Ion-pair Reversed-phase High-performance Liquid Chromatography Research Articles

Retention of nucleic acids in ion-pair reversed-phase high-performance liquid chromatography depends not only on base composition but also on base sequence.

The study on nucleic acid retention in ion-pair reversed-phase high-performance liquid chromatography mainly focuses on size-dependence, however, other factors influencing retention behaviors have not been comprehensively clarified up to date. In this present work, the retention behaviors of oligonucleotides and double-stranded DNAs were investigated on silica-based C18 stationary phase by ion-pair reversed-phase high-performance liquid chromatography. It is found that the retention of oligonucleotides was influenced by base composition and base sequence as well as size, and oligonucleotides prone to self-dimerization have weaker retention than those not prone to self-dimerization but with the same base composition. However, homo-oligonucleotides are suitable for the size-dependent separation as a special case of oligonucleotides. For double-stranded DNAs, the retention is also influenced by base composition and base sequence, as well as size. This may be attributed to the interaction of exposed bases in major or minor grooves with the hydrophobic alky chains of stationary phase. In addition, no specific influence of guanine and cytosine content was confirmed on retention of double-stranded DNAs. Notably, the space effect resulted from the stereostructure of nucleic acids also influences the retention behavior in ion-pair reversed-phase high-performance liquid chromatography.

Reversed‐phase ion‐pair high‐performance liquid chromatography assay of polyprenyl diphosphate oligomer homologues

A reversed-phase ion-pair high-performance liquid chromatography procedure was developed for the separation of polyprenyl diphosphate oligomer homologues obtained chemically from plant polyprenols. Tetrabutylammonium phosphate was used as the ion-pair reagent, and the dependence of the separation quality on pH of ion-pair reagent was investigated for the first time. The procedure is applicable for the control of commercial available polyprenyl monophosphates (the active components of veterinary drugs Phosprenyl and Gamapren) for the possible presence of polyprenyl diphosphate byproducts.

Structural peculiarity and antithrombin binding region profile of mucosal bovine and porcine heparins

The major compositional differences between bovine mucosal heparin (BMH) and the currently employed porcine mucosal heparin (PMH) have been reported to essentially consist of reduced 6-O-sulfation of the glucosamine residues in BMH and somewhat lower 2-O-sulfation of the iduronate residues in PMH. The present work is based on direct comparison of several BMH and PMH commercial preparations. A combined study by 2D (heteronuclear single quantum coherence, HSQC) NMR and ion-pair reversed-phase high performance liquid chromatography (IPRP-HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) on the heparins, extended to the analysis of their heparinases digests and fractions separated by affinity chromatography on antithrombin (AT), confirmed the previously reported lower degree of 6-O-sulfation and showed lower 3-O-sulfated glucosamine content in BMH. More detailed studies allowed the identification of structural variants of AT-binding region (ATBR) structural variants, showing higher content of the N-sulfated components in BMH than in PMH.

Fragment profiling of low molecular weight heparins using reversed phase ion pair liquid chromatography-electrospray mass spectrometry

Low molecular weight heparins (LMWHs) are linear and highly charged carbohydrate polymers prepared by chemical or enzymatic depolymerization of heparin. Compared to unfractionated heparin (UFH), LMWHs are prevalently used as clinical anticoagulant drugs due to their lower side effects and better bioavailability. The work presented herein provides a rapid and powerful fragment mapping method for structural characterization of LMWHs. The chain fragments of two types of LMWHs, enoxaparin and nadroparin, were generated by controlled enzymatic digestion with each of heparinase I (Hep I, Enzyme Commission (EC) # 4.2.2.7), heparinase II (Hep II, no EC # assigned) and heparinase III (Hep III, EC # 4.2.2.8). Reversed phase ion pair high performance liquid chromatography (RPIP-HPLC) coupled with electrospray ion trap time-of-flight mass spectrometry (ESI-IT-TOF-MS) was used to profile the oligosaccharide chains ranging from disaccharides to decasaccharides. A database containing all theoretical structural compositions was established to assist the mass spectra interpretation. The six digests derived by three enzymes from two types of LMWHs exhibited distinguishable fingerprinting patterns. And a total of 94 enoxaparin fragments and 109 nadroparin fragments were detected and identified. Besides the common LMWH oligosaccharides, many components containing characteristic LMWH structures such as saturated l-idopyranosuronic acid, 2,5-anhydro-d-mannitol, 1,6-anhydro-d-aminopyranose, as well as odd number oligosaccharides were also revealed. Quantitative comparison of major components derived from innovator and generic nadroparin products was presented. This approach to profile LMWHs' fragments offers a highly reproducible, high resolution and information-rich tool for evaluating the quality of this category of anticoagulant drugs or comparing structural similarities among samples from various sources.

PH Oscillations and Mechanistic Analysis in the Hydrogen Peroxide–Sulfite–Thiourea Reaction System

A new pH oscillator has been constructed by combining the pH clock reaction H2O2-SO3(2-)-H(+) with thiourea (Tu, (NH2)2CS) as a proton-consuming species. The system exhibited oligo-oscillatory behavior in a closed system, and large amplitude oscillations in a continuous-flow stirred tank reactor (CSTR) were observed in a narrow range of input concentrations, flow rate, and temperature. For the purpose of constructing the kinetic model, a reversed-phase ion-pair high-performance liquid chromatography (HPLC) and mass spectrometer (MS) were used to track and determine intermediate species during the oxidation of thiourea by hydrogen peroxide. Experimental results illustrated that the four species: thiourea monoxide (TuO), formamidine disulfide (Tu2(2+)), thiourea dioxide (TuO2), and thiourea trioxide (TuO3) were formed during the oxidation process. A ten-step mechanistic model was proposed, where TuO was another key species participating in two proton feedback loops in addition to bisulfite. Numerical simulations based on this model agreed well with the experimental results.

Speciation of [PtIVCl6-nBrn]2- (n = 0-6) and some of their mono-aquated [PtIVCl5-nBrn(H2O)]- (n = 0-5) anions in solution at low concentrations by means of ion-pairing reversed-phase ultra-high-performance liquid chromatography coupled to electrospray ion

The speciation of the purely inorganic [PtCl6-n Brn](2-) (n = 0-6) anions and their corresponding mono-aquated [PtCl5-n Brn (H2O)](-) (n = 0-5) anions is of considerable importance to the precious metal refining and recycling industry, to ensure optimum recovery and separation efficiencies. Speciation of platinum complexes present in precursor solutions used for the preparation of precious metal nano-crystals of defined size and morphology appears also to be important. The various possible Pt(IV) complex anions in dilute aqueous can be characterized using ion-pairing reversed-phase high-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOFMS). Ion-pairing reversed-phase ultra-high-performance LC separation of the Pt(IV) complex anions present in aqueous solutions prior to detection by means of high-resolution ESI-Q-TOFMS using a low ESI source cone voltage (5 V) allows for the clear identification of all the platinum complexes from the characteristic pattern of fragment ions (m/z), presumably generated by 'reductive conversion' in the ESI source of the mass spectrometer. Sufficient chromatographic resolution for the series of Pt(IV) complexes is achieved using the (n-butyl)3 NH(+) ion generated in a formic acid/water/methanol (pH ~3.5) mobile phase. This mobile phase composition facilitates a low-background for optimal ESI-Q-TOFMS detection with enhanced sensitivity. Direct-infusion mass spectrometry of the inorganic platinum complexes in aqueous solution is impractical due to their low volatility, but more importantly as a result of the very extensive series of fragment ions generated in the ESI source, which leads to virtually uninterpretable mass spectra. However, with prior separation, and by using low ESI cone voltages (5 V), the mass spectra of the separated analyte ions show simpler and systematic fragmentation patterns [Pt(IV) X5](-) → [Pt(III) X4 ](-) → [Pt(II) X3](-) → [Pt(I)X2 ](-) (X = Cl(-) and Br(-)), resulting in clear assignments. This methodology facilitates the characterization of the partially aquated [PtCl5-n Brn (H2O)](-) (n = 0-5) anions derived from the homo- and heteroleptic [PtCl6-n Brn](2-) (n = 0-6) anions, in equilibrated solutions at low concentrations. Speciation of homo- and heteroleptic [PtCl6-n Brn](2-) (n = 0-6) anions, together with some of their partially aquated [PtCl5-n Brn (H2O)](-) (n = 0-5) species in dilute solution, can successfully be carried out by means of prior ion-pairing reversed-phase LC separation coupled to high-resolution ESI-Q-TOFMS at low ESI cone-voltage settings.

Determination of spectinomycin and related substances by HPLC coupled with evaporative light scattering detection

An improved ion-pairing reversed-phase high-performance liquid chromatography method coupled with evaporative light scattering detection (HPLC-ELSD) was developed to determine spectinomycin and its related substances in commercial samples. The method was validated in accordance with International Conference on Harmonization (ICH) guidelines. The specificity of the HPLC-ELSD method was similar to that of the European Pharmacopoeia (Ph. Eur.) method, and repeatability and robustness were markedly improved relative to other reported methods due to our empirical evaluation of separation columns. Indeed, it is a more specific assay of spectinomycin than traditional microbiological techniques. The HPLC-ELSD method was used to evaluate the impurity profiles of eight compounds in seven spectinomycin batches from five different companies. Liquid chromatography coupled with electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was employed to characterize the structures of these compounds. Though the HPLC...

Structural features of glycol-split low-molecular-weight heparins and their heparin lyase generated fragments

Periodate oxidation followed by borohydride reduction converts the well-known antithrombotics heparin and low-molecular-weight heparins (LMWHs) into their "glycol-split" (gs) derivatives of the "reduced oxyheparin" (RO) type, some of which are currently being developed as potential anti-cancer and anti-inflammatory drugs. Whereas the structure of gs-heparins has been recently studied, details of the more complex and more bioavailable gs-LMWHs have not been yet reported. We obtained RO derivatives of the three most common LMWHs (tinzaparin, enoxaparin, and dalteparin) and studied their structures by two-dimensional nuclear magnetic resonance spectroscopy and ion-pair reversed-phase high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. The liquid chromatography-mass spectrometry (LC-MS) analysis was extended to their heparinase-generated oligosaccharides. The combined NMR/LC-MS analysis of RO-LMWHs provided evidence for glycol-splitting-induced transformations mainly involving internal nonsulfated glucuronic and iduronic acid residues (including partial hydrolysis with formation of "remnants") and for the hydrolysis of the gs uronic acid residues when formed at the non-reducing ends (mainly, in RO-dalteparin). Evidence for minor modifications, such as ring contraction of some dalteparin internal aminosugar residues, was also obtained. Unexpectedly, the N-sulfated 1,6-anhydromannosamine residues at the enoxaparin reducing end were found to be susceptible to the periodate oxidation. In addition, in tinzaparin and enoxaparin, the borohydride reduction converts the hemiacetalic aminosugars at the reducing end to alditols. Typical LC-MS signatures of RO-derivatives of individual LMWH both before and after digestion with heparinases included oligosaccharides generated from the original antithrombin-binding and "linkage" regions.

Retraction Note: Analysis of Risedronate and Related Substances by Ion-Pair Reversed-Phase High-Performance Liquid Chromatography with Evaporative Light-Scattering Detection

This article published in Anal. Sci., 2010, 26(3), 325 was withdrawn by the Editor on June 7, 2013, because it was concluded that the author has copied significant parts of an earlier paper (Y. Jiang and Z. Xie, Determination of Ibandronate and its Degradation Products by Ion-Pair RP LC with Evaporative Light-Scattering Detection, Chromatographia, 2005, 62, 257, doi: 10.1365/s10337-005-0618-4). We regret that this was not detected during the reviewing process.

Simultaneous determination of nicotine and cotinine in serum using high-performance liquid chromatography with fluorometric detection and postcolumn UV-photoirradiation system

A simple and rapid method for the simultaneous determination of serum nicotine and cotinine using high-performance liquid chromatography (HPLC)-fluorometric detection with a postcolumn ultraviolet-photoirradiation system was developed. Analytes were extracted from alkalinized human serum via liquid-liquid extraction using chloroform. The organic phase was back-extracted with the acidified aqueous phase, and the analytes were directly injected into an ion-pair reversed-phase HPLC system. 6-Aminoquinoline was used as an internal standard. Nicotine, cotinine, and 6-aminoquinoline were separated within 14min. The extraction efficiency of nicotine and cotinine was greater than 91%. The linear range was 0.30-1000ng for nicotine and 0.06-1000ng for cotinine. In serum samples from smokers, the concentrations of nicotine and cotinine were 8-15ng/mL and 156-372ng/mL, respectively.

Determination of Fluorescent Whitening Agents in Paper Materials by Ion-Pair Reversed-Phase High-Performance Liquid Chromatography

A simple method was developed for the analysis of seven stilbene-type fluorescent whitening agents (FWAs) in paper materials by ion-pair reversed-phase high-performance liquid chromatography with fluorescence detection. These stilbene-type FWAs included two disulfonate, two tetrasulfonate, and three hexasulfonate compounds. After optimization of chromatographic conditions, the FWAs were satisfactorily separated using a reversed-phase column (RP-18) with the following isocratic mobile phase: methanol-water (60:40) containing 17.5 mM TBABr and 10 mM citrate buffer (pH = 7.0). The calibration plot was linear in the range from 5 to 500 ng/mL for two disulfo-FWAs and from 1 to 500 ng/mL for the other five FWAs. Precision levels of the calibration curve as indicated by RSD of response factors were 1.2 and 8.1%. Limits of quantitation (LOQ) ranged from 1.2 to 11 ng/mL.

Separation of Polyprenyl Phosphate Oligomerhomologues by Reversed-Phase Ion-Pair High-Performance Liquid Chromatography

A simple and rapid method of reversed-phase ion-pair high-performance liquid chromatography (HPLC) with UV-detection on a common C(18) column in an isocratic mode with the addition of tetrabutylammonium phosphate for the separation of polyprenyl phosphate oligomerhomologues has been developed. The method was successfully applied to assay the composition of polyprenyl phosphates obtained from polyprenols isolated from mulberry (Morus alba) leaves and fir (Abies sibirica) needles (the active components of veterinary drugs Gamapren and Phosprenyl correspondingly).

Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC

Nucleotides and nucleosides are not only involved in cellular metabolism but also act extracellularly via P1 and P2 receptors, to elicit a wide variety of physiological and pathophysiological responses through paracrine and autocrine signalling pathways. For the first time, we have used an ion-pair reversed-phase high-performance liquid chromatography ultraviolet (UV)-coupled method to rapidly and simultaneously quantify 12 different nucleotides and nucleosides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, uridine triphosphate, uridine diphosphate, uridine monophosphate, uridine, guanosine triphosphate, guanosine diphosphate, guanosine monophosphate, guanosine): (1) released from a mouse renal cell line (M1 cortical collecting duct) and (2) in human biological samples (i.e., urine). To facilitate analysis of urine samples, a solid-phase extraction step was incorporated (overall recovery rate ≥ 98 %). All samples were analyzed following injection (100 μl) into a Synergi Polar-RP 80 Å (250 × 4.6 mm) reversed-phase column with a particle size of 10 μm, protected with a guard column. A gradient elution profile was run with a mobile phase (phosphate buffer plus ion-pairing agent tetrabutylammonium hydrogen sulfate; pH 6) in 2–30 % acetonitrile (v/v) for 35 min (including equilibration time) at 1 ml min−1 flow rate. Eluted compounds were detected by UV absorbance at 254 nm and quantified using standard curves for nucleotide and nucleoside mixtures of known concentration. Following validation (specificity, linearity, limits of detection and quantitation, system precision, accuracy, and intermediate precision parameters), this protocol was successfully and reproducibly used to quantify picomolar to nanomolar concentrations of nucleosides and nucleotides in isotonic and hypotonic cell buffers that transiently bathed M1 cells, and urine samples from normal subjects and overactive bladder patients.Electronic supplementary materialThe online version of this article (doi:10.1007/s11302-012-9321-8) contains supplementary material, which is available to authorized users.

A Simple and High Resolution Ion-Pair HPLC Method for Separation and Simultaneous Determination of Nitrate and Thiocyanate in Different Water Samples

An ion-pair reversed-phase high-performance liquid chromatography method with isocratic elution and ultraviolet detection was developed and validated for the separation and simultaneous determination of nitrate and thiocyanate. The separation was performed on a C18 analytical column with mobile phase containing 0.08 mM hexadecyltrimethylammonium bromide as an ion-pair reagent, 40 mM of acetate buffer and 30% methanol at pH 3.2. The detection was monitored at 206 nm. The response was linear from 1 to 10 mM for nitrate, with a detection limit of 0.05 mM, and 1 to 10 mM for thiocyanate, with a detection limit of 0.31 mM. Calibration curves were found to be linear in these concentration ranges with correlation coefficient better than 0.99.

Mitochondrial DNA Variant for Complex I Reveals a Role in Diabetic Cardiac Remodeling

Myocardial remodeling and dysfunction are serious complications of type 2 diabetes mellitus (T2DM). Factors controlling their development are not well established. To specifically address the role of the mitochondrial genome, we developed novel conplastic rat strains, i.e. strains with the same nuclear genome but a different mitochondrial genome. The new animals were named T2DN(mtFHH) and T2DN(mtWistar), where the acronym T2DN denotes their common nuclear genome (type 2 diabetic nephropathy (T2DN) rats) and mtFHH or mtWistar the origin of their mitochondria, Fawn Hooded Hypertensive (FHH) or Wistar rats, respectively. The T2DN(mtFHH) and T2DN(mtWistar) showed a similar progression of diabetes as determined by HbA1c, cholesterol, and triglycerides with normal blood pressure, thus enabling investigation of the specific role of the mitochondrial genome in cardiac function without the confounding effects of obesity or hypertension found in other models of diabetes. Echocardiographic analysis of 12-week-old animals showed no abnormalities, but at 12 months of age the T2DN(mtFHH) showed left ventricular remodeling that was verified by histology. Decreased complex I and complex IV but not complex II activity within the electron transport chain was found only in T2DN(mtFHH), which was not explained by differences in protein content. Decreased cardiac ATP levels in T2DN(mtFHH) were in agreement with a lower ATP synthetic capacity by isolated mitochondria. Together, our data provide experimental evidence that mtDNA sequence variations have an additional role in energetic heart deficiency. The mitochondrial DNA background may explain the increased susceptibility of certain T2DM patients to develop myocardial dysfunction.

A novel ion-pair RP-HPLC method for determination of five components in compound α-ketoacid tablets

A sensitive and reliable ion-pair reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed and validated for the simultaneous determination of D,L-α-hydroxymethionine calcium (HMACa), D,L-α-ketoisoleucine calcium (KILCa), α-ketovaline calcium (KVCa), α-ketoleucine calcium (KLCa) and α-ketophenylalanine calcium (KPACa) in the compound α-ketoacid tablets using tetrabutylammonium hydroxide as an ion-pair reagent. The analytes were separated on a C(18) column (250 mm × 4.6 mm, 5 μm) with the mobile phase of methanol-potassium dihydrogen phosphate buffer (pH 3.0; 50mM) (37:63, v/v) at a flow rate of 0.6 mL min(-1). The UV detection wavelength was set at 210 nm. Good linearity with correlation coefficients larger than 0.9990 (n=6) for all analytes were achieved. The average recoveries were within the range of 99.6-100.9%, and the RSDs of the results were within the acceptable limit of 2.0%, which showed that this method was accurate and precise. The limits of detection were 10.44, 5.94, 3.44, 3.60 and 1.63 ng mL(-1), and the limits of quantification were 34.80, 19.80, 10.32, 12.00 and 5.44 ng mL(-1) for HMACa, KILCa, KVCa, KLCa and KPACa, respectively. The method is simple and accurate for quality control of the compound α-ketoacid tablets.

The analysis of major impurities of lipophilic-conjugated phosphorothioate oligonucleotides by ion-pair reversed-phase HPLC combined with MALDI-TOF-MS

A simple and rapid ion-pair reversed phase high-performance liquid chromatography (IP-RP-HPLC) method was developed to analyse the major impurities of lipophilic-conjugated phosphorothioate oligonucleotides (ODNs), which provided better separation performance than capillary gel electrophoresis and ion exchange chromatograph methods. The study showed that covalent conjugations of lipophilic group (docosanyl, C(22)) to ODN at 5'-termini (denoted as 5'C(22)-Flu) or 3'-termini (denoted as 3'C(22)-Flu) exhibited similar chromatographic retention behavior. Some important analytical conditions of IP-RP-HPLC, including column type, ion-pairing buffer composition, and separation temperature, were investigated for the effects on the separation of crude 5'C(22)-Flu. As expected, the method developed was successfully applied to the analysis of crude 3'C(22)-Flu and both purified products. Furthermore, the related impurities derived from the synthetic process were identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrum. These MS results are of benefit to understanding the major process-related impurities in lipophilic-ODN conjugates synthesis, thereby elevating the quality of target products.

Synthesis of mixed coating with multi-functional groups for in-tube hollow fiber solid phase microextraction–high performance liquid chromatography–inductively coupled plasma mass spectrometry speciation of arsenic in human urine

A novel method based on in-tube hollow fiber-solid phase microextraction (in-tube HF-SPME) on-line coupled with ion pair reversed phase high performance liquid chromatography (IP-RP-HPLC)–inductively coupled plasma mass spectrometry (ICP-MS) was developed for arsenic speciation. Partially sulfonated poly(styrene) (PSP) and mixed-sol of 3-mercapto propyltrimethoxysilane (MPTS) and N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AAPTS) were prepared and immobilized in the pores and the inner surface of polypropylene hollow fiber (HF). The prepared MPTS–AAPTS/PSP immobilized HF was characterized by FT-IR spectroscopy and scanning electron microscope (SEM). With arsenite (As(III)), arsenate (As(V)), monomethylarsonic acid (MMA), dimethylarsenic acid (DMA), arsenobetaine (AsB) and arsenocholine (AsC) as model arsenic species, a series of factors that influence the extraction of target arsenic species by in-tube HF-SPME, including pH value, sample volume and flow rate, elution conditions and interference of co-existing ions were investigated in details, and the conditions for subsequent HPLC–ICP-MS determination were also optimized. Under the optimal conditions, the sampling frequency was 6.5h−1, the detection limits for six target arsenic species were in the range of 0.017–0.053μgL−1 with the relative standard deviations (cAs(V),MMA=0.1μgL−1, cAs(III),DMA,AsB,AsC=0.5μgL−1, n=5) ranging in 3.1–8.7%, and the enrichment factors were varied from 4 to 19-fold. To validate the accuracy of this method, certified reference materials DORM-2 (dogfish) and CRM No. 18 (human urine) were analyzed, and the determined values were in good agreement with the certified values. The proposed method was also successfully applied for arsenic speciation in human urine samples, and the recoveries for the spiked samples were in the range of 92.6–107%. The self-designed in-tube HF-SPME–HPLC–ICP-MS system shows high efficiency and good stability, and the proposed method is sensitive and suitable for simultaneous speciation of organic and inorganic arsenic species (including anions and cations) in biological samples.

Characterization of side reactions during the annealing of small interfering RNAs

Small interfering RNAs (siRNAs) are emerging as a novel therapeutic modality for the specific inhibition of target gene expression. The development of siRNA-based therapeutics requires in-depth knowledge of the manufacturing process as well as adequate analytical methods to characterize this class of molecules. Here the impurity formation during the annealing of siRNA was investigated. Two siRNAs containing common chemical RNA modifications (2′- O-methyl, 2′-deoxy-2′-fluoro, 2′-deoxy-ribose, and phosphorothioate linkages) were used to determine major side reactions—such as 2′,3′-isomerization, strand scission, and HF elimination—depending on annealing parameters such as RNA concentration, presence of cations, temperature, and time. Individual impurities were characterized using analytical size exclusion chromatography, denaturing and nondenaturing ion-pair reversed-phase high-performance liquid chromatography, differential scanning calorimetry, and ultraviolet spectrometry. The degradation pathways described in this work can lead to significantly reduced product quality and compromised drug activity. The data reported here provide background to successfully address challenges associated with the manufacture of siRNAs and other nucleic acid therapeutics such as aptamers, spiegelmers, and decoy and antisense oligonucleotides.

Isomeric separation and identification of tetra-, tri-, and di-β-sulphonic phthalocyanine zinc complexes

The synthesis, isomeric separation, and identification of β-sulphonic phthalocyanine zinc complexes were reported. While the sulphonic phthalocyanines have been studied extensively, the development of separation technology may enable deeper insights into their isomeric constitution. An ion-pair reversed-phase high performance liquid chromatography (IP-RP-HPLC) method was developed to separate the sulphonic phthalocyanine isomers. The results showed that the product of the condensation reaction is a mixture of all possible isomers with statistical distribution. Several isomers were obtained and structural determination was undertaken by NMR. Based on the IP-RP-HPLC elution sequence of these well-identified isomers, a relationship between the structure and efficiency was deduced: closely spaced intervals of sulphonic groups lead to higher hydrophobicity and shorter retention times on HPLC. Based on this relationship, each HPLC peak was assigned to the corresponding isomeric structure.