Gelatin as a complete,endogenous source of calcium for Serratia marcescens protease activity

Growth of Serratia marcescens in gelatin-containing medium is associated with proteolytic activity in the centrifuged supernatants. This activity requires calcium at a minimum concetration of 10⁻⁴M. Commercial gelatin, naturally contaminated with calcium, satisfies this minimum cation requirements in full. This finding can also help to explain a number of inconsistencies in the literature regarding the use of gelatin.     

THE COMBINATION OF GELATIN WITH HYDROCHLORIC ACID : II. NEW DETERMINATIONS OF THE ISOELECTRIC POINT AND COMBINING CAPACITY OF A PURIFIED GELATIN.

1. Cooper''s gelatin purified according to Northrop and Kunitz exhibited a minimum of osmotic pressure and a maximum of opacity at pH 5.05 ±0.05. The pH of solutions of this gelatin in water was also close to this value. It is inferred that such gelatin is isoelectric at this pH and not at pH 4.70. 2. Hydrogen electrode measurements with KCl-agar junctions were made with concentrated solutions of this gelatin in HCl up to 0.1 M. The combination curve calculated from these data is quite exactly horizontal between pH 2 and 1, indicating that 1 gm. of this gelatin can combine with a maximum of 9.35 x 10^–4 equivalents of H⁺. 3. Conductivity titrations of this gelatin with HCl gave an endpoint at 9.41 (±0.05) x 10^–4 equivalents of HCl per gram gelatin. 4. E.M.F. measurements of the cell without liquid junction, Ag, AgCl, HCl + gelatin, H₂, lead to the conclusion that this gelatin in 0.1 M HCl combines with a maximum of 9.4 x 10^–4 equivalents of H⁺ and 1.7 x 10^–4 equivalents of Cl^- per gram gelatin.     

COMBINATION OF SALTS AND PROTEINS : III. THE COMBINATION OF CuCl2, MgCl2, CaCl2, AlCl3, LaCl3, KCl,AgNO3AND Na2SO4WITH GELATIN.

1. The combination of Cu⁺⁺, Ca⁺⁺, Mg⁺⁺, Al⁺⁺⁺, La⁺⁺⁺, K⁺, Ag⁺, and Cl^- with gelatin has been determined. 2. The equivalent combining value for copper is about 0.9 millimols per gm. of gelatin and is therefore the same as that of hydrogen. The value for copper with deaminized gelatin is about 0.4 to 0.5, again the same as that of hydrogen. The sum of the hydrogen and copper ions combined in the presence of an excess of either is 0.9 millimols showing that there is an equilibrium between the copper hydrogen and gelatin and that the copper and hydrogen are attached to the same group. 3. The equivalent combining value of La⁺⁺⁺ and Al⁺⁺⁺ is about 0.5 millimols per gm. of gelatin. This value is not significantly different with deaminized gelatin so that it is possible these salts combine only with groups not affected by deaminization. 4. No calcium is combined on the acid side of pH 3. The value rises rapidly from pH 3 to 4.7 and then remains constant. 5. No combination of K, Li, Na, NO₃ or SO₄ could be detected. 6. Cl combines less than the di- and trivalent metals so that the protein is positive in CaCl₂ but negative in KCl.     

A Gelatin Fixative for Paraffin Sections

Mayer's albumen fixative, of which the active principle is white of egg, is used almost universally for affixing paraffin ribbons to the slide. About eight years ago the writer's attention was called to a gelatin fixative which has proved to be so superior to albumen that he has used it almost exclusively ever since in the making of a great variety of botanical preparations, and has recommended it to a number of other workers whose experience with it subsequently has been just as satisfactory. The gelatin method was first described by Szombathy¹ and later discussed by Artschwager,² but it does not seem to have received the attention in the literature which its importance deserves. It certainly merits a wide spread use among both botanists and zoologists.     

Injectable and moldable hydrogels for use in sensitive and wide range strain sensing applications

Recently, the use of hybrid double network (DN) hydrogels has become prominent due to their enhanced mechanical properties, which has opened the door for new applications of these soft materials. Only a few of these gels have demonstrated both injectable and moldable capabilities. In this work, we report the mechanical properties, gauge factor (GF) values and demonstrate both the injectability and moldability of a gelatin/polyacrylamide DN hydrogel. We optimized several parameters, such as, gelatin to polyacrylamide ratio, reactant concentrations and metal ion concentration, to produce a gelatin/polyacrylamide hydrogel with superior mechanical properties. The highest water content gel was capable of withstanding strains of 5000% before failure. These gels were facilely injected into molds where they effectively changed shape and maintained similar properties prior to remolding. When 20 mM calcium was doped into a similar gel, a tensile strength of 1.71 MPa was achieved. Aside from improving the mechanical properties of the gels, both Ca²⁺ and Mg²⁺ also improved their conductivity, so they were tested for use as strain sensors. The sensitivity of the hydrogel strain sensors were measured using the GF. For the 20 mM Ca²⁺ hydrogel, these GF values ranged from 1.63 to 6.85 for strains of 100% to 2100% respectively. Additionally, the sensors showed good stability over continuous cyclic stretching, demonstrating their long term reliability for strain sensing.     

Immobilization of enzymes on crosslinked gelatin particles activated with various forms and complexes of titanium(IV) species

A number of methods of activating the surface of glutaraldehyde crosslinked gelatin beads with titanium(IV) compounds, for subsequent enzyme coupling, have been investigated. Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) was so immobilized using titanium(IV)-urea, -acrylamide, -citric acid and -lactose complexes; however, immobilized enzyme preparations with low activities were obtained (0.36–1.28 U g^?1). Activation with uncomplexed titanium(IV) chloride, however, of both moist and freeze-dried crosslinked gelatin particles resulted in highly active immobilized glucoamylase preparations (1.74–26.6 U g^?1). Dual immobilized enzyme conjugates of glucoamylase and invertase (β-d-fructofuranosidase, EC 3.2.1.26) were also prepared using this method. Invertase was served on the entrapped enzyme while glucoamylase was coupled on the surface of titanium(IV)-activated gelatin pre-entrapped invertase particles. A dual gelatin coupled glucoamylase/gelatin entrapped glucoamylase was prepared (3.8 U g^?1) and ～72.5% of the total combined activity was due to the surface bound enzyme.     

A LB film morphological study with reference to biopolymer–surfactant interaction taking gelatin–CTAB system as a model

The interaction of gelatin with cetyltrimethylammonium bromide (CTAB) studied at pH 9 and an ionic strength of 0.005, produces an interfacial surface active gelatin–CTAB (monomer) complex (GS_n^I), a surface inactive gelatin–CTAB (micelle) complex in bulk (GS_m^B), followed by coacervation, and its solubilization in micellar solution of CTAB. We have herein attempted to probe the interfacial morphological changes of gelatin and its CTAB complexes, and not the bulk properties like coacervation and/or micellar solubilization. The morphologies of pure gelatin and CTAB films and that of gelatin–CTAB interacted complex at the interface have been investigated using LB, SEM, AFM and ellipsometric techniques. The stability of the gelatin monolayer at varied concentrations with and without CTAB has been examined. The SEM images of stabilized films of gelatin and gelatin–CTAB complex have witnessed compact smooth as well as rough surfaces with formation of distinct domains. Drastic morphological change in the film before the critical aggregational concentration of CTAB (T₂) has been in line with an initial abrupt decrease in surface tension. This has been corroborated by AFM measurements, which along with morphology demonstration has provided information on the diameter of the ensembles formed and roughness of the LB films constituted of pure components and their complexes. Thickness of the film was at its maximum in the domain region, as corroborated by ellipsometric technique. Such an elaborate interfacial monolayer and film morphology study of biopolymer-amphiphile system has been rarely documented in literature.     

Complexing of fibronectin glycosaminoglycans and collagen

Collagen-fibronectin complexes, formed by binding of fibronectin to gelatin or collagen insolubilized on Sepharose, were found to bind 20–40% of radioactivity in [³⁵S]heparin. Fibronectin attached directly to Sepharose also bound [³⁵S]heparin, while gelatin-Sepharose without fibronectin did not. Unlabeled heparin and highly sulfated heparan sulfate efficiently inhibited the binding of [³⁵S]heparin, hyaluronic acid and dermatan sulfate were slightly inhibitory, while chondroitin sulfates and heparan sulfate with a low sulfate content did not inhibit.The interaction of heparin with fibronectin bound to gelatin resulted in complexes which required higher concentrations of urea to dissociate than complexes of fibronectin and gelatin alone. Heparin as well as highly sulfated heparan sulfate and hyaluronic acid brought about agglutination of plastic beads coated with gelatin when fibronectin was present. Neither fibronectin nor glycosaminoglycans alone agglutinated the beads.It is proposed that the multiple interactions of fibronectin, collagen and glycosaminoglycans revealed in these assays could play a role in the deposition of these substances as an insoluble extracellular matrix. Alterations of the quality or quantity of any one of these components could have important effects on cell surface interactions, including the lack of cell surface fibronectin in malignant cells.     

Factors Affecting the Toxic Effect of Tin on Estuarine Microorganisms

Inorganic tin (SnCl₄·H₂O) is toxic to microbial populations obtained from estuarine sediments plated on nutrient medium solidified with either agar or purified agar. The use of gelatin as a gelling agent decreased the apparent toxicity of tin, and toxicity was markedly reduced in medium solidified with silica gel. There was no evidence that toxic agar-tin complexes were involved. Cd, Cu, Pb, Ni, and Zn exhibited similar toxicity patterns; therefore, toxicity levels determined in the laboratory should be extrapolated to the environment with caution. The addition of cysteine to the medium had no effect on tin toxicity. Serine or 3-hydroxyflavone enhanced toxicity, while humic acids or gelatin inhibited toxicity. Replacement of SO₄²⁻ with NO₃⁻ did not alter tin toxicity, but replacement of Cl⁻ with NO₃⁻ decreased tin toxicity. Thus, the toxic effect(s) of tin depend as much on the chemical speciation of the metal as on the total concentration of the metal in the medium.     

Let It Catch: A Short‐Branched Protein for Efficiently Capturing Polysulfides in Lithium–Sulfur Batteries

Uncovering the key contributions of molecular details to capture polysulfides is important for applying suitable materials that can effectively restrain the shuttle effect in advanced lithium–sulfur batteries. This is particularly true for natural biomolecules with substantial structural and compositional diversities strongly impacting their functions. Here, natural gelatin and zein proteins are first denatured and then adopted for fabrication of nanocomposite interlayers via functionalization of carbon nanofibers. From the results of experiment and molecular dynamic simulations, it is found that the lengths of the sidechains on the two proteins play critical roles. The short‐branched gelatin shows significantly stronger adsorption of polysulfides, as compared with zein comprising many long‐chain residues. The gelatin‐based interlayer, along with its good porous structures/electrical conductivity, greatly suppresses the shuttle effect and yields exceptional electrochemical performance. Furthermore, the implementation of proteins as functional binder additives further supports the finding that gelatin enables stronger polysulfide‐trapping. As a result, high‐loading sulfur cathodes (9.4 mg cm^?2) are realized, which deliver a high average areal capacity of 8.2 mAh cm^?2 over 100 cycles at 0.1 A g^?1. This work demonstrates the importance of sidechain length in capturing polysulfides and provides a new insight in selecting and design of desired polysulfide‐binding molecules.     

Microcarrier culture of recombinant Chinese hamster ovary cells for production of human immune interferon and human tissue-type plasminogen activator

Summary Recombinant Chinese hamster ovary cells were successfully cultured semi-continuously on microcarriers of gelatin or modified dextran under non-selective conditions for up to three weeks. High and constant production rates for human immune interferon and tissue-type plasminogen activator were obtained. For cells that produced interferon, the highest cell concentration and interferon production was obtained with gelatin microcarriers though the specific production when grown in the presence of 0.2% fetal calf serum was slightly higher for cells cultured on dextran microcarriers (0.12 U/cell day versus 0.11 U/cell day). For cells that produced plasminogen activator, a slightly higher cell concentration was obtained for cells grown on dextran microcarriers (9x10⁵ cells/ml versus 7x10⁵ cells/ml). However, the specific and total production rates were significantly higher for cells cultured on gelatin microcarriers (6.7 pg/cell day versus 2.1 pg/cell day). The maximum cell concentration and specific production rate could be increased to 2.3x10⁶ cells/ml and 3.4 pg/cell day for dextran microcarriers by adding 6-aminohexanoic acid to the medium. For gelatin microcarriers, the addition of 6-aminohexanoic acid increased the specific production rate to 14.4 pg/cell day. Cell growth, however, was inhibited.     

ELECTRIFICATION OF WATER AND OSMOTIC PRESSURE

1. Amphoteric electrolytes form salts with both acids and alkalies. It is shown for two amphoteric electrolytes, Al(OH)₃ and gelatin, that in the presence of an acid salt water diffuses through a collodion membrane into a solution of these substances as if its particles were negatively charged, while water diffuses into solutions of these electrolytes, when they exist as monovalent or bivalent metal salts, as if the particles of water were positively charged. The turning point for the sign of the electrification of water seems to be near or to coincide with the isoelectric point of these two ampholytes which is a hydrogen ion concentration of about 2 x 10^–5 N for gelatin and about 10^–7 for Al(OH)₃. 2. In conformity with the rules given in a preceding paper the apparently positively charged water diffuses with less rapidity through a collodion membrane into a solution of Ca and Ba gelatinate than into a solution of Li, Na, K, or NH₄ gelatinate of the same concentration of gelatin and of hydrogen ions. Apparently negatively charged water diffuses also with less rapidity through a collodion membrane into a solution of gelatin sulfate than into a solution of gelatin chloride or nitrate of the same concentration of gelatin and of hydrogen ions. 3. If we define osmotic pressure as that additional pressure upon the solution required to cause as many molecules of water to diffuse from solution to the pure water as diffuse simultaneously in the opposite direction through the membrane, it follows that the osmotic pressure cannot depend only on the concentration of the solute but must depend also on the electrostatic effects of the ions present and that the influence of ions on the osmotic pressure must be the same as that on the initial velocity of diffusion. This assumption was put to a test in experiments with gelatin salts for which a collodion membrane is strictly semipermeable and the tests confirmed the expectation.     

Recombinant collagen for animal product-free dextran microcarriers

Manufacturers of vaccines and other biologicals are under increasing pressure from regulatory agencies to develop production methods that are completely animal-component-free. In order to comply with this demand, alternative cell culture substrates to those now on the market, primarily collagen or gelatin, must be found. In this paper, we have tested a number of possible substitutes including recombinant collagen, a 100-kDa recombinant gelatin fragment and a peptide derived from a cell-binding region of type I collagen. The small 15-amino acid peptide did not support attachment of human fibroblasts in monolayer culture. The 100-kDa gelatin fragment supported cell attachment in monolayer culture, but was significantly less active than intact porcine gelatin. Recombinant type I collagen was as successful in promoting cell attachment as native collagen, and both were more effective than porcine gelatin. Based on these data, dextran microspheres were treated with the same attachment proteins—porcine gelatin, native collagen, or recombinant collagen. The same trends were observed as in monolayer culture. Concentrations of the recombinant collagen (as well as native collagen) supported cell attachment on dextran microspheres at concentrations as low as 0.01 μg/cm². Treatment of the dextran with a low level of polyethylenimine, a cationic moiety, further enhanced attachment when used in conjunction with the low concentration of recombinant collagen. Where there was increased cell attachment, increased proliferation followed. We are confident, based on these findings, that a fully recombinant substitute could replace gelatin in current microcarrier preparations without losing the cell growth benefits provided by the native protein.     

Dynamic mechanical and rheo-optical studies of collagen and gelatin

The frequency dependence of dynamic mechanical properties of rat tail tendon (RTT), enzyme-solubilized collagen membranes (ESC), AKM-23 dialysis membranes, and gelatin film have been measured at 110, 11, and 3.5 Hz from - 160 to 220°C. RTT and AKM-23 are devoid of a rubbery region; there are as many as six mechanical loss transitions. Gelatin and ESC membranes behave as rubbery materials above room temperature; only three tan δE peaks can be resolved for these materials. Strain birefringence was used to measure the crystalline and amorphous contribution of orientation induced by strain. Both the birefringence and the strain optical coefficient are sensitive to the amount of water in a sample. The effect of chemical swelling agents and of annealing on birefringence are described. Stress relaxation data on gelatin film were analyzed with the rubber elasticity theory to give the average number of chains per unit volume, the specific polarizability, the stress-birefringence ratio and the average molecular weight between hydrogen bonds were calculated. The intrinsic amorphous birefringence for “wet” gelatin film is 1.25 × 10^?2; it is estimated to be about 6 × 10^?2 for “dry” gelatin film.     

Collagenase-Released Non-Collagenous Proteins of Cortical Bone Matrix

Two distinct groups of non-collagenous components were isolated from rat cortical bone gelatin which had previously been digested with purified bacterial col-lagenase. One component was disulfide-bonded, strongly acidic, trypsin-labile glycoprotein aggregate with a molecular mass of more than 100, 000 daltons. When reduced with β-mercaptoethanol this protein disaggregated into subunits with a molecular mass of about 60, 000 daltons. The other components consisted of a group of polypeptides with a molecular mass of about 5, 000 daltons. The latter group was present in collagenase digests prepared from normal bone gelatin but was hardly detectable or absent in digests of gelatin prepared from either autolyzed, trypsinized or lathyritic bone, or from the residue of neutral salt extracted rat tail tendon.

A recently discovered group of non-collagenous proteins is tightly bound to the highly crosslinked insoluble structure of collagen fibrils of bone and dentin. Dische et al ¹ predicted the existence of these proteins in bone by analyzing the products of KOH hydrolysis of the EDTA-insoluble residues of cortical bone collagen. Comparable products were separated following digestion of bone matrix with collagenase by Herring², and oxidation of dentin matrix by alkaline sodium metaperiodate by Shuttleworth and Veis.³ Leaver et al ⁴ proposed the name collagenase-released proteins (CRP) for non-collagenous proteins obtained from the EDTA-insoluble residue of bone and dentin. There are at least two limitations in information about CRP in the above-cited reports. The first is that the collagenases were not certified to be protease-free. The second is that EDTA-insoluble residues of cortical bone were only partly digestable by bacterial collagenase. We report here isolation and partial characterization of CRP from a special preparation of bone matrix gelatin which is quantitatively digested by a protease-free bacterial collagenase-gelatinase purified by the method of Peterkofsky and Diegelmann.^{5, 6}     

A Layered‐Nanospace‐Confinement Strategy for the Synthesis of Two‐Dimensional Porous Carbon Nanosheets for High‐Rate Performance Supercapacitors

A general approach is developed for the synthesis of 2D porous carbon nanosheets (PCNS) from bio‐sources derived carbon precursors (gelatin) by an integrated procedure of intercalation, pyrolysis, and activation. Montmorillonite with layered nanospace is used as a nanotemplate or nanoreactor to confine and modulate the transformation of gelatin, further leading to the formation of 2D nanosheet‐shaped carbon materials. The as‐made 2D PCNS exhibits a significantly improved rate performance, with a high specific capacitance of 246 F g^?1 and capacitance retention of 82% at 100 A g^?1, being nearly twice that of microsized activated carbon particulates directly from gelatin (131 F g^?1, 44%). The shortened ion transport distance in the nanoscaled dimension and modulated porous structure is responsible for such an enhanced superior rate capability. More importantly, the present strategy can be extended to other bio‐sources to create 2D PCNS as electrode materials with high‐rate performance. This will also provide a potential strategy for configuring 2D nanostructured carbon electrode materials with a short ion transport distance for supercapacitors and other carbon‐related energy storage and conversion devices.     

Preparation and characterization of polyvinyl alcohol-gelatin hydrogel membranes for biomedical applications

The purpose of this research was to design and develop hydrogels by esterification of polyvinyl alcohol (PVA) with gelatin. The membranes were characterized by Fourier Transform Infrared (FTIR) spectroscopy, x-ray diffraction (XRD), and differential scanning calorimetry. The viscosity of the esterified product (as solution) was compared with the mixture of PVA and gelatin of the same composition. The mechanical properties of the hydrogels were characterized by tensile tests. Swelling behavior and hemocompatibility of the membrane were also evaluated. The diffusion coefficient of salicylic acid (SA), when the receptor compartment contained Ringer's solution, through the membrane was determined. SA was used as a model drug. FTIR spectra of the membranes indicated complete esterification of the free carboxylic groups of gelatin. XRD studies indicated that the crystallinity of the membranes was mainly due to gelatin. The comparison of viscosity indicated an increase in segment density within the molecular coil. The membrane had sufficient strength and water-holding capacity. Hemocompatibility suggested that the hydrogel could be tried as wound dressing and as an implantable drug delivery system. The diffusion coefficient of SA through the membrane was found to be 1.32×10⁻⁵ cm²/s. The experimental results indicated that the hydrogel could be tried for various biomedical applications. Published: March 16, 2007 Formerly College of Pharmacy, University of Delhi, Pushp Vihar, New Delhi-110017 India     

Selection of a biopolymer based on attachment,morphology and proliferation of fibroblast NIH/3T3 cells for the development of a biodegradable tissue regeneration template: Alginate,bacterial cellulose and gelatin

Material selection is one of the most important aspects to construct a novel template for tissue regeneration. In this research alginate, bacterial cellulose (BC) and gelatin were selected based on available material and published data to prepare membranes and then the morphology of Swiss mouse embryo fibroblast NIH/3T3 cells on the surface of these membranes was examined to select the best material for the development of a biodegradable skin tissue regeneration template. The cells on alginate membrane crosslinked with Ca²⁺ (AGM_Ca) showed a spherical morphology and growth retardation, probably due to high calcium content on and in the surface of membrane. This has been confirmed in control experiments in which calcium was added to the culture medium (DMEM medium). The NIH/3T3 cells grown on the BC membrane (BC_M) and membrane of glutaraldehyde (GTA) crosslinked gelatin (GTA_GM) had a polygonal morphology. The proliferation rate of cells on the GTA_GM was faster than that on the BC_M. Therefore the GTA_GM is better than the AGM_Ca and BC_M to apply as a skin tissue regeneration template. Cytotoxic effects of GTA were studied in GTA_GMs prepared using gelatins obtained from cow bones, pork skins, and fish skins. It was found that molar ratio of GTA to gelatin for preparation of membrane should not be higher than 0.033. Cytotoxic effects of GTA were observed on the GTA_GMs prepared with molar ratio higher than 0.033, except pork skin gelatin membrane with a molar ratio, 0.033, which showed the cytotoxic effects on fibroblast cells. The physical morphology of the membranes of cow bone gelatin and fish skin gelatin is stronger and more flexible than that of pork skin gelatin in wet forms. According to these results, it can be suggested that pork skin gelatin might have had less crosslinked points, NH₂-sites of lysine molecules than cow bone gelatin and fish skin gelatin. These two gelatins are selected for further development of a template for skin tissue regeneration.     

Potential application of immobilized streptokinase extracted from Streptococcus equinus VIT_VB2

Streptokinase purified from Streptococcus equinus VIT_VB2 isolated from bovine milk sample was immobilized in various solid supports namely entrapment in agarose gel, calcium alginate beads and gelatin gel by cross-linking with formaldehyde. Immobilization of streptokinase in calcium alginate beads showed maximum efficiency (81.8?±?1.06%) when compared with entrapment with agarose gel (55.6?±?2.17%) and cross-linked gelatin formaldehyde gel (71.0?±?1.54%). The purified SK activity was expressed maximum in calcium alginate (1%) and gelatin gel (0.25%) with 1292.68?±?1.33 and 1121.9?±?1.2?U?mL^?1, respectively. Similarly, SK entrapped in gelatin gel and calcium alginate showed maximum in vitro blood clot lysis activity with 77.67?±?2.64% and 76.16?±?2.72%, respectively. The immobilized SK in gelatin gel showed complete clot lysis within 15?min; hence, this application of the study could be used in the treatment of superficial thrombophlebitis, phlebitis, and venous thrombosis. These beads were used for three repeated cycles to check the conversion of substrates into their products, and we concluded that SK can be immobilized in the suitable matrices. Therefore, this helps in the drug-delivery strategies in highly efficient way, moreover, economically competent process in the pharmaceutics.     

Eco‐friendly synthesis of gelatin‐capped bimetallic Au–Ag nanoparticles for chemiluminescence detection of anticancer raloxifene hydrochloride

This study described the utility of green analytical chemistry in the synthesis of gelatin‐capped silver, gold and bimetallic gold–silver nanoparticles (NPs). The preparation of nanoparticles was based on the reaction of silver nitrate or chlorauric acid with a 1.0 wt% aqueous gelatin solution at 50°C. The gelatin‐capped silver, gold and bimetallic NPs were characterized using transmission electron microscopy, UV–vis, X‐ray diffraction and Fourier transform infrared spectroscopy, and were used to enhance a sensitive sequential injection chemiluminescence luminol–potassium ferricyanide system for determination of the anticancer drug raloxifene hydrochloride. The developed method is eco‐friendly and sensitive for chemiluminescence detection of the selected drug in its bulk powder, pharmaceutical injections and biosamples. After optimizing the conditions, a linear relationship in the range of 1.0 × 10^–9 to 1.0 × 10^–1 mol/L was obtained with a limit of detection of 5.0 × 10^–10 mol/L and a limit of quantification of 1.0 × 10^‐9 mol/L. Statistical treatment and method validation were performed based on ICH guidelines. Copyright © 2016 John Wiley & Sons, Ltd.