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Current analytical methodology for iodopropynyl butylcarbamate (IPBC) analysis focuses on the use of liquid chromatography and mass spectrometer (LC-MS), but the high instrumentation and operator investment required has resulted in the need for a cost effective alternative methodology. Past publications investigating gas chromatography with electron capture detector (GC-ECD) for IPBC quantitation proved largely unsuccessful, likely due to the preservatives limited thermal stability. The use of pulsed injection techniques commonly used for trace analysis of thermally labile pharmaceutical compounds was successfully adapted for IPBC analysis and utilizes the selectivity of GC-ECD analysis. System optimization and sample preparation improvements resulted in substantial performance and reproducibility gains. Cosmetic formulations preserved with IPBC (50-100ppm) were solvated in toluene/isopropyl alcohol and quantified over the 0.3-1.3μg/ml calibration range. The methodology was robust (relative standard deviation 4%), accurate (98% recovery), and sensitive (limit of detection 0.25ng/ml) for use in routine testing of cosmetic formulation preservation.

An analytical method has been developed for the determination of alcohol ethoxylate (AE) components in sewage sludge. The method has been extensively ring tested in several industrial laboratories and concentrations in sludge samples from a number of EU countries have been obtained. The method is based on a methanol soxhlet extraction of centrifuged sludge, which is then cleaned up using an alumina column, followed by derivatisation with naphthoyl chloride and a further alumina column clean-up. The extract is analysed using high performance liquid chromatography (HPLC) with fluorescence detection. The method is capable of determining alcohol ethoxylate components in the range of C12–C18 alkyl chain lengths with an ethoxylate chain of EO4 up to approximately EO20 in sludge samples. The detection limit is approximately 20–30 mg kg−1 of total AEs in dry weight of sludge. Using the method, sludges from several European Sewage Treatment Plants (STP) were analysed. The concentration of the AEs, which are primarily linear, in digestor inlet averaged 1164 mg kg−1 (550–2947 mg kg−1) and in outlet sludges the mean value obtained was 167 mg kg−1 (−1). At those plants in which concentrations were monitored in both inlet and outlet sludges, removal of the AEs by anaerobic digestion at the STP averaged 82% (range 61–93%). The interlaboratory relative standard deviation of the procedure was around 40% for the digester sludges analysed. The method developed provides a more accurate estimate of the environmental level of AE components compared to existing colorimetric approaches, but the method will over-estimate the concentration of alcohol ethoxylates in sludges due to the non-specific nature of the detection. However, it is sufficiently robust and accurate to estimate alcohol ethoxylates in sludges and hence concentrations that could be applied to soil.

Using phenanthrene as a model analyte, we show that extraction from aqueous solution is possible via adsorption onto the surface of a poly(tetrafluoroethylene)-coated magnetic stir bar. Desorption from the stir-bar via ultrasonic agitation in acetonitrile solution allows quantitation of the analyte in solution. The technique is presented as a simple strategy, using commonly available laboratory apparatus, for the extraction of analytes from solution based on a surface adsorption processes: stir-bar adsorptive extraction (SBAE).

The supercritical fluid extraction (SFE) of metal ions was assessed in terms of the ability of the process to extract, solvate, transport and collect metal species. The extraction efficiency (per cent removed from the sample matrix), collection efficiency (per cent recovered in the collection solvent) and SFE residue (unextracted metal complex or metal ion recovered from the apparatus by flushing with an organic or acidic solvent, respectively) were determined to ascertain whether a mass balance for the metal could be achieved. Using this approach, it was demonstrated that the amount of metal extracted from the sample matrix did not necessarily correspond to the concentration of metal recovered in the collection solvent, as the chelated metal complex could partially degrade and/or dissociate in the supercritical fluid prior to collection. Typically, a discrepancy of 1 to 15% could be obtained. The non-fluorinated metal β-diketones and dithiocarbamates had a greater tendency to dissociate and/or degrade in a supercritical fluid (i.e., ca. 14 and 5%, respectively) than the corresponding fluorinated metal complex (i.e., ca. 6 and 1%, respectively). As for the collection solvents, a range of suitable fluids were found to be capable of obtaining quantitative metal complex recoveries, including methanol, chloroform, isobutyl methyl ketone or 10% nitric acid. A radioactive isotope, 177Lu, was used to trace the location of the metal residue in the SFE apparatus.

The retention behavior in capillary supercritical fluid chromatography (SFC) of a range of ferrocene derivatives is demonstrated. Prediction of retention times and hence elution order of the ferrocene derivatives based on a knowledge of solute molar volume, solute solubility parameter and the physical parameters of the chromatography column system is shown to be feasible using this procedure.

Supercritical fluid extraction of cobalt ions was evaluated in terms of the ability of the process to extract, solvate, transport and to collect the metal species. The aim of the study was to ensure that a mass balance could be obtained for the metal before and after extraction with supercritical carbon dioxide (CO2). The stability of the ligand and resulting metal complex were assessed as was the synergistic effect of the presence of water in the extraction. Two widely used β-diketones, namely, acetylacetone (AcAc) and hexafluoroacetylacetone (HFAcAc) were chosen to extract spiked cobalt from the surface of stainless steel and >90% of the metal was extracted and collected using the HFAcAc ligand with supercritical CO2.

The solubility of 49 metal-containing compounds and 15 “free” ligands in supercritical fluid carbon dioxide is reviewed. Solubilities were found to range over eight orders of magnitude, with the highest value of solubility being 56 g l−1. Metals complexed with fluorine substituted ligands were found to be the most soluble, and metals complexed with phenyl-substituted ligands the least soluble. A general trend for increasing solubility with increasing oxidation state is observed and this is correlated with the increased number of coordinating ligands protecting the metal center from interaction with the supercritical fluid and also due to the increased number of solvation interactions with the increased number of ligand groups.

The deacidification of high-acidity oils from Black cumin seeds (Nigella sativa) was investigated with supercritical carbon dioxide at two temperatures (40 and 60°C), pressures (15 and 20 MPa) and polarities (pure CO2 and CO2/10% MeOH). For pure CO2 at a relatively low pressure (15 MPa) and relatively high temperature (60°C), the deacidification of a highacidity (37.7 wt% free fatty acid) oil to a low-acidity (7.8 wt% free fatty acid) oil was achieved. The free fatty acids were quantitatively (90 wt%) extracted from the oil and left the majority (77 wt%) of the valuable neutral oils in the seed to be recovered at a later stage by using a higher extraction pressure. By reducing the extraction temperature to 40°C, increasing the extraction pressure to 20 MPa, or increasing the polarity of the supercritical fluid via the addition of a methanol modifier, the selectivity of the extraction was significantly reduced; the amount of neutral oil that co-extracted with the free fatty acids was increased from 23 to 94 wt%.

Real world samples which contain high concentrations of water and/or extractable material frequently cause intermittent or irreversible plugging of the flow control device during off-line supercritical fluid extraction (SFE). A supercritical fluid (SF) flow control/collection device has been developed which can simultaneously maintain the extraction flow-rate of the SF (±0.1 ml/min) and quantitatively (>90%) collect analytes as volatile as n-octane directly into an organic solvent. With this device, the extract is partially depressurized through a heated capillary restrictor and into a pressurized collection solvent, so that both temperature and pressure are used to maintain the solubility of the extract in the SF. The pressurized mixture is finally depressurized to atmospheric conditions using a backpressure regulator, so the extract can be recovered in the collection vial. Depending on the sample matrix, a restrictor heater temperature of 200 °C and a backpressure regulator with a heated (>5 wt% water in sample) or unheated (

Supercritical fluid extraction of metals in the presence of complexing agents for environmental monitoring, cleanup, and metals processing is now being extensively researched, and there is a need for solubility data of metal complexes. In this paper, the method used is the combination of a study of chromatographic retention over a wide range of pressures at 40, 50, 60, and 70 `C, followed by selected direct solubility measurements, to provide a comprehensive set of solubility results obtained with minimum effort. The method relies on the assumption that an inverse relationship exists between chromatographic retention and solubility in the mobile phase. This assumption is checked in this study. Results are presented for the solubility of ferrocene in supercritical carbon dioxide and a nickel complex, (5,7,12,14-tetramethyl-2,3:9,10-dibenzo[b,i][1,4,8,11]tetraazacyclotetradecine)nickel(II), in supercritical carbon dioxide modified with 10 vol % methanol.

A coupled supercritical fluid extraction-gas chromatography (SFE-GC) method has been developed for the quantitative extraction and analysis of gasoline and diesel range organics from real world environmental samples. Petroleum-contaminated samples containing gasoline- to diesel- and motor oil-range hydrocarbons (total hydrocarbon content typically ranging from 2 to 26 mgg) could be quantitatively extracted by a 15-min SFE-GC extraction using 400 atm (1 atm = 101 325 Pa), 60°C CO2. The SFE-GC hydrocarbon recoveries from real-world samples were comparable to those obtained by sonicating the samples in methylene chloride for 14 h, except for the gasoline recovery which was higher by SFE-GC analysis due to the more efficient collection of the more volatile analytes. Reproducibilities for replicate SFE-GC extractions and analyses were typically

Of 21 potential drying agents investigated, five (anhydrous and monohydrated magnesium sulfate, molecular sieves 3A and 5A and Hydromatrix) were able successfully to prevent restrictor plugging by water during off-line supercritical fluid extraction (e.g., 400 atm CO2 at 60°C) by retaining the majority of the water (but generally not the analytes of interest) in the extraction cell. Increasing the extraction temperature (e.g., to 150°C) or adding a polar modifier [10% (v/v) methanol] to the CO2 extraction fluid greatly reduced the amount of water the drying agents retained. However, when 10% (v/v) toluene was used for the extraction, the drying agents were able to retain the majority of the water (ca. 80% w/w). Polar and non-polar pollutants were quantitatively extracted from the wet drying agents (i.e., water present), but nearly all of the drying agents selectively retained at least one of the polar analytes if used dry (i.e., no water present), thus demonstrating the need for a spike recovery study to determine the potential for analyte loss. The successful drying agents eliminated restrictor plugging when used with moderately wet [ca. 20% (w/w) water at a 1:1 reagent-to-sample ratio] and very wet [ca. 90% (w/w) water at 4:1 reagent-to-sample ratio] samples without the need to heat the restrictor or the collection solvent.

Fused-silica restrictors used for off-line supercritical fluid extraction (SFE) frequently break when extractions are performed with polar supercritica

The development of quantitative supercritical fluid extraction (SFE) methods for the recovery of organic pollutants from environmental samples requires three steps: quantitative partitioning of the analytes from the sample into the extraction fluid, quantitative removal from the extraction vessel, and quantitative collection of the extracted analytes. While spike recovery studies are an excellent method to develop the final two steps, they are often not valid for determining extraction efficiencies from complex real-world samples such as soils and sediments, exchaust particulates, and sludges. SFE conditions that yield quantitative recoveries of spiked analytes may recover < 10% of the same analytes from real-world samples, because spiked pollutants are not exposed to the same active sites as the native pollutants. Because of the heterogeneous nature of environmental samples, the partitioning step may be controlled by analyte solubility in the extraction fluid, kinetic limitations, and/or the ability of the extraction fluid to interrupt matrix-analyte interactions. While the interactions that control SFE rates from heterogenous environmental samples are not well understood, a generalized scheme for developing quantitative SFE methods is proposed based on interactive considerations of the collection efficiencies after SFE, fluid flow parameters in the extraction cell, analyte solubility, extraction kinetics, and analyte-matrix-extraction fluid interactions. The proposed development scheme includes increasing SFE extraction rates by the use of more polar fluids than CO2 such as CHClF2, the addition of organic modifiers to CO 2, and the use of high temperature extractions with pure CO2. Validation of quantitative extractions based on multiple extraction methods (SFE followed by liquid solvent extractions) is also described.

The relative extraction rates of native polycyclic aromatic hydrocarbons (PAHs) ranging from naphthalene (M = 128) to benzo[b]fluoranthene (M = 252) and those of spiked deuterated PAHs (d-PAHs) from heterogeneous environmental samples including petroleum waste sludge, urban air particulate matter (SRM 1649), and railroad bed soil were compared using sequential extractions with pure supercritical CO[sub 2] or modified (10% v/v methanol) supercritical CO[sub 2] and using sonication with methylene chloride. Regardless of the spiking method (injection of the spike or suspension of the sample in the spiking solution) or aging time (up to 14 h), the extraction rates of most of the spiked d-PAHs were substantially (up to 10-fold) higher than those of the same native PAHs. Differences in extraction rates of the spiked and native PAHs were most dramatic for the lower molecular weight PAHs, indicating that relatively volatile species such as naphthalene must be tightly bound in order to remain associated with a real-world sample. In most cases, 30-min extractions with pure CO[sub 2] quantitatively recovered (>90%) the spiked deuterated-PAHs, but only extracted ca.25-80% of the native PAHs. Similar differences were observed using conventional methylene chloride sonication, demonstrating that spike recovery studies are not valid for developing quantitativemore » extraction methods for heterogeneous environmental samples. 21 refs., 8 figs., 4 tabs.« less

The High Operating Temperature Auger suppressed infrared detector concept is being pursued using the high density vertically integrated photodiode (HDVIP ® ) architecture and an n + -p device structure. Dark current densities as low as 2.5 mA/cm 2 normalized to a 5 µm cutoff at 250K have been demonstrated on these diodes. These dark currents imply minority carrier lifetimes in excess of 300µsec. 1/f noise in these devices arises from the tunneling of charge into the passivation interface, giving rise to a modulation in the surface positive charge and hence to the width of the depletion region in the p-side of the device and a modulation in the total dark current. The measured 1/f noise is in agreement with the predictions of this model, with very low noise being observed when the lifetimes are high. Keywords: Mercury cadmium telluride, infrared detector, focal plane array, high operating temperature, HOT 1. INTRODUCTION 1.1 Motivation In the past, most photon detecting infrared Focal Plane Arrays (FPAs) have operated at a temperature of 77K or below. There is a considerable system advantage if the operating temperature of the FPA can be increased, namely that the resulting Dewar and Cooler package into which the FPA is integrated will be considerably lighter, smaller, and thus cheaper. In addition the power required to cool the FPA can be decrease by a factor of ~5-10, as illustrated in Figure 1. Implementation of this technology enables new system concepts and functionality.

Supercritical carbon dioxide was used to extract the sesquiterpene lactone parthenolide, which is reported to be the active component of the medicinal herb feverfew ( Tanacetum parthenium ), from dried plant material for gas-liquid chromatographic analysis. The extracts also contained significant amounts of camphor and chrysanthenol acetate. The pressure and densities required for quantitative extraction and the methods of sample collection were studied. The addition of methanol or acetonitrile as modifiers in the carbon dioxide gave higher yields of parthenolide bu the extractions were less selective and considerable amounts of co-extractives were obtained. By employing a trapping column made of cellulose or silica the purity of the extracts could be improved and a practical method for the rapid isolation of parthenolide is described.

Real-world environmental samples which contain high concentrations of water and/or extractable matrix components frequently cause intermittent or irreversible plugging of capillary flow restrictors during off-line supercritical fluid extraction. Heating the entire restrictor at 50°C produces a constant extraction flow-rate (± 0.03 ml/min) for such samples, but poor collection efficiencies (30–65% recovery for polycyclic aromatic hydrocarbons) were obtained because the supercritical fluid could not be depressurized directly into the collection solvent. While the collection efficiency was improved (80–90%) by nebulizing an organic solvent with the restrictor effluent, a simpler method was to heat all but the last 3 cm of the restrictor and to depressurize the extract directly in the collection solvent. Depending on the sample matrix, restrictor heater temperatures ranging from 50 to 200°C were required to avoid restrictor plugging. With proper heating, constant extraction flow-rates (± 0.07 ml/min) and high collection efficiencies (90–100%) for polycyclic aromatic hydrocarbons and n -alkanes as volatile as n -octane were achieved.

Supercritical fluid extraction (SFE) recoveries for native pollutants including PCBs from a standard reference material sediment (SRM 1939), PAHs from a petroleum waste sludge, and PAHs from railroad bed soil were compared using supercritical CHCIF 2 (Freon-22), N 2 O, and CO 2 . While SFE with pure CO 2 yielded the lowest recoveries for each sample, CHCIF 2 consistently yielded the highest extraction efficiencies, most likely because of its high dipole moment

α-Cellulose has been used as a model plant matrix to investigate the conditions required to optimize the supercritical fluid extraction of typical plant constituents, limonene, caryophyllene, carvone, eugenol and santonin, using carbon dioxide as the extraction medium. The conditions required for the successful recovery of the analytes were monitored by gas-liquid chromatography. Timed recovery studies enabled differences in the rates of extraction to be determined to ensure that sufficiently long extraction runs were used. Subcritical and supercritical extractions over the ranges −10 to 80°C and 50–250 bar were examined and 250 bar and 40°C were chosen as the optimum conditions. The effects of the addition of modifiers to the supercritical fluid were also examined. The work also demonstrated that increased selectivity for polar analytes such as lactones could be obtained by trapping the extract on a silica column coupled with selective elution.

Supercritical fluid extraction (SFE) collection efficiences of 66 compounds with a wide range of volatility and polarity were examined. Good collection efficiencies required efficient partitioning of the analyte into the collection solvent after depressurization, and factors including collection solvent polarity and temperature were found to be more important than collection solvent volume and height. Heating the collection solvent with a heat gun to avoid plugging of the outlet restrictor resulted in 20−50% losses of the more volatile analytes, while > 90% trapping of all test analytes could be attained by controlling the solvent temperature at 5°C. Extraction cell geometry (“long, narrow” versus “short, broad” vessel) at constant internal volume and the orientation of the extraction cell were found to have negligible effects on the extraction rates of polycyclic aromatic hydrocarbons (PAHs) from railroad bed soil and flavor and fragrance compounds from lemon peels. The supercritical fluid flow-rate also had little effect on the extraction rate of native PAHs provided that it was sufficient to sweep the cell dead volume every ca. 3 min.

Conventional supercritical fluid extraction (SFE) system are constructed using stainless steel due to its relative inertness, ready availability, tensile strength and low cost. Recently, metal complex solubilities and metal extraction using chelating agents have been investigated using a stainless steel SFE system. Metal complexes are usually soluble in supercritical carbon dioxide (SC-CO2) and so this was the fluid of choice for the experiments. During the chelation step, temperature and pressure have great effect on the extraction efficiency. Using a metal salt as a sample, and hexafluoroacetylacetone (HFAcAc) as a ligand, the most efficient extraction conditions were found to be 60°C and 400atm. However, when contaminated soil was used as a sample, extraction efficiency fell. This was thought to be related to the corrosion of the stainless steel SFE system by the ligand HFAcAc. During the 30min extraction period, 1678μg of iron was obtained. The corrosion of the SFE system was much greater during the soil extraction than during both the metal salt extraction and metal complex solubility experiments. Copyright (C) 2000 Elsevier Science B.V.

Sample preparation is often proved the most time consuming part of a chromatographic assay because it usually involves an extraction procedure with organic solvents and the subsequent need to evaporate the solvent to concentrate the extract.

A model for dynamic supercritical fluid extraction (SFE) is proposed, which contains the steps of: rapid fluid entry into the matrix; a reversible release process such as desorption from matrix sites or penetration of a biological membrane; transport, by diffusion or otherwise to the edge of a matrix particle; and removal by biological membrane; transport, by diffusion or otherwise to the edge of matrix particle; and removal by solvation in the fluid. It can explain the following kinetic features of dynamic SFE : (i) a slow initial extraction; (ii) a more rapid extraction phase; and (iii) a slow final phase, which can be so slow that it appears that extraction is complete when only a fraction of the solute present in the matrix has been recovered. The model may be used to extract values for the parameters quantifying the individual steps, to understand further the SFE process.

1. Evidence for the occurrence of a rapidly polymerizing equilibrium system in freshly prepared aqueous solutions of porcine neurophysin has been obtained by frontal and zonal analysis of gel-filtration elution patterns. 2. Elution volumes increased with decreasing protein concentration and derivative analysis of fronts suggested the existence of different polymerizing forms varying in relative proportion with protein concentration. 3. In the presence of lysine vasopressin, the results suggested a shift of the equilibrium in favour of higher polymerizing forms. 4. In the presence of Ca2+ elution patterns became characteristic of a monodisperse system of relatively high molecular weight. The effect of Ca2+ did not survive removal of the ion and was not reproduced by Mg2+. 5. Evidence for a slowly developing polymerization of neurophysin in aqueous solution has been obtained in experiments with solution prepared 24 hr before subjection to gel-filtration. 6. A model is proposed for the description of the binding of lysine vasopressin to porcine neurophysin.

A method is described for the rapid separation of cells from their suspending medium with only 1 5 th to 1 30 th the amount of contamination from the medium as with conventional centrifuging. The principle is to interlayer between the cells and the medium upon centrifuging an immiscible liquid whose density lies between that of the cells and medium. Quantitative measurement of the amount of contamination of the cells by the medium which does occur is achieved by adding a nonpermeable radioactively labeled substance to the suspension prior to centrifugation.

1. The concentration of Bromophenol Blue used as tracking dye in polyacrylamide-gel electrophoresis affected the resolution of rat neurophysins. 2. A final dye concentration of 1μg/ml in the tris–glycine running buffer was found to give the best results. 3. The presence of two major and one minor neurophysin(s) in the rat was confirmed. 4. The two major proteins were found to re-run as single discrete bands, which had the same mobilities in the absence of dye and different mobilities in its presence.

Publisher Summary This chapter presents three basic methods for the analysis of (Na, K, Mg, Ca, Mn, Fe, Co, Cu, Zn) elements. First, a rapid, sensitive colorimetric procedure is given that can be readily utilized for routine analyses of medium precision. A volumetric method is included for requirements of precision of an order of magnitude greater than that available by colorimetry, and gravimetric methods are presented because they are applicable to radioactive tracers. In a number of cases the selection of three such methods is not necessary, because one may have little advantage over another. One special instrumental method of analysis has been included as a separate section in view of its wide usage and applicability to elements that are otherwise difficult to determine— namely, the flame photometry of the alkali metals and alkaline earths. Flame attachments for spectrophotometers have become so widespread that it is rare to find a laboratory which does not somewhere have a flame attachment available. Certainly, when the equipment is available, this becomes the method of choice for the alkali metals and alkaline earths.

Substances that have not exhibited properties as excitants of ectopic activity by intracoronary injection into normal hearts often produce rapid ectopic activity and sometimes ventricular fibrillation upon slow injection through the vascular bed of an infarcted area, particularly upon the first one or two injections. Sodium lactate has reduced and stopped ectopic activity for brief periods. Excesses of both potassium and calcium have increased ectopic activity in infarction and produced it in normal hearts. No antagonism between these two ions, with respect to ectopic activity, has been demonstrated.

The change in the radioactivity of vasopressin–neurophysin in the rat neurohypophysis after an intracisternal injection of [35S]cysteine was fitted to several mathematical models. The data fitted best a model in which there is a linear input of radioactive protein into one pool of the neurohypophysis, from which it is either released by an exponential process or transferred to a second pool from which it is released by a second exponential process with a rate constant much lower than the first. This model is compatible with the existence of a ‘readily releasable’ pool first postulated by Sachs et al. (1967). Data for the change in radioactivity of vasopressin also gave a good fit in this model. Calculation of the rate constants suggested that the first pool represented about 2% of the total hormone.

1. Rat neurohypophysial extracts have been examined by polyacrylamide-gel electrophoresis. 2. Three of the proteins were tentatively identified as neurophysins by their acidic nature and their disappearance after dehydration of the animals. 3. These proteins were radioactive 24h after intracisternal injection of [35S]cysteine. 4. Two of the proteins were present in much greater quantities than the third, and these two were present in the gland in the same ratio as the hormones vasopressin and oxytocin. 5. One of these proteins was absent from glands of rats homozygous for diabetes insipidus but present in heterozygous animals. 6. It is suggested that these two proteins are the vasopressin–neurophysin and oxytocin–neurophysin of the rat.

1. 1.|The object of the investigation was to determine the molecular weight of neurophysin polypeptide chains and to test the hypothesis that these exist in polymerising equilibrium. 2. 2.|Polyacrylamide gel electrophoresis of a denatured porcine neurophysin preparation in the presence of detergent revealed three components, two major and one minor, of molecular weights ⩽ 10 000, ⩽ 12 000 and ≈ 14 000, respectively. 3. 3.|The weight-average molecular weight of the porcine neurophysin preparation was estimated by analytical ultracentrifugation and with the denatured protein was 11 000, and independent of protein concentration. With undenatured protein the weight-average molecular weight increased (at pH 5.8) with concentration from approx. 10 000 to approx. 35 000 and the influence of protein concentration was pH dependent. 4. 4.|In the presence of Ca 2+ (1 · 10 −5 M at pH 5.8) the weight-average molecular weight of porcine neurophysin was 21 000 and was independent of protein concentration. 5. 5.|It is concluded that neurophysin monomers consist of single polypeptide chains, which in aqueous solution, exist in polymerising equilibrium and that equilibrium is perturbed by Ca 2+ .