Direct UV Spectrophotometry Research Articles

A simple and rapid method for measuring total free sulfides in marine sediments

AbstractThe quantitatively most important process by which organic matter in marine sediments is mineralized is performed by sulfate‐reducing bacteria, resulting in the accumulation of total dissolved (free) sulfide (S2− = H2S + HS− + S2−) in porewater. S2− is toxic to benthic animals and vascular plants and measurements serve as a proxy for the deleterious effects of organic enrichment on benthic habitat, biodiversity, and ecosystem function. Methodologies for measuring S2− in water have been pursued for at least a century, and standard approaches employ colorimetry (methylene blue and iodometric titration) and potentiometry. These standard methods require between 1 and 200 mL of porewater, which can be laborious to obtain. The ion‐selective electrode method is widely employed as a practical approach for sediment S2− analysis but lacks analytical robustness and is highly prone to measurement biases that misinform research and environmental management decisions. A technically simple method is described, based on direct UV spectrophotometry, for the near real‐time field analysis of small porewater samples. The procedure prevents known measurement biases associated with particulate sulfide interference, S2− volatilization and oxidation, and represents a practical approach for monitoring organic enrichment and classifying benthic ecological quality status. Porewater concentrations between 200 and 15,000 μmol L−1 can be measured and instrument calibration is highly stable. The method has the capacity to rapidly process and analyze sediment samples at low cost, which helps resolve the problem of chronic under‐sampling associated with the use of traditional S2− methods.

Current aspects of Quality Control and Standardization of <I>Eleutherococcus senticosus</I> Medicines

Introduction. Nowadays, herbal medicines are becoming more and more popular. In this regard, herbal medicinal preparations based on rhizomes and roots of Eleutherococcus senticosus (Rupr. & Maxim.) Maxim., syn. Acanthopanax senticosus (Rupr. & Maxim.) Harms, are of particular interest. Its have an adaptogenic, immunomodulatory effects mainly due to the presence of phenyl propane compounds: eleutherosides B, D, E, E1. The current pharmacopeial monographs FS.2.5.0053.15 "Eleutherococcus senticosus rhizomes and roots" and FS.3.4.0009.18 "Eleutherococcus senticosus rhizomes and roots liquid extract" in The State Pharmacopoeia of the Russian Federation XIV Edition, in our opinion, does not allow to fully objectively and uniformly assess the quality of raw materials and preparations.Aim. The aim is substantiation of new approaches to quality control of raw materials and preparations of Eleutherococcus senticosus, consisting in changing the conditions of sample preparation and analysis of active biological compounds in comparison with pharmacopoeial methods.Materials and methods. Samples of the medicinal preparation "Eleutherococcus extract liquid" of a domestic manufacturer (5 lots) and experimental preparation "Eleutherococcus syrup". When performing high performance liquid chromatography (HPLC) analysis, a reference sample of syringin was used that met the requirements of pharmacopeial monograph VFS 42-2088-92. Methods of UV-spectroscopy and HPLC were used to determine the quantity of biologically active substances.Results and discussion. As a result of the research, other methodological and methodological approaches have been developed to determine the content of the total of phenylpropanoids in terms of eleutheroside B (syringin) by direct UV-spectrophotometry using a standard sample of syringin or the value of its specific absorbance. New conditions for HPLC analysis of eleutheroside B (syringin) in preparations of Eleutherococcus senticosus have been selected.Conclusion. As a result of the phytochemical study, a spectrophotometric method was developed for the determination of the total of phenylpropanoids in terms of syringin and an HPLC method for the determination of eleutheroside B (syringin) using an external standard after preliminary purification of the total extracts by filtration through a layer of aluminum oxide.

The Main Aspects of End-to-End Standardisation of Herbal Drugs, Herbal Drug Preparations, and Herbal Medicinal Products of Eucalyptus viminalis

The principle of consistent, end-to-end standardisation builds upon the fact that qualitative and quantitative analysis of bioactive compounds in herbal drugs, herbal drug preparations, and corresponding herbal medicinal products involves the same methods of analysis. The possibility of such standardisation should be considered at the stage of specification development. The aim of the study was to explore approaches to analysing of Eucalyptus viminalis leaves and chlorophyllipt medicinal products, observing the principle of end-to-end standardisation. Materials and methods : the authors analysed Eucalyptus viminalis as a herbal drug (leaves), a herbal drug preparation (chlorophyllipt soft extract), and herbal medicinal products (Chlorophyllipt®, 1% alcohol solution for oral, cutaneous, rectal, and vaginal use; 2% oil solution for cutaneous and vaginal use; and 25 mg orodispersible tablets) by direct UV spectrophotometry, thin-layer chromatography (TLC), and qualitative reactions. Results : the study demonstrated the possibility of using a herbal reference standard of eucalimin to analyse the Eucalyptus viminalis herbal drug and chlorophyllipt preparations and medicinal products in different dosage forms by TLC. Conclusions : procedures for determination of total phenolic aldehydes, if included into specifications and production technology of chlorophyllipt preparations and products, ensure compliance with the principle of end-to-end standardisation and allow for quantitative determination and control of active ingredients throughout the product lifecycle: from the acceptance of raw materials, through in-process controls, to testing and monitoring of finished products’ quality.

Aquaculture organic enrichment of marine sediments: assimilative capacity, geochemical indicators, variability, and impact classification

Benthic organic enrichment at 2 high-flow Atlantic salmon Salmo salar farms and at a low-flow mussel Mytilus edulis farm was studied to assess the capacity of local physical and biological processes to assimilate organic waste inputs. Geochemical metrics served as proxies for detecting potential biological effects. High-flow sites are generally predicted to assimilate organic enrichment by flow- and wave-induced waste dispersion and metabolic processes. However, a decrease in porewater dissolved oxygen was detected out to 100 m at the salmon farm with cohesive sediments and to approximately 1000 m outside the farm with permeable sediment. Sediment oxygen consumption was responsive to the vertical flux of organic matter, resulting in hypoxic conditions. An increase in total free sulfides (H2S + HS- + S2-) in porewater was restricted to the immediate vicinity of both salmon farms. Despite exhibiting a high degree of small-scale patchiness, benthic effects were greatest at the fish farms during the pre-harvest period, regardless of season. Natural organic enrichment at the mussel farm constrained the assimilative capacity for biodeposition, resulting in substantial free sulfide accumulation. Sediment free sulfide analysis at a wide array of fish and shellfish farms showed that the ion-selective electrode method that is widely prescribed for regulatory aquaculture monitoring gave biased readings relative to methylene blue colorimetry and direct UV spectrophotometry. The ecological quality status classification system was extended to include quantitative relationships between a wide range of geochemical and biological variables employed worldwide to monitor and regulate the effects of benthic organic enrichment.

Development and Validation of Dimeric Macrocyclic Tannin Assay Method in Dosage Forms

Introduction. Quantitative assessment of the active substance is necessary and perhaps the most significant part of the drug quality control. Validation of the analytical methods of quantitative assessment ensures their compliance with high requirements. The present study describes the development and validation of a spectrophotometry method for the quantitative evaluation of the active substance in the drug form of the national antitumor and antiangiogenic drug «Dimeric macrocyclic tannin (DMT) lyophilizate for solution for injection, 100 mg».Aim. The development and validation of the assay method for the standardization of «DMT lyophilizate for solution for injection, 100 mg».Materials and metods. The study used «DMT lyophilizate for solution for injection, 100 mg» and the active substance DMT. Method – spectrophotometry.Results and discussion. The methodof the quantitative assessment of the active substance in the DMT lyophilized drug by direct UV spectrophotometry was developed and the validation characteristics of the method were defined as a result of the study.Conclusion. The validation results showed that the assay method of DMT in the drug form has the appropriate accuracy, precision and linearity. The obtained results correspond to the approved criteria that allow the use of the developed methodology for evaluating the quality of the drug.

Chemical characterization of liposomes containing nutraceutical compounds: Tyrosol, hydroxytyrosol and oleuropein

Tyrosol, hydroxytyrosol and oleuropein are among the major phenolic compounds in fruits, leaves and oils from Olea europaea L. These natural antioxidants molecules revealed several beneficial effects on human health, but a low bioavailability and accessibility to targeted site. Liposomes are drug/nutraceutical delivery carriers, used for driving bioactive molecules to desired target tissues, decreasing potential side effects and protecting the encapsulated molecule from enzymatic metabolic processes.In this study, zwitterionic liposomes containing tyrosol, hydroxytyrosol and oleuropein were synthesized and characterized for their size and surface charge. Particular attention was devoted to the determination of encapsulation efficiency (EE%), quantifying the loaded Tyr, HTyr and Ole amount, by using three different techniques: direct UV spectrophotometry, High Performance Liquid Chromatography and Trolox Equivalent Antioxidant Capacity assay. The results revealed higher EE% for oleuropein. Cyto-toxicity and cyto-compatibility of liposomes were also tested on human chondrocyte cells.

Development and Validation of Spectrophotometric Methods for the Determination of Amoxicillin trihydrate and Dicloxacillin sodium in Their Binary Mixture

Three sensitive and cost-effective spectrophotometric methods are introduced for the determination of amoxicillin and dicloxacillin in their binary mixtures with minimum sample pretreatment, with or without the presence of their common impurity; 6-aminopenicillanic acid (6-APA). In this work, dicloxacillin is determined by dual wavelength, derivative ratio and isoabsorptive spectrophotometric methods. However, amoxicillin is determined by direct UV spectrophotometry at 273.6 nm and by the first derivative ratio spectrophotometric method (DD method). For method (A); the dual wavelength method, the wavelengths selected for the determination of dicloxacillin were 223.6 and 237 nm. Method (B); the isoabsorptive spectrophotometric method, comprises measuring the total content of the mixture at their isoabsorptive point (230.4 nm), and then the content of dicloxacillin can be calculated by subtraction. Method (C) is the first derivative of ratio spectra for the determination of amoxicillin (using 8 μg/mL of dicloxacillin as a divisor at 231 nm) and dicloxacillin (using 20 μg/mL of 6-APA as a divisor at 234.6 nm) in presence of their common impurity; 6-APA. Statistical comparison of the results obtained from the suggested methods with those of the reported HPLC method showed no significant difference with respect to accuracy and precision. The developed methods can be used for quality control analysis of the studied drugs in laboratories lacking HPLC qualifications.Kay words: Amoxicillin, Dicloxacillin, Dual wavelength, Isoabsorptive point, First derivative ratio

An Innovative Method, Interference Subtraction, for Determination of Drugs in Syrups in Presence of Potential Interfering Excipients

Pharmaceutical dosage forms contain both active ingredients and inactive materials called exipients, most of which cause high interference specially in direct UV-spectrophotometry. This problem is highly obvious in syrups which may contain preservatives, coloring agents, thickening agents and sweetening agents. Thus, there is an urgent need to develop and validate a simple, accurate and selective method named interference subtraction method for determination of drugs in syrups in presence of these excipients without pre-separation by solving this interference. This newly designed method was successfully applied to; Oxybutynin hydrochloride in Detronin® syrup, Ambroxol hydrochloride in Muco® syrup and Ibuprofen in both Brufen® syrup and Megafen N® oral suspension. These chosen syrups contain different excipients causing different types of interference. This innovative method was validated according to ICH guidelines. The results obtained by interference subtraction method were statistically compared with those obtained by the official and /or reported methods of the proposed drugs.

Methods for Analytical Control of Darunavir Tablet Dosage Forms. Part II

Previously, targeted development of special procedures regulating analytical control methods for darunavir (DRV) dosage forms showed that the use of expensive imported reference standards (RSs) could be avoided. Part I discussed such procedures and their validation results. They were intended for sections Identity; Talc, Silica, and Titania; and Dissolution of the corresponding monographs. Part II continued the examination of procedures not requiring the use of RSs that were proposed for inclusion into sections of the monographs Side Impurities and Quantitative Determination. HPLC in gradient mode was proposed for estimating the contents of side impurities. The lower limit of quantitation of a separate impurity relative to DRV was 0.02%. The relative standard deviation (RSD) of the total impurity content was less than 10%. The DRV concentration in the section Quantitative Determination was proposed to be determined by direct UV spectrophotometry of the analyte in solution (pH ~ 9) using a procedure that required preliminary experimental determination of the specific absorption coefficient ( $$ {A}_{1\mathrm{cm}}^{1\%} $$ ) of the wavelength maximum at 267 nm for DRV in aqueous MeOH solutions (pH ~ 9). The experimental $$ {A}_{1\mathrm{cm}}^{1\%} $$ ) = 393.4 cm–1 (RSD < 1% at confidence level a = 0.05). Therefore, $$ {A}_{1\mathrm{cm}}^{1\%} $$ ) was a physicochemical constant. The RSD of results could be reduced to 1% and the analysis time shortened by 2 – 3 times because the preparation of RS solutions and measurements of their optical densities were obviated if $$ {A}_{1\mathrm{cm}}^{1\%} $$ ) was used for quantitative determinations of DRV by direct UV spectrophotometry.

Background Correction Method for Determination of Ascorbic Acid in Baobab Fruit Pulp Using Direct UV Spectrophotometry

Background Correction Method for Determination of Ascorbic Acid in Baobab Fruit Pulp Using Direct UV Spectrophotometry

Selection and verification of the method for phynelefrine hydrochloride assay in Simanovsky ointment

Phenylephrine hydrochloride is a sympathomimetic with expressed local vasoconstrictor effect. Its ability to reduce swelling and hyperemia of the nasal mucosa is used in the Simanovsky ointment which is preparing for stock in the pharmacies of Ukraine. For the assay of phenylephrine hydrochloride in many single and multicomponent dosage forms, it is suggested to use a spectrophotometric method. However, in literary sources there are no examples of the use of spectrophotometric method for quantitative determination of phenylephrine hydrochloride in the studied ointment. Aim. The purpose of the research was to select and verify the optimal method for phenylephrine hydrochloride assay in the Simanovsky ointment with the possibility of its further use for the stability analysis of the dosage form during storage. Methods. Direct UV-spectrophotometry method for assay of phenylephrine hydrochloride in the studied ointment. Results. For the quantitative determination of phenylephrine hydrochloride in the Simanovsky ointment, the method of direct spectrophotometry was selected after its extraction from the ointment base with 0.1 M hydrochloric acid. In order to prove the possibility of its use in the ointment analysis, the determination of the validation characteristics was made. The obtained results indicate that the requirements for the specificity of the method (δ noise , %=0.47≤1.02), the parameters of linear dependence, the accuracy (δ,%=0.20≤1.02) and the precision (ΔZ=0.39≤3.20) are met. The study of the robustness of the technique shows the stability of solutions within an hour. The method was tested on the researched ointment. The metrological characteristics of methods for calculating the quantitative content of phenylephrine hydrochloride by the standard method and specific absorption index were determined. The obtained results indicate the possibility of using both methods. Conclusions. For the assay of phenylephrine hydrochloride in the Simanovsky ointment, the method of direct spectrophotometry was chosen. Validation characteristics of the method indicate the possibility of its use for medicine analysis and study of its stability during storage. It was proved that the calculation of the quantitative content of phenylephrine hydrochloride in the ointment can be carried out using both the standard method and the method of specific absorption index

Determination of the content of benzocaine and penicillin-g-procaine in combined finished dosage forms using tropaeolin o azo dye

The paper presents the results of developing the method for quantitative determination of benzocaine and penicillin-G-procaine in seven combined medicines and veterinary medicinal products of different dosage forms – ointments, suppositories and tablets. The content of the medicinal substances was determined spectrophotometrically according to the absorbance intensity of the coloured product of diazotized amine azocoupling with the acid monoazo dye tropaeolin O. The presence of other biologically active substances (streptomycin, neomycin, prednisolone, nitrofuran, chloramphenicol, bismuth subgallate, papaverine, extracts of buckthorn and belladonna, vitamin A, menthol, xeroform, and zinc oxide), which are often found in medicines, does not prevent determination of benzocaine and penicillin-G-procaine by of the method developed. The results obtained using the method developed are consistent with those received by the direct UV-spectrophotometry and nitritometric titration after the preliminarily extraction separation of the components.

Direct UV Spectrophotometry and HPLC Determination of Triton X-100 in Split Virus Influenza Vaccine.

One of the most commonly used surfactants in the production of split virus influenza vaccine is nonionic surfactant Triton X-100. After splitting of the virus is accomplished, Triton X-100 is removed from the vaccine by subsequent production steps. Because of toxicity of Triton X-100, which remains in the vaccine in residual amounts, a sufficiently sensitive method for its detection and quantification needs to be defined. Two methods for determination of Triton X-100 residuals were developed: the UV-spectrophotometry and HPLC methods. For both methods, preparation of vaccine samples and removal of proteins and virus particles were crucial: samples were treated with methanol (1:1) and then centrifuged at 25 000 × g for 30 min. After such treatment, the majority of vaccine components that interfered in the UV region were removed, and diluted samples could be directly measured. The chromatographic system included C18 column, step methanol gradient, and detection at 225 nm with a single peak of Triton X-100 at 12.6 min. Both methods were validated and gave satisfactory results for accuracy, precision, specificity, linearity, and robustness. LOQ was slightly lower for the HPLC method. Hence, it was shown that both methods are suitable for analysis of residual amounts of Triton X-100, with the advantages of the UV method being its simplicity and availability in most laboratories.

Synthesis and Molecular-Weight Characteristics of N-Oxidized Copolymers of N-Vinylpyrrolidone and 2-Methyl-5-Vinylpyridine

Aseries of N-oxides of N-vinylpyrrolidone and 2-methyl-5-vinylpyridine copolymers with various degrees of oxidation were prepared via oxidation of the copolymers by peracetic acid. The N-oxidation kinetic rate constants were calculated using UV spectrophotometry. The temperature dependence of the limiting N-oxidation was determined. The molecular-weight characteristics of the N-oxidized copolymers were established using 13C NMR methods. An approach based on direct UV spectrophotometry was proposed for practical monitoring of the reaction completion (degree of conversion).

Phytochemical Study of the North Caucasian Rosa Spinossima L. Fruit

Qualitative composition and content of anthocyanins in the Rosa spinossima L. fruit were studied. The anthocyanin sum was isolated by talc adsorption with subsequent desorption. The presence of anthocyanins in the resultant fraction was confirmed by thin layer chromatography. Three spots corresponding to anthocyanins were found. Anthocyanins were identified by mass spectrometry. Ion peaks corresponding to glycoside cyanidin and delphinidin were found. Position of sugar in the glycoside structure was determined by UV spectrophotometry with aluminum chloride. The content of anthocyanins was measured by direct UV spectrophotometry by the anthocyanins absorption. Anthocyanin content in raw material in conversion to cyanidin-3-glucoside was 1.46±0.03%.

Estimation of Memantine Hydrochloride Using Ultraviolet-Visible Spectrophotometry in Bulk Drug and Formulation

Memantine HCl (ME-HCl) is a novel class of Alzheimer's disease medication acting on glutamatergic system by blocking NMDA glutamate receptors. ME-HCl is lacking of chromophores or auxochromes, thus it shows no distinct absorption in UV-VIS region. Therefore, no direct UV spectrophotometry method is available for its analysis. Thus, main aim of present study was to develop and validate simple and sensitive UV spectrophotometric method for estimation of ME-HCl and application of validated method for routine analysis of ME-HCl. Me-HCl was converted to free base with ammonia and allowed to react with iodine. The complex formed, shows UV absorptions bands at two wavelengths, 291 nm and 365 nm, but method was validated using 291 nm as λ max . The Lambert-Beer's law is obeyed in range of (10-30 μg/ml). Developed method was found to be linear (r 2 = 0.9999), accurate (102.2 %), precise (% RSD<1 %) and sensitive (LOD = 2.51 μg/ml, LOQ = 7.62 μg/ml). The method was successfully applied to marketed formulation and % purity was found to be 97.17 % w/w.

Comparative study of analytical methods by direct and first-derivative UV spectrophotometry for evaluation of losartan potassium in capsules

Losartan potassium is an antihypertensive non-peptide agent, which exerts its action by specific blockade of angiotensin II receptors. The aim of the present study was the validation and application of analytical methods for the quality control of losartan potassium 50 mg in pharmaceutical capsules, using direct and first-derivative UV spectrophotometry. Based on losartan potassium spectrophotometric characteristics, a signal at 205 nm of the zero-order spectrum and a signal at 234 nm of the first-derivative spectrum, were found adequate for quantification. The results were used to compare these instrumental techniques. The linearity between the signals and concentrations of losartan potassium in the ranges of 3.0-7.0 mg L-1 and 6.0-14.0 mg L-1 for direct and first-derivative spectrophotometry in aqueous solutions, respectively, presented a correlation coefficient (r) of 0.9999 in both cases. The methods were applied for losartan potassium in capsule dosage obtained from local pharmacies, and were shown to be efficient, easy to apply and low cost. These methods do not use polluting reagents and require relatively inexpensive equipment.

Quantitative aspects of the analysis of the monoclonal antibody trastuzumab using high-performance liquid chromatography coupled with electrospray mass spectrometry

The analytical possibilities of quantification of the intact monoclonal antibody trastuzumab by high-performance liquid chromatography coupled with electrospray mass spectrometry (HPLC–ESI-MS) were investigated. To clarify the results obtained by LC–MS, complementary experiments were performed using direct UV-spectrophotometry and high-performance liquid chromatography coupled with ultraviolet detection (HPLC–UV). A polystyrene–divinylbenzene (POROS) column was applied with gradient elution using formic acid 0.08% (v/v) in water and formic acid 0.08% (v/v) in acetonitrile as mobile phase for chromatographic analysis. Quantification on LC–MS was performed by using the peak area of the total ion current (TIC) chromatograms of one charge state. Non-linearity and sensitivity loss were the major limitations observed with the LC–MS method, of which the non-linearity is most likely caused by detector saturation. The sensitivity loss during analysis could be reduced by lowering the MS source temperature. This parameter is critical in creating a robust LC–MS system for the quantitative analysis of trastuzumab.

A Comparative Study of the Kinetics of Cytarabine Hydrolytic Deamination in Aqueous Solutions

Cytarabine (1-D-arabinofuranosylcytosine, I) is a drug belonging to the group of nucleoside antimetabolites [1]. At present, the drug is produced abroad both in dry (lyophilized) and liquid forms. The production of liquid forms is less labor-consuming and more economically effective. At the same time, it was reported [2 – 8] that cytarabine is subject to hydrolytic deamination in aqueous media with the formation of a therapeutically inactive 1-D-arabinosyluracil (II) and ammonia (Fig. 1). Our task was to study the stability of cytarabine in aqueous solutions during a preset storage time. For this purpose, it was necessary to select a method that would be (i) capable of studying the kinetics and rate of the cytarabine hydrolytic deamination reaction and (ii) sufficiently simple and optimum from the standpoint of sensitivity and reproducibility. Various methods were developed for determination of the content of I in medicinal preparations and for the study of hydrolytic deamination of this compound. These include TLC [9], RIA [10 – 13], mass spectrometry [14], gas chromatography in various modes, including those combined with other techniques [15, 16], UV spectrophotometry [17], extraction methods [18], and liquid chromatography at low [19] and high pressures [20 – 35]. The method of direct UV spectrophotometry [17] is based on the difference between the absorption spectra of I and its decomposition product II (having absorption peaks at 280 and 260 nm, respectively, in 0.1 N HCl). This technique is most simple and was widely used in the early investigations of the hydrolytic deamination reaction [2 – 4, 6 – 8]. In recent years, compounds I and II are more frequently determined by HPLC techniques offering some advantages over the other methods. Compounds belonging to arabinosyl nucleosides were predominantly studied by inversed-phase HPLC (IPHPLC) [20 – 22], although ion-exchange HPLC [23] and combined techniques can be used as well [27]. In application to nucleosides, IPHPLC can be used in several different modes [36], including chromatography under socratic conditions (most frequently used for the analysis of arabinosyl nucleosides) [20 – 22], gradient-elution chromatography, and ion-pair chromatography [29, 34, 35]. As is known [3], the maximum extinction coefficient of cytosine nucleosides is observed at low pH. Therefore, using these conditions we may reach a maximum sensitivity of spectrophotometric detection. At the same time, all the molecules of cytosine nucleosides studied under these conditions are ionized and, hence, exhibit weak adsorption on the immobile (nonpolar) phase that results in lower resolution. Thus, optimum possibilities for controlling the interaction of partitioned components with the immobile phase are provided by the method of ion-pair chromatography.

Comparative Studies on Physically Adsorbed and Chemically Bound IgG to Carboxylated Latexes, II

The aim of the present work is to compare the behavior of physically (passively) adsorbed and of chemically (covalently) bound IgG to carboxylated latexes (CLs). In this work we have tried to determine the advantages and disadvantages of covalent coupling of antibodies to CLs against the passive adsorption procedure. Physically adsorbed IgG was measured by direct UV spectrophotometry at 280 nm. Even when the protein has the same charge sign as its sorbent, adsorption occurs spontaneously, and this suggests an adsorption caused by hydrophobic forces. IgG chemically bound using the carbodiimide method was both directly measured using the bicinchoninic acid reaction (BCA method) and indirectly calculated using UV spectrophotometry. The covalent coupling had an efficiency rate of more than 70%. The different electrophoretic behavior shown by the IgG covalently bound to the CL particles versus that of the IgG passively adsorbed on these particles is due to the presence of the carbodiimide used in those that are chemically bound. Mobility values were dealt with using the most recent theoretical approaches, given by Ohshima and Kondo, to obtain information about the protein layer. The chemical coupling provides a better immunological response of the IgG–CL complexes.