An overview of sequential injection chromatography

New generation of sequential injection analysis (SIA) called sequential injection chromatography (SIC) has already been consolidated as a good alternative of high performance liquid chromatography (HPLC) for fast analysis of simple samples. Benefits of flow methods are automation, miniaturization and low sample and mobile phase consumption. Implementation of short monolithic chromatographic column into SIA opens new area—on-line chromatographic separation of multi-compound sample in low-pressure flow system, with the advantage of flow programming and possibility of sample manipulation. In the presented review the potential of SIC and its comparison with HPLC for determination of pharmaceutical mixtures is discussed and outlines past and recent trends focused on separation with SIC.     

A novel application of Onyx™ monolithic column for simultaneous determination of salicylic acid and triamcinolone acetonide by sequential injection chromatography

A novel and fast simultaneous determination of triamcinolone acetonide (TCA) and salicylic acid (SA) in topical pharmaceutical formulations by sequential injection chromatography (SIC) as an alternative to classical high performance liquid chromatography (HPLC) has been developed. A recently introduced Onyx™ monolithic C18 (50 mm × 4.6 mm, Phenomenex^®) with 5 mm monolithic precolumn were used for the first time for creating sequential injection chromatography system based on a FIAlab^® 3000 with a six-port selection valve and 5.0 mL syringe pump in study. The mobile phase used was acetonitrile/water (35:65, v/v), pH 3.3 adjusted with acetic acid at flow rate 0.9 mL min⁻¹. UV detection provided by fibre-optic DAD detector was set up at 240 nm. Propylparaben was chosen as suitable internal standard (IS). There is only simple pre-adjustment of the sample of topical solution (dilution with mobile phase) so the analysis is not uselessly elongated. Parameters of the method showed good linearity in wide range, correlation coefficient >0.999; system precision (relative standard deviation, R.S.D.) in the range 0.45-1.95% at three different concentration levels, detection limits (3σ) 1.00 μg mL⁻¹ (salicylic acid), 0.66 μg mL⁻¹ (triamcinolone acetonide) and 0.33 μg mL⁻¹ (propylparaben) and recovery from the pharmaceutical preparations in the range 97.50-98.94%. The chromatographic resolution between peaks of compounds was more than 4.5 and analysis time was 5.1 min under the optimal conditions. The advantages of sequential injection chromatography against classical HPLC are discussed and showing that SIC can be a method of option in many cases.     

Determination of pesticides fenoxycarb and permethrin by sequential injection chromatography using miniaturized monolithic column

Sequential injection chromatography system equipped with miniaturized 10 mm monolithic column was used for fast simultaneous determination of two pesticides—fenoxycarb (FC) and permethrin (PM). The system was composed of a commercial sequential injection analysis (SIA) system (FIAlab^® 3000, 6-port selection valve and 5.0 mL syringe pump), commercially available column Chromolith™ RP-18e (10 mm × 4.6 mm i.d.) (Merck^®, Germany) and CCD UV-vis detector (USB 2000, Ocean-optics) with 1.0 cm Z-flow cell, absorbance was monitored at 225 nm. The mobile phase used for analysis was acetonitrile/water (60:40, v/v), flow rates were 0.6 mL min⁻¹ for elution of fenoxycarb and 1.2 mL min⁻¹ for elution of permethrin. For each analysis 4.8 mL of mobile phase was used. The chromatographic resolution between both compounds was >8 and analysis time was <6.5 min under the optimal conditions. Limits of detection were determined at 2.0 μg mL⁻¹ for fenoxycarb and 1.0 μg mL⁻¹ for permethrin. Samples were prepared by diluting with mobile phase and injected volume was 10 μL for each analysis. Developed method was applied to analysis of both pesticides in veterinary pharmaceutical foams and sprays ARPALIT^® Neo (Aveflor, Czech Republic). SIC method was compared with validated method (HPLC, reverse phase 100 mm monolithic column, gradient elution).     

Sequential injection spectrophotometric determination of trace amounts of iodide by its catalytic effect on the 4,4'-methylenebis(N,N-dimethylaniline)-chloramine-T reaction

A simple and sensitive sequential injection spectrophotometric procedure is proposed for the determination of trace amounts of iodide in pharmaceutical preparations. The method is based on the catalytic effect of iodide on the (tetra base) 4,4′-methylenebis(N,N-dimethylaniline)-chloramine-T reaction in acidic solution. The method involves a sequential aspiration of 255 μl sample/standard followed by 170 μl tetra base and then 128 μl chloramine-T solutions into a carrier stream to be stacked inside a holding coil and flow reversed through a reaction coil towards a detector. The resulting colored compound is measured at 600 nm using an UV/Vis-spectrophotometer. All the parameters that affect the reaction were evaluated and the calibration curve is linear over a range of 0.1–6.0 μg l⁻¹ of iodide concentration with detection limit of 0.05 μg l⁻¹. A sample throughput of 80 samples per hour and relative standard deviation of less than 2.0% was achieved. The method is successfully applied for the determination of iodide in three different samples (tablets).     

Simple automated generation of gradient elution conditions in sequential injection chromatography using monolithic column

The paper deals with the concept of simple automated creation of gradient profile of the mobile phase for gradient-elution sequential injection chromatography (GE-SIC). The feasibility and merits of this concept are demonstrated on the separation and simultaneous assay of indomethacin as active principle and of its two degradation products (5-methoxy-2-methylindoleacetic acid and 4-chloro-benzoic acid) in a topical pharmaceutical formulation.The GE-SIC separation was performed with a FIAlab^® 3000 SIC set-up (USA) equipped with an Onyx™ Monolithic C18 (25 mm × 4.6 mm, Phenomenex^®) column, a six-port selection valve, a 5-mL syringe pump and a fiber-optics UV CCD detector. Ketoprofen was used as an internal standard (IS). The gradient elution was achieved by automated reproducible mixing of acetonitrile and aqueous 0.2% phosphoric acid in the holding coil of the SIC system. Different profiles of the gradient elution were tested. The optimal gradient using two mobile phases 30:70 and 50:50 of acetonitrile/0.2% phosphoric acid (v/v) was achieved under the optimum flow rate 1.2 mL min⁻¹. The chromatographic resolution R between the peaks of all solutes (including the IS) was >2.00. The repeatability of retention times was characterized by the RSD values 0.18-0.30% (n = 6). Net separation time was 3.5 min and the mobile phase consumption was 4.5 mL for a single GE-SIC assay. The figures of merit of the novel GE-SIC method compared well with those of conventional HPLC.     

Flow injection catalytic spectrophotometric simultaneous determination of nitrite and nitrate

A new flow injection catalytic spectrophotometric method is proposed for the simultaneous determination of nitrite and nitrate based on the catalytic effect of nitrite on the redox reaction between crystal violet and potassium bromate in phosphoric acid medium and nitrate being on-line reduced to nitrite with a cadmium-coated zinc reduction column. The redox reaction is monitored spectrophotometrically by measuring the decrease in the absorbance of crystal violet at the maximum absorption wavelength of 610 nm. A technique of inserting a reduction column into sampling loop is adopted and the flow injection system produces a signal with a shoulder. The height of shoulder in the ascending part of the peak corresponds to the nitrite concentration and the maximum of the peak corresponds to nitrate plus nitrite. The detection limits are 0.3 ng ml⁻¹ for nitrite and 1.0 ng ml⁻¹ for the nitrate. Up to 32 samples can be analyzed per hour with a relative standard deviation of less than 2%. The method has been successfully applied for the simultaneous determination of nitrite and nitrate in natural waters.     

Possibilities and limitations of the sequential injection chromatography technique for the determination of anticoccidial agents in water, pharmaceutical formulations and feed

This paper explores the potential of applying reversed-phase sequential injection chromatography (SIC) to determine the anticoccidial agents Lasalocid and Toltrazuril in various matrices including ground water, pharmaceutical formulations and feed. SIC was performed by connecting a 25 × 4.6 mm monolithic C18 column to a 2 m long pathlength capillary flow cell, where the usage of a flow cell lowers the detection limit compared to a conventional short-distance flow cell, providing a simple detection system for these two compounds which are initially poorly UV absorbents.The proposed set-up provides a high injection throughput of 12 h^{− 1}, as well as a limit of detection of 0.019 and 0.010 mg/L for Toltrazuril and Lasalocid, respectively. The repeatabilities obtained (n = 10) were lower than 2% and 4% for Toltrazuril and Lasalocid, respectively.     

Sequential injection lab-on-valve simultaneous spectrophotometric determination of trace amounts of copper and iron

A sequential injection (SI) method in a lab-on-valve (LOV) format for simultaneous spectrophotometric determination of copper and iron has been devised. The detection chemistry is based on the complex formation of 2-(5-bromo-2-pyridylazo)-5-[N-n-propyl-N-(3-sulfopropyl)amino]aniline (5-Br-PSAA) with copper(II) and/or iron(II) at pH 4.6. Copper(II) reacts with 5-Br-PSAA to form the complex which has an absorption maximum at 580 nm but iron(III) does not react. In the presence of a reducing agent only iron(II)-5-Br-PSAA complex is formed and detected at 558 nm. Under the optimum experimental conditions, the determinable ranges are 0.1-2 mg l⁻¹ for copper and 0.1-5 mg l⁻¹ for iron, respectively, with a sampling rate of 18 h⁻¹. The limits of detection are 50 μg l⁻¹ for copper and 25 μg l⁻¹ for iron. The relative standard deviations (n = 15) are 2% for 0.5 mg l⁻¹ copper and 1.8% for 0.5 mg l⁻¹ iron when determined in standard solutions. The recoveries range between 96 and 105% when determining 0.25-2 mg l⁻¹ of copper and 0.2-5 mg l⁻¹ of iron in artificial mixtures at copper/iron ratios of 1:10 to 5:1. The proposed SI-LOV method is successfully applied to the simultaneous determination of copper and iron in multi-element standard solution and in industrial wastewater samples.     

顺序注射催化动力学光度法测定织物和室内空气中痕量甲醛

在磷酸介质中 ,甲醛可以催化溴酸钾氧化　花青的反应使其退色 ,据此建立了顺序注射催化动力学光度法测定织物和室内空气中痕量甲醛的新方法。方法的线性范围 0 .5～ 7.0 μg mL ,检出限 0 .1 μg mL。用于树脂整理特殊织物和室内空气中痕量甲醛的测定 ,回收率在 97.0 %～ 1 0 5 .0 %之间。     

Automation of simultaneous release tests of two substances by sequential injection chromatography coupled with Franz cell

This presented paper deals with a methodology for the separation and simultaneous determination of two active substances in topical pharmaceutical formulation composed of lidocaine (L) and prilocaine (P). The methodology described is based on the sequential injection chromatography (SIC) with UV detection. Monolithic Column Chromolith Flash RP-18, 25 mm × 4.6 mm (Merck, Germany) was used. Separation was performed using elution with binary mobile phase composed of acetonitrile-phosphate buffer 0.05 M (40:80 (v/v)) + 0.01% triethylamine (adjusted to pH 7.1 with H₃PO₄) at a flow rate of 0.6 ml min⁻¹. The analysis duration was <7 min. The method was linear over the range of 2.5-200 mg l⁻¹ with a detection limit of 0.25 mg l⁻¹ for both substances.The system was then coupled with Franz cell. Fully automated system for the in vitro release testing of semisolid dosage forms based on sequential injection analysis (SIA) was developed. Simultaneous measurement of L and P release was done by this system. Samples were taken in 10.5 min intervals during 4 h of the release test. Each test was followed by calibration with five standard solutions. Receiving medium was replenished automatically by the system.     

Flow injection spectrophotometric determination of europium using chlortetracycline

A flow injection (FI) spectrophotometric determination of europium (III) is described, based on the complexation between europium (III), and chlortetracycline (CTC) in a Tris-buffer pH 8.0 medium. The resulting yellow-coloured complex is measured at its absorption maximum of 400 nm after 100 μl of sample or standard solution containing europium (III) are injected into the merged streams of CTC and Tris-buffer solutions. Optimum conditions for determining μg amounts of europium (III) are achieved by univariate method. Various types of reactors are also investigated. It is shown that the use of a single bead string reactor gives rise to the enhancement of peak height. A linear calibration curve over the range of 0.10-0.60 μg ml⁻¹ europium (III) is established with the regression equation (n=6) Y=34.93X+0.01 and the correlation coefficient of 0.9994 is obtained. A detection limit (3σ) of 0.01 μg ml⁻¹ of europium (III) and the relative standard deviation (R.S.D.) of 4.32% for determining 1.0 μg ml⁻¹ of europium (III) (n=7) are obtained. The recommended method has been applied to the quantitation of europium (III) in spiked water and stream sediment samples with average recoveries of 99.9 and 97.5%, respectively. The sampling rate is found to be 85 h⁻¹.     

Successive determination of urinary protein and glucose using spectrophotometric sequential injection method

A new sequential injection (SI) system with spectrophotometric detections has been developed for successive determination of protein and glucose. The protein assay is based on ion-association of protein with tetrabromophenolphthalein ethyl ester (TBPE) in the presence of Triton X-100 at pH 3.2. The blue product is monitored for absorbance at 607 nm. For glucose, hydrogen peroxide, generated by the oxidation of glucose in the presence of glucose oxidase immobilized on glass beads packed in a minicolumn, is monitored using iron-catalyzed oxidation reaction of p-anisidine to form a red colored product (520 nm). The SI procedure takes advantage in performing the protein assay during the incubation period for glucose oxidation. Linear ranges were up to 10 mg dL⁻¹ human serum albumin (HSA) with a limit of detection (LOD) (3σ) of 0.3 mg dL⁻¹, and up to 12.5 mg dL⁻¹ glucose with LOD of 0.08 mg dL⁻¹. R.S.D.s (n = 11) were 2.7% and 2.5% (for 1 mg dL⁻¹ and 5 mg dL⁻¹ HSA) and 1.4% (9 mg dL⁻¹ glucose). Sample throughput for the whole assay of both protein and glucose is 6 h⁻¹. The automated system has been demonstrated for the successive assay of protein and glucose in urine samples taken from diabetic disease patients, with good agreement with the other methods. This developed SI system is an alternative automation for screening for diabetic diagnosis.     

Reversed flow injection and sandwich sequential injection methods for the spectrophotometric determination of copper(II) with cuprizone

Two new flow methods, flow injection analysis (FIA) and sequential injection analysis (SIA), for the spectrophotometric determination of Cu(II) in water at trace levels have been developed and optimised. Both methods are based on the reaction with oxalic acid bis(cyclohexylidene hydrazide) (cuprizone) in alkaline media. The two procedures have been developed for the final aim to compare their performances and to offer new rapid heavy metals analysis tools, avoiding the use of extraction steps. A detailed study of the physico-chemical parameters affecting the systems performances has been carried out. The reversed FIA and sandwich SIA approaches offered the best sensitivity. In both cases, an extremely good linearity has been obtained within the range 0.06-4 μg ml⁻¹ (correlation coefficient r=0.9999), whereas the observed detection limits were 0.013 and 0.004 μg ml⁻¹, for FIA and SIA, respectively. Furthermore, due to the great similarity of the diffusion zones in the reaction slugs, our approach offers the opportunity to compare the two methods in analogous conditions. This SIA method, besides keeping its typical reagent saving features, offered analytical performances equivalent to those of FIA. To obtain these results, an original “stop-flow like” method was successfully employed in the SIA approach. Both methods were validated by analysis of real water samples, after copper addition, and certified reference samples of fortified and waste waters.     

Implementing stepwise solvent elution in sequential injection chromatography for fluorimetric determination of intracellular free amino acids in the microalgae Tetraselmis gracilis

The concept of sequential injection chromatography (SIC) was exploited to automate the fluorimetric determination of amino acids after pre-column derivatization with o-phthaldialdehyde (OPA) in presence of 2-mercaptoethanol (2MCE) using a reverse phase monolithic C₁₈ stationary phase. The method is low-priced and based on five steps of isocratic elutions. The first step employs the mixture methanol: tetrahydrofuran: 10 mmol L⁻¹ phosphate buffer (pH 7.2) at the volumetric ratio of 8:1:91; the other steps use methanol: 10 mmol L⁻¹ phosphate buffer (pH 7.2) at volumetric ratios of 20:80, 35:65, 50:50 and 65:35. At a flow rate of 10 μL s⁻¹ a 25 mm long-column was able to separate aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), glycine (Gly), threonine (Thr), citruline (Ctr), arginine (Arg), alanine (Ala), tyrosine (Tyr), phenylalanine (Phe), ornithine (Orn) and lysine (Lys) with resolution >1.2 as well as methionine (Met) and valine (Val) with resolution of 0.6. Under these conditions isoleucine (Ile) and leucine (Leu) co-eluted. The entire cycle of amino acids derivatization, chromatographic separation and column conditioning at the end of separation lasted 25 min. At a flow rate of 40 μL s⁻¹ such time was reduced to 10 min at the cost of resolution worsening for the pairs Ctr/Arg and Orn/Lys. The detection limits varied from 0.092 μmol L⁻¹ for Tyr to 0.51 μmol L⁻¹ for Orn. The method was successfully applied to the determination of intracellular free amino acids in the green alga Tetraselmis gracilis during a period of seven days of cultivation. Samples spiked with known amounts of amino acids resulted in recoveries between 94 and 112%.     

Rapid and simultaneous determination of imidazolium and pyridinium ionic liquid cations by ion-pair chromatography using a monolithic column

A method for rapid and simultaneous determination of imidazolium and pyridinium ionic liquid cations by ion-pair chromatography with ultraviolet detection was developed.Chromatographic separations were performed on a reversed-phase silica-based monolithic column using 1-heptanesulfonic acid sodium-acetonitrile as mobile phase.The effects of ion-pair reagent and acetonitrile concentration on retention of the cations were investigated.The retention times of the cations accord with carbon number rule.The method has been successfully applied to the determination of four ionic liquids synthesized by organic chemistry laboratory.     

Sequential injection chromatographic determination of ambroxol hydrochloride and doxycycline in pharmaceutical preparations

A new separation method based on a novel reversed-phase sequential injection chromatography (SIC) technique was used for simultaneous determination of ambroxol hydrochloride and doxycycline in pharmaceutical preparations in this contribution.The coupling of short monolith with SIA system results in an implementation of separation step to until no-separation low-pressure method.A Chromolith^® Flash RP-18e, 25-4.6 mm column (Merck, Germany) and a FIAlab^® 3000 system (USA) with a six-port selection valve and 5 ml syringe were used for sequential injection chromatographic separations in our study. The mobile phase used was acetonitrile-water (20:90, v/v), pH 2.5 adjusted with 98% phosphoric acid, flow rate 0.48 ml min⁻¹, UV detection was at 213 nm.The validation parameters have shown good results: linearity of determination for both compounds including internal standard (ethylparaben) >0.999; repeatability of determination (R.S.D.) in the range 0.5-5.4% at three different concentration levels, detection limits in the range 0.5-2.0 μg ml⁻¹, and recovery from the pharmaceutical preparation in the range 99.3-99.9%. The chromatographic resolution between peak compounds was >5.0 and analysis time was <9 min under the optimal conditions. The method was found to be applicable for routine analysis of the active compounds ambroxol hydrochloride and doxycycline in various pharmaceutical preparations.     

On-line separation, simultaneous dilution and spectrophotometric determination of zinc in fertilisers with a sequential injection system and xylenol orange as complexing agent

A dialyser unit, equipped with a passive neutral membrane, was incorporated into the conduits of a sequential injection (SI) system for the on-line removal of suspended solids and simultaneous dilution of the analyte before reaction and detection of the analyte. The system was applied to the determination of zinc(II) in fertilisers. The fully automated system is able to analyse zinc at a sampling frequency of ten samples per hour at a %R.S.D. of better than 0.55. The calibration graph was linear between 10 and 50 mg l⁻¹. The detection limit was found to be 4.75 mg l⁻¹. The results obtained with the proposed SI analyser compared favourably with the standard manual flame atomic absorption spectrometric method.     

顺序注射阻抑动力学光度法测定环境水样中的间苯二酚

在酸性条件下,利用间苯二酚能抑制罗丹明B和溴酸根反应速率的原理,联用顺序注射技术建立了非平衡态快速测定痕量间苯二酚的新方法.优化了顺序注射流路参数、试剂用量,考察了共存物的影响.在最佳条件下,该法测定间苯二酚的线性范围为0.20～3.00 μg/mL,检出限为0.09 μg/mL,每小时可测定40个样.     

Spectrophotometric determination of magnesium in pharmaceutical preparations by cost-effective sequential injection analysis

A simple and rapid, inexpensive spectrophotometric method was proposed for magnesium assay in pharmaceutical preparations by sequential injection analysis (SIA). The method is based on the reaction between o-cresolphthalein complexone (CPC) and Mg(II) in alkaline media, yielding a pink colored complex with absorption maximum at 570 nm. Since the formation constant between Ca-CPC and Mg-CPC is similar, initially a sample/standard solution was aspirated into the holding coil followed by a mixture of masking-buffer solutions. This was done because masking of calcium should be accomplished before Mg-CPC complexation. Then the reagent was introduced into the reaction coil to produce a colored complex, which is measured spectrophotometrically at 570 nm. In this way the interference of calcium was reduced. Furthermore, all the parameters that affect the reaction were evaluated. The calibration curve is linear over a range of 0-20 mg l⁻¹ of Mg(II) with a detection limit of 0.24 mg l⁻¹. A sample throughput of 80 samples per hour and relative standard deviation <2.0% were achieved. The proposed method was successfully applied for the assay of magnesium in three different compositions of pharmaceutical preparations (tablets). The results were found to be in good agreement with the manual flame atomic absorption spectrophotometry (FAAS) and UV-Vis spectrophotometry methods and with the claimed values by the manufactures. The t-test shows no significant difference at 95% confidence level.     

Two-column Sequential Injection Chromatography—New approach for fast and effective analysis and its comparison with gradient elution chromatography

This work presents novel approach in low-pressure chromatography flow systems—two-column Sequential Injection Chromatography (2-C SIC) and its comparison with gradient elution chromatography on the same instrument. The system was equipped with two different chromatographic columns (connected to selection valve in parallel design) for isocratic separation and determination of all components in composed anti-inflammatory pharmaceutical preparation (tablets). The sample was first injected on the first column of length 30 mm where less retained analytes were separated and then the sample was injected on the second column of length 10 mm where more retained analytes were separated. The SIC system was based on a commercial SIChrom™ manifold (8-port high-pressure selection valve and medium-pressure syringe pump with 4 mL reservoir) (FIAlab^®, USA) with two commercially available monolithic columns the “first column” Chromolith^® Flash RP-18e (25 mm × 4.6 mm i.d. with guard column 5 mm × 4.6 mm i.d.) and the “second column” Chromolith^® RP-18e (10 mm × 4.6 mm i.d.) and CCD UV-vis detector USB 4000 with micro-volume 1.0 cm Z flow cell. Two mobile phases were used for analysis (one for each column). The mobile phase 1 used for elution of paracetamol, caffeine and salicylic acid (internal standard) was acetonitrile/water (10:90, v/v, the water part of pH 3.5 adjusted with acetic acid), flow rate was 0.9 mL min⁻¹ (volume 3.0 mL of mobile phase per analysis). The mobile phase 2 used for elution of propyphenazone was acetonitrile/water (30:70, v/v); flow rate was 1.2 mL min⁻¹ (volume 1.5 mL of mobile phase per analysis). Absorbance was monitored at 210 nm. Samples were prepared by dissolving of one tablet in 30% acetonitrile and 10 μL of filtered supernatant was injected on each column (2 × 10 μL). The chromatographic resolution between all compounds was >1.45 and analysis time was 5.5 min under the optimal conditions. Limits of detection were determined at 0.4 μg mL⁻¹ for paracetamol, at 0.5 μg mL⁻¹ for caffeine and at 0.7 μg mL⁻¹ for propyphenazone. The new two-column chromatographic set-up developed as an alternative approach to gradient elution chromatography shows evident advantages (time and solvent reduction more than one-third) as compared with single-column gradient SIC method with Chromolith^® Flash RP-18 (25 mm × 4.6 mm i.d. with guard column 5 mm × 4.6 mm i.d.).