Generalized Electrical Substitution Methods and Detectors for Absolute Optical Power Measurements.

We have developed generalized methods for electrical substitution optical measurements, as well as cryogenic detectors which can be used to implement them. The new methods detailed here enable measurement of arbitrary periodic waveforms by an electrical substitution radiometer (ESR), which means that spectral and dynamic optical power can be absolutely calibrated directly by a primary standard detector. Cryogenic ESRs are not often used directly by researchers for optical calibrations due to their slow response times and cumbersome operation. We describe two types of ESRs with fast response times, including newly developed cryogenic bolometers with carbon nanotube absorbers, which are manufacturable by standard microfabrication techniques. These detectors have response times near 10 ms, spectral coverage from the ultraviolet to far-infrared, and are ideal for use with generalized electrical substitution. In our first tests of the generalized electrical substitution method with FTS, we have achieved uncertainty in detector response of 0.13 % (k=1) and total measurement uncertainty of 1.1 % (k=1) in the mid-infrared for spectral detector responsivity calibrations. The generalized method and fast detectors greatly expand the range of optical power calibrations which can be made using a wideband primary standard detector, which can shorten calibration chains and improve uncertainties.

Development of electrical substitution Fourier transform spectrometry for absolute optical power measurements.

We have demonstrated the first continuous-scan electrical substitution Fourier transform spectrometer (ES-FTS), which serves initially as an apparatus for absolute spectral responsivity calibrations of detectors over the wavelength range from 1.5 µm to 11 µm. We present data on the realization of a spectral detector-comparator system with high accuracy, high dynamic range, high spectral resolution and fast measurement in the infrared region, which is tied directly to an absolute power scale through electrical substitution. The ES-FTS apparatus employs a commercial Fourier transform spectrometer and a custom electrical substitution bolometer detector to enable spectrally-resolved absolute optical power measurements. A generalization of electrical substitution techniques enables determination of the voltage waveform that must be applied to the bolometer's electrical heater to cancel the optical signal from a Michelson interferometer in order to quantify the time-dependent optical power incident on the bolometer. The noise floor of the electrical substitution bolometer is on the order of 10 pW/Hz½ and its response is expected to be linear from the noise floor to 1 mW. A direct comparison between a pyroelectric standard detector and the ES-FTS has been performed, and experimental results reported here show great potential for this technique.

Wideband infrared trap detector based upon doped silicon photocurrent devices.

We have designed, fabricated, and measured infrared trap detectors made from arsenic-doped silicon (Si:As) blocked impurity band (BIB) photodetectors. These trap detectors are composed of two detectors in a wedge geometry, with an entrance aperture diameter of either 1 or 3 mm. The detectors were calibrated for quantum efficiency against a pyroelectric reference detector using a Fourier transform spectral comparator system, and etalon effects and spatial uniformity of the traps were also quantified. Measurements of the traps at a temperature of 10 K show that nearly ideal external quantum efficiency (>90%) can be attained over much of the range from 4 to 24 µm, with significant responsivity from 2 to 30 µm. The traps exhibited maximum etalon oscillations of only 2%, which is about 10 times smaller amplitude than those of the single Si:As BIB detectors measured under similar conditions. Spatial nonuniformity across the entrance apertures of the traps was about 1%. The combination of high detectivity, wideband wavelength coverage, spectral flatness, and spatial uniformity make these trap detectors an excellent reference detector for spectrally resolved measurements and radiometric calibrations over the near- to far-infrared wavelength range.

[Femur fracture in a patient with dog-leg deformity secondary to arthrogryposis. Description of a unique osteosynthesis method]. / Fractura de fémur en un paciente con deformidad «dog-leg¼ secundaria a artrogriposis. Descripción de un método único de osteosíntesis.

The concept of arthrogryposis encompasses several conditions that share the presence of multiple congenital contractures. The knee is frequently involved and is an important cause of morbidity in these patients. Flexion contractures of the knee are the most common ones and have a worse prognosis than extension contractures. Different approaches are available to treat flexion contractures of the knee. Distal femoral extension osteotomy effectively corrects fixed flexion, but may lead to residual deformity. This iatrogenic deformity disrupts the anterior convexity of the femoral shaft and leads to serious problems in the subsequent management of orthopedic conditions. This is a case report of a patient with arthrogryposis and a femur deformity who sustained a supracondylar fracture. Managing the fracture was challenging due to a disruption in the normal architecture of the femur. This is a description of the osteosynthesis approach used to treat the fracture and the patients pre-existing deformity.

El concepto «artrogriposis¼ agrupa varias enfermedades cuya característica común es la presencia de múltiples contracturas congénitas. La rodilla se afecta con frecuencia y es causa importante de morbilidad en estos pacientes. Las contracturas en flexión de la rodilla son más comunes y tienen peor pronóstico que las contracturas en extensión. Existen diferentes técnicas de tratamiento para la contractura en flexión de la rodilla; la osteotomía distal de extensión femoral corrige eficazmente la flexión fija, pero puede dar lugar a una deformidad residual. Esta deformidad iatrogénica altera la convexidad anterior de la diáfisis femoral y conlleva problemas graves en el manejo posterior de patología ortopédica. Se presenta el caso de una paciente con artrogriposis y una deformidad en fémur con una fractura en la región supracondílea. El manejo de la fractura fue dificultoso por la presencia de una distorsión de la arquitectura normal del fémur. En nuestro trabajo se describe el método de osteosíntesis utilizado para hacer frente a la fractura y a la deformidad previa de la paciente.

Spectrally Tunable Sources for Advanced Radiometric Applications.

A common radiometric platform for the development of application-specific metrics to quantify the performance of sensors and systems is described. Using this platform, sensor and system performance may be quantified in terms of the accuracy of measurements of standardized sets of source distributions. The prototype platform consists of spectrally programmable light sources that can generate complex spectral distributions in the ultraviolet, visible and short-wave infrared regions for radiometric, photometric and colorimetric applications. In essence, the programmable spectral source is a radiometric platform for advanced instrument characterization and calibration that can also serve as a basis for algorithm testing and instrument comparison.

Exploring the folding funnel of a polypeptide chain by biophysical studies on protein fragments.

We are examining possible roles of native and non-native interactions in early events in protein folding by a systematic analysis of the structures of fragments of proteins whose folding pathways are well characterised. Seven fragments of the 110-residue protein barnase, corresponding to the progressive elongation from its N terminus, have been characterised by a battery of biophysical and spectroscopic methods. Barnase is a multi-modular protein that folds via an intermediate in which the C-terminal region of its major alpha-helix (alpha-helix1, residues Thr6-His18) is substantially formed as is also its anti-parallel beta-sheet, centred around a beta-hairpin (residues Ser92-Leu95). Fragments up to, and including, residues 1-95 (fragment B95), appeared to be mainly disordered, although a small amount of helical secondary structure in each was inferred from far-UV CD experiments, and fluorescence studies indicated some native-like tertiary interactions in B95. The largest fragment (residues 1-105, B105) is compactly folded. The secondary structure in alpha-helix1 in the seven fragments was found by NMR to increase with increasing chain length faster than the build-up of tertiary interactions, indicating that alpha-helix1 is being stabilised by non-native interactions. This behaviour contrasts with that in fragments of the 64-residue chymotrypsin inhibitor 2 (CI2), in which tertiary and secondary structures build up in parallel with increasing length. CI2 consists of a single module of structure that folds without a detectable intermediate. The largest fragment of barnase, B105, has interactions that resemble its folding intermediate, whereas one of the largest fragments of CI2 (residues 1-60) resembles the folding transition state. The folding pathways of both proteins are consistent with a scheme in which there are low levels of native-like secondary structure in the denatured state that become stabilised by long-range interactions as folding proceeds. Neither protein forms a stable fold when lacking the last ten residues at the C terminus. Since at least 20 amino acid residues are bound to the ribosome during protein biosynthesis, these small proteins do not fold until they have left the ribosome, and so the studies of the folding of such proteins in vitro may be relevant to their folding in vivo, especially as the molecular chaperone GroEL binds only weakly to denatured CI2 and does not discernibly alter the folding mechanism of barnase.

Acquisition of native-like interactions in C-terminal fragments of barnase.

We have characterised a series of C-terminal fragments of barnase by different biophysical techniques to find out when they acquire secondary and tertiary native-like structure. Fragments B96-110 (which comprises the last 15 residues of the intact protein) up to B37-110 (which involves most of the protein except the two first helices and a loop) were mainly disordered. Only fragment B23-110, which lacks alpha-helix1, showed native-like near and far-UV CD and fluorescence spectra. The intensities of these spectra were lower than those of the full-length protein, which suggests the absence of complete side-chain packing. Urea denaturation followed by fluorescence, far-UV CD and gel-filtration chromatography techniques indicated a co-operative transition only for B23-110. None of the fragments melted co-operatively with temperature. Thus, the formation of secondary and tertiary structure requires most of the polypeptide chain to be present, that is, secondary and tertiary structure are formed in parallel. This agrees with the proposed model for barnase folding, where the residual structure in small fragments is weak and flickering, and it is only consolidated when there are enough tertiary interactions. Thus, the development of structure in the series of C-terminal fragments follows a similar behaviour to that observed in the series of N-terminal fragments of barnase.

An N-terminal fragment of barnase has residual helical structure similar to that in a refolding intermediate.

A fragment of barnase comprising amino acids 1 to 36 (B(1-36)) that encompasses the region containing the two large helices (residues 6-18 and 26-34) of the native protein has been obtained by cleavage of the barnase mutant Val36----Met with cyanogen bromide. The circular dichroism (c.d.) spectrum of B(1-36) in the far ultraviolet indicates that the fragment is only weakly structured in water at neutral pH. The two-dimensional 1H nuclear magnetic resonance spectrum of B(1-36) shows, however, that a fraction of the population does have helical structure, spanning amino acid residues 8 to 18. B(1-36) becomes more helical in 35% trifluoroethanol. This is indicated by the c.d. spectrum and the increase from 6.6 to 7.0 in the pKa of His18, which is known to interact with the dipole of helix 6-18 in native barnase. The helical region of B(1-36) in 35% trifluoroethanol extends to residue 6. It is calculated from extrapolation of a trifluoroethanol titration of the ellipticity at 222 nm that B(1-36) exhibits in water approximately 6% of helical structure, calculated for a 36 residue alpha-helical peptide. This corresponds to approximately 20% of that expected for an 11-residue alpha-helical region. In trifluoroethanol, c.d. measurements indicate that approximately 30% of the 36-residue peptide is helical. It has been shown from extensive studies of the refolding of barnase that there is a folding intermediate that contains residues 8 to 18 in a helical conformation and that residue 6 is mainly unfolded. The experiments on the conformation of B(1-36) show that a small, but significant fraction, of its population in water adopts the conformation of the major alpha-helix during the barnase folding pathway, in the absence of tertiary interactions. Thus, in the folding of native barnase, secondary structure formation can precede the docking of the major alpha-helix onto the beta-sheet.

Hydrogen exchange in ribonuclease A and ribonuclease S: evidence for residual structure in the unfolded state under native conditions.

Two-dimensional NMR spectroscopy has been used to monitor the exchange of backbone amide protons in ribonuclease A (RNase A) and its subtilisin-cleaved form, ribonuclease S (RNase S). Exchange measurements at two different pH values (5.4 and 6.0) show that the exchange process occurs according to the conditions of the EX2 limit. Differential scanning calorimetry measurements have been carried out in 2H2O under conditions analogous to those used in the NMR experiments in order to determine the values of DeltaCp, DeltaHu and Tm, corresponding to the thermal denaturation of both proteins. For the amide protons of a large number of residues in RNase A, the free energies at 25 degreesC for exchange competent unfolding processes are much lower than the calorimetric denaturation free energies, thus showing that exchange occurs through local fluctuations in the native state. For 20 other protons, the cleavage reaction had approximately the same effect on the exchange rate constants than on the equilibrium constant for unfolding, indicating that those protons exchange by global unfolding. There is a good agreement between the residues to which these protons belong and those involved in the putative folding nucleation site identified by quench-flow NMR studies. The unfolding free energies of the slowest exchanging protons, DeltaGex, as evaluated from exchange data, are much larger than the calorimetric free energies of unfolding, DeltaGu. Given the agreement between DeltaDeltaGex(A-S), the difference in free energy from exchange for a given proton of the two proteins, and DeltaDeltaGu(A-S), the difference in the calorimetric free energy of the two proteins, the discrepancy indicates that the intrinsic exchange rates in the unfolded state of those protons cannot be approximated by those measured in short unstructured peptides and, consequently, exchange for those protons in RNase A and S must occur through a rather structured denatured state.

Hydrogen exchange in chymotrypsin inhibitor 2 probed by denaturants and temperature.

Hydrogen exchange of chymotrypsin inhibitor 2 has been measured in the presence of low concentrations of GdmCl and at different temperatures. The study of exchange at different temperatures allows us to obtain the activation enthalpies for the local exchange processes, and the change in enthalpy between the closed, exchange-incompetent, forms and the open, exchange-competent, forms. From the GdmCl dependence of exchange, an m-value, which is a measure of the new surface area exposed to solvent in the equilibrium between open and closed forms, can be determined for individual protons. This parameter therefore provides information about the structural nature of the opening reactions. In the absence of denaturant, exchange from native and native-like states dominates. As GdmCl concentration is increased, opening reactions that involve global unfolding are selectively promoted for the majority of amide protons. Three classes of protons emerge: for one set of protons, there is a linear and weak dependence on denaturant, indicating that the dominant opening reaction is the same throughout the range of GdmCl concentrations and involves local fluctuations with exposure of little new surface. For another set of protons, the most slowly exchanging residues, a linear, but much stronger, denaturant dependence is observed. For these protons, global unfolding dominates, and the m-values are similar to that obtained by equilibrium GdmCl denaturation measured by fluorescence under identical conditions. For the remaining protons, the GdmCl-dependence is weak at low GdmCl concentrations and increases at higher GdmCl concentrations. No segment of sub-global unfolding could be identified. Rather, all protons appear to merge together at high GdmCl concentrations to the global unfolding reaction.

High-resolution three-dimensional structure of ribonuclease A in solution by nuclear magnetic resonance spectroscopy.

High-resolution three-dimensional structures of bovine pancreatic ribonuclease A in aqueous solution have been determined by nuclear magnetic resonance (NMR) spectroscopy combined with restrained molecular dynamics calculations. The structures are based on: (1) 464 interproton distance constraints with accurate upper and lower limits, determined from build-up rates of nuclear Overhauser effects (NOE) by using the complete relaxation matrix; (2) 999 more approximate upper limits for interproton distances; and (3) 42 dihedral angle constraints (37 for phi and 5 for chi 1). A total of 16 structures were calculated, which show a root-mean-square (r.m.s.) deviation of 0.66 A for the backbone atoms and 1.68 A for all heavy-atoms. The converged structures are highly similar to those found in the crystal state. r.m.s. deviation of backbone atom positions in the crystal as compared to those in the average solution structure is 0.92 A. Observed differences are concentrated in loop regions and in the neighborhood of His119 and His48 side-chains. Dynamic aspects, such as H/D amide proton exchange and side-chain mobility have been examined.

Search for nucleation sites in smaller fragments of chymotrypsin inhibitor 2.

There is a region of well-ordered structure in the transition state of folding of chymotrypsin inhibitor 2 (CI2) that consists of N-terminal residues in the unique alpha-helix (residues 12 to 24) plus some long range interactions, in particular those of Ala16 with Ile57 and Leu49 in the hydrophobic core. This is proposed to be a nucleation site. A crucial question for understanding the initiation of protein folding is: when is the nucleation site formed? Is the alpha-helix pre-formed in the nominally unfolded state, or does it require long-range interactions to be stabilized? To answer this question, we have characterized a series of N-terminal fragments of CI2, each containing an increasing number of subsets of the regular secondary structure. Four small fragments have been examined by circular dichroism and two-dimensional 1H and 15N NMR spectroscopy. The smallest, [1-5], comprises the sequence corresponding to the first beta-strand of the intact protein; the second, [1-13], contains also a type III reverse turn, the second beta-strand, and a type II reverse turn; the third [1-25], consists additionally of the sequence corresponding to the alpha-helix (residues 12 to 24); the fourth, [1-28], contains, in addition, the turn following the alpha-helix. All the fragments have disordered, non-compact structure in aqueous solution. In the structure-promoting co-solvent, trifluoroethanol, alpha-helical structure is stabilized in [1-25] and [1-28] in the region corresponding to the alpha-helix in the intact protein; however, the helix is frayed at both ends and is only fractionally populated, being in dynamic equilibrium with extended conformations. These observations indicate that there is little drive for independent formation of local secondary structure in CI2, and this is reflected in the highly concerted nature of the folding reaction of this protein. The nucleation site of folding of CI2 does not accumulate in the starting state for the folding reaction, but remains embryonic until there are sufficient long range interactions to stabilize it.

Hydrogen exchange in chymotrypsin inhibitor 2 probed by mutagenesis.

Two-dimensional NMR spectroscopy has been used to monitor hydrogen-deuterium exchange in chymotrypsin inhibitor 2. Application of two independent tests has shown that at pH 5.3 to 6.8 and 33 to 37 degrees C, exchange occurs via an EX2 limit. Comparison of the exchange rates of a number of mutants of CI2 with those of wild-type identifies the pathway of exchange, whether by local breathing, global unfolding or a mixture of the two pathways. For a large number of residues, the exchange rates were unaffected by mutations which destabilized the protein by up to 1.9 kcal mol(-1), indicating that exchange is occurring through local fluctuations of the native state. A small number of residues were found for which the mutations had the same effect on the rate constants for exchange as on the equilibrium constant for unfolding, indicating that these residues exchange by global unfolding. These are residues that have the slowest exchange rates in the wild-type protein. We see no correspondence between these residues and residues involved in the nucleation site for the folding reaction identified by protein engineering studies. Rather, the exchange behaviour of CI2 is determined by the native structure: the most protected amide protons are located in regions of hydrogen bonding, specifically the C terminus of the alpha-helix and the centre of the beta-sheet. A number of the most slowly exchanging residues are in the hydrophobic core of the protein.

Conformational pathway of the polypeptide chain of chymotrypsin inhibitor-2 growing from its N terminus in vitro. Parallels with the protein folding pathway.

We have obtained a series of fragments growing from the N terminus of the protein chymotrypsin inhibitor-2 (C12) in order to study the development of structure on elongation of the polypeptide in solution. We present an extensive biophysical characterization of ten fragments using different conformational probes. Small fragments up to residue 40 of the 64-residue protein are disordered. Fragment (1-40) has non-native local hydrophobic clusters, but nevertheless does not bind 8-anilinonaphthalene-1-sulphonate (ANS). Hydrophobic regions in longer fragments become gradually more capable of binding ANS as the chain grows to completion, with a tendency to form native structures. Major changes in secondary structure and accessibility to hydrophobic sites occur in parallel, between (1-40) and (1-53), together with changes in hydrodynamic volume and flexibility. NMR studies of (1-53), the first fragment displaying tertiary interactions, show that a subcore is fully formed and the alpha-helix (residues 12 to 24) is of fluctuating structure. Fragments (1-53) and (1-60) share many properties with molten globule-like structures, with varying degrees or order. Fluorescence properties of the native fold are gradually recovered from fragments (1-60) to full-length C12, together with a decrease in hydrophobic exposure. A small degree of co-operativity of formation of structure appears when residue 60 is added, gradually increasing as residue 62 is added, but a full two-state co-operative transition appears only on addition of Arg62 and Val63. We believe this is the result of correct side-chain packing of the hydrophobic core, capping the major elements of secondary structure in C12 at this late stage, which is probed by the complete recovery of the fluorescence of the unique Trp5. The structures that develop as the polypeptide chain increases in length parallel the structural features present in the nucleus for the folding of intact protein, which develops in the transition state. The folding nucleus consists of much of the helix and the interactions made by Ala16 in the helix with residues in the core, especially with Leu49 and Ile57, with the rest of the structure being formed only very weakly in the transition state.

Following co-operative formation of secondary and tertiary structure in a single protein module.

We have prepared a family of peptide fragments of the 64 amino acid protein chymotrypsin inhibitor (CI2), corresponding to progressive elongation from the N terminus, in order to elucidate the basis of conformational preferences in single-domain proteins and to obtain insights into their conformational pathway. Structural analysis of the fragment comprising the first 50 residues, CI2(1-50), indicates that it is mainly disordered, with patches of hydrophobic residues exposed to the solvent. Structural characterisation of the fragment CI2(1-63) which lacks only the C-terminal glycine, Gly64, shows native-like structure in all regions of the fragment. The study provides insights into the contribution of specific residues to the stability and co-operativity of the intact protein. We define a phiNMR value, derived from chemical shift analysis, which describes the build-up of structure at the level of individual residues (protons). All the macroscopic probes used to study the growth of structure in CI2 on elongation of the chain (circular dichroism, fluorescence and gel filtration) are in agreement with the residue-by-residue description by NMR. It is seen that secondary and tertiary structure build up in parallel in the fragments and show similar structures to those developed in the transition state for folding of the intact protein.

Towards a complete description of the structural and dynamic properties of the denatured state of barnase and the role of residual structure in folding.

The detailed characterization of denatured proteins remains elusive due to their mobility and conformational heterogeneity. NMR studies are beginning to provide clues regarding residual structure in the denatured state but the resulting data are too sparse to be transformed into molecular models using conventional techniques. Molecular dynamics simulations can complement NMR by providing detailed structural information for components of the denatured ensemble. Here, we describe three independent 4 ns high-temperature molecular dynamics simulations of barnase in water. The simulated denatured state was conformationally heterogeneous with respect to the conformations populated both within a single simulation and between simulations. Nonetheless, there were some persistent interactions that occurred to varying degrees in all simulations and primarily involved the formation of fluid hydrophobic clusters with participating residues changing over time. The region of the beta(3-4) hairpin contained a particularly high degree of such side-chain interactions but it lacked beta-structure in two of the three denatured ensembles: beta(3-4) was the only portion of the beta-structure to contain significant residual structure in the denatured state. The two principal alpha-helices (alpha1 and alpha2) adopted dynamic helical structure. In addition, there were persistent contacts that pinched off core 2 from the body of the protein. The rest of the protein was unstructured, aside from transient and mostly local side-chain interactions. Overall, the simulated denatured state contains residual structure in the form of dynamic, fluctuating secondary structure in alpha1 and alpha2, as well as fluctuating tertiary contacts in the beta(3-4) region, and between alpha1 and beta(3-4), in agreement with previous NMR studies. Here, we also show that these regions containing residual structure display impaired mobility by both molecular dynamics and NMR relaxation experiments. The residual structure was important in decreasing the conformational states available to the chain and in repairing disrupted regions. For example, tertiary contacts between beta(3-4) and alpha1 assisted in the refolding of alpha1. This contact-assisted helix formation was confirmed in fragment simulations of beta(3-4) and alpha1 alone and complexed, and, as such, alpha1 and beta(3-4) appear to be folding initiation sites. The role of these sites in folding was investigated by working backwards and considering the simulation in reverse, noting that earlier time-points from the simulations provide models of the major intermediate and transition states in quantitative agreement with data from both unfolding and refolding experiments. Both beta(3-4) and alpha1 are dynamic in the denatured state but when they collide and make enough contacts, they provide a loose structural scaffold onto which further beta-strands pack. The beta-structure condenses about beta(3-4), while alpha1 aids in stabilizing beta(3-4) and maintaining its orientation. The resulting beta-structure is relatively planar and loose in the major intermediate. Further packing ensues, and as a result the beta-sheet twists, leading to the major transition state. The structure is still expanded and loops are not well formed at this point. Fine-tuning of the packing interactions and the final condensation of the structure then occurs to yield the native state.

HPr as a model protein in structure, interaction, folding and stability studies.

The small size and lack of disulphide bonds or cofactors in the Histidine-containing phosphocarrier protein (HPr) makes it an attractive system with which to study structure, interaction to its enzymatic partners, and its stability and folding. Here we give an overview on the immense work that has been performed on this protein and we will show that HPr has been widely used as a model protein to study important aspects in modern Structural Biology.

Determination of cadmium in hair and blood by tungsten coil electrothermal atomic absorption spectrometry with chemical modifiers.

Three chemical modifiers ((NH(4))(2)HPO(4), NH(4)H(2)PO(4), and Pd as Pd(NO(3))(2)) were evaluated for the determination of Cd in acid-digested solutions of hair and blood using electrothermal atomic absorption spectrometry in a tungsten coil atomizer (TCA). All modifiers caused some thermal stabilization of Cd when compared to the behavior observed in nitric acid medium. The best effects were observed in 15 mug ml(-)(1) Pd medium; the characteristic mass of Cd was 0.3 pg and the method detection limits were 0.009 mug g(-)(1) in hair and 0.2 mug l(-)(1) in blood. In addition to a slight thermal stabilization effect, Pd also increased the sensitivity for Cd by ca. 40% and the tungsten coil lifetime by 20% (i.e. from 300 to 360 heating cycles), reduced background signals, and eliminated condensed phase interferences caused by concomitants. The accuracy (3.2% as mean relative error in the Pd modifier) was checked for the determination of Cd in acid-digested solutions of certified reference materials of human hair and blood and by recoveries of Cd in spiked hair and blood samples by both TCA and a graphite furnace procedure. All results obtained in chemical modifiers are in agreement at a 95% confidence level.

Determination of cadmium and lead in mussels by tungsten coil electrothermal atomic absorption spectrometry.

In this work it was evaluated the determination of Cd and Pb in mussels by tungsten coil electrothermal atomic absorption spectrometry (TCA-AAS). A critical and comprehensive study of the effects caused by Pd, Mg, ascorbic acid, and binary mixtures of these compounds on the atomization of Cd and Pb in acid digested solutions of mussels was performed. Palladium and mixtures containing it were useful to increase sensitivity and thermal stability of Cd and Pb. Additionally, the coil lifetime was increased and the background signals were decreased in these modifiers. All these favorable effects were analytically exploited to determine Cd and Pb in samples of mussels. The proposed methodology was validated using two certified reference materials (oyster tissue and mussel). No statistical difference was observed between determined and certified values at a 95% confidence level. Cadmium and Pb in Chilean bivalve mussels were determined by TCA and by graphite furnace-AAS. Again, the results showed statistical agreement.