then five groups of F344 rats were fed isocalorically either one of four low-fat human diets or rat chow; in order to investigate influences of diet both on B[a]P and endogenous mutagens, half of each group was tested at 2 weeks with this PEI microcapsule/[14C]B[a]P protocol and then at 3 weeks, PEI-CPTS microcapsules (two gavages). So as to provide a cross-over comparison, the other half of each group was first tested with PEI-CPTS microcapsules followed by PEI microcapsules/[14C]B[a]P 1 week later. The human diets were prepared from cooked British foods so as to simulate the adequate intake of all nutrients required by humans; but with 3-fold differences in intake levels of beef and dietary fibre non-starch polysaccharide (NSP), while ensuring the same intake of available energy, protein, fat and calcium. They gave very similar body-weight gains in the four groups but greatly reduced faecal weight, protein and total faecal enzyme activity compared with chow; the extraction pattern of microcapsule-trapped B[a]P metabolite radioactivity was not significantly altered. However, human diet consumption caused a 2- to 6-fold increase in B[a]P metabolite binding to microcapsules and reductions in microcapsule recovery, net 70-h B[a]P excretion, faecal protein and total activities for beta-glucuronidase and beta-galactosidase; these effects were more pronounced after 3 weeks, presumably due to prolonged dietary adaptation. Increased NSP in human diets significantly increased the B[a]P metabolite excretion and marginally reduced the microcapsule binding. The increase in microcapsule binding of B[a]P metabolites, interpreted as reflecting an increased amount of reactive metabolites encountered, was related to the dietary intake weight ratio of beef/NSP.(ABSTRACT TRUNCATED AT 400 WORDS)

Copper phthalocyanine labelled magnnetic microcapsules: preparation, and binding properties in vitro and in vivo for mutagens having planar molecular structure

Copper phthalocyanine tetrasulphonic acid (CPTS) functions were introduced into magnetic semi-permeable polyethyleneimine (PEI) microcapsules in order to create a recoverable scavenging system for trapping and biomonitoring, within the gastrointestinal cavity, of mutagens having a planar molecular structure. Stable ionic CPTS and covalent (thionylated CPTS, TCPTS) adducts to the microcapsule PEI were produced and shown to trap benzo[a]pyrene (B[a]P) in vitro in relation to the porphyrin/B[a]P ratio employed. 3-hydroxy B[a]P and B[a]P 3,6-dione from a crude B[a]P metabolite mixture, and a set of planar mutagens from crude opium/morphine pyrolysate mixtures could also be recovered in 7-86% yields after shaking with modified microcapsules followed by methanol/ammonia (50:1) desorption. Tetraols derived from B[a]P 7,8-diol-9,10 epoxide could also be recovered. Modified microcapsules were recovered magnetically from faeces of rats treated with [14C]B[a]P, and 45-51% of trapped radioactivity could be directly desorbed for HPLC assay compared with 30% for unmodified microscapsules. The relative extent of trapping by unmodified or CPTS- or TCPTS-modified microcapsules was different for various substrates, and it appears that the copper phthalocyanine tetrasulphonic acid moiety competes with another unidentified absorption/desorption structure in the microcapsules. These results show that selective and reversible trapping of carcinogens/mutagens having planar molecular structure can be achieved within the gastrointestinal tract.

Pulsed irradiation of water-soluble porphyrins

Nanosecond laser flash photolysis and pulse radiolysis have been used to characterise the excited states and radicals of meso-tetraphenylporphyrin tetrasulphonic acid as the tetrasodium salt (Na4TPPS) and of meso-tetra-N-methylpyridylporphyrin as the tetra-p-toluenesulphonate (TMPyrP). After 347 nm irradiation, aqueous Na4TPPS forms triplet states (ϕT= 0.76) and photoionises monophotonically (ΦI= 0.029), while TMPyrP forms only triplets (ΦT= 0.92) and does not photoionise. The difference and corrected spectra of the triplet states and the semi-oxidised and semi-reduced radicals of these molecules have been obtained.

Aromatic sulphonation. Part 66. Sulphonation of some biphenyl derivatives

The sulphonation of a number of biphenyl derivatives, viz. 2,2′-Me2, 3,3′-Me2, 4,4′-Me2, 3,5-Ph2, 2,2′-(SO3H)2, 2,4′-(SO3H)2, 4,4′-(SO3H)2-, 2,2′-SO2-, 2,2′-(NO2)2, and 2,4,6-(NO2)3 in sulphuric acid at 25 °C has been studied; product analysis was by multicomponent 1H n.m.r. analysis. Sulphonation of 2,2′-dimethylbiphenyl resulted in a 5 : 19 : 76 mixture of the 3,3′-, 3,5′-, and 5,5′-disulphonic acids. Further sulphonation yielded the 3,3′,5,5′-tetrasulphonic acid. 3,3′-Dimethylbiphenyl yielded a 52 : 48 mixture of the 4,4′- and 4,6′-disulphonic acid. Further sulphonation of each of the disulphonic acids yielded the 4,4′,6,6′-tetrasulphonic acid. 4,4′-Dimethylbiphenyl yielded a 34 : 66 mixture of the 2,3′- and 3,3′-disulphonic acids. Further sulphonation of the latter yielded the 3,3′,5,5′-tetrasulphonic acid. 1,3,5-Triphenylbenzene gave exclusively the 4′,4″,4‴-trisulphonic acid, which could not be further sulphonated. Biphenyl-2,4′- and -4,4′-disulphonic acid both yielded the 2,2′,4,4′-tetrasulphonic acid, which is not obtained from biphenyl-2,2′-disulphonic acid. Dibenzothiophen 5,5-dioxide gave the 3,7-disulphonic acid. 2,2′-Dinitrobiphenyl yielded the 4,4′-disulphonic acid, but 2,4,6-trinitrobiphenyl only a mixture of monosulphonic acids which contained 88% of the 4′-acid.

Aromatic sulphonation. Part 70. The sulphuric acid sulphonation of α,ω-diphenylalkanes, biphenylene, fluorene, 9,10-dihydrophenanthrene, 10,11-dihydro-5H-benzo[a,d]cycloheptene, and triptycene

The sulphonic acid isomer distribution for the sulphonation of α,ω-diphenylalkanes, biphenylene (2a), fluorene (2b), 9,10-dihydrophenanthrene (2c), 10,11-dihydro-5H-benzo[a,d]cycloheptene (2e), and triptycene (3) with concentrated aqueous and with fuming sulphuric acid at 25 °C has been determined by multicomponent 1H-n.m.r. analysis. With Ph[CH2]nPh in 95.4% H2SO4 the degree of ortho-substitution increases with increasing length of the alkylidene chain to reach for n 4 the same limiting value as observed for the 1-phenylalkanes (Ph[CH2]nMe) with n 3. Further sulphonation of the α,ω-bis(sulphophenyl)alkanes yields the o,o′,p,p′-tetrasulphonic acids.Sulphonation of (2a) and biphenylene-1-sulphonic acid in 98.4% H2SO4 yields the 2,6- and 1,6-(or 1,7-)di-sulphonic acid respectively.The sulphonation of (2b), (2c), and (2e) in 95.3–98.3% H2SO4 leads to a mixture of disulphonic acids, that of (3) to a mixture of trisulphonic acids, all containing one sulpho-group per phenylene. The isomer distribution for the monosulphonation of (2b) has been estimated from the disulphonic acid isomer composition. For (2c), (2e), and (3) the substitution ratio per phenylene has been estimated.The isomer distributions of the tri- and of the tetrasulphonic acid mixtures obtained from (2b), and of the tetrasulphonic acid mixture formed from (2e) have also been determined.