Synthesis of Copper-Chelates Derived from Amino Acids and

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Synthesis of Copper-Chelates Derived from Amino Acids and Evaluation of Their Efficacy as Copper Source and Growth Stimulator for Lactuca Sativa in Nutrient Solution Culture Narongpol Kaewchangwat, Sattawat Dueansawang, Gamolwan Tumcharern, and Khomson Suttisintong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03809 • Publication Date (Web): 26 Oct 2017 Downloaded from http://pubs.acs.org on October 27, 2017

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Journal of Agricultural and Food Chemistry

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Synthesis of Copper-Chelates Derived from Amino Acids and Evaluation of

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Their Efficacy as Copper Source and Growth Stimulator for Lactuca Sativa

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in Nutrient Solution Culture

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Narongpol Kaewchangwat, Sattawat Dueansawang, Gamolwan Tumcharern

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and Khomson Suttisintong*

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National Nanotechnology Center (NANOTEC), National Science and Technology

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Development Agency (NSTDA), 111 Thailand Science Park, Thanon Phahonyothin,

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Tumbon Khlong Nueng, Amphoe Khlong Luang, Pathum Thani, 12120, Thailand

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[email protected]

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Abstract

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Five tetradentate ligands were synthesized from L-amino acids and utilized for the synthesis

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of Cu(II)-chelates 1-5. The efficacy of Cu(II)-chelates as copper (Cu) source and growth

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stimulator in hydroponic cultivation was evaluated with Lactuca sativa. Their stability test

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was performed at pH 4–10. The results suggested that Cu(II)-chelate 3 is the most pH

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tolerant complex. Levels of Cu, Zn and Fe accumulated in plants supplied with Cu(II)-

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chelates were compared with those supplied with CuSO4 at the same Cu concentration of 8.0

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µM. The results showed that Cu(II)-chelate 3 significantly enhanced Cu, Zn and Fe content

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in shoot by 35%, 15% and 48%, respectively. Application of Cu(II)-chelate 3 also improved

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plant fresh and dry matter yield by 54%. According to the results, Cu(II)-chelate 3

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demonstrated the highest stimulating effect on plant growth and plant mineral accumulation

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so that it can be used as an alternative to CuSO4 for supplying Cu in nutrient solutions and

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enhancing the plant growth.

ACS Paragon Plus Environment


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Keywords: nutrient solution, copper chelate, amino acid derived chelator, plant copper

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uptake, hydroponics

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Introduction

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Copper (Cu) is one of essential micronutrients that play a significant role in a number of

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plant physiological processes such as photosynthesis and respiratory electron transport

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chains.1, 2 It also acts as a cofactor or a part of the prosthetic group of many key enzymes

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involved in different metabolic pathways, including ATP synthesis.3 When copper is not

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available, plants develop specific deficiency symptoms, most of which affect young leaves

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and reproductive organs.4 Thus, copper deficiency can alter essential functions in plant

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metabolism resulting in low crop productivity. Recently, polymer-encapsulated copper

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nanoparticles have been applied to solve copper deficiency symptoms in plants,5 and to

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improve plant crop productivity. Moreover, encapsulation of copper cations and Trichoderma

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viride in chitosan/alginate microcapsule were used as biological and chemical agents in plant

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nutrition and protection.6

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Higher plants accumulate copper from soil solution mainly as Cu(II). In nutrient solution

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cultures, Cu ion is prepared at a desirable concentration (0.5-12.5 µM)7-12 of this element for

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the plant.13 The most common Cu source used in nutrient solutions is Cu(II) ion from CuSO4.

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When CuSO4 is added to a nutrient solution which contains an abundance of anions such as

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phosphate (PO43-) or hydroxide (OH-), its precipitation can occur.14-17 The precipitation

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causes a lower concentration of free Cu ion in nutrient solutions and results in insufficient

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amount of Cu for plant uptake. This contributes to plant disorder.18-22

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To maintain sufficient amount of certain micronutrients (e.g. Fe, Zn, Mn, Cu) in nutrient

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solutions, free ligands or chelators are required to form a stable and soluble complex (chelate)

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of micronutrients.23-33 This also helps translocation of micronutrient to most parts of the


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plants.34-37 To date, many synthetic chelators such as EDTA,23 EDDHA,24-27 IDHA,28-29 or

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DTPA30 have been used commercially in hydroponic cultivation or spray nutrient solution

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especially for iron (Fe)23-30 and manganese (Mn)31, 32. Application of Cu-chelates such as Cu-

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EDTA and Cu-DTPA in soil culture demonstrates a significant improvement of nutritional

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value to the crop.38 However, their application in hydroponic cultivation is limited.

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Amino acids are cheap and abundant sources for chelates and have been widely used as

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agrochemical products. It has been shown that uses of some amino acids in nutrient solutions

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improves micronutrient uptake by crops.39 Amino acids have the ability to coordinate metal

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ions via their carboxyl and amino groups.40, 41 They are less sensitive to photodegradation or

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their degradation is completely biological.42 There are some results that indicate negligible

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degradation of amino acids in nutrient solutions,43 however the degradability of metal-amino

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acid complexes is still less than free amino acids.44, 45 In addition, amino acids are sources of

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nitrogen for plant nutrition46 and most plants can directly absorb amino acids and use them in

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their physiological structures and processes.43, 47, 48 This may result in less accumulation of

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free ligand in the media and help maintain other micronutrient balance.

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Although a free amino acid (bidentate ligand) can be a good chelator, it may form an

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unstable metal-chelate complex in a hydroponic nutrient solution which consists of high

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concentration of anions such as OH-, PO43- or HPO42-. The first objective of this research is,

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therefore to synthesize tetradentate ligands from amino acids and evaluate their efficiency as

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copper chelators. To achieve this goal, four L-enantiomers (natural form in plants) of amino

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acids including L-phenylalanine (Phe), L-leucine (Leu), L-isoleucine (Ile) and L-valine (Val)

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were chosen as starting materials to synthesize Cu chelators. These amino acids were selected

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because of their significance in plant and human nutrition. It has been shown that systemic

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application of L-phenylalanine increases plant resistance to vertebrate herbivory because this

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significantly increases the phenolic pool of oilseed rape.49 Additionally, L-phenylalanine is a



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precursor for many substance including ubiquinone which is an essential respiratory

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cofactor.50 Valine, leucine and isoleucine together form a unique group of so-called

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branched-chain amino acids (BCAAs) needed for human nutrition. These essential amino

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acids are critical for protein synthesis and normal plant growth, while also providing

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precursors for a number of secondary metabolites such as; cyanogenic glycosides,51

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glucosinolates,52 and acyl-sugars.53 In addition, valine has been reported to act as a metabolite

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for the respiration of higher plants and yield CO2.54 Our next objectives are to form Cu(II)-

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chelates (from tetradentate ligands), characterize and confirm their structures, investigate

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their stability under various pH conditions, and evaluate their efficacy against Lactuca sativa

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L., a red oak leaf lettuce commercially grown hydroponically in Thailand. To the best of our

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knowledge, there are only a few studies showing the effect of Cu(II)-chelates on plant growth

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in hydroponic cultivation.12 Thus, the investigation of stimulating effects of Cu(II)-chelates

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on plant growth and mineral accumulation is our ultimate target.

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Materials and Methods

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Chemicals

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L-phenylalanine, L-leucine, L-isoleucine and L-valine were purchased from Acros

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Organics. Ethylene glycol, ethylenediaminetetraacetic acid and p-toluenesulfonic acid were

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purchased from Aldrich Chemical Co. Pyrocatechol was purchased from Tokyo Chemicals.

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Copper sulfate pentahydrate (CuSO4·5H2O), calcium nitrate (Ca(NO3)2), iron sulfate

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heptahydrate (FeSO4·7H2O), potassium nitrate (KNO3), magnesium sulfate (MgSO4),

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ammonium dihydrogen phosphate (NH4H2PO4), boric acid (H3BO3), manganese sulfate

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(MnSO4), zinc sulfate (ZnSO4), ammonium heptamolybdate ((NH4)6Mo7O24), sodium

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hydroxide (NaOH) and hydrochloric acid (HCl) were purchased from Carlo Erba. Acetone

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and toluene were purchased from Fisher Scientific. Seeds of red oak leaf lettuce (Lactuca


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sativa L.) were purchased from Chai Tai Co., Ltd. All reagents received were analytical grade

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and used as received unless stated otherwise. Deionized water (DI water) was used in

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experiments. All stock solutions were kept in the refrigerator at 4 °C prior to use at room

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temperature.

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Instrumentation Fourier Transform Infrared (FT-IR) spectra were recorded using a Thermo Scientific

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Nicolet 6700 spectrometer in ATR mode. 1H NMR (500 MHz) spectra and

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MHz) with complete proton decoupling spectra were recorded by a Bruker AV-500 in

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Fourier transform mode. Spectra were obtained in D2O solutions and chemical shifts in ppm

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(part per million) are quoted relative to the residual signals of D2O (δH 4.79 ppm). Data are

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reported as follows: chemical shifts, multiplicity, coupling constant. Multiplicities in the 1H

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NMR spectra are described as: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet,

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br = broad; coupling constants (J) are reported in Hz. High resolution mass spectra (HRMS)

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were recorded using Bruker micrOTOF mass spectrometer (LC-ESI-TOF) and reported with

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ion mass/charge (m/z) ratios.

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Methods

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General Procedure for the Synthesis of Amino Acid-Derived Chelators

C NMR (125

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To a stirred suspension of 8.00 mmol L-amino acid (L-phenylalanine, L-leucine, L-

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isoleucine or L-valine) and 4.00 mmol ethylene glycol (or pyrocatechol) in 15 mL toluene

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was added 8.00 mmol p-toluenesulfonic acid and the resulting mixture was heated to 115 °C

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with stirring for overnight. The reaction mixture was cooled to room temperature and 20 mL

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acetone was added. The precipitate was filtered, washed with acetone and dried under

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reduced pressure to give the corresponding chelator which was used in the next step without

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purification.



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General Procedure for the Synthesis of Cu(II)-Chelate Complexes

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Cu(II)-chelate complexes (Cu(II)-chelate 1-5) were prepared using amino acid-derived

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chelators 1-5 as complexing agents. (Their characterization is available in the Supporting

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Information, SI). A solution of 1.25 mmol chelator 1 in 100 mL DI water was slowly added

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to a solution of 1.25 mmol CuSO4 in 100 mL DI water. The mixture was heated to 45°C and

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stirred for 2 h. The mixture was cooled to room temperature and water was removed by

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freeze-drying technique to yield blue solid of Cu(II)-chelates. The products were washed with

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cold ethanol followed by diethyl ether and air-dried. All complexes were characterized by

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NMR and FT-IR.

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Hydroponics Cultivation

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Seeds of red oak leaf lettuce (Lactuca sativa L.), most commonly grown hydroponically in

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Thailand, were thoroughly rinsed with DI water and germinated on moist sponge for

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hydroponic cultivation in a half-filled water tray which was placed in incubator at 28 °C.

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After 7–10 days, uniform-size seedlings which had two first leaves (primary leaves) were

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transferred to PVC lids that fit tightly over 1-L glass bottle containers which were placed in a

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greenhouse under controlled conditions, with a 12-h light period and 32/29 °C day/night

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temperature, and 65-75% relative humidity. The bottles were wrapped with black

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polyethylene to prevent light from reaching the roots and nutrient solution. One plant was

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grown in each bottle. A basic nutrient solution was prepared in DI water (electrical resistivity

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= 18 MΩ cm-1). The basic nutrient solution contained 4.0 mM Ca(NO3)2, 0.055 mM Fe-

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EDTA, 8.0 mM KNO3, 2.0 mM MgSO4, 1.3 mM NH4H2PO4, 0.046 mM H3BO3, 9.5 µM

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MnSO4, 0.77 µM ZnSO4, 20.2 nM (NH4)6Mo7O24 adjusted to pH 6.0 with NaOH or HCl as a

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buffer. Copper was supplied from six different sources: CuSO4 and Cu(II)-chelate 1-5 and

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the Cu concentration in all treatments was 8.0 µM (2 ppm). Plants were harvested


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approximately 4 weeks after seeding and divided into shoot and root. Fresh and dry matter

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yields of shoot and root in each bottle were determined.

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Remaining Concentration (ppm) of Copper in Nutrient Solutions

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The remaining concentration of Cu (in ppm) in the CuSO4 solution and the Cu(II)-chelate

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3 solution were investigated to confirm the absorption of Cu into the plants. 5.0 mL of

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nutrient solutions were sampling at day 4, 7, 14, 21, and 28 after the seedlings were planted

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in the nutrient solutions. At day 18, 1 ppm of CuSO4 or Cu(II)-chelate 3 was added to each

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bottle. The calibration curve was prepared using standard CuSO4 solutions at 0.05, 0.25, 0.75,

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1.25 and 2.50 ppm. Analyses of Cu contents were carried out with an atomic absorption

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spectrometer (AAS) model PinAAcle 900F, Perkin-Elmer.

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Elemental Analyses of Cu, Zn and Fe content in Dry Plant Matter

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Plant materials were dried in a vacuum-dried oven at 50 °C to a constant weight and

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ground to a fine powder form in a Milestone ETHOS labstation. Dry samples (0.10 g) were

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placed in TFM vessels. 65% HNO3 (14 mL) and 30% H2O2 were added dropwise to the

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vessels, then the vessels were gently swirled to homogenize the samples. The vessels were

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put under microwave digestion at 1000 watt, 200 °C for 10 minutes. The final solution was

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diluted to meet the range requirements of the analytical procedures. Analyses of Cu, Zn and

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Fe contents in shoots and roots were carried out with an inductively coupled plasma atomic

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emission spectrometer (ICP-AES) model Optima 7300DV, Perkin Elmer.

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Statistical Analysis

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Analysis of variance (using one-way ANOVA) and Tukey test were applied to the data of

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sample set at 95% confidential interval (p < 0.05). Statistical processing was carried out using

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Minitab Release 17.2.1 software (Minitab Inc., State College, PA, USA).



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Results and Discussion

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Synthesis of Chelators 1-5

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The amino acid-derived chelators 1-5 were successfully synthesized as outlined in

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Scheme 1 and 2. Chelator 1, 3, 4 and 5 were designed based on different amino acids to

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investigate the effect of amino acid types on chelation efficacy. Chelator 2 was synthesized to

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compare the chelation efficacy when bridging moiety between two amino acid units is

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changed from diethylene glycol (Scheme 1) to pyrocatechol (Scheme 2). All products were

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white solid with the reaction yields of 23-88%. The difference in reaction yields may result

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from different solubility of amino acid starting materials in the reaction mixtures. All

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chelators were characterized using FT-IR, NMR and HRMS to confirm the structures. The

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FTIR spectra of chelator 1, 3, 4 and 5 showed an absorption band in the range of 1735-1750

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cm-1 indicating the stretching vibration of the newly formed ester carbonyl group (C=O).

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Moreover, the strong absorption band at 1100-1200 cm-1 derived from the stretching mode of

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the C-O bond also confirmed the presence of an ester moiety. The bands in these regions are

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distinguished from simple amino acid starting materials and this ensures the ethylene linkage

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on the chelator molecules. In addition, the 1H NMR spectra of chelators 1, 3, 4 and 5

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exhibited the signals of the ethylene linkage moiety at around δ 3.80-4.20 ppm with the

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integration of four protons. This ester formation was also confirmed by

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DEPT135 experiments which show extra ethylene CH2-carbons in the δ 60-70 ppm region.

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The FTIR spectra of chelator 2 showed an absorption band of the ester carbonyl (C=O)

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stretching at 1747 cm-1 and ester C-O stretching at 1122 cm-1. Besides, the 1H- and 13C-NMR

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spectra of chelator 2 displayed the proton signals at δ 7.05-7.15 (4H) and carbon signals at δ

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125-130 ppm. These signals indicate the presence of pyrocatechol aromatic moiety. The high

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resolution mass spectra of all chelators were relevant to the molecular weight of the

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structures.


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C-NMR and

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Scheme 1 Synthesis of chelator 1, 3-5

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Scheme 2 Synthesis of chelator 2

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Characteristics of Cu(II)-chelate 1-5 Complexes

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Infrared Spectroscopy

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The FTIR spectra of the Cu(II)-chelate 1-5 complexes showed an absorption in the 4,000-

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500 cm-1 region similar to the chelators, which generally act as tetradentate ligands.

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Predominant vibrations for the complexes are associated with ν(C=O), ν(C-O), ν(NH2). The

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observed vibrational bands for –NH2 groups appear around 3,100-3,500 cm-1 as very broad

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signals due to the effect of intermolecular interaction in the solid state. The carbonyl group

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(C=O) of the chelates has approximately the same frequency to the chelators at around 1,590

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– 1,690 cm-1.

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1

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Fig. 1 Structure of chelator 4. a) Chelator 4 with numbering system; b) Proposed structure of

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Cu(II)-chelate 4 complex

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The formation of Cu(II)-chelates in aqueous solution was demonstrated using chelator 4 as

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a model study and the experiments were performed using 1H- and 13C-NMR titration in D2O.

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Chelator 4 was selected for this experiment because its 1H NMR and

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uncomplicated with enough structural detail. The results revealed that the suitable ratio of

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Cu(II) to chelator 4 to form the chelate complex is 1:1. The proposed structure of Cu(II)-

H- and 13C-NMR tritration of CuSO4 and Chelator 4


13

C NMR signals are


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chelate 4 complex was also shown in Fig. 1 based on the NMR experiments. The NMR

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method to confirm the geometry of the chelate complex has been studied and the report

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suggested that Cu(II) tended to form a square planar geometry.55 In our study, the 1H-NMR

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spectra of chelator 4 show signals of protons at C1 and C1’ position (Fig. 1a) at 3.81 and

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4.31 ppm as triplets (Fig. 2). These triplet signals are eventually broader after the ratio of Cu

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increased to 1:1 ratio. The multiplet signals at around 4.00 ppm (1H) and 4.15 ppm (1H)

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indicate the differentiation of protons at C3 and C3’. When the ratio of Cu increased to 1:1

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ratio, these proton signals collapsed to a new broad peak at 4.13 with the integration of 2H,

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revealing a lack of distinction of C3-H and C3’-H after the formation of Cu(II)-chelate 4

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complex. This can be illustrated by structure of Cu(II)-chelate 4 (Fig. 1b) in which a C2

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symmetry is present in the molecule after the complex was formed. The protons at C3 and

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C3’ or so called Hc were in the C2 environment resulting in the collapsing of the initial

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signals to a broad peak at 4.13 (2H).

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Fig. 2 1H-NMR spectra of Cu(II)-chelate 4 (δ 3.70 – 4.40 ppm)

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The

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C-NMR spectra of chelator 4 contain signals of C4 and C4’ at 38.7 and 38.9 ppm

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(Fig. 3). After the ratio of Cu increased, these two carbon signals collapsed to a single peak at

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38.7 ppm with doubled intensity. This can also be explained by the structure of Cu(II)-

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chelate 4 (Fig. 1b) where the molecule has a C2 symmetry. The carbon signals of chelator 4

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at 51.4 and 51.6 ppm show the distinction of carbon at C3 and C3’. These two signals

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eventually collapsed to a peak at 51.4 ppm after the ratio of Cu increased to 1:1 ratio.

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The changes in chemical shifts of chelator 4 in 1H NMR and

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C NMR spectra after

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addition of CuSO4 indicate the formation of Cu(II)-chelate 4 at 1:1 ratio of chelator 4 to

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CuSO4.


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Fig. 3 13C-NMR spectra of Cu(II)-chelate 4 (δ 20.0 – 70.0 ppm)

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pH Tolerance of Cu(II)-chelate 1-5 (Formation of Cu(OH)2)

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Since pHs of nutrient solutions significantly affect bio-availability of Cu in hydroponic

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nutrient solution, the stability of Cu(II)-chelate 1-5 was investigated by means of pH

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tolerance test at pH 4 – 10 (data shown in the SI Fig. S2). CuSO4 solution started

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precipitating at pH 5 (picture not shown) while Cu(II)-chelate 2 and Cu(II)-chelate 4

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solutions began precipitating at pH 4. Cu(II)-chelate 1 precipitated in the pH ranging from 6

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– 10 while Cu(II)-chelate 5 could tolerate basic condition up to pH 8. Cu(II)-chelate 3 gave

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no precipitation under the whole pH range (4 – 10). The results obtained from this test

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suggested that Cu(II)-chelate 3 and Cu(II)-chelate 5 were stable in pH ranging from 5 – 8

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which is the pH range used for hydroponic cultivation. This stability may result from a strong

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interaction of Cu(II) ion and chelator 3 or 5 that prevents the complexes from reacting with

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hydroxide ion.

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Gravimetric analysis (Measurement of Cu(OH)2 precipitate)

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The stability of Cu(II)-chelate complexes were relevant to gravimetric analysis of

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Cu(OH)2 where Cu(II)-chelate 3 and 5 gave only small amount of the precipitate from the

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solution in the whole range of tested pH (pH 5 – 8) (Fig. 4). The results revealed that basicity

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of the solution had a great impact on precipitation of Cu(OH)2. When pH increased, more

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Cu(OH)2 precipitated out from each solution. The culprit was from high concentration of

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hydroxide ion (OH-) at higher pH reacting with the Cu(II) ion and give precipitation (eq. 1

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and eq. 2). Cu(II)-chelate 1, Cu(II)-chelate 2 and Cu(II)-chelate 4 gave as much precipitate



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as CuSO4 at pH 8. Cu(II)-chelate 5 gave about 5 times less precipitate than CuSO4. Cu(II)-

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chelate 3 gave superior result by generating a minimal amount of precipitation throughout

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the whole range of tested pH. These results suggested that Cu(II)-chelate 3 and 5 are the best

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candidates suitable for nutrient solution that can tolerate a changing in pH.

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CuSO4 (aq) + chelator (aq)

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Cu(II)-chelate (aq) + 2NaOH (aq)

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----------------------------------------------------------------------------------------------------------------

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Fig. 4 Gravimetric analysis of Cu(OH)2 from Cu(II)-chelate solutions compared with CuSO4

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at various pH (*Asterisks above the bars indicate a significance of p

Synthesis of Copper-Chelates Derived from Amino Acids and

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