TXRF Spectrometry as a Powerful Tool for the Study of Metallic Traces in Biological Systems by Shirley Wang

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TXRF Spectrometry as a Powerful Tool for the Study of Metallic Traces in Biological Systems Ramón Fernández‐Ruiz Laboratorio de TXRF. Servicio Interdepartamental de Investigación (SIdI). Facultad de Ciencias. Universidad Autónoma de Madrid (UAM), Cantoblanco 28049, Madrid, Spain. ramon.fernandez@uam.es Received 31 March 2014; Accepted 17 April 2014; Published 15 December 2014 © 2014 Science and Engineering Publishing Company Abstract Perhaps because of this, the TXRF is a technique little known and extended when compared with more This work describes the basic principles of the Total‐ conventional techniques such as atomic absorption reflection X‐Ray Fluorescence (TXRF) spectrometry, as well as some of their most excellent applications, in the study of (AAS) or plasma (ICPS) spectroscopies. The geometric metal traces in different materials but mainly in biological variation of the Source‐Sample‐Detector system, which systems. The TXRF is an analytical technique that combines differentiates the TXRF with respect to the the versatility and potentiality of the elemental analysis by conventional EDXRF, introduces drastic consequences means of X‐ray energy dispersion with an extremely low in physical and analytical aspects which differentiate it detection limits, in the order of picograms. The from the spectroscopic conventional X‐ray techniques. microanalytical capability of the TXRF allows us to analyze Working in condition of total reflection implies the very small sample quantities. Both factors are of great generation of a field of X‐ray standing waves (XSW) importance in the research of metallic traces in biological systems because it allows a fast, precise and accurate on the surface of the sample‐carrier reflector that is the characterization of the elemental fingerprint at trace level. reason of the unique characteristics that TXRF Their successful application in studies of coordination presents. This work tries to show a brief introduction kinetic of the new platinum based anti‐tumor drugs with the to its physical fundaments as well as its analytical nuclear DNA, its application in the study of the metal profile benefits and limitations with a better criterion. The of healthy and cancerous human tissues, or even its second goal is to show a brief summary of the more application in the study of the processes of metal diffusion relevant applications developed in the field of biology through cellular membranes, turns the TXRF into a powerful tool, still to discover, within the field of biomedical and biomedicine. sciences and general biosystems. So, the main objective of this review is to show a briefly panorama of the TXRF spectrometry and some of its more relevant applications developed up to now, mainly in the biological field, with the purpose that reader can evaluate its potential future applications. Keywords TXRF; Microanalysis; Trace Metals; Biomedicine; Biomaterials; Biosystems; Atomic Analysis

Introduction Total‐reflection X‐Ray Fluorescence (TXRF), is an X‐ ray spectrometry technique that derives from the classic energy dispersive X‐ray fluorescence (EDXRF) (Klockenkämper, R., 1997). This implies that both techniques are confuse and associates as equivalent.

Historical Background In 1923, Compton discovered the phenomenon of total reflection X‐ray (Compton, A. H. , 1923) which showed that the X‐ray reflectivity of a flat material increased rapidly below a certain critical angle around 0.1 degrees. It was not until 1971, almost 50 years later, when Yoneda and Horiuchi (Yoneda, Y. and Horiuchi, T., 1971) had the ingenious idea of applying total reflection geometry to produce excitation emission X‐ ray fluorescence of the atoms in a small amount of material deposited over a reflector (FIG. 1). This idea materialized in a few, but innovative work during the 70ʹs and early 80ʹs (Aiginger, H. and Wobrauschek P., 1974, Aiginger, H. and Wobrauschek, P., 1975, Schwenke, H. and Knoth, J., 1982).

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Th hese studies assumed thee formal app pearance of the tottal reflection n X‐ray fluorrescence (TX XRF) in the field f of X‐ray specttrometry. Th he appearancce of the TX XRF tecchnique allo owed that the X‐ray spectromeetry, rellegated sincee the decad de of the 600 mainly to the analysis of major m elemen nts, could be reintroducced, with more success, in the ffield of chem mical analysiis at traace and ultrra‐trace leveels. But thiis developm ment occcurred abou ut a century y after the groundbreak g king ressearch of Bun nsen and Kirrchhoff regarrding the usse of flame techniqu ues for spectrroscopic anallysis.

FIG. 1. BASIC DIAGRAM OF F TOTAL REFLE ECTION X‐RAY Y UORESCENCE ((TXRF) GEOME ETRY FLU

he continu uous tech hnological developm ment, Th insstrumental cost c minimizzation, stand dardization and rou utine incorrporation of o almost all analyttical meeasurements at the micro (0.01 ‐ 1% %), trace (0.1‐‐100 pp pm, mg/kg) aand ultra tracce (less than 100 ppb, μg//kg) thee techniquess of Atomicc Absorption n Spectroscopy (AAS) and Ind ductively Coupled Plasm ma Spectroscopy (IC CPS) during g this long period, cou upled with the errroneous asso ociation of TX XRF with con nventional X XRF, and additionally the intelleectual challen nge posed to o the ressearcher to break with h 100 yearrs of scientific bib bliography, have h caused d that the in ncorporation n of thee TXRF has b been slow an nd, even tod day, little kno own and widespreaad for the biiomedicine and a much otther sciientific fieelds. Moreover, thee continu uous im mprovement of the ICP‐M MS techniqu ue, in particu ular with the development of th he collision ccell to minim mize intterfering speecies, togetheer with an elevated e cosst of TX XRF instrum mentation, bu ut not for its i instrumen ntal maaintenance, h have contribu uted to the stagnation off the nu umber of TXRF T instru uments arou und the wo orld (Feernandez‐Ru uiz, R., 2008)). Until receently, the major draawback of th he TXRF tech hnique was itts high cost. But thaat barrier, w which has beeen perhaps the t main reaason forr not be present at the same s level as a AAS or IC CPS

hniques, hass begun to b be reduced. The emergence tech of n new generattion technolo ogies as X‐ra ay microsourrces witth high‐flux x, policapillaar X‐ray op ptics and SD DD dettectors with h high reso olution and counting rate r (Sillicon Drift Detector) D hav ve allowed the t progresssive min nimization o of costs of th he TXRF instrrumentation n. In fact, compact T TXRF instrum ments have a appeared in the ma arket using th hese new tecchnologies, su uch as TXRF F S2 PiccoFox (Brukeer‐Nano, Gerrmany), TXR RF NanoHun nter (Rigaku, Japan n) or the ʺWO OBISTRAXʺ TXRF vacu uum cha amber (Atom mInstitut, X‐‐Ray Lab, Vienna, V Austria) at a cost from 60.000 € deepending on n its equipm ment lev vel (Tsuji, K. et al, 2004). Nowadays, these costs are alreeady compeetitive with cconventional AAS or IC CPS instruments so,, this fact open the doorss to the TXRF F in all scientifc an nd industriaal fields wh here the meetal tracces analysis are necessary y. In parallel, thee TXRF specctrometry ha as been ablee to ma ature due mainly m to th he celebration of bienn nial con nference monographicss in TXRF F and relaated meethods. The in nternational TXRF confeerences, held up to now, have been as fo ollows: 1986 in Geesthaacht (Geermany), 19988 in Do ortmund (G Germany), 1990 Vieenna (Austria), 1992 in G Geesthacht (G Germany), 1994 in T Tsukuba (Jap pan), 1996 in n Dortmund (Germany) aand Ein ndhoven (N Netherlands),, 1998 in Austin, Teexas (US SA), 2000 in n Vienna (A Austria), 2002 in Madeeira (Po ortugal), 20033 in Awaji IIsland, Hyog go (Japan), 2005 in Budapest (H Hungary), 20007 Trento (Italy), 20099 in Goteborg (Swed den), 2011 in n Dortmund (Germany) aand 201 13 in Osaka ((Japan). To date, TXRF has begun to o be applied d successfully y to solv ve many pro oblems in in nherently diffficult materials in many scienttific fields su uch as medicine (Carvallho, M.L L. et al., 20007), environm mental sciencces (Fernánd dez‐ Ruiz, R. et al., 22002), archaeeology (Fern nández‐Ruiz, R., and d Garcia‐Heeras, M., 20008), physiccs of materials (Feernández‐Ruiiz, R. and d Bermudeez, V., 20005), electronics (Neeumann, C. and Eichin nger, P., 19991), nan notechnology y (Fernándeez‐Ruiz, R. et e al, 2008), art (Kllockenkämpeer, R. et al, 2000) and biolo ogy (Feernández‐Ruiiz, R. et al, 1999) among others. But sstill a lo ong way rem mains to be d done within the scope off its pottential appliccations. As w we can see in i FIG. 2, since thee pioneering article by Yo oneda and H Horiuchi in 1971 (Yo oneda, Y. an nd Horiuchii, T., 1971), the numberr of scieentific publications relaated with TXRF T or th heir app plications waas very low,, around zerro, until 19855. It wa as from this year when the first TXR RF instrumeents were commerccialized. Thee pioneer comercial TX XRF instruments weere developeed in Germany by Rich h &

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Seifert and, from this time, the new TXRF technology began to be more and more recognized in the international scientific field. 100 1400 80

1200

Papers per year

1000 60 800 40

600

n1 cos 1  n2 cos  2 (1) where n1 and n2 are the refractive indices of media 1 and 2 respectively (Casas, J., 1994). For X‐rays, any medium is optically less dense than vacuum and any solid optically less dense than air, ie nsolid < nair ≈ 1. If the angle α2 is zero, the refracted beam will emerge tangentially to the interface. Therefore, there must be a critical angle for the incident beam, 1 = c, where the refracted beam acquires an angle α2 = 0. n 1

400 20 200 0

1

1 *

1

0 1972 1976 1980 1984 1988 1992 1996 2000 2004 2008 2012

Year

1 * 2

2

FIG. 2. NUMBER OF PAPERS PUBLISHED BY YEAR (BARS) AND ITS ACCUMULATED SUM (LINE), WHERE “TXRFʺ HAS BEEN USED AS KEYWORDS (SCIFINDER 2014).

The database of technical literature is at present around 1407 international scientific publications. This value indicates that there are still many scientific fields where TXRF can demonstrate its potential. FIG. 2 also shows the annual number of scientific publications recorded in the same period. As we can see, the overall annual scientific productivity increased with a positive slope from 1985 to 2000, being stable since that year with a value around 60 international publications per year. The punctual biannual increase that are visible in the FIG. 2 coincides with the years after the celebrations of the international TXRF conferences, because the scientific contributions presented at these conferences usually have been compiled and published in special issues of the journal, Spectrochimica Acta Part B: Atomic Spectroscopy. Today, these are the more important and extended technical literature (Von Bohlen A., 2009) alongside the excellent monograph published by Reinhold Klockenkämper (Klockenkämper, R., 1997). Physical Background A X‐ray beam follows a straight path through a homogeneous medium. But, just like visible light, if the X‐ray beam finds, in its path, a new medium, its path varies over the original. This means that part of the X‐ray beam is reflected toward the first half and the remainder is refracted into the second half, as shown in FIG. 3. Incidence angle α1 and refracted angle α2 are defined by the X‐ray beam with respect to the interface, following the Snellʹs law

FIG. 3. REFLECTION AND REFRACTION OF AN X‐RAY BEAM AS A FUNCTION OF REFRACTIVE INDEX N MEANS THAT IT CROSSES. LEFT N2> N1 AND RIGHT, N2 <N1

According to Snellʹs law (1) and taking into account that nair1 cos  c  nsolid . (2)

The refractive index of the solid reflector has the complex value nsolid  1    i  (3)

where i is the imaginary unit and the parameters δ and β are the parameters of scattering and absorption of the material respectively (Van Grieken, R. and Markowicz, A., 2002). From equations (2) and (3) we can estimate the value of the critical angle of any material in the form

 c  2 (4) For incidence angles less than c, Snellʹs law does not give real values for the angle of refraction 2. In this case, the X‐ray beam does not penetrate in the second medium and the interface behaves like a mirror close to the ideal, fully reflecting the incident beam to the first medium. Thus, for angles below the critical angle, the incident beam interferes constructively with the reflected beam, generating a field of X‐ray standing waves (XSW) as FIG. 4 shown. The phenomenon of constructive interference generated in the XSW field implies that the inside intensity is amplified. Thus, on average, the excitation

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in TXRF geom metry is app proximately twice that for con nventional XRF. X If theree is a depositing materiaal in thee XSW, an nother way y of undeerstanding this am mplification is considering that the attoms are excited by both the inccident beam and the refleected beam. The cro oss section o of the station nary wave fieeld is known n as “m magic trianglee”.

deffined as the d depth that X X‐ray beam p penetrates in the meedium such aas its intensitty is reduced d by a factor 1/e. No ote that in tottal reflection condition , the penetrattion of tthe evanesceent wave is o only of few n nanometers aand rem mains practiically constaant for ang gles below the crittical angle. FIG. F 5 show ws the intenssity distributtion for a sample with a typ pical thickneess of 500 nm dep posited on a Si reflector and the fluo orescence sig gnal of tthe Si reflecto or with respeect to the angle of incidence of tthe primary b beam.

FIIG. 4. CROSS SE ECTION OF TH HE FIELD OF ST TANDING WAV VES O ON THE SURFA ACE OF A REFL LECTOR BELOW W THE CRITICA AL ANGLE OF TOTA AL REFLECTIO ON.

Inttensity of thee TXRF Signal Bedzyk et al deduced d in 1989 1 a generral equation for thee distribution n of X‐ray intensity, i I(α α,z) in the XSW X field in terms of their ang gular depend dence α and d its position z abo ove the reflector (Bedzy yk, M. J. ett al, 19889). The shaape of this distribution d is given by the exp pression

   2 z I  , z   I 0 1  R    2 R   cos        (5)  W     XSW wh here I0 is th he intensity of the prim mary beam, the arg gument of cosine c is thee phase difference betw ween inccident and reflected r wav ves, broken down into two com mponents, a a spatial disttance 2z/λXSW X and a ph hase shiift (α). Finallly, R(α) is th he reflectivity y of the mateerial useed as reflector, or samp ple carrier, fo or each angle of inccidence α. If a a sample of thickness s is introducced within XSW X field, the atom ms will be ex xcited and the t fluoresceence em missions inteensity will be propo ortional to the inttensity of th he XSW field d. Now assuming that the con ntribution to o the spectraal backgroun nd of the sam mple carrrier is in nfinitely th hick, opticaally flat and homogeneous, the intensity of the ev vanescent wave w field that peneetrates into the t reflector is given by the equ uation  z  (6) I  , z   I 0 1  R    2 R   cos      exp e      z e 

wh here the new w parameter zze, or depth o of penetration n, is

FIG. 5. INTENSIT TY OF THE FLU UORESCENCE SIGNAL (BLUE E) COMBINED W WITH THE BACK KGROUND SIG GNAL (BLACK)) REFLECTOR S SPECIMEN OF SI IN THE VICIINITY OF THE C CRITICAL ANG GLE CONDITION

As FIG. 5 sho ows, below the criticall angle of the refllector c, TXR RF measurem ment region, the intensity y of thee fluorescen nce emission n (blue) is approximattely twiice that for h higher angless, XRF measu urement regiion. In the case of the t backgrou und intensity y (black) bellow thee critical angle, a the contribution n of spectral bacckground fro om the reflecctor decreasees very quicckly beccause the intensity i of incident X‐ray X beam is refllected almosst entirely an nd the rema aining intenssity pen netrates only y a few naanometers in n the reflecctor ma aterial. For angles abo ove the crittical angle the con ntribution off spectral bacckground in ncreases quicckly and d in a linearr fashion. Th he decrease in the spectral bacckground in a factor 5000 is mainly due d to work k in tota al reflection region or n not. If this reeduction in the bacckground iss convoluteed with th he factor two t asssociated with h the ampllification of the elemen ntal fluo orescence signal below critical angle, the signall to noiise ratio ob btained on condition of TXRF over o con nventional XRF X is up tto 3 orders of magnitu ude hig gher. Thus, the resultin ng increase in sensitiv vity allo ows to the TXRF T detectt masses deeposited on the surrface of the reflector of on nly a few piccograms. In tthis wa ay, if the meaasurement an ngle αmeas is ffixed about 770% of the critical angle, the T TXRF condittion is assu ured

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with a high counting rate capacity (SDD) and all next with a source of Cr, or a synchrotron line, in order to optimize the excitation of low energy transitions, it is possible to analyze by TXRF light elements like B, C, N, O, F, Na and Mg, with detection limits in the nanogram range (Streli, C. et al, 2004). Sr

14000

Analytical Background

Elements with atomic numbers below Z=13 can not be determined in conventional instruments of EDXRF or TXRF, because their fluorescence emissions are of so low energy that are absorbed quickly on their way from the sample to the detector. In order to increase the range of elements in the TXRF to lighter elements, there have been several instrumental approaches, mainly by the group of Peter Wobrauschek and Christina Streli in the AtomInstitut of Vienna. This group has recently succeeded in the developing of a special TXRF camera, known as “Wobi‐module”, which is capable of working under vacuum. With this module, using an ultrathin window and a detector

10000 Ga Zn

8000

4000 2000

Cu Ni

Fe Mn Cr

Compton

6000

Si K (Reflector)

Intensity (cts)

TXRF is a ʺmicro‐analyticalʺ technique, because only small amounts of sample, around 0.1 to 10 micrograms, properly deposited on a reflector are analysed. The sample is deposited on a thin layer with a thickness between 0.1 and 10 μm thick depending on the type of matrix analyzed (Klockenkämper, R. and Von Bohlen, A., 1989). Under these conditions, the effects of absorption and secondary excitation are negligible and the model of infinitely thin film can be applied. The great advantage inherent is that in the process of quantification by TXRF can be neglected, in most cases of troublesome matrix effect. From the analytical point of view, this is by far the biggest advantage that TXRF has over conventional XRF. As a result of being able to bypass the matrix effect, the relative sensitivities in TXRF have a universal character, regardless of the analysed matrix. Assess the relative sensitivities of a TXRF instrument is a relatively simple task. FIG. 6 shows a TXRF spectrum of the deposition on a quartz reflector of 10 microliters of a solution of multielement pattern where the concentration of each of its elements is 1 ppm (ng/μL). Thus, the net mass of each item deposited is 10 nanograms. From certified standards, where the absolute mass of deposited element can be known, it is easy to calculate the relative sensitivities of each element. Regarding the elemental range of TXRF, all elements with Z >13 can be analyzed if the appropriate excitation sources is used.

12000

Ti Sc Ca

Rayleigh Mo K (Source)

and, as consequence, the spectral measurements obtained for any material deposited over the surface of a reflector, allow the analysis of the present elements with the best signal to noise ratio and lower detection limits than the X‐ray spectrometry is able to get today. From the physical point of view, this is the main differentiating factor of TXRF over conventional XRF.

0 2

Energy (keV)

FIG. 6. TXRF SPECTRUM OF A MULTIELEMENT PATTERN SOLUTION WHERE THE MASS DEPOSITED IN EACH OF THE ELEMENTS IS 10 NANOGRAMS FOR AN INCIDENT BEAM OF MO KΑ X‐RAY AND ACQUISITION TIME OF 300 S. TRANSITIONS CORRESPOND TO K LINES FOR EACH ONE OF THE ELEMENTS

From an analytical point of view, TXRF has several strong advantages with respect to other more conventional techniques. The more important is that, once relative sensitivities curves are known, their values remain invariant for years and their update is only necessary when the detection system or the geometry is varied. Under these conditions the TXRF shows all its potential in the atomic chemical analysis, due mainly to its simplicity of understanding, quantification and interpretation of the TXRF spectra. The X‐ray elemental analysis within angular region of total reflection simplifies the main and no‐trivial problem of the conventional XRF, the non‐linear matrix effect, in a simple and linear relation (Van Grieken, R. and Markowicz, A., 2002). So, the master equation for quantification by TXRF is given by the linear relationship

Cx  Cref

N x Sref (7) N ref S x

where Cx and Cref are the concentrations of element x and the internal standard ref, Nx and Nref are their net intensities and finally, Sx and Sref are their relative sensitivities respectively. As shown in equation 7, the most suitable quantification method for TXRF technique is the internal standard. This method is based on the addition of an aliquot of a known concentration of monoelemental pattern that is not

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present in the problem sample. So, the simple deposition of an aliquot of the standarized sample on a reflector, as shown in FIG. 7, is enough to quantify the elements present in the problem sample solution.

FIG. 7. FINAL DEPOSITION OF AN ALIQUOT OF THE STANDARDIZED SAMPLE ON A QUARTZ

The volume of sample deposited over reflector can be increased or decreased, within certain limitations imposed by the technique (Klockenkämper, R. and von Bohlen, A., 1989), to get the best counting ratio in the process of acquiring the spectrum. Under these conditions, the measurable concentration range of TXRF can vary from a few ppbʹs to thousands of ppmʹs, which imply an analytical concentration range of 105, that is five order of magnitude from the higher to the lower concentration of the elements present in the problem sample. Comparison with Other Techniques TXRF is a competitive technique when compared with AAS or ICP‐OES because detection limits (DL) for liquid samples are equivalent and vary from hundreds of ppt (ng/L) to tens of ppb (μg/L). ICP‐MS is even more sensitive for liquid samples, with DL´s around 1 ppt (ng/L), and as consequence is more adequate for ultratrace analysis than any other technique. For the analysis of solid samples by TXRF, as in the AAS and ICPS techniques, the conventional treatment way is the acid digestion of the sample but, in addition, TXRF allows the quick and direct qualitative and mass ratio evaluation of the elements present in the analized sample with only some micrograms of sample and, more important, without chemical distortion via acid digestion. This is the more valuable analytical characteristic that TXRF posses, its inherent ʺMicroanalytical capabilityʺ.

One of the biggest advantages of analysis by TXRF is that it allows simultaneous qualitative inspection of the atomic fingerprint of the material studied. This feature allows easily monitoring processes through the comparative study of their spectra, in each of its phase. Moreover, the ability to analyze liquid or solid phase directly, allows tracking the analysis process in a comprehensive manner. As consequence allows easily evaluating the loss of elements during the processes of digestion or the induction of impurities during the preparation process. On the other hand, the microanalytical character, inherent to the TXRF, can be an advantage for certain types of studies, but also a drawback, to adequately assess a heterogeneous sample. In these kinds of samples, like any other analytical technique, the importance of proper sampling and homogenization in the preparation process is essential. One of the great drawback of TXRF with respect to techniques such as ICP‐MS, is its limitation in the analysis of light elements Z<13. Nevertheless, in the process of quantifying, techniques such as AAS or ICPS require the performance of external calibration curves while TXRF technique only requires the addition of an element as internal standard of known concentration and of course, that it is not present in the studied sample. This simplification of the quantification process implies an important savings of time and analytical cost. From the instrumentation cost point of view; the ʺflameʺ techniques can be grouped in three levels. First, the Atomic‐Absorption level (AAS), which includes graphite chamber, the cost oscillates around 50.000 euros. Second, the Optic‐Plasma level (ICP‐ OES), where the cost oscillates around 90.000 euros. Finally, the Mass‐Plasma level (ICP‐MS) where the prices oscillate around 140.000 euros. In the case of TXRF instrumentation for analytical aplications, prices also are at different levels. The first level is constituted by the more cheaper ʺWOBIʺ TXRF chambers from the AtomInstitut in Vienna, with prices around 60.000 euros. Second level is constituted by the robust TXRF spectrometer S2 PicoFox, from Bruker‐nano, with prices around 90.000 euros and finally, a third level, constituted by the NANOHUNTER spectrometer from Rigaku, where additional grazing incidence studies can be carried out, with prices around 120.000 euros. Nowadays, TXRF instrumentation only requires electrical supply, while the flame or plasma techniques mainly require high purity gas, whose price greatly increases the cost per sample of the flame/plasma techniques compared with the TXRF technique. This is

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the more significative difference, if cost per sample is compared, because for the sample preparation, standardization and cleaness, the necessity of reactives and material is similar. Main Fields of Application of the TXRF In the environmental field, the TXRF has been applied in many matrices, to studies of pollution control (Fernández‐Ruiz, R. et al., 2006). In this field, the TXRF technique is very suitable for analyzing all types of water. Almost without prior preparation and to ppb levels it is possible to analyze drinking water (Prange, A. et al., 1987), river (Prange A. et al., 1993) or rain (Stößel R. P. and Prange A., 1985). Sea water or contaminated sludge can also be analyzed directly to levels of ppmʹs, or ppbʹs, conducting a pre‐removal of matter in suspension or removal the majority salts composition (Prange, A., 1985). Moreover, the analysis of air pollution due to suspended particles in aerosol form is a section where TXRF has begun to be applied with satisfactory results. Aerosol particles can be collected on environmental survey stations on filter paper with different pore size, which are easily digested in acid. But more recent applications are based on the use of cascade impactors that allow direct deposition of the different size fractions of the aerosol, on TXRF sample holder (Streit, Niklaus et al., 2000). A recent work presents the application of TXRF, among other techniques, to study the problem of induction of uranium in the crops grown on a mining site in Hungary (Alsecz, A. et al., 2007). Also, TXRF has been applied to the study of different species of trees as bioindicators of pollution (Sirito de Vives, A. E. et al., 2007). The industrial applications of the TXRF is where more has been introducing so far. Particularly in the field of microelectronics by developing techniques for thin film characterization as well as in the study of impurities in the wafers used in microelectronics, both directly (Hellin, David et al., 2005) or using vapor decomposition (VPD‐TXRF) (Shimazaki, Ayako et al., 2006). The industry of synthesis of new materials, such as mixed‐phase lithium niobate tantalate (LNT) (Fernández‐Ruiz, R. and Bermudez, V., 2005) or solid solutions of CuInSe:Ga (Fernández‐Ruiz, R. et al., 2001) also has benefited from the versatility of TXRF for the stoichiometric studies of such compounds. Microparticles and nanoparticles systems with high interest as new catalytic materials (Fernández‐Ruiz, R. et al., 2010b), luminiscent materials (Fernández‐Ruiz, R. et al., 2010a) or voltaic pile (Fernández‐Ruiz, R. et

al., 2009) also has been studied by TXRF. On the other hand, TXRF has also addressed the analysis of products related with consumer industry. There are applications developed in the wine industry for the analysis of metal contaminants in wines (Carvalho, M. L. et al., 1996) or in the oil industry for the analysis of the crude fractionation processes (Ojeda, Nelson et al., 1993). In the field of archaeological applications, the TXRF has also been incorporated into very diverse facets. A very interesting review, written by Alex Von Bohlen, shows the versatility of TXRF to tackling complex archaeological problems (Von Bohlen, A., 2004). In particular in the field of archaeological ceramics, the first applications of TXRF were developed in our own laboratory (García‐Heras, M. et al., 1997). Biomedical and Biological Applications As a summary we can say that TXRF has the following distinguishing features: microanalytical capability, easy sample quantification by the simple addition of an internal standard, analysis of concentrations in the range of ppbʹs, reduced analysis time, minimum cost of analysis and maintenance of equipment because today only a plug is necessary and finally, capacity for analysis of solids and liquids directly. For this reason, the field of biomedical knowledge can find a powerful ally in the TXRF spectrometry. In this way, TXRF has also made an unobtrusive appearance in the biomedical field. The number of applications is few but is increasingly widespread. One of the first applications of TXRF in the biomedical field was the hability of direct analysis of microtome sections of biomedical and vegetables tissues, developed by Klockenkämper et al. (Klockenkämper, R. et al., 1989). Samples were prepared by means of a freezing microtome with a thick around 10 microns and a mass around 200 micrograms directly deposited on a quartz reflector and spiked with 10 nanograms of Ga. The method was applied to the analysis of vegetable and animal foodstuff (nuts, mushrooms, shrimps) and to the analysis of various tissues (liver, human lung). Quantitative results, in the range from 200 ppb to 700 ppm, were achieved for Zn, Mn, Fe, Cu, Br, As, Sr, Rb, Se, and Ni. The analysis of Pb in blood was investigated by Ayala et al. (Ayala, R. E. et al., 1991) for two kind of donors; one ocuppationally exposed to lead contamination in a car battery factory and other unexposed. In this case

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the analysis was performed by mean of the addition of 5 ppm of Sr as internal standard, the deposition of 2 uL on a quartz reflector and the ashed in a low temperature oxygen plasma. The elements detected in blood were K, Ca, Ti, Cr, Fe, Ni, Cu, Zn, Pb, Rb and Sr. The detection limit achieved for Pb was of 30 ppb. Occupationally exposed donors clearly showed higher lead concentrations, between 230 and 680 ppb, than unexposed individuals with concentrations of 100 ppb or less. Savage et al. (Savage, I. et al., 1998) also investigated the effect of chemical modifiers in the analysis of blood plasma by TXRF. They analyzed the elements As, Br, Cd, Ca, Cl, Co, Cu, I, Fe, Pb, Mn, Mo, Ni, Se, Sn and Zn in a concentration range from 1 ppb to 270 ppm. An interesting application was developed by Günther et al. (Günther, Klaus et al., 1995) where TXRF was used for the simultaneous determination of Ca, Cu, Fe, K, Mn, Rb, Sr and Zn in 12 different vegetable foodstuffs and their cell fractions after mechanical cell breakdown. The homogenates were separated into citosols (liquid fraction) and pellets (solid fraction) by centrifugation. Before their analysis by TXRF, samples were digested with nitric acid and spiked with Ga as internal standard. In the study, the elemental distributions of each one of the fractions were determined, obtaining that, on average Sr, Ca and Fe were mainly bound to pellet components. Wittershagen el al. (Wittershagen, A. et al., 1998), developed a procedure for the determination of metal cofactors in respiratory chain complexes. The two terminal oxidades, cytochrome c oxidase and quinol oxidase, isolated from the soil bacterium Paracoccus denitrificans, were transferred from their usual salyne buffer into a solution of 100 mmol/L tris(hydroxymethyl)aminometano acetate (TRIS) with 0.02 % of Triton X. By this procedure an improved signal/noise ratio was obtained. Without any decomposition, elements Fe, Ni, Cu, Zn, Mn and Mo could be determined with high accuracy. Besides also sulfur could be determined in protein samples. Fernández‐Ruiz et al. (Fernández‐Ruiz, R. et al. 1999) applied TXRF for monitoring the molecular introduction kinetic of the Pt‐Berenil and cis‐DDP compounds that took place when crossing the biological barrier of the HeLa cells. The medicines which interacted with the DNA on the nucleus were also quantified. This work opened the intracellular metal studies in conjunction with an adequate technique of subcellular constituent separation by

TXRF. In this work, a sample volume of only 100 μL with Pt concentrations from 3 to 30 ng/mL was determined with a relative estandard deviation between 2% and 8%. Gonzalez et al. (Gonzalez, Mauricio et al., 1999) used for first time the TXRF technique for the determination of Cu, Fe, Zn, Ca and S in different types of tumoral mammalian cultured cells; clonal human cell lines HepG2, Caco‐2 and HeLa, clonal cell lines from mice NIH 3T3 and N2A and finnally, clonal cell line from rats B12. The goals of the work were as follows;the evaluation of their elemental distribution, intracellular concentrations and the changes induced in their proportions when cells were chronically exposed to copper. Results indicated that TXRF allowed the detection of total trace metal contents using a minimum amount of cells (1‐2)x106 while (4‐6)x106 cells were sufficient to determine their cytosol/pellet distribution. Carvalho et al. (Carvalho, M. L. et al., 2001) applied TXRF to the study of human amniotic fluid and placenta with the aims to correlate the metal contents with the newborn infants weigth and maternal age. Very low levels of Ni and Sr were found in the amniotic fluid samples and their contents were independent of the age of the mother and weight of the child. Zn was not significantly different in the samples analyzed; however, it was weakly related to birth weigth. Ca and Fe were significantly correlated with mother´s age and newborn weight. Zarkadas et al. (Zarkadas, Ch. et al., 2001) developed a procedure to evaluate U in human urine. The method utilized open vessel digestion with nitric acid and uranium preconcentration by using sodium dibencyldithiocarbamate (NaDBDTC) as the complexing agent. By means of the developed procedure, detection limits around 1 ppb were obtained with recoveries around 100 % and uncertainties lower than 10 %. Zheludeva et al. (Zheludeva, S. I. et al., 2003) took advantage for first time of the angle dependence of the XSW field to study molecular monolayers. This enabled to localize ions in the monolayer with respect to the film‐liquid interface by analyzing the angular curves of their characteristic fluorescence radiation. The possibilities of this method were considered on phthalocyanine, cyclolinear polyorganoxilanes and phospholipid of Langmuir layers on the surface of a liquid and a protein‐lipid film based on Ca‐ATPase on a solid substrate. This work opened a new field of

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application of TXRF with angular variation for the study of diffusion of metals in systems interfaces. Mages et al. (Mages, Margarete et al., 2004) analyzed by TXRF biofilm as bioindicators of polluted water. They found differences in concentration of several orders of magnitude between biofilms grown in polluted water and grown in unpolluted waters. The elements evaluated were K, Ca, Cr, Mn, Fe, Ni, Cu, Zn, As and Sr with masses of sample between 1 and 500 μg. Woelfl et al. (Woelfl, Stefan et al., 2004) applied TXRF to the study of trace metals in planktonic microcrustacean. The mass of sample evaluated was lower to 50 μg in any case and the elements Mn, Fe, Ni. Cu, Zn and As were studied at ppm order. Pepponni et al. (Pepponi, G., 2004) applied TXRF to the study of pollen as an indicator for atmospheric pollution for the C. avellana L. (hazel) pollen colected in five sites of the province of Trento, Italy with different antrophic impact. The elements Al, P, S, K, Ca, Ti, V, Cr, Mn, Fe, Ni, Cu, Zn, Br, Rb, Sr, Ba, and Pb were evaluated and detection limits in the ppb range were obtained. Magalhaes et al. (Magalhães, T. et al., 2006) investigated the elemental distribution of P, S, Cl, K, Ca, Cr, Mn, Fe, Ni, Cu, Zn, Se, Br, Rb, Sr, I and Pb in normal and cancerous tissues of the same individual along several contigous thin sections of each tissue. Carcinoma tissues of colon, breast and uterus on a total of 7 citizen from German population, were analysed directly by TXRF. By the same processes, 10 carcinoma samples of 10 Portuguese citizens from rectum, sigmoid, thyroid, kidney, larynx and lung were evaluated in order to find out a similar correlation pattern in the studied elements in carcinoma tissues. A similar pattern for all the analysed tissues was obtained: increased or constants levels of P, S, K, Ca, Fe and Cu and decreased levels of Zn and Br were found in carcinoma tissues, when compared with the corresponding healthy ones. Ostachowicz et al. (Ostachowicz, B. et al., 2006) studied the trace metal of cerebrospinal and serum human fluids in amyotrophic lateral sclerosis patients by TXRF. The elements Na, Mg, Cl, K, Ca, Cu, Zn and Br were determined in both fluids. For the serum samples higher values for Br were found in the ALS group, for the cerebrospinal fluid lower values of Na, Mg and Zn as well as higher Ca values were found in the ALS group compared to the control group.

Carvalho et al. (Carvalho, M. L. et al., 2007) conducted an interesting summary of studies on elemental profiles between healty and cancerous human for breast, lung, serum, intenstinal, prostate and uterus tissues carried out by TXRF. Suspension of magnetic nanoparticles in aqueous media is nowadays attracting much interest because of their potential for many applications such as biomedicine (Pankhurst, Q. A. et al., 2009) including biomolecules separation, magnetic resonance imaging or drug delivery. Fernandez‐Ruiz et al. (Fernández‐ Ruiz, R. et al., 2008) developed a procedure for the analysis of Fe and traces metals in these systems without sample digestion and in a direct solid way by TXRF. Szoboszlai et al. (Szoboszlai, Norbert et al., 2008) developed a procedure for the direct elemental analysis of cancer cell lines by TXRF by means of the direct deposition of cancer cells in the TXRF reflector. In these conditions the method was applied to human colon adenocarcinomes and the levels of Cu, Zn and Fe were evaluated. Wellenreuther et al. (Wellenreuther, G. et al., 2008) investigated the best deposition conditions for the analysis by TXRF of the followings metalloproteins; hELAC1, insulin, concavalin A, thermilysin and glucose isomerase. The main conclusion of the study was that elaborate sample decomposition was not neccesary in general; instead a direct analysis of proteins samples was feasible. Szoboszlai et al. (Szoboszlai, Norbert et al., 2009) have published a paper compilation of biological applications of TXRF showing a comprehensive overview of the research conducted in this field. In clinical service laboratories, one of the most common analytical task with regard to inorganic traces is the determination of the nutrition‐relevant elements Fe, Cu, Zn and Se. Stonach and Mages (Stosnach, Hagen and Mages, Margarete, 2009) showed that TXRF can be applied in human blood and serum only by dilution and internal standarization of the samples for the routine analysis of the nutrition‐ relevant elements. Espinoza‐Quiñones et al. (Espinoza‐Quiñones, F. R. et al., 2009) studied the bioaccumulation kinetic of lead by living aquatic macrophyte Salvinia auriculata by TXRF. According to the experimental data, both adsorption and bioaccumulation mechanism are present and a competition between phosphorus

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macronutrient and lead for plant growth was observed when great concentration of lead in roots was present. Abraham et al. (Abraham, J. A. et al., 2010) carried out an interesting study of the elemental composition of oral fluids by TXRF and their relation with smoking. The most significant differences in concentration between smokers and non‐smokers were found in saliva samples for S, K and Ca. In addition, a significant difference in the concentration of Cl in gingival crevice fluid was observed between both groups. Fernández‐Ruiz et al. (Fernández‐Ruiz, R. et al., 2011) applied TXRF spectrometry to evaluate the kinetic behavior of the Cr(VI) bioaccumulation process of the bacterium ANCR (Acinetobacter beijerinckii type). The results demonstrate that this new strain of Acinetobacter bacterium is able to reduce the chromium present in the culture medium and, as consequence, it can be used as a promising microorganism for Cr(VI) bioremediation from polluted wastewaters. Additionally this work shows clearly the versatility, potential and sensitivity of TXRF spectrometry for the analytical evaluation of metals in this kind of microbiological systems. Camejo et al. (Camejo, M. I. et al., 2011) applied for the first time TXRF in the research of the human sperm. In particular, to determine the concentrations of Se, Cu, and Zn in semen of patients with varicocele and the relationship with seminal parameters. As main conclusion, a decrease in selenium concentration was associated with detriment of seminal parameters: spermatozoa concentrations, motility and morphology. Antosz et al. (Antosz, F. J. et al., 2012) published for first time an introduction of the TXRF technique applied to the pharmaceutical industry community. The work showed that the results obtained by TXRF were comparable with those obtained by ICP‐MS for the same samples for Pd and Cu measurement, and statistical analysis indicated that the results obtained by the two technologies are equivalent at the 95% confidence level.

Conclusions Currently, TXRF is an instrumental technique which is still continuously expanding, from the standpoint of development, implementation and application in all fields of scientific knowledge. Their simple and basic principlesmake TXRF a very versatile technique, capable of approaching the analysis of complex matrices with relative ease. There are still many fields of application where TXRF can provide its analytical ability. In particular, its application to solving biomedical, biological or biochemical problems, is an open field where TXRF can still make an important contribution to understanding problems of cytotoxicity, drug delivery, processes of chemical balance in cellular systems or metallomic among others. Finally, TXRF is initiating the complex way of quality assurance according to ISO standards (Fernández‐ Ruiz, R., 2008). This road is still ahead and in the future will be a field that must be seriously developed to try to unify criteria of normalization, standardization and traceability of the TXRF results. REFERENCES

Abraham, J. A., Sánchez, H. J., Valentinuzzi, M. C. and Grenón, M. S. ʺInfluence of smoking on the elemental composition of oral fluids: a TXRF approachʺ. X‐Ray Spectrometry, 39, 6, 372–375, 2010. Aiginger, H. and Wobrauschek, P. ʺA method for quantitative X‐ray fluorescence analysis in the nanogram region. Nucl. Instrum. Methods. 114, 157, 1974. Aiginger, H. and Wobrauschek, P. ʺTotal‐reflection x‐ray fluorescence spectrometric determination of elements in nanogram amountsʺ. Anal. Chem. 47, 852, 1975. Alsecz, A., Osan, J., Kurunczi, S., Alfoeldy, B., Varhegyi, A. and Toeroek, S. ʺAnalytical performance of different X‐ ray spectroscopic techniques for the environmental monitoring of the recultivated uranium mine siteʺ. Spectrochim. Acta Part B. 62, 769‐776, 2007. Antosz, F. J., Xiang, Y., Diaz, A.R., Jensen, A. J. ʺThe use of total reflectance X‐ray fluorescence (TXRF) for the

As one of the last applications developed by means of TXRF, Leitão et al. (Leitão, R.G. et al., 2014) has used TXRF in the evaluation of the elements P, S, K, Ca, Fe, Cu, Zn and Rb in human prostate tissues with and without cancer with the aim of finding diagnosed strategies.

determination of metals in the pharmaceutical industryʺ. J. Pharm. Biomed. Anal. 25, 62, 17‐22, 2012. Ayala, R. E., Alvarez, E. M., Wobrauschek, P. ʺDirect determination of lead in whole human blood by total reflection X‐ray fluorescence spectrometryʺ. Spectrochim.

Development in Analytical Chemistry Volume 1, 2014 www.seipub.org/dac

Acta Part B, 46, 1429‐1432, 1991.

Bedate, C.A. ʺQuantification of Pt bound to DNA using

Bedzyk, M. J., Bommarito, G. M. and Schildkraut, J. S. ʺX‐ ray standing waves at a reflecting mirror surfaceʺ. Phys. Rewiev Letters, 62, 1376, 1989.

total‐reflection X‐ray fluorescence (TXRF)ʺ. Analyst. 124, 583‐585, 1999. Fernández‐Ruiz, R. and Bermudez, V. ʺDetermination of the

Camejo, M. I., Abdala, L., Vivas‐Acevedo, G., Lozano‐

Ta and Nb ratio in LiNb1−xTaxO3 by total reflection X‐ray

Hernández, R., Angeli‐Greaves, M., Greaves, E. D.

fluorescence spectrometryʺ. Spectrochimica Acta Part B.

ʺSelenium, copper and zinc in seminal plasma of men

2005, 60, 231‐235.

with varicocele, relationship with seminal parametersʺ. Biol. Trace Elem. Res., 143(3), 1247‐54, 2011.

Fernández‐Ruiz, R., Furió, M., Cabello Galisteo, F., Larese, C., López Granados, M., Mariscal, R. and Fierro, J. L. G.

Carvalho, M. L., Barreiros, M. A., Costa, M. M., Ramos, M.

ʺChemical Analysis of Used Three‐Way Catalysts by

T., Marques, M. I. ʺStudy of heavy metals in Madeira

Total Reflection X‐ray Fluorescenceʺ. Anal. Chem. 74,

wine by total reflection X‐ray fluorescence analysisʺ. X‐

5463‐5469, 2002.

Ray Spectrometry. 25(1), 29‐32. 1996.

Fernández‐Ruiz, R. and Garcia‐Heras, M. ʺAnalysis of

Carvalho, M. L., Magalhães, T., Becker, M., von Bohlen, A.

archaeological

ceramics

total‐reflection

X‐ray

ʺTrace elements in human cancerous and healthy tissues:

fluorescence: Quantitative approachesʺ. Spectrochimica

A comparative study by EDXRF, TXRF, synchrotron

Acta Part B. 63, 975–979, 2008.

radiation and PIXEʺ. Spectrochimica Acta Part B. 62, 1004– 1011, 2007.

Fernández‐Ruiz, R., Larese, C., Cabello Galisteo, F., López Granados, M., Mariscal, R. and Fierro, J. L. G.. ʺTXRF

Carvalho, M. L., Custodio, P. J., Reus, U. and Prange, A. ʺElemental analysis of human amniotic fluid and

analysis of aged three way catalystsʺ. Analyst, 131, 590‐ 594, 2006.

placenta by total‐reflection X‐ray fluorescence and

Fernández‐Ruiz, R., Cabañero, J. P., Hernandez, E. and León,

energy‐dispersive X‐ray fluorescence: child weight and

M. ʺDetermination of the stoichiometry of CuxInySez by

maternal age dependenceʺ. Spectrochim. Acta Part B. 56,

total‐reflection XRFʺ. Analyst, 126, 1797‐1799, 2001.

2175‐2180, 2001.

Fernández‐Ruiz, Ramón, Andrés Román, de Jesús, Ernesto

Casas, J., ʺÓpticaʺ, 7ª Ed, (Ed. Justiniano Casas Pelaez), Zaragoza, 1994.

and Terreros, Pilar. ʺOptimization of the quantitative direct solid total‐reflection X‐ray fluorescence analysis of

Compton, A. H. ʺThe total reflexion of X‐raysʺ. Philosophical Magazine, 45, 1121,1923.

glass

microspheres

functionalized

with

organometallic compoundsʺ. Spectrochim. Acta Part B. 65,

Espinoza‐Quiñones, F. R., Módenes A. N., Thomé L. P.,

450–456, 2010b.

Palácio S. M., Trigueros D. E. G., Oliveira A. P. and

Fernández‐Ruiz, R., Rodríguez‐Ubis, Juan Carlos, Salvador

Szymanski N. ʺStudy of the bioaccumulation kinetic of

Álvaro, Brunet Ernesto and Juanes Olga. ʺEu and Tb

lead by living aquatic macrophyte Salvinia auriculataʺ.

quantitation

Chemical Engineering Journal, 150, 316–322, 2009.

compounds by TXRF direct solid procedureʺ. J. Anal. At.

Fernandez‐Ruiz, R. ʺAplicación de la Fluorescencia de rayos X por reflexión total (TXRF) al análisis composicional de cerámicas

arqueológicasʺ.

ISBN

978‐84‐8344‐132‐9.

Ediciones de la UAM, Madrid, 2008. Fernández‐Ruiz, R., Costo, R., Morales, M. P., Bomati‐

luminescent

g‐ZrP‐organometallics

Spectrom., 25, 1882–1887, 2010a. Fernández‐Ruiz, R., Ocon, P. and Montiel Manuel. ʺFirst approximation to the analysis of Ru and Se in carbon nanoparticles as a new voltaic pile system by TXRFʺ. J. Anal. At. Spectrom., 24, 785‐791, 2009.

Miguel, O. and Veintemillas‐Verdaguer, S. ʺTotal‐

Fernández‐Ruiz, R. ʺUncertainty in the Multielemental

reflection X‐ray fluorescence: An alternative tool for the

Quantification by Total‐Reflection X‐ray Fluorescence:

analysis of magnetic ferrofluidsʺ. Spectrochimica Acta Part

Theoretical and Empirical Approximationʺ. Analytical

B, 63, 1387–1394, 2008.

Chemistry, 80, 8372–8381, 2008.

Fernández‐Ruiz, R., Tornero, J.D., González, V.M. and

Fernández‐Ruiz, R., Malki Moustafa, Morato Ana I. and

www.seipub.org/dac Development in Analytical Chemistry Volume 1, 2014

Marin Irma. ʺStudy of bioaccumulation kinetics of chromium(VI)

Acinetobacter

beijerinckii

Magalhães, T., von Bohlen, A., Carvalho, M. L. and Becker,

type

M. ʺTrace elements in human cancerous and healthy

bacterium by Total Reflection X‐Ray Fluorescence

tissues from the same individual: A comparative study

Spectrometryʺ. J. Anal. At. Spectrom., 26, 511‐516, 2011.

by TXRF and EDXRFʺ. Spectrochim. Acta Part B. 61, 1185‐

García‐Heras, M., Fernández‐Ruiz, R. and Tornero, J. D.

1193, 2006.

ʺAnalysis of Archaeological Ceramics by TXRF and

Mages Margarete, Óvári Mihály, Tümpling Jr Wolf V.,

Contrasted with NAAʺ. Journal of Archaeological Science,

Kröpfl Krisztina. ʺBiofilms as bio‐indicator for polluted

24, 1003–1014, 1997.

waters?ʺ. Anal. Bioanal. Chem., 378, 1095–1101, 2004.

Gonzalez, Mauricio, Tapia, Lucia, Alvarado Milton, Tornero

Neumann, C. and Eichinger, P. ʺUltra‐trace analysis of

Jesús, Fernández‐Ruiz, R. ʺIntracellular determination of

metallic contaminations on silicon wafer surfaces by

elements in mammalian cultured cells by total‐reflection

vapour phase decomposition/total reflection X‐ray

X‐Ray Fluorescenceʺ. J. Anal. At. Spectrom., 44, 885‐888,

fluorescence (VPD/TXRF)ʺ. Spectrochimica Acta Part B, 46,

1999.

1369–1377, 1991.

Günther Klaus, von Bohlen Alex, Strompen Christoph. ʺElement

determination

total‐reflection

Norbert Szoboszlai, Zsófia Polgári, Victor G. Mihucz, Gyula

X‐ray

Záray. ʺRecent trends in total reflection X‐ray

fluorescence spectrometry at the initial step of element

fluorescence spectrometry for biological applicationsʺ.

speciation in biological matricesʺ. Anal. Chim. Acta, 309,

Analytica Chimica Acta, 633, 1‐18, 2009.

327‐332, 1995.

Ojeda Nelson, Greaves Eduardo, D., Alvarado Jose, Sajo‐

Hellin David, De Gendt Stefan, Rip Jens and Vinckier Chris.

Bohus Laszlo. ʺDetermination of V, Fe, Ni and S in

ʺTotal reflection X‐ray fluorescence spectrometry for the

petroleum

introduction of novel materials in clean‐room production

fluorescenceʺ. Spectrochim. Acta Part B. 48, 247‐253, 1993.

environmentsʺ. IEEE Transactions on Device and Materials

Ostachowicz, B., Lankosz, M., Tomik, B., Adamek, D.,

Reliability, 5(4), 639‐651, 2005.

crude

oil

total‐reflection

X‐ray

Wobrauschek, P., Streli, C. and Kregsamer, P. ʺAnalysis

Klockenkämper, R., ʺTotal Reflection X‐Ray Fluorescence

of some chosen elements of cerebrospinal fluid and

Analysisʺ. Chemical Analysis Series. Vol. 140, Wiley‐

serum in amyotrophic lateral sclerosis patients by total

Interscience, New York, 1997.

reflection X‐ray fluorescenceʺ. Spectrochim. Acta Part B.

Klockenkämper, R., von Bohlen, A. and Moens, L. ʺAnalysis

61, 1210‐1213, 2006.

of pigments and inks on oil paintings and historical

Pankhurst, Q. A., Thanh, N. K. T., Jones, S. K. and Dobson, J.

manuscripts using total reflection x‐ray fluorescence

ʺProgress in applications of magnetic nanoparticles in

spectrometryʺ. X‐Ray Spectrom. 29, 119–129, 2000.

biomedicineʺ. J. Phys. D: Appl. Phys., 42, 224001, 2009.

Klockenkämper, R. and von Bohlen, A. ʺDetermination of

Pepponi, G., Lazzeria, P., Coghea, N., Bersania, M.,

the critical thickness and the sensitivity for thin‐film

Gottardinib, E., Cristofolinib, F., Clauserc, G. and

analysis

fluorescence

Torboli, A. ʺTotal reflection X‐ray fluorescence analysis

spectrometryʺ. Spectrochim. Acta Part B. 44, 461–469, 1989.

of pollen as an indicator for atmospheric pollutionʺ.

total

reflection

X‐ray

Klockenkämper, R., Von Bohlen, A., Wiecken, B. ʺQuantification in total reflection X‐ray fluorescence

Prange, A., Knoth, J., Stößel, R. P., Böddeker, H. and Kramer,

analysis of microtome sectionsʺ. Spectrochim. Acta Part B.

K. ʺDetermination of trace elements in the water cycle by

44, 511‐517, 1989.

total‐reflection x‐ray fluorescence spectrometryʺ. Anal.

Leitão, R.G., Palumbo, Jr A., Souza, P.A.V.R., Pereira, G.R.,

Chim. Acta. 195, 275‐287, 1987.

Canellas, C.G.L., Anjosa, M.J., Nasciutti, L.E., Lopes, R.T.

Prange, A., Böddeker, H. and Kramer, K. ʺDetermination of

ʺElemental concentration analysis in prostate tissues

trace elements in river‐water using total‐reflection X‐ray

using total reflection X‐ray fluorescenceʺ. Radiation

fluorescenceʺ. Spectrochim. Acta Part B, 48, 207‐215, 1993.

Physics and Chemistry, 95, 62–64, 2014.

Spectrochim. Acta Part B. 59, 1205‐1209, 2004.

Prange, A., Knöchel, A., Michaelis, W. ʺMulti‐element

Development in Analytical Chemistry Volume 1, 2014 www.seipub.org/dac

determination of dissolved heavy metal traces in sea

analysis of cancer cell lines by total reflection X‐ray

water

fluorescenceʺ. Spectrochim. Acta Part B. 63, 1480‐1484,

total‐reflection

x‐ray

fluorescence

spectrometryʺ. Anal. Chim. Acta, 172, 79‐100, 1985.

2008.

Savage, I. and Haswell, S. J. ʺThe development of analytical

Tsuji, K., Injuk, J. and Van Grieken, R. ʺX‐Ray Spectrometry:

methodology for simultaneous trace elemental analysis

Recent Technological Advancesʺ. Wiley, England. 2004.

of blood plasma samples using total reflection X‐ray

Von Bohlen, A. ʺTotal reflection X‐ray fluorescence and

fluorescence spectrometryʺ. J. Anal. At. Spectrom., 13,

grazing incidence X‐ray spectrometry — Tools for micro‐

1119‐1122, 1998.

and surface analysis. A reviewʺ. Spectrochimica Acta Part

Schwenke, H. and Knoth, J. ʺA highly sensitive energy‐

B. 64, 821–832, 2009.

dispersive X‐ray spectrometer with multiple total

Von Bohlen, A. ʺTotal reflection X‐ray fluorescence

reflection of the exciting beamʺ. Nucl. Instrum. Methods.

spectrometry – A versatile tool for ultra‐micro analysis of

193, 239, 1982.

objects of cultural heritageʺ. e‐Preservation Science. 1, 23‐

Shimazaki Ayako, Miyazaki Kunihiro, Matsumura Tsuyoshi

34, 2004.

and Ito Shoko. ʺAnalysis of Low Metallic Contamination

Van Grieken, R. and Markowicz, A. ʺHandbook of X‐Ray

on Silicon Wafer Surfaces by Vapor‐Phase Treatment and

Spectrometryʺ. 2ª Edición, Practical Spectroscopy Series,

Total Reflection X‐ray Fluorescence Analysisʺ. Jap. J.

Vol. 29. Marcel Dekker, New York, 2002.

Appl. Phys., Part 1, 45, 9037‐9043, 2006.

Wellenreuther, G., Fittschen, U.E.A., Achard, M.E.S., Faust,

Sirito de Vives, A. E., Moreira, S., Brienza, S., Medeiros, J. G.,

A., Kreplin, X. and Meyer‐Klaucke, W. ʺOptimizing total

Tomazello Filho, M., Zucchi, O., do Nascimento Filho, V.,

reflection X‐ray fluorescence for direct trace element

Barroso, R. ʺSpecies arboreal as a bioindicator of the

quantification in proteins I: Influence of sample

environmental pollution: Analysis by SR‐TXRFʺ. Nuclear

homogeneity and reflector typeʺ. Spectrochim. Acta Part B.

Instruments & Methods in Physics Research A, 579, 494‐498,

63, 1461‐1468, 2008.

2007.

Wittershagen, A., Rostam‐Khani, P., Zickermann, V.,

Streli, C., Wobrauschek, P., Pepponi, G. and Zoeger, N. ʺA

Zickermann, I., Gemeinhardt, S., Ludwig, B., Kolbesen ,

new total reflection X‐ray fluorescence vacuum chamber

B. O. ʺDetermination of metal‐cofactors in respiratory

with sample changer analysis using a silicon drift

chain complexes by total‐reflection x‐ray fluorescence

detector for chemical analysisʺ. Spectrochim. Acta Part B.

spectrometry (TXRF)ʺ. Fresenius J. Anal. Chem, 361, 326‐

59, 1199‐1203, 2004.

328, 1998.

Stößel, R. P. and Prange, A. ʺDetermination of trace elements

rainwater

total‐reflection

x‐ray

fluorescenceʺ. Analytical Chemistry, 57, 2880‐2885, 1985. Stosnach Hagen, Mages Margarete. ʺAnalysis of nutrition‐

Woelfl Stefan, Mages Margarete, Mercado Susana, Villalobos Lorena,

Óvári

Mihály

and

Encina

Francisco.

ʺDetermination of trace elements in planktonic microcrustaceans

using

total

reflection

X‐ray

relevant trace elements in human blood and serum by

fluorescence (TXRF): First results from two Chilean

means of total reflection X‐ray fluorescence (TXRF)

lakesʺ. Anal. Bioanal. Chem., 378, 1088–1094, 2004.

Spectroscopyʺ. Spectrochim. Acta Part B, 64, 345‐356, 2009.

Yoneda, Y. and Horiuchi, T. ʺOptical flats for use in X ray

Streit Niklaus, Weingartner Ernest, Zellweger Christoph,

spectrochemical microanalysisʺ. Rev. Sci. Instrum. 42,

Schwikowski

Margit,

Gaggeler

Heinz

and

1069, 1971.

Baltensperger Urs. ʺCharacterization of size fractionated

Zarkadas, Ch., Karydas, A. G. and Paradellis, T.

aerosol from the Jungfraujoch (3580 m asl) using total

ʺDetermination of uranium in human urine by total

reflection X‐ray fluorescence (TXRF)ʺ. Int. J. Environ.

reflection X‐ray fluorescenceʺ. Spectrochim. Acta Part B.

Anal. Chem. 76‐1, 1‐16, 2000.

56, 2505‐2511, 2001.

Szoboszlai Norbert, Réti Andrea, Budai Barna, Szabó Zsuzsa,

Zheludeva, S. I., Novikova, N. N., Konovalov, O. V.,

Kralovánszky Judit and Záray Gyula. ʺDirect elemental

Kovalchuk, M. V., Stepina, N. D., Yur’eva, E. A.,

www.seipub.org/dac Development in Analytical Chemistry Volume 1, 2014

Myagkov, I. V., Godovski, Yu. K., Makarova, N. N., Rubtsov, A. M., Lopina, O. D., Erko, A. I., Tolstikhina, A. L., Gainutdinov, R. V., Lider, V. V., Tereshchenko, E. Yu. and Yanusova, L. G. ʺPossibilities of X‐ray Fluorescence in the Region of Total External Reflection for Studying Langmuir Monolayers on the Surface of a Liquid and Solid Substrateʺ. Crystallography Reports, 2003, 48, S25‐ S36. Dr. Ramón Fernández‐Ruiz is the head of the TXRF laboratory of the Interdepartamental Research Service (SIdI) of the Autonomous University of Madrid (UAM). He began its labor in the

UAM (TXRF/XRD Labs) as laboratory assistant in 1988 under the direction of Prof. J.D. Tornero. He receives the Chemical Assistant degree from Polytechnique Institute of Cartagena (Murcia, Spain) in 1988 and the Theoretical Physics degree from UAM in 2003. In 2006, he obtains master degree in Materials Physics by a novel neutron diffraction research on Lithium Niobate at low temperatures. In 2008, he obtains the PhD degree in Chemistry by the application of the TXRF spectroscopy to the Archaeometry field. During his trajectory, he has developed and published more than 42 scientific papers mainly related with TXRF applications to fields as diverse as biotechnology, biomedicine, new materials, nanotechnology or archaeometry, in several of the more important international scientific journals in the spectroscopy and analytical chemistry fields.