Cresol Purple Research Articles

Enzymatic decolorization of sulfonphthalein dyes

The white rot fungus (WRF) Pleurotus ostreatus produced manganese peroxidase (MnP) and manganese-independent peroxidase (MIP) activities during solid state fermentation of wheat straw, a natural lignocellulosic substrate. Most of the sulfonphthalein (SP) dyes were decolorized by MnP at pH 4.0. The higher K m for meta-cresol purple (40 μM) and lower K m for ortho-cresol red (26 μM) for MnP activities explained the preference for the position of methyl group at ortho than at meta on chromophore. Bromophenol blue decolorizing activity was higher at pH 3.5 and decreased as the concentration of Mn II was increased. SP-decolorizing activity was associated not only with MnP but also with MIP. Additional bromine group along with the methyl group on SP chromophores decreases the rate of decolorization. Bromination of sulfonphthalein chromophore makes them the poorer substrate for MnP. This is evident from the higher K m for bromocresol green (117 μM) when compared to bromocresol purple (36 μM) and bromophenol blue (78 μM). The order of preference for the SP dyes as substrate for the MnP-catalyzed decolorizing activity is phenol red > ortho-cresol red > meta-cresol purple > bromophenol red > bromocresol purple > bromophenol blue > bromocresol green and the order of preference for the SP dyes as substrate for the MIP-catalyzed decolorizing activity is bromocresol green > bromophenol blue > bromocresol purple > bromophenol red > meta-cresol purple > ortho-cresol red > phenol red. Inhibition of PR decolorizing activity by NaN 3 provided the evidence of decolorizing activity as an oxidative process.

Spectrophotometric pH measurement in estuaries using thymol blue and m-cresol purple

The conditional acid dissociation constants (p K a′) of two sulfonephthalein dyes, thymol blue (TB) and m-cresol purple ( mCP), were assessed throughout the estuarine salinity range (0< S<40) using a tris/tris–HCl buffer and spectrophotometric measurement. The salinity dependence of the p K a′ of both dyes was fitted to the equations (25 °C, total proton pH scale, mol kg soln −1): p K a ′ ( TB ) = 9.1427 − 0.3105 ⁢ S 1 / 2 + 0.0783 ⁢ S − 0.01135 ⁢ S 3 / 2 + 0.000684 ⁢ S 2 p K a ′ ( m CP ) = 8.6353 − 0.3238 ⁢ S 1 / 2 + 0.0807 ⁢ S − 0.01157 ⁢ S 3 / 2 + 0.000694 ⁢ S 2 The estimated accuracy of pH measurements using these calculated p K a′ values is considered to be comparable to that possible with careful use of a glass electrode (±0.01 pH unit) but spectrophotometric measurements in an estuary have the significant advantage that it is not necessary to calibrate an electrode at different salinities. pH was measured in an estuary over a tidal cycle with a precision of ±0.0005 pH unit at high ( S>30) salinity, and ±0.002 pH unit at low ( S<5) salinity. The pH increased rapidly in the lower salinity ranges (0< S<15) but less rapidly at higher salinities.

Hydrophobicity-induced deprotonation of dye in dye–submicellar surfactant systems

Interaction of aqueous sulfonephthalein dyes, viz., phenol red, cresol red and cresol purple with alkyltrimethylammonium and alkylpyridinium halide surfactants in submicellar concentration ranges of the surfactants has been studied by UV–VIS spectroscopy. The spectral changes of the dyes observed on addition of the surfactants at concentrations far below critical micelle concentration (c.m.c.) indicate the formation of doubly deprotonated dye anions, which otherwise are formed only at much higher pH in the absence of the surfactant. The equilibrium constants for the interactions were determined. The deprotonation has been found to increase considerably with increase in hydrophobicity of the surfactant. It has been suggested that combined electrostatic–hydrophobic interaction increases acidity of the sulfonephthalein dyes in their closely packed dye–surfactant ion-pairs, formed at concentrations of surfactant far below c.m.c., causing deprotonation of the dye.

Spectral curve deconvolution in micellar systems with H-point curve isolation and H-point standard addition methods

A method for spectral curve deconvolution is described, evaluated and applied to micellar systems. The technique is based on combination of H-point curve isolation method (HPCIM) and H-point standard addition method (HPSAM). HPCIM is used for extracting the spectrum of solute in micellar pseudophase and HPSAM is used for calculation of equilibrium concentrations of solute in aqueous phase for each sample. The outputs of procedure are spectrum of dye molecules in micellar pseudophase free from contribution of the dye in the aqueous phase, and partition coefficients of considered solutes between micelle and water phase. The effects of noise and extent of solute partitioning on the reliability of the method are evaluated using model data. The applications of the method to the study of interaction of two different solutes, m-cresol purple (m-CP) and Azure C with Brij-35 and 2-amino-cyclopentene-1-dithiocarboxylic acid (ACDA) with cetyltrimethylamonium bromide (CTAB) micellar systems are presented.

Monitoring emissions from cellulose nitrate and cellulose acetate costume accessories: An evaluation of ph indicator dyes on paper, cotton tape and cotton threads

Abstract This article reports on an evaluation of simple and inexpensive techniques for monitoring ‘off‐gassing’ from costume accessories, which may be made with cellulose nitrate (CN) and cellulose acetate (CA). As they degrade, CN and CA produce acidic emissions, which not only accelerate the decomposition process of the objects themselves but also promote the fading of dyes and the degradation of nearby objects. pH indicator dyes, namely cresol red, cresol purple, Congo red, methyl orange and cochineal, applied to paper, cotton tape and threads, were tested for their suitability and then for their usefulness in monitoring a collection of 15 hair accessories, one of CA, one of tortoiseshell and 13 of CN, and three fans. As well as a cresol type recommended by another author (Julia Fenn) for the lower pH emissions from CN, it is suggested that Congo red or methyl orange for the higher pH emissions from CA should be used simultaneously. It was noted that cotton tape and cotton thread exhibited better resi...

Spectrophotometric flow-injection analysis of the total base number in lubricants by using acid–base buffers

A spectrophotometric FIA method for the determination of the total base number (TBN) in a lubricant was proposed, which involved using an acid–base buffer solution prepared with a nonaqueous solvent. This method is based on measurements of the absorbance change of an indicator contained in the acid–base buffer solution, which is generated due to a neutralization reaction of base in the lubricant with the buffer acid. The sample injected into a stream of a nonaqueous solvent, toluene/H 2O/2-propanol (52/1/47 v/v/v), was merged with a stream of perchloric acid (HClO 4) solution, and then neutralized with HClO 4. An excess of HClO 4 was subsequently merged with a stream of tetrabutylammonium salt of trifluoroacetic acid (TFA·TBA) solution containing an indicator ( m-cresol purple), which p K a value was found to be similar to p K a value of TFA in the present mixed nonaqueous solvent. The reaction of the excess HClO 4 with TFA·TBA gave rise to the composition change of the acid–base buffer solution, TFA/TFA·TBA. Since the indicator, m-cresol purple, behaves similarly to the buffer component, the change in the concentration ratio of TFA/TFA·TBA can be determined by the measurement of the absorbance change of the indicator. The absorbance changes were monitored with a spectrophotometric detector. The sensitivity of the proposed method for several kinds of basic compounds, which were often added to lubricant, was nearly identical irrespective of the basicity of the compounds. The proposed method was successfully applied to the determination of the total base number in lubricant.

Color transitional dyes as indicators of ionic boundaries in sodium dodecylsulfate polyacrylamide gel electrophoresis.

Several dyes were evaluated for their potential use as indices of ionic boundaries and pH discontinuities during polyacrylamide gel electrophoresis (PAGE). Dye selection was based largely upon color transition over the pH intervals present in the commonly used Tris-HCl-glycine buffer systems. In the absence of sodium dodecylsulfate (SDS) nitrazine yellow, cresol purple, cresol red, and phenol red had relative mobilities (relative to the chloride boundary; RC1) of 1.00 at 20% polyacrylamide concentration (% T) compared to bromophenol blue (BPB) for which RC1=0.77. The homologue of BPB, 3, 4, 5, 6-tetrabromophenolsulfonephthalein (TBPS), exhibited a useful color transition over the pH 6.6-8.2 range but was an equally poor indicator of the chloride boundary at 20% T in the absence of SDS (RC1=0.73). The RC1 of other dyes was increased in the presence of SDS, particularly the cationic dye neutral red which complexes with micellar but not monomeric SDS. In the presence of SDS, all dyes except neutral red exhibited RC1 of 1.00 at 15% T, whereas only neutral red and nitrazine yellow had RC1 values of 1.00 at lower gel concentrations. Partial characterization of SDS, in terms of RC1 and zone broadening, was performed in a system devoid of extraneous SDS in the gel or electrolytes.

The role of pH T measurements in marine CO 2-system characterizations

Spectrophotometric pH T measurements can routinely be obtained with an imprecision on the order of ±0.0005 pH T units. This level of imprecision is equivalent to an imprecision in total hydrogen ion concentration of ±0.1% (where pH T=−log[H +] T and [H +] T≅[H +]+[HSO 4 −]). At this level of precision, pH T measurements provide an important tool in quality control assessments of other CO 2-system parameters (CO 2 fugacity, total inorganic carbon, total alkalinity). CO 2 fugacities and total alkalinities calculated using measured pH T and total inorganic carbon, for the large data set considered in this work, have relative precisions on the order of 0.15 and 0.1%, respectively. The precision of total inorganic carbon calculated via pH T and salinity-normalized-alkalinity is on the order of 0.02% or better. In this work, using the NOAA 1992 boreal autumn Equatorial Pacific (EqPac) dataset, it is shown that CO 2-system variables calculated via pH T can be used to enhance both the precision and accuracy of directly measured parameters. Through the procedures described in this work significant problems were revealed in the initial version of the 1992 NOAA dataset, and the dataset was greatly improved. Additionally, in this work, we revisit CO 2-system thermodynamic consistency issues in view of changes in the pH T values assigned to tris seawater buffers and consequential changes in the calibration of sulfonephthalein pH T indicators. As the principal result of a +0.0047 increase in the pK of meta cresol purple, CO 2 fugacity calculations and measurements are in very good agreement for the NOAA 1992 boreal autumn EqPac dataset. We note, as well, that due to a reassignment of the titrant acid concentration used in the NOAA 1992 boreal autumn dataset, measured total alkalinities are in good agreement with total alkalinities calculated from total carbon and pH T.

A flow-batch titrator exploiting a one-dimensional optimisation algorithm for end point search

A flow-batch hybrid system is proposed as a novel approach to automated titration. Sampling and signal processing are done as in usual flow systems, whereas chemical reactions take place in a reaction chamber similar to those found in batch systems. End point is found by means of the Fibonacci method, adapted to act as a one-dimensional optimisation algorithm for fast titration. A model system involving the spectrophotometric NaOH/HCl titration was initially studied, and the feasibility of the approach was further demonstrated in the titration of white wines with NaOH using m-cresol purple as indicator. About 20 samples are run per hour and ca. 0.6ml of sample and titrant are consumed per determination. Within the 5.2–7.3gl−1 acidity (expressed as tartaric acid) range, relative standard deviations of results were estimated as about 0.7% after six-fold processing of typical wine samples, and results are in agreement with those obtained by a classical potentiometric titration, with relative errors <0.8%.

Influence of m-cresol purple indicator additions on the pH of seawater samples: correction factors evaluated from a chemical speciation model

Abstract The perturbation of the indicator m -cresol purple on the pH in seawater is illustrated in diagrams, representing measurements in 1-cm and 5-cm cells. The diagrams apply to a measured pH interval of 7.4–8.4 using a 2-mM stock solution of m -cresol purple sodium salt dissolved in seawater. The magnitude of the perturbation is described as correction values, i.e., the change in seawater pH caused by the indicator. The diagrams are based on calculations made by using the equilibrium speciation programme, MARINHALT. From these calculations, and least squares fitting methods, pH correction values are described in terms of the pH difference between each seawater sample and the pH of an indicator stock solution. Calculations are performed for a typical high latitude water and a north Pacific deep water. Diagrams are presented for a salinity of 35 and a temperature of 15°C. Responses to salinities between 32 and 36 and temperatures 15–25°C are illustrated as well. A ±0.05 pH difference between a seawater sample and an indicator stock solution gives a correction of less than 0.001 pH unit for a 1-cm cell. For a 5-cm cell, pH differences between the indicator stock solution and a seawater sample as large as ±0.3 cause corrections smaller than ±0.001 pH unit. Calculations demonstrate that the five-fold lower indicator concentration used with 5-cm cells decreases the perturbation effect by approximately a factor of five relative to 1-cm cells.

Proton and electron transfer during the reduction of molecular oxygen by fully reduced cytochrome c oxidase: a flow-flash investigation using optical multichannel detection.

Proton and electron transfer events during the reaction of solubilized fully reduced bovine heart cytochrome c oxidase with molecular oxygen were investigated using the flow-flash technique. Time-resolved spectral changes resulting from ligand binding and electron transfer events were detected simultaneously with pH changes in the bulk. The kinetics and spectral changes in the visible region (450-750 nm) were probed by optical multichannel detection, allowing high spectral resolution on time scales from 50 ns to 50 ms. Experiments were carried out in the presence and absence of pH-sensitive dyes (carboxyfluorescein at pH 6.5, phenol red at pH 7.5, and m-cresol purple at pH 8.5) which permitted separation of spectral changes due to proton transfer from those caused by ligand binding and electron transfer. The transient spectra recorded in the absence of dye were analyzed by singular-value decomposition and multiexponential fitting. Five apparent lifetimes (0.93 microseconds, 10 microseconds, 36 microseconds, 90 microseconds, and 1.3 ms at pH 7.5) could consistently be distinguished and provided a basis for a reaction mechanism consistent with our most recent kinetic model [Sucheta, A., Szundi, I., and Einarsdóttir, O. (1999) Biochemistry 37, 17905-17914]. The dye response indicated that proton uptake occurred concurrently with the two slowest electron transfer steps, in agreement with previous results based on single-wavelength detection [Hallén, S., and Nilsson, T. (1992) Biochemistry 31, 11853-11859]. The stoichiometry of the proton uptake reactions was approximately 1.3 +/- 0.3, 1.4 +/- 0.3, and 1.6 +/- 0.5 protons per enzyme at pH 6.5, 7.5, and 8.5, respectively. The electron transfer between heme a and CuA was limited by proton uptake on a 90 microseconds time scale. We have established the lower limit of the true rate constant for the electron transfer between CuA and heme a to be approximately 2 x 10(5) s-1.

An automated flow-injection titrator for spectrophotometric determinations of total acidity in wines, using a single standard solution and gradient calibration

An automated flow-injection titrator to perform spectrophotometric determinations of acidity in wine samples is described. The proposed titrator exploits concentration gradients generated in the flow-injection system and, in contrast to the automated flow titrators previously proposed, it does not require analytical curves based on several standard solutions of tartaric acid. Only one single standard solution, the proper titrant, is directly used for gradient calibration and calculating of the analyte concentrations. A simple laboratory-made photometer that uses an LED (Light-Emitting Diode) as light source and a phototransistor as detector was built in to this titrator. Yellow LED was usually used for spectrophotometric measurements because m-cresol purple (7.4 < pH < 9.0 yellow–purple transition) was selected as acid–base indicator. The system is fully controlled by microcomputer and the software for control and for acquisition and treatment data was written in C language. A good agreement between results yielded by the proposed titrator and those produced by a reference method of the Association of Official Analytical Chemistry (AOAC) was obtained and no statistical difference between results at the 95% probability level was confirmed by applying the paired t-test. An overall relative accuracy of about 0.9% and a mean relative standard deviation lower than 1.5% were obtained with nine replicate titrations. The proposed titrator allows 72 determinations per hour to be carried out, consuming just 200 µl of the sample and 1.5 ml of the titrant.

Phase Fluorometric Optical Carbon Dioxide Gas Sensor for Fermentation Off-Gas Monitoring.

We demonstrated an optical carbon dioxide gas sensor suitable for replacement of gas chromatographs and mass spectrometers for the measurement of carbon dioxide in the off-gas of a bioreactor for fermentation and cell culture applications. The sensor is based upon the change in lifetime of a donor fluorophore, sulforhodamine 101 (SR101), induced by fluorescence resonance energy transfer to a pH-sensitive, nonfluorescent acceptor, m-cresol purple (MCP). Carbon dioxide diffusing into the sensor produces carbonic acid, changing the absorbance spectrum of the MCP, and thus its spectral overlap with the SR101, changing its lifetime. This lifetime change was measured in the frequency, rather than the time domain, as a change in the phase angle of the fluorescence relative to the modulated excitation light. The sensor was calibrated by correlating the phase response to carbon dioxide concentrations. The calibration remained valid over the life of the sensor, which has been shown to be greater than 2 weeks. The sensor was most sensitive at low CO2 concentrations and responded to concentration changes in seconds. The sensor film is very inexpensive to produce and the light source is an inexpensive light-emitting diode. Furthermore, lower cost detection electronics can be developed since only one modulation frequency is required. In addition, this sensor can potentially be used in vivo, with a fiber optic both delivering the excitation light and collecting the emission.

A Lifetime-Based Optical CO 2 Gas Sensor with Blue or Red Excitation and Stokes or Anti-Stokes Detection

We describe the fabrication and characterization of an optical CO 2 sensor based on the change in fluorescence lifetimes due to fluorescence resonance energy transfer from a pH-insensitive donor, sulforhodamine 101, to a pH-sensitive acceptor, either m-cresol purple or thymol blue, entrapped in an ethyl cellulose film. A phase transfer agent allows incorporation of the dyes and water into the film, while providing an initially basic environment for the acceptor. Diffusion of CO 2 into the water entrapped in the film produced carbonic acid, causing a pH-dependent decrease in the spectral overlap of the acceptor absorbance with the donor emission, and decreased energy transfer, resulting in increased SR101 donor lifetimes. The lifetime changes were detected as a change in the phase of the emission, relative to the modulated excitation, and were insensitive to excitation intensities and emission signal levels. In addition to an externally modulated 442-nm light source, we excited the sensor with a directly modulated 635-nm laser diode and detected the anti-Stokes emission. The CO 2 sensor is not fragile and can provide stable readings for weeks. The use of fluorescence resonance energy transfer, along with the simple entrainment procedure, allows facile change of the CO 2 response range through change of the acceptor dye and the use of laser diode excitation sources.

The role of pH measurements in modern oceanic CO 2-system characterizations: Precision and thermodynamic consistency

In May 1992, surface seawater samples were collected along an equatorial transit (130 to 100°W) and analyzed for total hydrogen ion concentration (expressed as spectrophotometric pH T) total dissolved inorganic carbon (coulometric C T), and total alkalinity (potentiometric A T and spectrophotometric A T). This data set, which presents a striking view of the chemical signature of tropical instability waves, is also unusual in that its “overdetermination” of the CO 2-system in seawater includes both potentiometric A T data and the first spectrophotometric A T data collected at sea using a double-wavelength, one-step acid addition method. Our data set indicates that spectrophotometric A T measurements ( A Tspec) are both precise (±1.8 μmol/kg) and accurate; the mean observed difference between A Tspec and A T obtained potentiometrically in this study ( A Tpot) is 1 μmol/kg. As only two of the three measured parameters are required to characterize the CO 2-system in seawater, this analytical redundancy of our analyses ( C T, A T, pH) afforded an opportunity to evaluate, in terms of thermodynamic consistency, a recent calibration of m-cresol purple, a pH indicator. Using various carbonic acid dissociation constants, measured parameters were combined in pairs ( C T- A T, pH- C T, and pH- A T) to calculate a third parameter for comparison with the shipboard measurements. Depending on the selected set of carbonic acid dissociation constants, the average offset between directly measured and predicted values of A T and C T was as small as ±1 μmol/kg. The results of this study indicate that the present 25°C mCP calibration, paired with the 25°C combined dissociation constants of Hansson and Mehrbach, produces accurate predictions of A T and C T. Extensive replication of the shipboard spectrophotometric measurements made it possible to examine directly the sensitivity of derived parameters to variations or errors in input A T and pH. In accord with earlier theoretical treatments of this question, the results presented demonstrate that pH is imprecisely predicted from A T and C T, whereas imprecision in measured pH—at the level typical of spectrophotometric measurements (±0.0004)—contributes negligibly (±0.3 μmol/kg) to imprecision in derived A T and C T. In view of the high precision of pH Tspec measurements and both the precision and reliability of C T measurements (supported by the use of certified SIO reference materials) pH Tspec and C Tcoul constitute a particularly useful pair of parameters for shipboard study of the oceanic carbon dioxide system. The results indicate, as well, that the A T-pH Tspec pairing is of particular interest in ocean regions where simple A T vs salinity relationships are observed. In light of recent advances, the role of pH measurements in CO 2-system characterizations should be re-evaluated. Spectrophotometric measurements of pH have much to contribute in documenting the oceans' evolving response to anthropogenic C0 2.

Colorimetric polymer film sensors for dissolved carbon dioxide

Plasticized and unplasticized polymer colorimetric film sensors for gaseous CO 2, containing the dye m-cresol purple, are tested as sensor for dissolved CO 2. The plasticized polymer m-cresol purple film sensor develops a measurable degree of opacity when exposed to aqueous solution, especially in netural, compared with alkaline, solution. However, it is shown that a presoaked, fogged plasticized polymer m-cresol purple film does function as a quantitative sensor for dissolved CO 2 over the range 0–4% CO 2. An unplasticized polymer m-cresol purple film remains largely clear upon exposure to aqueous solution and also function as a quantitative sensor for dissolved Co 2 over the range 0–4% CO 2. However, in both of films the dye interacts with electrolytes present in solution; invariably the dye appears to be converted from its initial deprotonated form (blue) to its protonated form (yellow) and the rate of this process appears to increase with increasing ionic strength, anionic charge and decreasing pH. The 90% response and recovery times for an unplasticized film are determined as 19 s (CO 2:O→5%) and 21 s (CO 2:5→3.6%), respectively.

Synthesis of 14C‐labelled bromocresol green and metacresol purple

Abstract[methyl‐14C]‐Bromocresol green 1 was synthesized from [14C] ‐ methyl iodide in four steps via 3‐[methyl‐14C]‐methylanisole 3, [methyl‐14C]‐metacresol 4 and [methyl‐14C]‐metacresol purple 6. The overall yield of 1 from [14C] methyl iodide was 5.3% and the specific activity 112 mCi/mmol.

Spectrophotometric seawater pH measurements: total hydrogen ion concentration scale calibration of m-cresol purple and at-sea results

Spectrophotometric open-ocean seawater pH measurements are simple, fast and precise. The sulfonephthalein indicator m-cresol purple ( mCP) is recommended for open-ocean surface-to-deep pH measurements. The salinity- and temperature-dependence of such measurements (293⩽ T⩽303 and 30⩽ S⩽37) is given as: pH T (sw) = 1245.69 T + (2.11 × 10 −3)(35 − S) + log( R − 0.0069 2.222 0 − R0.133 0 ) on the total hydrogen ion concentration scale ([H +] T = [H +] f + [HSO 4 −]), in units of mol·(kg-soln) −1. R is the ratio of indicator absorbances at molar absorptivity maxima (i.e. R + 578 A/ 434 A). The at-sea analytical precision of the technique, evaluated on two recent NOAA cruises, is approximately 0.0004 pH units.

Multiple-indicator fiber-optic sensor for high-resolution pCO 2 sea water measurements

A multiple-indicator fiber-optic sensor was employed to detect low level CO 2 changes in sea water. p K a values of these indicator systems at high ionic strength were determined, and the bicarbonate concentrations were added as calculated by the Henderson-Hassellbach equation. The 7-hydroxycoumarin-4-acetic acid (HCA)— meta-Cresol Purple (MCP) indicator system provides a resolution of 7 μg ml −1 CO 2 in the 200–800 μg ml −1 CO 2 range, the CO 2 range of sea water.

Simultaneous determination of lead, nickel, and copper in Orchard Leaves by high performance liquid chromatography followed by solvent extraction

High performance liquid chromatography (HPLC) was applied for the determination of lead, nickel, and copper in Orchard Leaves followed by solvent extraction. Ammonium pyrrolidinedithiocarbamate (APDC) was employed as the chelating agent for the above heavy metals. Orchard Leaves (500mg) sample was ashed at 450°C for (1520) h and dissolved with 2ml of 2N hydrochloric acid, then the solution was put into the separatory funnel and diluted up to 50ml with water. Ten milliliters of 1.0M ammonium citrate, dibasic solution (pH9.3) and two drops of 0.1% metacresol purple solution were added. Ammonia. water (1+1) was added until purple color was developed. Then 2.0ml of 0.01M APDC and 1.0ml of chloroform were added accurately. After shaking the contents for 15min, a 3μl portion of chloroform layer was injected into the HPLC column (Cosmosil 5C18, 4.6mm × 150mm). The mixed solution of acetonitrile-water-0.01M APDC (67 : 33 : 0.15) was used as the eluent. The proposed method permitted simultaneous determination of lead, nickel, and copper in Orchard Leaves. Analytical results were in good agreement with the certified values of standard sample.