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ADVANCES IN ATOMIC SPECTROSCOPY

Volume5

9 1999

This Page Intentionally Left Blank

ADVANCES IN ATOMIC SPECTROSCOPY Editor: JOSEPH SNEDDON Department of Chemistry McNeese State University Lake Charles, Louisiana

VOLUME5

9 1999

JAI PRESS INC.

Stamford, Connecticut

Copyright 91999 by JAI PRESSINC 1O0 Prospect Street Stamford, Connecticut 06904-0811 All rights reserved. No part of this publication may be reproduced, stored on a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, filming, recording, or otherwise without prior permission in writing from the publisher. ISBN: 0-7623-0502-9 ISSN: 1068-5561 Manufactured in the United States of America

CONTENTS

LIST OF CONTRIBUTORS

vii

PREFACE

Joseph Sneddon

ix

SPECIATION STUDIES BY ATOMIC SPECTROSCOPY M. de la Guardia, M.L. Cervera, and

A. Morales-Rubio

NEW TYPES OF TUNABLE LASERS

Xiandeng Hou, Jack X. Zhou, Karl X. Yang, Peter Stchur, and Robert G. Michel

99

DEVELOPMENTS IN DETECTORS IN ATOMIC SPECTROSCOPY

Frank M. Pennebaker, Robert H. Williams, John A. Norris, and M. Bonner Denton

145

GLOW DISCHARGE ATOMIC SPECTROMETRY

$ergio Caroli, Oreste Senofonte, and Gianluca Modesti

173

LASER-INDUCED BREAKDOWN SPECTROMETRY

Yong-II! Lee and Joseph Sneddon

INDEX

235 289

This Page Intentionally Left Blank


Sergio Caroli

Applied Toxicology Department Istituto Superiore di Sanit~ Rome, Italy

M.L. Cervera

Department of Analytical Chemistry University of Valencia Valencia, Spain

M. Bonner Denton

Department of Chemistry University of Arizona Tuscon, Arizona

M. de la Guardia

Department of Analytical Chemistry University of Valencia Valencia, Spain

Xiandeng Hou

Department of Chemistry University of Connecticut Storrs, Connecticut

Yong-lllLee

Department of Chemistry Changwon National University Changwon, Korea

Robert G. Michel


Gianluca Modesti


A. Morales-Rubio

Department of Analytical Chemistry University of Valencia Valencia, Spain vii

viii


John A. Norris


Frank M. Pennebaker


Oreste 5enofonte


Joseph Sneddon

Department of Chemistry McNeese State University Lake Charles, Louisiana

Peter Stchur


Robert H. Williams


Kad X. Yang

Wandsworth Research Center New York State Department of Health Albany, New York

Jack X. Zhou

CVI Lasers Corporation Putnam, Connecticut

PREFACE Element speciation determines the different forms or species a chemical metal can have within a given compound. It is well known that different forms of a metal have different toxicity effects. Chapter 1 by Miguel de la Guardia and coworkers describes the use of various atomic spectroscopic methods and applications of speciation studies in atomic spectroscopy. The emphasis is on combining atomic spectroscopy with gas and liquid chromatography. While the laser has been around for close to 40 years, new lasers are becoming available which have the potential to directly impact atomic spectroscopy. In Chapter 2, Bob Michel and coworkers describe new developments in tunable lasers for use in atomic spectroscopy. Traditional methods of detection such as photography and the photomultiplier tube are being replaced by new detectors which have potential for multielement detection and more. Chapter 3 describes the developments in detectors in atomic spectroscopy from M. Bonner Denton and coworkers. Chapter 4 is on the very active area of glow discharge atomic spectrometry presented by Sergio Caroli and coworkers. After a brief introduction and historical review, the use of glow discharge lamps for atomic spectrometry and mass spectrometry is discussed. This includes a discussion on the geometry and the use of radiofrequency power. A discussion on recent applications including metals and alloys, nonconductive solid materials, and liquid and gaseous samples is presented. Finally the future of this source in atomic spectrometry is discussed.

x

PREFACE

Chapter 5 covers the use of a laser-induced or laser-ablated plasma as a spectrochemical source for atomic emission spectrometry. The technique is referred to as laser-induced breakdown spectrometry (LIBS). A brief introduction is followed by a description of the instrumentation, in particular the laser and detection device. A brief outline of the plasma physics is followed by a description of the applications of LIBS, particularly where it is advantageous over conventional atomic emission techniques. Joseph Sneddon Editor

SPECIATION STUDIES BY ATOMIC SPECTROSCOPY

M. de la Guardia, M.L. Cervera, and A. Morales-Rubio

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Definition and Several Approaches . . . . . . . . . . . . . . . . . . . . . . . Importance of Speciation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Factors Affecting Speciation . . . . . . . . . . . . . . . . . . . . . . . . . . . . Speciation in Waters: The Methodology . . . . . . . . . . . . . . . . . . . . . Metal Speciation in Biological Fluids: Some Specific Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Speciation in Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Speciation in Urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Speciation in Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Speciation of Miscellaneous Biological Fluids . . . . . . . . . . . . . . . VI. Speciation in Solid Samples: The Challenge . . . . . . . . . . . . . . . . . . . . A. Speciation of Soil and Sediment Samples . . . . . . . . . . . . . . . . . . B. Speciation of Solid Biological and Food Materials . . . . . . C. Speciation of Miscellaneous Solid Samples . . . . . . . . . . . . . . . . . VII. Speciation Studies of Different Metals . . . . . . . . . . . . . . . . . . . . . . A. Aluminium . . . . . . . . . . . ....................... B. Antimony . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 2 4 6 8

I. II. III. IV. V.

Advances in Atomic Spectroscopy Volume 5, pages 1-98. Copyright 9 1999 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0502-9

. ......

15 18 25 28 30 41 41 49 56 57 58 68

2

M. DE LA GUARDIA, M.L. CERVERA, and A. MORALES-RUBIO C. Arsenic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Cadmium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Calcium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E Chromium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Copper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Germanium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Iodine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. Iron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. Lead . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L. Mercury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. Nickel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. Platinum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . O. Selenium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. Tin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Q. Zinc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

70 71 72 72 74 75 76 76 77 79 81 81 82 83 85 85

ABSTRACT The term speciation is used to describe the oxidation state or chemical form of a metal in a sample. The importance of speciation, particularly in clinical or biological and environmental samples, is described. Sample preparation and preservation is considered to be the key to accurate determination of the species and is discussed in detail. The use of various atomic spectroscopy techniques, coupled with both gas and liquid chromatography, to allow speciation studies of natural or real samples is presented. The application of these hyphenated techniques to selected metals is further presented.

I. A D E F I N I T I O N A N D SEVERAL APPROACHES The term speciation is commonly employed in geochemistry to differentiate dissolved metals from particulated solid forms. In the past it has been used to describe the different oxidation states of a metal in the same sample. Furthermore, speciation has been used to interpret the results obtained in the electrochemical analysis of complex samples as a function of their chemical forms. This is achieved through the availability of metals to be reduced on the electrodes. In the analysis of soils, the use of sequential extraction schemes based on different reagents provide an excellent way to discriminate the chemical form of metals as a function of their suitability to be extracted by plants. In clinical analysis and toxicological studies, it was clear that the availability of metals to be absorbed by humans is a function of the specific chemical form of the metal. This is used to explain the biogeochemical cycle of trace metals and dramatic accidents, such us the 1954 methyl mercury intoxication in Minamata (Japan) (Smith and Smith,

Speciation Studies

3

1975). A series of different approaches, based on toxicity, bioavailability, bioaccumulation, mobility, or biodegradability of metals are of interest to analytical chemists for speciation studies. Recently, the International Union of Pure and Applied Chemistry (IUPAC) has defined speciation as the process yielding evidence of the atomic or molecular form or configuration in which a metal can occur in a compound or a matrix. From this point of view it is clear that to perform actual speciation studies, it is absolutely necessary to quantitatively determine the amount of a chemical form of a metal in a sample (Krull, 1991). The analytical process offers tremendous possibilities to perform speciation studies, based on the use of selective detection systems, chemometrics, and the exploitation of analytical data. Additionally, it is possible to improve speciation during the sampling and sample pretreatment by introducing specific collector devices or appropriate chemistries, as indicated in Figure 1. Using the IUPAC definition, it is clear that selectivity in front of the determination of a species is the key problem. Conventional atomic spectroscopy, because of the high temperatures used in the measurement cells, offers only limited applicability for speciation. The atomic signals are based on the presence of free atoms in the fundamental or the excited states, or on the previous formation of free gaseous ions. It makes it difficult to obtain different signals as a function of the chemical form of the metal to be determined. However, it is possible to improve the performance of atomic spectroscopy, from the ability to accurately determine the total metal content to the specific determination of species at trace levels by means of hyphenation between the atomic detector and some separation processes. The main objective of this chapter is to provide an analytical perspective about the state-of-the-art of speciation by atomic spectroscopy. Furthermore, a look at the

SAMPLING

l

SAMPLE PRETREATMENT

l l

SEPARATION DETECTION

l

CHEMOMETRICS

* Specific collection

and/or preconcentration of species

* Selective leaching * Derivatization

* E x t r a c t i o n in specific conditions * V o l a t i l i z a t i o n of compounds

* Chromatographic separations

* Specific molecular m e t h o d s * Selective atomic spectrometry methods hyphenated with separation procedures

* data deconvolution

Figure 1. Possibilitiesoffered by the analytical process to do speciation studies.

4

M. DE LA GUARDIA, M.L. CERVERA, and A. MORALES-RUBIO

future of speciation by considering the tremendous possibilities offered by flow injection analysis (FIA)-microwave-assisted sample treatments, and the combination of different approaches will be presented. It has been proposed that the hyphenation between chromatography and the most sensitive of atomic detectors is the main approach to achieve speciation studies. However, other approaches, such as the old practice of sequential extraction schemes, and some specific leaching or chemical treatments, could also be suitable to improve atomic measurements. They must be considered in order to make speciation available for complex samples and eventually control laboratories. In fact, the challenge is to provide suitable methodologies, far from the reduced perspective of pure academic studies. We will try to harmonize the rigorous application of the IUPAC definition with a touch of imagination in order to incorporate as many possible analytical tools from both classical chemical knowledge and modern instrumentation.

II.

IMPORTANCE OF SPECIATION

The main characteristics of the behavior of chemical metals, such as their solubility, mobility, availability or toxicity, are strongly dependent on their specific form. It is clear that for life science studies, it is necessary to know the concentration of each species, as well as determination of the total metal content. The interest in speciation studies has been growing from the 1950s and particularly during the last two decades. A series of conferences have specifically focused on speciation and related topics. In April in 1983 in Gtittingen (Germany), a symposium on "Forms of Binding of Chemical Elements in Environmental and Biological Materials" was organized by Schwedt (1983). In September 1984 in Berlin (Germany), a new congress organized by the Association of Sponsors of German Science was devoted to "The Importance of Chemical Speciation in Environmental Processes," thus highlighting the tremendous interest on this aspect of environmental pollution. In September 1988 in Karlsruhe (Germany), EH. Frimmel organized a symposium about "Elements and their Chemical State in the Environment." In 1989, a North Atlantic Treaty Organization (NATO) workshop organized in Izmur (Turkey) focused "International Conference on Metal Speciation in the Environment." Since 1990, BCR have organized several meetings and workshops on speciation. These include those at Arcachon (France) on "Improvements in Speciation Analysis in Environmental Materials," in 1992 at Sitges (Spain) on "Sequential Extraction of Trace Metals in Soils and Sediments" and in February 1994 in Rome (Italy) on "Trends in Speciation Analysis." In March 1993, Schwedt organized a new meeting in Clausthal on "Advances in Elemental Species Analysis: Concepts, Findings and Evaluation" which focused on the methodological problems of speciation. In June 1991 in Loen (Norway) it was organized as a post-symposium of the XXVII Colloquium Spectroscopic International (C.S.I), entitled "Speciation of Elements in Environmental and Biological Sciences." In June 1994,

Speciation Studies

5

the 2nd International Symposium on "Speciation of Elements in Toxicology and Biological Sciences" assured the continuity of international conferences on this topic. From the pioneering German interest in this field, the international scientific community has been quick to accept speciation as a major topic of current analytical chemistry. This is not only due to its extreme importance in evaluating the environmental impact and mobility of metals and their toxicological behavior, but also from the methodological point of view in order to assure the accuracy, repeatability and ruggedness of methods of speciation. A good parameter for the evaluation of interest in a research topic is the number of published books. The first publication was in 1983 of the book edited by Leppard on "Trace Element Speciation in Surface Water and its Ecological Implications" (Leppard, 1983). Since that time several books have been published: 1984 (Kramer and Duinker), 1986 (Bernhard et al.), 1987 (Lander), 1988 (Buffle; Kramer and Allen), 1989 (Batley; Harrison and Rapsomanikis), 1990 (Patterson and Passino), 1991 (Krull), and 1996 (Caroli). This regular appearance is a strong indication of the continuous attention paid to this topic by major scientific publishers. Figure 2 shows the accumulated number of published papers from 1980 until the present (late 1998). There is an exponential growth in this area, with the total number of papers published during the period of time considered (from Analytical Abstract Data Base) of more than 700. Considering the metals studied, it can be seen from Figure 3 that the major interest has focused on metals with different stable oxidation states such as As, Se, Cr, and Sb, or with highly stable specific organic forms, commonly used in industrial applications, such as Hg, Sn, and Pb.

Figure 2. Chronological evolution of the scientific literature about speciation (from Analytical Abstracts January 1980-March 1998).

6

M. DE LA GUARDIA, M.L. CERVERA,and A. MORALES-RUBIO As

Sb 2%

Se 8%

Pb

..........

Miscellaneous 19%

Cr 11%

~~N Sn 14%

g 12%

Figure 3. Distribution of the literature on speciation as a function of the elements determined.

Throughout this chapter the reader will find a guide to follow the scientific literature about speciation studies. It includes a final section providing a summary of speciation of individual metals.

III.

FACTORS AFFECTING SPECIATION

All analytical steps preceding speciation are extremely critical to assure the stability of a chemical species present in natural samples. Sampling, sample preservation, storing, and sample treatment must be carefully controlled in order not to disturb the equilibria established among the species. Therefore, practical studies on speciation must involve several protocols to ensure the accuracy and representivity of laboratory data. Additionally, typical problems which can occur during the determination of total concentration of trace metals, such as the contamination of samples from the material employed for sampling by reagents employed for sample preservation or losses during storing and sample pretreatment, must be taken into account. The instability of redox species changes in a sample erwironment, may also be significant. Due to theses concerns, it is necessary that changes involved by sample handling do not modify or change the ratio between species. A good example of this potential problem can be found during the speciation of Fe in deep lakes. The absence of 0 2 and the presence of high quantities of dissolved CO 2 involve a sample environment totally different than that found outside; Fe (II) being frequently oxidized to Fe (III) during the sampling step. Redox conditions, macroconstituents, ionic strength, pH, temperature, and pressure are some of the factors affecting speciation and species preservation. Additionally, synergistic and antagonistic effects between trace compounds present in the sample must also be taken into consideration. When biological fluids are to be analyzed, the conditions of a

Speciation Studies

7

minimum and soft pretreatment procedure must be applied to attain quick and accurate analysis, paying particular attention to the type of compound used for calibration (Dawson, 1986). During the sampling step, a number of potential problems should be considered during the collection of biological fluids. The major problem in such work is due to the contamination and losses of the trace metal. The effect of contaminants may alter the amount of analyte bound to a given fraction. For example, zinc and copper ions added in-vitro to a serum sample result in an increase in the albumin-bound fraction (Cornelis et al., 1993). It must also be considered that some factors, such as person-to-person differences, region-to-region variations, occupational exposure, and physiological state of the subject, would influence the chemical speciation of a given metal in body fluids (Negretti de Br~itter et al., 1995). Sampling of urine in studies devoted to metal speciation is commonly performed on urine collected during a 24-h period. One reason for this is that many constituents exhibit diurnal variation, with variable peak excretions as a result of variation in drinking patterns. At the beginning of the collection period (usually when the person awakens), the bladder should be emptied, the specimen discarded, and the time noted. All urine specimens passed during the next 24-h period are collected in a pre-cleaned polyethylene or polypropylene container. At the end of the collection period the bladder is emptied and the specimen is added to those already collected. Transfer of the urine from the body into the container may introduce contamination from clothes and skin (Robberecht and Deelstra, 1984). The timing of body fluids taken from a subject can have a significant influence on the concentration of total metal and species. Thus, the diurinal variation of zinc concentration in human blood plasma, and the history of the tissue before sampling, e.g. the use of cosmetic agents (shampoo or conditioner) in the treatment of hair, affects the result (Dawson, 1986). It has been shown that the concentration of protein-bound serum zinc in human blood plasma varies depending on whether the sample was taken from a patient who was standing or lying in a recumbent position (Behne, 1981). Based on the previous comments, it is absolutely necessary in the determination of speciation of metals in biological fluids to include a detailed description of both, the samples analyzed and the procedures for sampling and storage, in order to make possible a critical evaluation of the published literature. The key to successful speciation work is the preservation of the species. Often this is impossible and the integrity of an organometallic compound is violated (Woittiez et al., 1991). Nevertheless, collected samples are usually frozen at -20 ~ until required. Next, the frozen substance is allowed to thaw to room temperature. At this stage, pretreatment needs the addition of preservatives, stabilizers, and other additives. If these substances are complexing agents or their presence could change the pH, ionic strength, redox potential, and dielectric constant of the medium, it could result in some changes in the distribution of chemical species. For example, methylmercury may be lost from blood on freeze-drying (Horvat and Byrne, 1992).

8

M. DE LA GUARDIA, M.L. CERVERA,and A. MORALES-RUBIO

Lyophilization is a common process applied to natural or real samples. It converts the material into a form that can be easily stored for longer periods. It is necessary to ensure that the analytes (metals) are not lost and the material characteristics as well as the stability of the analyte species are maintained. In fact, it has been reported that lyophilization could cause the destruction of lipoproteins (Kroll et al., 1989) and also result in denaturation and aggregation of other proteins. Evidently, this will change the speciation of metals like selenium which is associated with lipoproteins (Gardiner, 1993). A problem, studied in depth, is the effect of storage conditions of sediments for species of Cd, Cu, Fe, Mn, Ni, Pb, and Zn. Typical methods of sample preservation available are (1) wet at room temperature, (2) wet at low temperature, (3) frozen, (4) freeze-drying, (5) oven-drying, (6) microwaveassisted drying, and (7) air-drying at room temperature. It was found that wet preservation methods produced a reducing soil or sediment environment, whereas drying procedures provided an oxidizing environment with important consequences for speciation. Drying at 90 ~ promotes the crystallization of amorphous oxides and the formation of new solid mineral phases. It appears that freeze-drying and microwave-assisted drying could be more appropriate for geochemical material preservation. During a study on preparation of reference materials of clays and sediments, it was concluded that microwave-assisted drying provided excellent precision but did not produce identical results than those found after conventional drying. The composition changes and sample instability could be produced during the microwave treatment (Beary, 1988). However, in spite of changes introduced by drying, due to the inevitable oxidation of samples, which can be dramatic in the case of oxidizing sediments, it is clear that drying inhibits further changes in speciation mediated by microbiological action. It was concluded, in a rigorous study, that air-drying preservation of soils and sediments facilitates sample handling, homogenization, and preservative subsampling without affecting chemical species (Ure, 1994). In order to improve the methodologies for species separation and determination, efforts must be made in order to assure correct protocols, including all the steps of the analytical process. This will lead to accurate results in speciation studies. Appropriate methodologies for sampling, which avoid species transformation, and a complete guide for sample preservation, homogenization, and subsampling must be developed, and accompanied by a careful check on their effect on species stability. In the following sections problems regarding speciation in complex liquid samples, such biological fluids, and particularly in solid samples, for which a drastic sample pretreatment is often required, will be presented. These areas will be discussed in practical applications of speciation studies.

IV.

SPECIATION IN WATERS: THE M E T H O D O L O G Y

All atomic spectrometric methods work for the direct analysis of liquid or dissolved samples. All the main atomic spectrometry detectors have been employed in

Speciation Studies

9

speciation studies. From the techniques indicated in Figure 4, cold vapor atomic absorption spectrometry (CVAAS) is preferred for Hg determination and microwave-induced plasma-atomic emission spectrometry (MIP-AES) can be applied to easy volatile compounds or easily derivatizable metals, from which a gaseous phase, free from the solvent, can be obtained. On the other hand, the lack of an appropriate commercially available atomic fluorescence spectrometry (AFS) instrumentation (this has been changed recently) requires a strong reduction of the metals to determine Hg, As, Se, Sb and Te (Corns et al., 1993). In fact, the evolution of the literature about speciation in atomic spectrometry clearly shows that, in the case of atomic emission measurements, the recent development of hot atomization systems such as plasma, has completely replaced flame emission procedures. The inductively coupled plasma (ICP) is the most commonly and widely employed plasma source. Regarding the use of atomic absorption techniques, flame atomic absorption spectrometry (FAAS) continues to be employed to a larger extent than electrothermal atomization atomic absorption spectrometry (ETAAS). This is due to the easily coupling between FAAS and dynamic separation systems. This is despite the reduced sensitivity of FAAS as compared with ETAAS. An important aspect to be considered in order to improve the sensitivity of speciation studies by FAAS is the possibility to generate on-line volatile derivatives, like covalent hydrides. This avoids problems related to the reduced nebulization efficiency of classical continuous introduction systems. This dramatically increases the sensitivity and will add

/EMISSION

[FLAME PHOTOMETRY(FP) I PLASMA EMISSION | MICROWAVEINDUCED PLASMA (MIP) | INDUCTIVELY COUPLED PLASMA (ICP) I DIRECT COUPLEDPLASMA (DCP)

/FLUORESCENCE I ATOMIC FLUORESCENCE (AFS)

I/ION COUNTING

i ICP.MASSSPECTROMETRY ~

lioBsORPTiON

LD VAPOURATOMICABSORPTION(OVAAS) AME ATOMICABSORPTIOM(FAAS) LECTROTHERMALATOMICABSORPTION(ETAAS)

Figure 4.

Atomic spectrometrymethods currently employed in speciation studies.

10


new variables. These can be suitable to improve the selective determination of different species, as a function of their ability to form hydrides and the kinetics of this process. However, as indicated previously, the low residence time of sample particles in the measurement zone and the high temperature of the atomization cells can cause problems in discriminating the chemical forms of a metal. As a consequence, the single use of atomic spectrometry is not convenient for "in situ" analysis of chemical species with only a few exceptions (Arpadjan and Krivan, 1986; de la Guardia, 1996). This has led to the general strategy for speciation by atomic spectrometry being based on its hyphenation with separation techniques. Figure 5 summarizes the techniques most often employed to preconcentrate selectively or to isolate a specific chemical form of a metal to be determined by atomic spectrometry. From these techniques, a general consensus has been reached that gas chromatography (GC) (Schwedt and Russel,1973; Fernandez, 1977; Van Loon, 1979; Segar, 1984; Ebdon et al., 1986; Chan and Wong, 1989) and liquid chromatography (LC) (Van Loon, 1981; Fuwa et al., 1982; Chau, 1986; Irgolic, 1987; Ebdon et al., 1987a and 1988) are the best alternatives to provide accurate determination of the different species of an metal. Recent studies have focused on the development of appropriate interfaces between chromatography and flame spectrometers (Aue and Hill, 1973; Kawaguchi et al., 1973; Jones and Managhan, 1976; Parris et al., 1977; Ebdon et al., 1988), electrothermal atomizers (Brickman et al., 1977; Ebdon et al., 1982 and 1987b) or plasmas (Beenakker, 1977; Windsor and Denton, 1979; Gast et al., 1979; De Jonghe et al., 1980; Krull and Jordan, 1980; Hansler and Taylor, 1981; Duebelbeis et al., 1986; Bushee, 1988; Bushee et al., 1989; Crews et al., 1989; Heitkemper et al., 1989). Gas chromatographic (GC) separation requires that the species to be determined are volatile and thermally stable under the conditions employed for separation.

Figure 5. Separation methods commonly employed in speciation studies classified as a function of the phases involved (reproduced from de la Guardia, 1996).

Speciation Studies

11

Speciation through GC-atomic spectrometry is limited to the analysis of volatile organic compounds such as lead or mercury alkylderivatives, which commonly occur in natural samples. Additionally, volatile derivatives can be prepared before the chromatographic separation, as was the case in the arsenic speciation through methyl thioglycolate formation (Haraguchi and Takatsu, 1987; Ebdon et al., 1988). Compared with GC, procedures based on liquid chromatography (LC) separation followed by atomic spectrometry detection are more simple and suitable to the direct speciation of many natural occurring species. In general, LC-AS speciation studies involve separations on C18 bonded silica columns, or the use of ionexchange resins. Porous gels based on the use of size exclusion mechanisms (Van Loon and Barefoot, 1992) are suitable to be applied to differentiate between inorganic and organic species, cationic and anionic species, and also species of the same type but with different molecular sizes. However, the simplest applicability of LC for speciation studies in atomic spectrometry is compounded by difficulties found in the development of appropriate interfaces between the LC and the atomic detector. The flow rate of the carrier gas or liquid phase through the chromatographic column must be adjusted to the gas or liquid uptake of the detector. A simple heated stainless steel tube, with minimal dimensions, can be employed in the case of GC. For LC separations, an auxiliary supply of the mobile phase is commonly required to supplement the column effluent. New nebulizers (Gustavsson and Nygren, 1987), jet separator interfaces (Gustavsson, 1987) and thermospray interfaces (Robinson and Choi, 1987) have been proposed for this reason. The time of species separation must be reduced to a minimum in order to save both time and gas consumption, particularly when inductively coupled plasma-atomic emission spectrometry (ICPAES) or inductively coupled plasma-mass spectrometry (ICP-MS) determination is performed. In the literature there are examples on fast chromatographic separation and atomic spectrometry detection of several species of a metal. Figure 6 depicts a gas chromatogram of seven organotin species separated after ethylation with NaBET 4 (Martin-Lecuyer and Donard, 1996). It can be seen in Figure 7 by an appropriate selection of both, the chromatographic column and the mobile phase, it is possible to clearly separate different mercury species, by liquid chromatography. This allows the quantitative analysis of traces of these species by using an extremely sensitive detector such as ICP-MS (Pastor, 1998). The hyphenation between chromatography and atomic spectrometry provides suitable tools for speciation in liquid and dissolved samples. However, additional efforts must be achieved in order to improve the multimetal capabilities of some atomic detectors, like ICP-AES, or ICP-MS in order to develop methodologies suitable for multimetal speciation studies in natural samples. There are some precedents on the use of typical single metal techniques, such as ETAAS, for multi-speciation analysis. These are based on the use of a gold trap for retention of Se and Te species and sequential leaching with H20, 1 M HC1 and 3 M HNO 3,

12

M. DE LA GUARDIA, M.L. CERVERA, and A. MORALES-RUBIO (MV) 20,0 19,5

-

19,0 18,5! 18,0 17,5~ 17,0 16,5

-

3

16,0 15,5 15,0 14,5 o

i

I

10

5

I

15

T i m e (min)

Figure 6. Gas chromatogram of seven organotin species separated by GC after ethylation with NABEt4 and detected by flame photometry (reproduced from MartinLecuyer and Donard, 1996). (1) BuSn 3+ , (2) Bu2Sn2 + , (3) PhSn3+ , (4) Bu3Sn+ , (5) Bu4Sn, (6) Ph2Sn2+, (7)Ph3Sn+, and (e) Internal Standard (Pr3Sn+). combined with ion chromatography (Muangnoicharoen et al., 1988) or based on extraction in CHC13 and CC14of As(III), Sb(III), Se(IV) and Te(IV) with 4% APDC followed by a separate treatment of another sample aliquot after an appropriate reduction of oxidized forms of these four metals (Chung et al., 1984a, b). However, multi-speciation studies has been achieved using ICP-AES (McCarthy et al., 1983; Gjerde et al., 1993; Sanz-Medel et al., 1994), MIP-AES (Sadiki and Williams, 1996), and ICP-MS (Haraldsson et al., 1993; Jantzen and Prange, 1995; Kumar et al., 1995; De Smaele et al., 1996; Krupp et al., 1996; Guerin et al., 1997). Gas chromatography has been employed in the multimetal speciation of organotin and organolead compounds. It is based on their extraction with pentane. This is followed by derivatization with pentylmagnesium bromide and butylmagnesiun

13

Speciation Studies 60000

HgCI2 2.56

50000 Ions/s 40000 EtHg 4.36

30000 -

20000 -

1 MeHg I~ ~3.15 JI

10000

PhHg

0 0

200

400

600

800

Time (s)

Figure 7. Liquid chromatogram of four mercury species separated by HPLC and direct ICPMS detection of 25 ng of each specie (from Pastor, 1998).

chloride, and MIP-AES (Sadiki and Williams, 1996). During the direct separation of organotin, organolead and organomercury species in environmental samples (De Smaele et al., 1996) and sediment samples (Jantzen and Prange, 1995), ICP-MS was used in the determination. Methylated and alkylated species of As, Sn, Sb and methylated derivatives of Bi, Hg, Ge and Te can be also separated and determined by GC-ICP-MS (Krupp et al., 1996). Supercritical fluid chromatography (SFC) with ICP-MS detection has been proposed for the separation of trimethylarsine, triphenylarsine, triphenyl arsenic oxide, triphenylantimony, and diphenylmercury (Kumar et al., 1995). Liquid chromatography coupled to ICP-AES (McCarthy et al., 1983; Gjerde et al., 1993; Sanz-Medel et al., 1994) and coupled to ICP-MS (Haraldsson et al., 1993; Guerin et al., 1997) provides an excellent way for multimetal speciation. Direct injection nebulization in ion chromatography-ICPAES provides a good way for As, Se and Cr multispeciation (Gjerde et al., 1993). Using a nucleosil dimethylamine column and a mobile phase of ammonium

14

M. DE LA GUARDIA, M.L. CERVERA,and A. MORALES-RUBIO

phosphate buffer with gradient elution from pH 4.6 to pH 6.9, it has been possible to separate AsO]-, SeO~-, AsO 3-, and SeO 2-. Absolute detection limits of 52, 140, 57, and 91 ng, respectively, were obtained in these studies (McCarthy et al., 1983). The use of a C18 bonded silica column, modified with didodecylodimethylammonium bromide in 50% methanol, has been proposed to achieve the separation of As, Se, Hg, and Sn species using surfactant vesicles mobile phase and ICP-AES determination (Sanz-Medel et al., 1994). The technique of ICP-MS provides a highly selective and sensitive detector for multimetal speciation. It has been proposed as a method for A1, Cd, Co, Cu, Pb, Mn, Mo, Ni, Zn, and Fe speciation in waters using Chelex 100, Fractogel TSK DEAE-650 and C18 to fractionate the free metal ions or easily dissociate complexes, humic complexes on nonpolar organic compounds (Haraldsson et al., 1993). Monomethylarsonic acid, dimethylarsinic acid, selenite, selenate, tellurate and antimonate can be separated using a PRP • 100 anion exchange columns and determined accurately by ICP-MS (Guerin et al., 1997). Figure 8 shows an example of the chromatograms which can be obtained for simultaneous speciation of arsenic and selenium compounds using a microcolumn of anion exchange for the quantitative separation of monomethylarsonic, As (III) and As (V) and that of Se (IV) and Se (VI) using ICP-AES as a detector. The excellent sensitivity of AFS and ICP-MS detection and, in the latter case, its exciting possibilities for multimetal and isotopic analysis offer excellent possibilities for a rigorous speciation of liquid samples. An additional effort is required in order to improve both analyte separations and detection, in order to be I

000-

S e +4

800 t--

600

MMA :~

Se+6

-

400
cold vapor atomic spectrometry (CVAAS) > hydride generation atomic absorption spectrometry (HGAAS) > FAAS > ICP-AES = ICP MS > direct current plasma-atomic emission spectrometry (DCP-AES) = MIP-AES. All these techniques are useful for the analysis of liquid samples (except MIP-AES which is more convenient for gaseous samples). In recent years, hyphenated methods like those involving chromatographic methods linked to atomic ones have emerged. All of the hyphenated methods have shown high promise for specific metal species in specific sample matrices, with different degrees of selectivity, specificity and sensitivity/detection limit. All these techniques have provided the analyst a choice of methods for trace metal species determination. Actually, there is no simple way to decide which specific hyphenated or direct method of analysis is the best for a particular metal species in a particular sample matrix. Perhaps that remains as a lacuna in the whole scheme of trace metal speciation. Urine

Milk 11%

Blood 36 %

~%~176 "/~o / -

~'*~.,@'~q'r162 e9

~ ~ LBr~176176

1%

..g

d Figure 10. Distribution of published papers about speciation in biological fluids as a function of the type of sample considered.

18

M. DE LA GUARDIA, M.L. CERVERA,and A. MORALES-RUBIO ETAAS 22.4%

ICPMS 1

8

~

.

1

~

~

i

.

~

x

~

DCPAES 1.7%

~:I~HGAFS

2.6%

i![,~,PAES

3.4*/.

FAAS 9.5% ICPAES 16.4% HGAAS 13.8%

2.1%

Figure 11. Atomic spectrometric methods applied to speciation of biological fluids.

In several cases, atomic spectrometric measurements can be employed for speciation purposes without requiring the use of chromatographic techniques (de la Guardia, 1996). In these cases, the procedures could be based on (1) the different atomization yields obtained for different chemicals, (2) the use of selective extraction, (3) derivatization procedures performed previously to the measurement step, (4) a selective volatilization of the different chemical forms of the elements to be determined, or, (5) other less commonly used separation methodsme.g, ultrafiltration, coprecipitation, electrodeposition, or electrophoresis. The development of automated procedures of analysis based on flow injection analysis (FIA) techniques offers new possibilities for the on-line treatment of samples. It can be expected to provide the availability of simple and low-cost procedures for the metal speciation based on on-line separation and atomic spectrometric determination (Luque de Castro, 1986; de la Guardia, 1996). Thus, by introducing FIA, a 5.5- to 60-fold increase in the sensitivity is obtained for FAAS and a 15- to 50-fold increase for ETAAS. Examples for other speciation methods proposed for the analysis of various trace metal species are based on high-performance liquid chromatography (HPLC) separation followed by determination by differential pulse anodic stripping voltammetry (DPASV) (Michalke and Schramel, 1990) or radiotracer labeling (Cornelis, 1992). To date, there is no generalized method for speciation of protein-bound trace metals (Cornelis et al., 1993). To provide an idea of such a method, an outline of procedures applied to trace metal speciation in blood is presented in Figure 12.

A. Speciation in Blood Blood is the most important indicator for humans and domestic animals as an illness diagnostic. For clinical purposes, serum is commonly used but in some cases

19

Speciation Studies

,I, I Se arat,on o. t.e prote,ns!

~On-l inemonitoring-1 |UV and~or

~ o., 0erm.a,,on-, .n,on-. ca,,on-, a,,in,,,-ch,oma,o0r,0hy..,o~ i

iii i

~1

9

!

$

ii

i M,no;,raot,on I

Major fraction for trace element analysis

~Atomicspectrometry I

,,

L Visible spectrometry

1

i

I Protein identification ~ Nephelometry 1

i

1

I Protein quantification i1

~. Electrophoresi~s

Figure 12. Scheme for trace element speciation in blood (reproduced from Das et al., 1996).

whole blood samples may be analyzed (Pais, 1994). Blood contains thousands of different compounds, although with little variability in total ionic strength. A series of procedures have been proposed in the bibliography for trace metal speciation involving aluminum, arsenic, chromium, copper, iron, mercury, lead, platinum, selenium, silicon and zinc. Most studies have been made with aluminum and mercury followed by selenium and chromium and then the other metals. There are only a few studies focused on simultaneous speciation of several metals (Br~itter et al., 1988a).

Aluminum Aluminum toxicity is now well recognized and gaining more and more interest. The only aluminum oxidation state in biological samples is (+III). Metal speciation is a crucial feature in directing the biological effects of aluminum. In blood plasma, citrate is the main small carrier and transferrin the main protein carrier for AI(III or +3). In fluids where the concentrations of these two ligands are low, nucleoside mono- and diphosphates become aluminum binders and when they are absent, then catecholamines are the major ligands. Double-stranded deoxynucleic acid (DNA) binds AI(III) weakly, and in general, is unable to compete with other ligands for its complexation. In the cell nucleus, AI(III) is probably bound to nucleotides or to phosphorylated proteins (Martin, 1992). Nevertheless, organically complexed forms of aluminum appear to be much less toxic than inorganic forms. An aluminum

20


species of particular concern are A13§ AI(OH) 2§ AI(O H)~, and AI(O H)4 (Gardiner et al., 1984). Aluminum species present in human blood serum have been separated by gel permeation chromatography (GPC) and by ultrafiltration (Blanco Gonzalez et al., 1989). The former procedure was applied for dialysis patients to fractionate aluminum-bound species. Aluminium determination was performed by ETAAS. Several inferences have been drawn by these authors including: (1) albumin and probably transferrin are the major proteins that bind aluminium; (2) added aluminium is taken up by serum constituents only after incubation and a slow exchange of aluminium between the species occurs in serum; (3) the low molecular mass fraction is made up of mainly inorganic aluminium complexes; and, (4) the pH of the serum determines the level of ultrafiltrable aluminium. In experiments with patients undergoing desferrioxamine (DFO), chelation therapy showed that the ultrafiltrable aluminium in their serum increased by up to 74% because of the formation of a relatively low molecular mass chelate with the DFO. HPLC separation of serum proteins was performed with an ion-exchange column ofTSK DEAE-3SW using a sodium acetate gradient (0-0.5 M) at pH 7.4 (Tris-HC1 buffer). Proteins were detected spectrophotometrically at 280 nm and the aluminium determined by ETAAS in 0.5 mL fractions collected from the HPLC column. Results obtained with this system suggests that transferrin is the only aluminium binding protein in normal serum, but in the presence of DFO in serum, most of the aluminium was found to be bound to this drug. Further work in this area using ultramicrofiltration and HPLC techniques by Sanz-Medel and Fairman (1992) has revealed that more than 90% of the aluminium in serum is bound to the protein transferrin. To have a clear idea about the presence of A1 species in blood the following studies at clinically relevant concentrations must be performed: (1) identification of the aluminium binding serum proteins; (2) quantification of the amount of these protein-bound aluminium as well as non-protein bound fraction; (3) study of the effect of the Fe-transferrin saturation on the transferrin binding of aluminium which is important in view of the large number of iron-depleted patients; and, (4) investigation of the effect of DFO on the binding of A1 and Fe to transferrin (Van Landeghem et al., 1994).

Mercury There has been a continuous work on mercury species present in biological fluids. For the determination of mercury one of the most selective and sensitive methods is the use of CVAAS based on the low boiling point of Hg ~ and the easy reduction of mercurials to the zero-oxidation state (Magos, 1971). From a number of published papers on mercury speciation in blood, it can be concluded that the reduction of mercurials has been selectively carried out by using different reduction steps with SnC12 and with a mixture of SnC12 + CdC12. Recently, most of the speciation methods for mercury are based on chromatographic separations with detection by means of atomic spectrometry. A procedure

Speciation Studies

21

for determination of organic and inorganic mercury in various biological materials including blood by ETAAS has been reported (Filippelli, 1987). Organic mercury was extracted as the chloride in benzene and reextracted by a thiosulphate solution. The organic mercury thiosulfate extract was next treated with cupric chloride, reextracted in the benzene layer, and analyzed by GLC for speciation. Inorganic mercury was converted into a methyl chloride derivative by methanolic tetramethyltin prior to extraction. Speciation of mercury in human whole blood by capillary gas chromatography with a MIP-AES system following complexometric extraction and derivatization has been described by Bulska et al. (1992). In this method, methyl- and inorganic mercury were extracted in toluene from whole blood samples as their diethyldithiocarbamate (DDTC) complexes. The product was butylated and the mercury species were then separated and detected.

Selenium The accurate speciation of selenium has been a major challenge for analytical chemists and knowledge of its pathways in the environment and living organisms is still limited. The metal can either be considered as essential; 10 to 40 lxg mL -1 in serum and 0.1 I.tg mL -1 in urine or can be toxic when it is in excess. Br~itter et al. (1988a) have developed a procedure for establishing profiles of selenium protein in various body fluids via the on-line coupling of gel permeation chromatography and ICP-AES after performing the acid digestion treatment and the formation of Se(IV) with the subsequent hydride generation in the connecting flow. Recovery studies have been performed to examine and optimize the wet ashing conditions with the aid of various selenium compounds. To demonstrate the usefulness of this technique for the speciation of selenium, its distribution in samples of human origin has been measured. In serum, selenium was found distributed among three different fractions. The location of these peaks seemed to be similar to those of zinc. The highest selenium peak at 90 + 15 kDa is in the same region but shows a broader shape when compared to zinc. This may be due to the presence of selenium containing enzyme glutathione peroxidase (molecular mass 88 kDa) which elutes in the same region. The selenium content bound to different fractions were as follows: 90 kDa fraction 77 + 4%, 200 kDa fraction 10 + 3% and the high molecular mass (>600 kDa) fraction 13 + 5%.

Chromium Speciation of chromium in blood has become an important area of research after it was known that patients with terminal renal failure treated with hemodialysis or continuous ambulatory peritoneal dialysis become iatrogenically loaded with chromium. The fact was revealed through observation of very high chromium concentrations (4.25 ng mL -1) in their serum in comparison to normal healthy persons (0.16 ng mL -1) (Wallaeys et al., 1986). Urasa and Nam (1989) developed a method for chromium speciation using both anion- and cation-separator columns. The

22


procedure used DCP-AES detection and required a preconcentration step to achieve a detection limit of 1 ng mL -1. The method was applied to human serum and other samples. The authors found 0.05 gg mL -1 of Cr(VI) and 0.06 l-tg mL -1 of Cr(III) in a standard reference material of human serum in freeze-dried form obtained from the National Institute of Standards and Technology (SRM 909); although the reducing capacity of saliva and stomach involves that any intake of Cr(VI) can be readily reduced to Cr(III) (De Flora and Wetterhahn, 1989). Chromium speciation in plasma was reported by Cornelis and her group. Chromium is known to be mainly bound to two plasma proteinsmtransferrin (molecular mass = 80 kDa) and albumin (molecular mass = 64.5 kDa). A suitable separation procedure was developed for these proteins with in vitro 51Cr(III) labeled plasma from healthy persons as well as from dialysis patients (Cornelis et al., 1992). Speciation studies were also undertaken with the aid of in vitro and in vivo 51Cr-labeled rat and rabbit plasma. It consisted of a combination of fast protein liquid chromatography with cation- and anion-exchange system, ensuring a complete resolution of both proteins and a total recovery of chromium. The metabolized Cr was measured by using NaI (T1) detector of the 51Cr label. On the other hand, identification and quantification of proteins were done by isoelectrofocusing and nephelometry, respectively (Cornelis and De Kimpe, 1994). The 51Cr has been found to distributed as follows: 85% is associated with the transferrin, 8% seems to be bound to albumin and 6% appears to be spread over the other components (Cornelis et al., 1992). Since transferrin is usually saturated at 30% only with iron, this protein should indeed be considered as a potential binding site for chromium (Wallaeys et al., 1987). Recent studies demonstrated that 51Cr can shift from albumin to an unidentified low molar mass complex in ambulatory peritoneal dialysis patients (Borguet et al., 1995).

Arsenic Arsenic occurs in both inorganic and organic forms which exhibit large differences in their metabolism and toxicity. Elimination kinetics has shown that arsenic is removed very quickly from blood to urine with a half-life in the body of about 30 h (Chana and Smith, 1987). The determination of inorganic arsenic and organoarsenicals in biological fluids was reviewed by Violante et al. (1989). This chapter emphasizes the necessity for distinguishing between As of nutritional origin and that from water or the environment and for guarding against possible interconversion of the inorganic oxidation states during sample treatment. Speciation studies for arsenic in blood are comparatively less numerous than in urine. Speciation of As (III) and As (V) in biological materials including blood was studied by HNO3-H2SO 4 digestion followed by hydride generation AAS technique. It was found that the extent of change of the original valency of As was not reproducible (Weigert and Sappl, 1983). Fast protein liquid chromatography cation and anion exchange separation scheme (Cornelis et al., 1993) were applied to serum incubated

Speciation Studies

23

in-vitro with carrier-free 74ASO43 for 24-hr. All 74As was completely eluted from the cation exchanger together with the negatively charged unbound proteins. The ultraviolet (UV) responses of the separated species in combination with the metalspecific responses can be used for correlating the arsenic species with the bulk amount of potential arsenic-binding partners in serum. The protein fractions were identified as asialotransferrin, sialotransferrin and albumin carrying, respectively, 17.7, 25.3, and 56.3 of total 74As radioactivity, i.e. arsenic is bound to these proteins in these proportions. However, when the albumin fraction was subjected to gel permeation chromatography on a Superose column for differentiating the molecules according to molecular mass instead of charge, the elution patterns of the albumin and the 74As did not coincide anymore.

Copper and Zinc Recently, several reports have appeared in the literature on the speciation of zinc. Faure et al. (1990) separated the human serum fractions by ultrafiltration with the use polyacrylonitrile membranes. Loosely bound zinc (bound to albumin and some other proteins) was separated with ultrafiltrable zinc after treatment of the serum with ethylene diamine tetraacetic acid (EDTA). The difference between the loosely bound and total zinc gave the content of strongly bound zinc, i.e. to t~2-macroglobulin. The zinc content in each fraction was measured by conventional ETAAS. Zinc in serum is bound to macroglobulin (720 kDa) and albumin (66.5 kDa) whereas Cu is bound to ceruloplasmin (160 kDa) and albumin (66.5 kDa) (Gardiner et al., 1981). Sch/3ppenthau and Dunemann (1994) have reported the separation of serum for characterization of metals (including copper and zinc) and nonmetal species by size-exclusion chromatography (SEC). The coupling of HPLC to ICP-AES was performed by connecting the column outlet of the chromatographic system with the nebulizer of the metal-specific detection systems of ICP-AES or ICP-MS. The metal distribution patterns in serum samples indicate a Cu maximum at 68 kDa which again correlates with the first major sulfur maximum at 75 kDa. Thus Cu may be bound to the albumin fraction. The Zn maximum has been recorded at the 49 kDa region. The absence of the high molecular mass proteins in the investigated samples was explained by the method of sample preparation (i.e. centrifugation and filtration through a 0.2 ~m membrane).

Iron In a pioneering study, for the determination of iron species in serum samples, an HPLC-ETAAS hyphenated method with an on-line metal scavenger for studying protein binding has been reported (Van Landeghem et al., 1994). Due to the currently introduced procedures for the treatment of anemia in dialysis patients involving a relative iron deficiency in these subjects, the study on the competition between A1 and Fe for binding to transferrin presents a renewed interest. As shown in this study, over 80% of the total serum A1 and over 97% of the total serum Fe is

24


bound to transferrin, indicating both elements do not dissociate from transferrin during gradient elution. The postelution Fe recovery was found to be 97.3 + 2.0%.

Lead The speciation of lead has attracted great deal of interest particularly with respect to organolead compounds. A high-resolution GC-ETAAS method for the determination of trimethyllead salts in human blood samples has been described by Nygren and Nilsson (1987). The method involves several steps including complexation with sodium diethyldithiocarbamate (pH 9), extraction with pentane, evaporation, and butylation by using a Grignard reagent in tetrahydrofuran.

Platinum In recent years, platinum compounds, especially cisplatin, are used for the treatment of cancer. In the analysis of biological fluids of patients treated with platinum salts, it is desirable to identify free platinum species and those bound to macromolecules. Ion exchange (Bannister et al., 1978) and ultrafiltration (Pinta et al., 1978) techniques coupled to traditional atomic spectrometry have been applied to study the different platinum species in blood plasma and serum. Cisplatin, its hydrolysis products, and two methionine-platinum complexes were studied by reversed phase ion-pair chromatography with on-line ICP-AES and applied to the analysis of (bio)transformation products originating from cisplatin in human and rat plasma in vitro and in vivo (De Waal et al., 1987). Free platinum (not bound to proteins) in plasma has been separated by ultrafiltration and the metal concentration was determined by ICP-AES (Dominici et al., 1986).

Silicon Silicon is becoming a biological trace metal of increasing scientific interest, particularly in connection with neurological disorders associated with aluminium in dialysis encephalopathy and in Alzheimer's disease. Relatively few analytical data exist on the concentration of Si in physiological fluids in health and disease (P6rez Paraj6n and Sanz-Medel, 1994). To provide evidence on the possible correlation between A1 and Si levels in the serum of renal failure patients, and the possibility of the reduction of aluminium bioavailability by the presence of silicon in biological fluids, the effects of different factors including storage conditions, administration of desferrioxamine, and kidney transplantation on the total A1 and Si contents and on their distribution in the same serum samples were examined and compared by Wr6bel et al. (1994). Ultramicrofiltration was used for the separation of low molecular mass and high molecular mass serum fractions and ETAAS for the determination of Si. Distribution of Si in serum proved to be affected only by the storage conditions. When the sample is stored properly (pH < 7.8), the ultrafiltrable Si content results were consistent and reproducible. It was found that 43 +

Speciation Studies

25

3% of total serum Si in the low molecular mass fraction was ultrafiltrable. Nevertheless, Si binding to serum proteins must be different from that observed for aluminium.

Various Metal Speciation Speciation of various metals in human serum by anion exchange and size exclusion chromatography (SEC) with detection by ICP-MS was reported by Shum and Houk (1993). A direct injection nebulizer was used with packed microcolumns for anion exchange chromatography and SEC. Proteins in human serum were separated by SEC without sample pretreatment. The metals present in each molecular mass fraction were determined by ICP-MS with detection limits of 0.5 to 3 pg of metal. Six metal-binding molecular mass fractions determined in human serum were assigned as follows: (1) >650 kDa fraction for Pb, Cd, Zn, Cu; (2) 300 kDa fraction for Pb, Zn, Cu; (3) 130 kDa fraction for Pb, Cd, Zn, Ba, Cu, Na; (4) 85 kDa fraction for Fe; (5) 50 kDa fraction for Zn, and (6) 15 kDa fraction for Pb and Zn. There was only one Fe-binding molecular mass fraction found at 85 kDa and this could be serum transferrin. The proteins responsible for the other molecular mass fractions required identification. In summary, speciation studies in blood must take into consideration the low metal contents in clinical samples, the presence of free ions and protein-bounded metals, and the effect of added compounds, like EDTA or DFO. Additionally, natural occurring organometalic species must be considered.

B. Speciation in Urine In the case of intoxication or to provide information on the balance between intake and output, the level of trace metals like arsenic, mercury, or selenium in urine is frequently taken as an indicator, since the kidney is an important feature in body homeostasis. Like blood, urine is a complex matrix often causing analytical problems because of its high salt content and a range of organic constituents. When applied to a chromatographic system, the urine matrix may create column overload problems. This can result in peak splitting or broadening of the analyte signals. Thus samples must be subjected to a desalting process, before subsequent separations, to allow control of the elution parameters. The major work that has been performed on metal speciation in urine samples concerns speciation of arsenic followed by selenium and then mercury. Reports on the speciation of other metals like tin, lead, cadmium, chromium, zinc, iron, and magnesium in this biological fluid have been also reported.

Arsenic The presence of As in the human body mainly occurs through food and/or occupational exposure. After absorption in the gastrointestinal tract or in the lungs,

26


this metal is eliminated through urine. Inorganic arsenic undergoes considerable biotransformation in the body; both monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) derivatives are formed. The proportion of inorganic and methylated species in urine may vary, although DMA is generally the major metabolite (Buchet et al., 1981). Although total urinary arsenic determinations are often used to assess occupational exposure to inorganic arsenic, specific measurements of DMA, MMA, and inorganic arsenic provide a more reliable indicator or exposure than total urinary arsenic levels (Chana and Smith, 1987). Again, organoarsenic compounds such as arsenobetaine have been found to be present in urine following the ingestion of seafood. The decreasing order of toxicity of arsenic compounds is now well known: arsenite > arsenate > MMA > DMA > As ~ > arsenobetaine. Different types of methods have been investigated for the speciation of arsenic compounds in urine. Initially for speciation studies of arsenic compounds by atomic spectrometry, a series of procedures based on the use of a selective liquid-liquid extraction or the use of chromatography were reported. Several reports deal with the separation and detection of arsenic species present in urine. This has been performed by HPLC coupled with hydride generation AAS for the determination of arsenobetaine (AB), arsenocholine (AC), and tetramethylarsonium cations in human urine (Momplaisir et al., 1991), and also for the determination of arsenite, arsenate, DMA, and MMA at Ixg L -l As level (Chana and Smith, 1987). There are several reports (Jimenez de Bias et al., 1994a; Le et al., 1994) on the determination and speciation of arsenic in human urine by ion-exchange chromatography-flowinjection analysis with hydride generation AAS. Eight arsenic compounds (four anionic, such as arsenite, arsenate, monomethylarsonate, and dimethylarsinate, and four cationic, such as arsenobetaine, trimethylarsine oxide, arsenocholine, and tetramethylarsonium ion) in urine were separated by anion- and cation-exchange HPLC and detected by ICP-MS at m/z = 75. Hexahydroxyantimonate(III) was used as an internal standard for their qualitative analysis. Arsenite was unstable in both urine samples and standard mixtures when diluted with a basic (pH 10.3) mobile phase used for anion chromatography. Interference due to 4~247 was eliminated by chromatographic separation of the chloride present in the sample from the arsenic analytes (Larsen et al., 1993a).

Selenium Nearly all information on selenium species in urine refers to rats and mice. Studies on selenium metabolites in human urine are less numerous. Trimethyl selenonium ion (TMSe§ a detoxification metabolite of selenium that is excreted in the urine, was first isolated and identified in rat urine (Palmer et al., 1969). The determination in human urine by cation exchange chromatography and ETAAS was reported by Tsunoda and Fuwa (1987). Fodor and Barnes (1983) were able to speciate selenate and selenite from urine samples by using different pH values for

Speciation Studies

27

complexation with a poly(dithiocarbamate) resin. It was found that for the urine of 11 healthy persons, the selenite content (8.6 I.tg L-1) was on average about three times more than the selenate concentration (3.1 lag L-l). Laborda et al. (1993) analyzed chromatographic effluents containing selenium species of urine samples by ETAAS using a sampling procedure based on fraction collection and hot injection into an electrothermal atomizer. The HPLC separation of TMSe § SeO32, SeO42 was performed by anion-exchange chromatography using 0.01M ammonium citrate at pH 3 and 7 as eluent.

Mercury For mercury speciation in urine samples similar methods have been suggested. In work on mercury speciation in urine and blood, the reduction of mercurials were selectively performed by using different reduction steps with SnC12 and with a mixture of SnC12+ CdC12. Several workers (Robinson and Wu, 1985; Seckin et al., 1986) have separated the organomercury species of urine primarily by gas chromatography followed by AAS detection. Samples of urine were injected directly on to a Chromosorb W AW-DMCS column interfaced with ETAAS. Inorganic Hg was the major form of mercury excretion. The results indicated the presence of unidentified nonvolatile Hg species in the samples (Robinson and Wu, 1985). Mercury and its species have been determined in urine samples from humans after breathing air in a dental workplace. The urine was treated with concentrated nitric acid and digested in a polytetrafluorethylen (PTFE) reactor at 140 ~ for 90 min. The resulting solution was mixed with SnC12 and the Hg vapor produced was analyzed by CVAAS. For GLC analysis of urine, samples were extracted with toluene and then with benzene. The extract was analyzed with temperature programming from 110 to 220 ~ at 15 ~ min -l. Nitrogen as carrier gas and 63Ni-electron capture detection was used (Seckin et al., 1986). Urine samples have also been analyzed by Shum et al. (1992) after separation with ion-pair liquid chromatography followed by ICP-MS detection with direct injection nebulization. A 24 h urine specimen was analyzed by this method and Hg 2§ methylHg§ and ethylHg§ species were found.

Other Metals Trialkyltins are the organotins having the greatest biocidal activity in mammalians. A purge and trap flame photometric gas chromatographic technique for speciation of trace organotin and organosulphur compounds in human urine standard reference material was reported by Olson et al. (1983). Urine samples were purged with nitrogen, with or without prior treatment with NaBH 4, and the volatile compounds were trapped on Tenax GC. These compounds were desorbed by heating the Tenax and were analyzed by GLC. The flame photometric detector was used in either the sulfur mode (394 nm) or the tin mode (600 to 2000 nm) with a hydrogen-rich flame. Neutral volatile organotin and organosulfur compounds did not require a pretreatment with NaBH4. This is necessary for alkylchlorotins.

28


Speciation studies of mono-, di- and tributyltin compounds in urine have been performed by extraction, GC separation, and ETAAS determination (Dyne et al., 1991). Preconcentration and determination of ionic alkyl lead compounds in human urine has been described by Neidhart and Tausch (1992). Preconcentration and cleanup steps were followed by HPLC. Ionic species were preconcentrated by solid-phase extraction and detection was performed on-line. The alkyllead species were eluted from the HPLC column, partially dealkylated by iodine solution to form dialkyllead species, and detected as [4-(2-pyridylazo) resorcinol] complexes after the reduction of excess iodine with thiosulfate. Various cationic species of lead as Pb 2§ Me3Pb § and Et3Pb § were separated as ion pairs by reversed-phase liquid chromatography-ICP-MS with a direct injection nebulization system (Shum et al., 1992). The method was tested for measurement of Pb species in National Institutes of Science & Technology-Standard Reference Material (NIST-SRM) 2670 freezedried human urine. Since this reference material contained only Pb 2§ and did not contain measurable levels of Me3Pb § and Et3Pb § these compounds were spiked in the NIST urine to test the suitability of the method. Reasonable chromatographic peaks were seen for Pb 2§ Me3Pb § and Et3Pb § species in the spiked urine sample. A thermospray-interfaced HPLC-flame AAS system was developed for studies of cadmium speciation in human body fluids by Chang and Robinson (1993). The cadmium compounds in urine were separated by HPLC on a Zorbax C 8 column with water as the mobile phase. The column eluate was fed directly to the thermospray interface and the analyte delivered directly into the base of the flame for effective atomization and detection by AAS. Successful separations were achieved for a large number of nonvolatile cadmium compounds.

C. Speciation in Milk Human milk, cow milk, or infant formulas are the main nutrient fluids for newborn infants. Assuming that the composition of breast milk may satisfy the growing demand of healthy babies during their early months of age, it could be a reference to evaluate the nutritional value of alternative milk formulas. The protein and composition as well as the kind of trace metal of breast milk have been known for the last several years. But the absorption and utilization of the trace metals depend not only on the total amount in the milk but also on the availability of the chemical form in which they occur. Hence the speciation of metals in milk is very meaningful. Few metal speciation studies have been performed in this body fluid. Only several reports on speciation of zinc, cadmium, selenium, mercury, lead, and a few other metals in milk may be found in the literature. Babies at their early ages are susceptible to selenium deficiency. This may arise due to intake of milk or infant formulas with low selenium content. Again, the total amount of this metal does not ensure the overall utilization or bioavailability of selenium. Thus the chemical forms of selenium and its distribution in food are

Speciation Studies

29

important factors of selenium bioavailability. The distribution patterns of selenium species in cow and human milk were compared by Van Dael et al. (1988). Milk samples were fractionated into fat, whey, and casein parts by ultracentrifugation. In order to separate the lipid components, milk fat globule membranes were solubilized with sodium dodecyl sulphate. Further centrifugation separated the outer and inner fat globule membranes from triglycerides. After separation all fractions were lyophilized and stored at -20 ~ Samples were digested with a mixture ofHNO 3 and HC10 4 and then analyzed for selenium by hydride generation AAS. The study revealed that the whey fraction represented 40 and 72% of the total selenium content of cow and human milk, respectively. The lipid fraction contained approximately 10% of either cow or human milk's total selenium. After solubilization of milk fat globule membranes, 61 and 80% of selenium was found in the outer fat globule membrane for cow and human milk, respectively. Determination of the selenium content in individual proteins of cow's milk revealed that the highest selenium concentrations were present in 13-1actoglobulin and in K-casein. Br~itter et al. (1988a) developed a procedure to determine selenium protein profiles in skimmed human breast milk via on-line coupling of gel permeation chromatography and ICP-AES after performing the acid digestion procedure and the formation of Se(IV) with the subsequent generation of selenium hydride in the connecting flow. In total, four selenium peaks (molecular mass at >600, 90, 25, and 10 kDa) have been detected in breast milk. A method for the preparative separation of human breast milk proteins was developed by Michalke (1993), keeping metal-protein complexes intact, especially with respect to zinc and cadmium species. Separations were performed on TSK columns, using HW 55 gel, with double distilled water as the mobile phase. The metals were determined in native human milk, the protein pellet, and supernatant (without fat fraction) as well as in peak related HPLC-fractions of protein pellet and supernatant with differential pulse anodic stripping voltammetry (DPASV) for cadmium and DCP-AES for zinc, respectively. Cadmium content of whole breast milk, the protein pellet, and the peak-fraction corresponding to metallothionein was determined to be 1 Ixg L -1, whereas no cadmium was found in the supernatant. On the other hand, the amount of zinc was found to be about 3.5 mg L -1 in human milk and only a small quantity (160 ~g L -1) could be detected in the protein pellet. Zinc content could be related to several breast milk proteins (e.g. metallothionein) in different amounts. In the case of the supernatant, zinc was related only to citrate. On-line combination of gel filtration chromatography and ICP-AES has been applied for the fractionation and identification of metal-containing species in skimmed human milk including background control and its subtraction. Subtraction yielded a selenium peak of lower intensity and its shift to position around 10 kDa. At this position, iron, manganese, and zinc elute exactly in the same fraction. Another chromatogram of defatted human milk showed the distribution of copper, iron, manganese, and zinc. Fromthis study, the binding of citrate was established

30


with zinc. There was also some evidence for the binding of iron to citrate (Br~itter et al., 1988a). The technique of ICP-AES coupled to a gel filtration chromatography system was used by Br~itter et al. (1988b) to characterize the species containing copper, zinc, iron, manganese, magnesium, and calcium in human milk and milk formulas based on cow's milk. The distribution profiles clearly indicate that the metal (Cu, Zn, and Fe)-protein binding in human milk and milk formulas are different. Investigations of protein-bound trace metals in human milk before and after the birth of a baby showed marked differences in zinc and iron bound to citrate. An increase of citrate concentration by a factor of 25 + 3 takes place after birth at the first day of milk secretion. About 80% Cs, 22% Sr, and 1.5% Eu were recovered in pectin when fresh pasteurized skimmed cow's milk spiked with the respective radionuclides (85Sr, 137Cs,and 152Eu) was shaken with a 4% aqueous solution of apple pectin at an initial milk/pectin volume ratio of 7:3. The recovery fraction was proportional to the abundance of radionuclides in milk. Extraction of the spiked milk was performed with Aerosol OT in isooctane (Mac~igek and Gerhart, 1994). Binding of added strontium by milk proteins under native conditions was also investigated using pectin of various degrees of esterification. Upon partition of Sr, Cs, and Eu in aqueous two-phase milk-pectin system performed by membraneless dialysis, it was compared with the distribution between cation exchanger and milk, milk formula, or pectin solutions. The low molecular mass fraction of added Sr in milk assessed from Dowex 50 x 8 sorption data was found to be 31% and that of Cs and Eu were 58 and 40%, respectively (Mac~igek et al., 1994).

D. Speciation of Miscellaneous Biological Fluids Some papers concerning different biological fluids in which speciation studies have been carried out other than blood, urine, and milk have been also published. Some examples are on blood cell lysate, sweat, saliva, cerebrospinal, seminal, tear, and bronchoalveolar fluids which have been developed in recent years are presented below. Several reports varying in manipulative complexity have been proposed for determining As, Fe, Zn, and Se species of blood cell lysate. The binding of arsenic in the red blood cells of rat were investigated by Cornelis and De Kimpe (1994) because it was thought that most of this metal binds to hemoglobin (Hb). The radiotracer 74As was used throughout the experiment. The red blood cells lysate was submitted to size exclusion chromatography (SEC) using Superose HR 12 column and 0.15 M NaCI + 25 mM Tris (pH 8) eluent and cation-exchange chromatography using Mono S HR 5/5 column and 10 mM malonic acid (pH 5.8) and 10 mM malonic acid + 0.3 M LiC1 (pH 5.8) eluents. The study of binding of As in the red blood cells lysate with SEC showed that 87.7% of the metal is associated with a protein with a relative molecular mass of around 60 kDa and a

Speciation Studies

31

strong absorption band at 420 nm. This compound is Hb. Cation exchange of the SEC fraction showed that the signal peak observed in the SEC chromatogram consisted of several Hb species, each carrying part of the 74As. On-line combination of gel filtration chromatography and ICP-AES for studying metal speciation in blood serum and human milk has already been discussed in previous sections. The same technique was also used for red blood cell lysate (Br~itter et al., 1988a). From the gel filtration chromatography of human erythrocyte-lysate on a TSK column, it may be found that selenium is eluted in two peaks which correspond to a molecular mass of 90 kDa and 33 kDa. The high molecular mass compound could be classified with the selenoenzyme glutathione peroxidase. Iron and zinc were also monitored with selenium. Fe indicates the position of Hb in erythrocytes and Zn being in the position of the enzyme carbonic anhydrase. Owing to the narrowness of these two markers, a clear identification of the selenium binding complex at 33 kDa is not possible. Accumulation of iron in the myocardium in circumstances of transferrin saturation is associated with heart failure in iron-loaded patients. To characterize the underlying causes of this phenomenon, Parks et al. (1993) measured the flux as well as the speciation of iron in normal and iron-loaded cultures of rat myocardiocytes. Iron loading of cultured myocytes induced shifts in iron speciation. Thus the ratio of iron bound in hemosiderin-like compounds to ferritin-bound iron increased twofold from a range of 0.84 to 1.44 in control cells to 1.96 to 3.3 in iron-loaded cells. Only few analytical methods are known for determining chemical species of mercury, cadmium and sodium in sweat (Robinson and Wu, 1985; Chang and Robinson, 1993). Calcium and magnesium species have been studied in saliva (Lagerlof and Matsuo, 1991). Speciation of iron, potassium, sodium, and calcium has been reported in cerebrospinal fluid (Gutteridge, 1992). Ferrous ion has been detected in cerebrospinal fluid by using bleomycin and DNA damage. Normal cerebrospinal fluid samples were centrifuged and the supernatant liquid was stored a t - 2 0 ~ For the determination of Fe(II) species the following reagents were added in order, into metal-free plastic tubes: (1) 0.4 mL of DNA solution (1 mg mL -1) stored over 0.05 volumes of Chelex-100 resin, (2) 0.1 mL of bleomycin sulphate (1.5 mg mL -1) stored over a solution of conalbumin (5%, w/v) where 120 mM sodium azide was added to inhibit the ferroxidase activity of ceruloplasmin, (3) 0.1 mL of cerebrospinal fluid, and (4) 0.4 mL of sodium phosphate buffer (pH 7) treated with conalbumin. The mixture was incubated at 37 ~ for 30 min to allow Fe2§ ent degradation of DNA and then treated with 0.5 mL of thiobarbituric acid (1%, w/v in 50 mM NaOH) and 0.5 mL of HC1 (25%, v/v). The mixture was heated at 100 ~ for 5 min, cooled and extracted with 1.5 mL of butan-1-ol. The mixture was centrifuged and the thiobarbituric acid-reactive material in the clear upper organic layer was determined spectrofluorometrically.

32


In seminal fluid, iron and zinc species have been studied by various authors (Caldini et al., 1986; Gavella, 1988). Atomic absorption techniques for direct determination of multimetal species in whole tear film were described by Giordano et al. (1983). Dissolved sodium, lithium, potassium, calcium, and magnesium were determined by flame AAS. No matrix effect was observed. ETAAS was used for determination of chromium, manganese, iron, lead, zinc, copper, nickel, cobalt, arsenic, and aluminium. These metals were present in the range between less than 0.01 to 1 l.tg mL -1 and probably, following this methodology the total content is determined. The reader is invited review Table 1 for the analytical details of the selected metal ion species in biological fluids. It suggests that chromatography is the method of choice for the preferential separation of various components in biological fluids. After separation, metals are usually analyzed by atomic spectrometry techniques suitable for trace analysis such as AAS (flame and electrothermal) inductively coupled plasma spectrometry (ICP-AES and ICP-MS). However, in some work, radiochemical methods like neutron activation analysis (NAA) and electrochemical techniques like DPASV have been used. It is clearly evident from Table 1 that serum and urine are the more commonly studied matrices. There has been more interest in speciation studies of arsenic, mercury, and selenium in different biological fluids. Aluminium in blood has also been studied widely. Only few reports are available on metals like cadmium, chromium, iron, lead, platinum, zinc, copper, and others in these matrices. An examination of Table 1 also reveals that the detailed information about the analytical performance of the developed methodologies is not very often reported. Speciation studies of biological fluids are very significant in clinical chemistry to understand the physiological behavior of trace elements in the living system. We have tried to highlight some of the achievements realized until now for the separation and quantification of the metal species which might be present below sub-lxg L -1 level in biological fluids. From the published literature it can be concluded that it is not possible to detect a general methodology for trace metal speciation in biological fluids. However, some important aspects of the most appropriate methodology could be taken into consideration, such as the importance of mechanical separation. This is a useful preliminary step to identify the different size of the chemical species. This provides information about the molecular mass of the proteins or the other molecules to which the metal ions are bound. This step, commonly performed by ultrafiltration or centrifugation, can be considered as a specific methodology for speciation studies in biological materials as a difference of similar studies carried out in other matrices, such as sediments, soils, or environmental samples. After the physical separation of different molecular mass fractions, the combination between chromatographic method and sensitive atomic spectrometric detection, as indicated for water analysis, seems to be the best alternative for the identification and quantitative analysis of different species. However, there is a lack

Table 1. Analytical Details of the Atomic Spectrometry Procedures Developed for Speciation in Biological Fluids

OJ

Element

Species

Matrix

Methodology

DetectionLimit

RSD

AI

Transferrin

Serum

HPLC-ETAAS

AI

Albumin, transferrin

Serum

GPC-ETAAS

AI

Proteins with different MM

Serum

SEC-ETAAS

AI

Albumin, transferrin

Serum

Ultrafiltration, HPLC-ETAAS

As

Urine

AS

AsC~3-,AsC)34-, MMA, DMA, AB, AC, TMA +, As-sugars AsO~3-,AsO34-, MMA, DMA, AB, AC AsC~3-,AsO34-, MMA, DMA

0.12 l~g L-1

8%

HPLC-HGAFS

--

--

Urine

HPLC-HGAAS

8-15 l~g L-1

2.5-5.3%

--

Blood

IEC-HGAAS

0.44-0.92 Ilg L-1

3.6-6.8%

90-110%

As

AsO~3-,AsO34-, MMA, DMA

Urine

--

--

94.9-107.7%

As

MMA, DMA, AB, AC

Serum

Extraction-ETAAS HPLC-ICPMS IEC-HGAAS

1-1.5 Mg L-1

4.2-4.8%

>90%

As

AsO~3-,AsO34-, MMA, DMA

Urine

HPLC-MIPAES

AsO~3-, DMA Toxic-As (AsC~3- + AsOa4- + MMA + DMA) Non-toxic-As (AB + AC)

Urine Urine

IC-ICPMS Microwave digestion-HGAAS

2.8% (AsC~-) 2.5% (AsO34-) 3.1% (MMA) 2.1% (DMA) -4-7%

--

AS As

1 ng mL-1 (AsO:]-) 5 ng mL-1 (AsCP4-) 6 ng mL -1 (MMA) 1.2 ng mL-1 (DMA) -4-6 I~g L-1

AS

Recovery

Ref.

101.1 + 15.3% Van Landeghem et al. (1994) -Gardiner et al. (1984) 60-125% Keirsse et al. (1987) Blanco Gonzalez et al. (1989) Wrobel et al. (1994) -Le et al. (1996)

---

Lopez Gonzalvez et al. (1996) Zhang et al. (1996a) Bavazzano et al. (1996) Zhang et al. (1996b) Costa Fernandez et al. (1995) Feldmann (1996) Lopez Gonzalvez et al. (1995)

(continued)

Table 1. Continued Element

Species

Matrix

Methodology

Detection Limit

RSD

As

AsO]3-, AsO~-, MMA, DMA

Urine

90-300 pg

3.4-5%

~

Ding et al. (1995)

As

Inorganic-As, MMA, DMA

Urine

Micellar LCICPMS IEC-HGAAS

0.9 ~g L-1 (Inorg-As)

3.5% (Inorg-As)

~

Jimenez de Bias et al. (1994b)

Urine

IC-ICPMS

1.3 l~g L-1 (MMA) 0.5 I~g L-1 (DMA) 0.22-0.44 l~g L-~

3.2% (MMA) 4.6% {DMA) 3.2-4.9%

Urine

HPLC-HGAAS

2 14g L-1

--

Chana and Smith (1987)

Blood

Digestion-HGAAS

m

5.1% (AsC~3-}; 3.8% (MMA); 5.3% (DMA); 7.2% (AsO34-) --

90-105%

m

--

Weigert and Sappl (1983) Dix et al. (1987)

As As

AsO]-, AsCP4-, MMA, DMA, AB, TMAO AsO]-, AsCP4-, MMA, DMA

,~o~-, , ~ -

4~

0.1-5 ng L-1 Blood, Derivatization-GC urine Urine HPLC-ICPMS 3-6 ng m1-1 (cations) 7-10 ng m1-1 (anions) Urine Extraction-ETAAS 10 l~g L-1

As

Inorganic and organic-As

As

AsO:]3-, AsO43-, MMA, DMA, AB, AC, TMAO, TMA+

As

Inorganic and organic-As

As

Urine

IEC-HGAAS

As

Inorganic, MMA, hydroxydimethylarsine oxide AB, AC, TMA +

Urine

HPLC-HGAAS

As

Inorganic, MMA and DMA

Urine

IEC-HGAAS

0.5 l~g L-1 I 2 l~g L-1

Recovery

Ref.

Inoue et al. (1994)

23% (AB}; 96-108% Larsenet al. (1993) 5.4-8.9% (others) 7.9% (inorg-As); 93% (inorg-As}; Fitchett et al. 3.6% (DMA} 88.4% (DMA) (1975) 3-6% 95-102% Buratti et al. (1984) --

85-97%

3.2-4.6%

85-93%

Momplaisir et al. (1991} Jimenezde Bias et al. (1994a)

As

AsO~3-, AsO34-, MMA, DMA

Urine

HPLC-ICPAES

10 I~g L-1 (AsO~3-, DMA, AB); L-1 (MMA); 15 l~g 20 llg L-1 (AsC~4-) 0.5 l~g L-1

As


Urine

HPLC-ICPMS

36-96 pg

As


Urine

HPLC-ICPMS

Cd

Proteins with different MM

HPLC-FAAS

Cr

Cr(lll), Cr(VI)

Cr

Cr(lli), Cr(VI)

Urine, sweat Blood, urine, serum Urine

63 pg (AsC~-); 37 pg (AsC~4-); 80 pg (MMA, DMA) 1 I~gL -1

Cr

Cr(lll), Cr(VI)

Urine

Cr

Cr(Vl)

Cr

Cr(lll), Cr(VI)

Blood, urine Serum

Fe

Transferrin

Hg Hg

As

M'I

AsO~3-, AsO43-, MMA, DMA, AB Urine

HPLC-ICPMS

Le et al. (1994)

Low et al. (1986 and 1987) Heitkemper et al. (1989) Sheppard et al. (1992)

10%

Chang and Robinson (199 3) Gaspar et al. (1996)

FAAS

24 l~g L-1 (Cr Itt) 75 llg L-1 (Crvl)

HPLC-ICPMS

3 pg

IC-ICPAES IC-ICPMS Extraction-AAS

12-15 I~g L-1 35-47 l~g L-1

IEC-DCPAES

3-15 l~g L-1

Serum

HPLC-ETAAS

0.17 l~gL-1

1.4%

Hg, MeHg

Urine

CV-ICPAES

4 ng mL-1

5%

Hg, MeHg

Urine

HPLC-MIPAES

0.15 ng mL-1 35 ng mL-1

6.7% 6.8%

Zoorob et al. (1995) Jensen and Bloedorn (1995)

0.9% (Crlll), 1.3% (Crvl) 0.3-1.6%

Devoto (1968)

97.3 + 2%

Urasa and Nam (1989) VanLandeghemet al. (1994) Menendez Garcia et al. (1996) Costa Fernandez et al. (1995)

(continued)

Table T. Continued

M,;

Element

Species

Matrix

Methodology

DetectionLimit

RSD

Recovery

Hg

MeHg, EtHg, PhHg

Urine

HPLC-CVAFS

5-7 ng

m

>95%

Hg

Hg, MeHg

Urine

HPLC-CVAAS

Hg

Hg, MeHg, MeEtHg, diEtHg

Blood

GC-CVAFS

0.3-0.6 ng g-1

6.9% 3.5% 5%

90-114% 89-103% m

Hg

Inorganic and organic-Hg

Hg Hg

Inorganic and organic-Hg Inorganic and MeHg, PhHg

1-8% 2.8-5.2%

86-106% 97-102%

Hg

Inorganic and MeHg

5-10%

Hg

Inorganic and organic-Hg

Urine Reduction-CVAAS

95.2 + 2.7% (MeHg); 99.5 + 4.3% (inorg-Hg)

Hg

Inorganic and organicoHg

Hg

. Inorganic and MeHg, EtHg, PhHg

Urine, sweat Urine

Hg Pb

Inorganic and organic-Hg TrimethyI-Pb

Pb

Ionic alkyI-Pb

O~

Blood, Reduction-CVAAS urine Blood Reduction-CVAAS Blood, GC-ETAAS milk Whole GC-MIPAES blood

3-5 ng L-1

GC-ETAAS

0.3 ng

GC-CVAAS

1.7 ~g L-1 (total); 12 ~g L-' (MeHg); 2.4 gg L-' (EtHg); 21 Mg L-1 (PhHg) 7 pg 3 l~g L-1

Urine Ion-pair LC-ICPMS Blood GC-ETAAS Urine

p u m p

(c) Figure 2. (a) An optical parametric generator (OPG) where a pump wave at frequency O)p is used to interact with a nonlinear optical medium, the BBO crystal, to generate tunable signal radiation at frequencies C0sand idler radiation at frequencies 0ai. (b) An optical parametric amplifier (OPA) where weak signal, or idler, seed radiation is amplified while an idler or signal, beam is generated simultaneously. (c) An optical parametric oscillator (OPO) in which a resonant cavity is employed for one or more of the waves that are generated, and tunable output results from the same nonlinear process as in (a) and (b).

New Typesof Tunable Lasers

105

Several commercial OPO lasers are available that are based on various permutations and combinations of OPDs (see Section II.B).

Three-Photon Optical Parametric Process The three-photon optical parametric process in OPDs is one of many nonlinear phenomena that result from second-order optical nonlinear susceptibility. In that process, the electrical field of a pump laser interacts directly with the charged species inside the nonlinear crystal, and to create oscillating dipoles. The negatively charged electrons respond with significant displacement, while the motion of the positively charged ion cores can be neglected due to their greater mass. If the pump laser fluence is strong, the response to the driving field is no longer linear. The oscillations take place in an anharmonic potential well, and nonlinear polarization is induced. The magnitude of the induced polarization P, per square volume, depends on the magnitude of the applied electric field E (in cgs electronic units). Macroscopically, the polarization P can be expressed as,

P = Z O) E + Z (2)E2+...

(I)

where Z Cm) are dimensionless coefficients termed "susceptibility" coefficients, which are dependent on frequency and temperature. The second-order nonlinear susceptibility7,42)is the foundation of many important nonlinear effectsincluding second harmonic generation (SHG) and the various three-photon parametric processes. It is only noncentrosymrnetric crystalsthat display second-order nonlinear susceptibilities,as second-order nonlinear susceptibilityreduces to zero for crystals with inversion symmetry. For a plane light wave that consists of two differentfrequencies, cos and COp,and amplitudes, E s and Ep, respectively,the electricfieldmay be expressed as: E = Ep cos ((apt) + E s cos (COst)

(2)

Combining Eqs. 1 and 2, the second-order term at an arbitrary point in space, becomes: 2 X(2)Ep E s cos

((apt) cos (COst)

(3)

Expression 3 can be rewritten as 2 ZC2)Ep E s {cos (COp+ (as) t + cos (cop - (as) t}

(4)

From the above expression it can be seen that the second-order nonlinear susceptibility will give rise to a nonlinear polarization, and reemit radiation at cop + (as (sum) and cop - COs (difference) frequencies when two primary beams, at frequencies (av and cos, interact with a nonlinear crystal. In a three-photon parametric process, a signal beam at (as can thus be amplified by the presence of a strong

106

HOU, ZHOU, YANG, STCHUR, and MICHEL

pump beam at C0p, while an idler beam at fop - fOs is generated simultaneously, as described in expression 4. The three-photon parametric process can be imagined as an energy conversion process in which a pump photon with wave vector Kp at a circular frequency fOp breaks down to two lower frequencies signal fOs and idler fOi with wave vectors K s and K i, respectively, as shown in Figure 3. The parametric interaction is considered between the pump photon and the second-order nonlinear susceptibility Z(2) and it couples the energy from the pump radiation into two new waves. The term "parametric" comes from the trigonometric relationship between the x and y coordinates of a moving point and a third parameter. Two parametric equations relate the coordinates to the third parameter.

Energy and Momentum Conservation Energy conservation is maintained during the three-photon parametric energy conversion process: fOp = COs + foi

(5)

This process can be represented by analogy with an optically pumped three-level laser (Figure lb). The upper level (Figure 1a) could be tuned by adjustment of the pump frequency, C0p, but the tuning is normally achieved by adjustment of the middle level. This implies the generation of infinite combinations of output frequencies, as numerous frequency pairs can satisfy Eq. 5. Consequently, OPO lasers can produce wavelengths that are difficult to generate by conventional laser sources that depend on specific transitions in a material. This infinite combination of output K,

Ki (gp = (Os + (Oi

and

Kp = K, + Ki

Figure 3. The three-photon parametric process, which is governed by energy and momentum conservation, is a process in which a pump photon with wave vector Kp, at a circular frequency cop, breaks up into two lower frequency signal and idler photons with wave vectors Ks and Ki at COsand CObrespectively.

New Typesof TunableLasers

107

frequencies is the origin of the broad tunability of OPDs. However, it is the momentum conservation, or phase matching, that governs the process to yield a specific frequency pair, Kp = K s + K i

(6)

where Kp, K s, and K i are the momentum vectors or wave vectors for the pump, signal, and idler waves, respectively, as defined previously. The magnitude of the vector K depends on refractive index, n0~ K= r

(7)

where n is the refractive index that the radiation at frequency co is experiencing, and c is the speed of the lightmfrom which, the momentum conservation, Eq. 6, can be rewritten as, np O)p-" n s ~s + ni 0~i

(8)

where np, n s, and n i are the refractive indices for pump, signal, and idler radiation, respectively. The phase-matching condition is said to be satisfied, when Eqs. 5 and 8 can be combined to give t.0p(np - ni)

(9)

COs --

ns - ni Equation 9 shows how the output of an OPD can be tuned, although it is a somewhat oversimplified model. There are two ways to tune the output frequencies. One is to change the pump frequency; the other is to manipulate one or more refractive indices. The latter is almost always used in practical OPO lasers, as the change of refractive indices through the manipulation of either the temperature or the orientation of the crystal can be achieved in a nonlinear, anisotropic medium, such as a BBO crystal. Moreover, the phase-matching characteristics and broad wavelength coverage of available nonlinear optical materials give OPO lasers a wide wavelength tuning range.

Angle Phase Matching The angle phase-matching method was first proposed by Maker et al. (1962) and, independently, by Giordmaine (1962). The approach employs the birefringence of a uniaxial crystal. If an object is viewed through such a birefringent crystal, in unpolarized light, two images are seen which result from different refractive indices experienced by light of different orthogonal polarizations. Manipulation of the angle of incidence of light on the crystal changes the refractive index experienced by one polarization of the light, called the extraordinary ray, but not the orthogonal polarization called the ordinary ray. Hence, one image moves when the crystal is

108


rotated while the other remains stationary. Consider the wave vector constructions for refraction shown in Figure 4. For the ordinary ray, the refractive index is independent of the direction of propagation, as shown in the solid locus of Figure 4, regardless of whether the material is isotropic or anisotropic. For the orthogonally polarized extraordinary ray in an isotropic material, the locus of the wave vector is also a sphere, and independent of the direction of propagation, but in an anisotropic material the locus is an ellipse depicted as a dotted line in Figure 4. For the extraordinary wave, the different refractive indices of an anisotropic material at different angles of incidence lead to the double-refraction phenomenon called "birefringence". It is the birefringence that is exploited to enable phase matching in an anisotropic material. If the value ofn e - n o is larger than zero, the birefringence is said to be positive; and for n e - n o smaller than zero, the birefringence is negative. The corresponding crystals are termed positive or negative uniaxial, respectively. Figure 5a illustrates a typical curve of refractive index as a function of wavelength. The dispersion causes the refractive index to drop with increased wavelength. As a result, the pump wave vector Kp is always too large and causes the phase mismatch, AK, that prevents conservation of momentum. However, for a nonlinear crystal, the refractive index for extraordinary rays varies elliptically, depending on the angle between the direction of propagation of the incident light, and the axis of symmetry, or optical axis, of the crystal. In a negative uniaxial nonlinear crystal, such as a BBO, the refractive index that an extraordinary ray experiences is always lower than, or at most equal to, that for an ordinary ray. The maximum index is reached when the extraordinary ray is propagated along the crystal's axis of symmetry. Such birefringence can be used to selectively vary the vector magnitude of the extraordinary wave to compensate for the dispersion and

i

optical axis q 4

i o

~o~.a1.

no . I

ss

p

Figure 4. In an anisotropic medium, such as BBO crystal, the wave vector of an ordinary ray follows the spherical locus, while the wave vector of an extraordinary ray follows the ellipsoidal locus.

109

N e w Types of Tunable Lasers

~

~K

e-

x" (D "10 _= ._> 15

Kp

t Ki

Ks

Q) n,"

UV

Vis

IR

(a)

t"

1

>~ r

~dinary

'1o

KpT I Ki

.>_ (D n,'

(op, np extraord~'na'01.:--_- . . . . . . . . . F-o~-r~ UV

Vis

IR

(b)

Figure 5. (a) Dispersion exists in any optical material and causes a phase mismatch, AK, which prevents conservation of momentum in an isotropic medium. (b) The phase mismatched condition can be obviated because the dispersion of the extraordinary pump beam can be slowed in an anisotropic medium.

allow phase matching (Figure 5b). If the pump is introduced as an extraordinary wave, the resultant signal and idler can emerge as ordinary waves, while the K vector for the pump, Kp, can be manipulated through the angle of incidence, (0 in Figure 6) which results in the required phase match (AK =~ zero). Another approach is to vary the crystal temperature and induce index changes in order to achieve the desired wave vectors. As depicted in Figure 6, phase matching occurs by variation of the vector magnitude of the pump wave until the point where the resultant extraordinary wave vector is equal to that of the signal plus idler wave vectors for the two ordinary rays. The corresponding phase matching condition, Kp(e) --~ Ks(~ + Ki(~ is termed Type I phase matching. If either the signal or the idler wave shares the same polarization as the pump wave, then it is called Type H phase matching.

110


S. ~f

i.==== l= ====Q ~

~,

optical axis

s ~'

pump(e-ray)

,-0-0-I-

signal + idler (o-rays) pump(e-ray)

Figure 6. Phasematching occurs at the intersection of the spheres that correspond to the combined spherical vector surfaces of the two ordinary rays, which are the signal and idler beams, with the ellipsoidal vector of the extraordinary ray, which is the pump beam. This figure is only an approximate representation of the phase matched vectors. B. Examples of Commercial Optical Parametric Oscillator Lasers With the advent of the BBO crystal and the maturity of spectrally narrow pump lasers, several OPO lasers have become commercially available in recent years. The dominant commercial narrow-band OPO lasers are based on diode injection-seeded Nd:YAG laser pumped BBO OPDs. These lasers are usually pumped with the third harmonic of Nd:YAG laser at 355 nm with a repetition rate of 10 Hz. The wavelength tuning range is in the range of 220 nm to 2 l.tm. Figure 7 shows the optical layout of one commercial pulsed OPO laser system (Johnson et al., 1995; Zhou et al., 1997). In this system, a frequency-tripled Nd:YAG laser, with a 6 ns pulse width and injection-seeded with a laser diode, is used to pump a tunable OPO equipped with a second harmonic generator (SHG). The OPO section consists of a master oscillator and a power oscillator. While the master oscillator is a true OPO, the power oscillator is no more than an OPA in a resonant cavity seeded by coherent monochromatic radiation from the master oscillator. It can also be thought of as a combination of an OPA and an OPO. Type I phase matching is used for the master and power oscillators in combination with a "collinear pump" configuration that aligns the pump and output beams colinearly. Typically, the 355 nm pump energy is in the range from 450 to 540 mJ, with 450 mJ being a threshold level below which the OPO does not oscillate efficiently. By use of a dichroic beam splitter about 30% of the pump energy is directed to the master oscillator, while the rest is directed to the power oscillator after delay in an optical delay line. In this design, a singly resonant scheme was adopted for both

New Types of Tunable Lasers

111

delayed 355 nm

i

+

,., ...... .i.I ............. }. ..... ii i I ' : : ' ; :;I "% rl BBOI ~" I::; I : " BS | BS v I ........................... ..... Pow Oscillator .. ..

,Y~ " I ,-

9i ; ; ............

;; .... i ...... ; ;

~~IBBOI

~.............. Master Oscillator

~,

i

' ......................... 3 (o[ BP BP. l ,~i \ / ~-?--;;--)~ i '-I -'~ to beam dump ,#, l ; ~ =~ ! I "5 ,";~ l;. ~ it',.... ...""",..""'"""",,.,, ";...,,~.:. ,:.i:.!": ,,,,,,I ,' D.._ ,.~, o

.a...,i

iBBOt_~BBOI.~[

- .............................. :355 nm

P.B.P" SHG

Figure 7. A schematic diagram of a Spectra Physics OPO laser system equipped with a second harmonic generator (SHG). This 10 Hz pulsed OPO laser consists of a master oscillator and a power oscillator (MOPO). BS = beam splitter, BP = beam steering prism, PR = beam steering and polarization rotation prism, GT = grating, TM = tuning mirror, PBP = Pellin-Broca prism. cavities, in which only one output, the signal, is resonant, which allows for relatively simple cavity designs. An intracavity grating set at grazing incidence is used in the master oscillator to achieve both a narrow linewidth and a wide tuning range. The master oscillator crystal is mounted on the same shaft as the power oscillator crystal, which allows the output wavelengths of the master oscillator and power oscillator to be tuned synchronously. Output from the power oscillator is directed into the SHG by use of beam steering and polarization rotation prisms. Polarization rotation is used to satisfy the phase-matching requirements of the SHG crystals. Two BBO crystals cut at 56 ~ and 36 ~ are used in the SHG to cover the wavelength range from 220 to 440 nm. The output wavelength of this laser system can be tuned, under computer control, from the UV to the NIR via synchronous rotation of the four BBO crystals and the tuning mirror. The tuning mirror is driven linearly by use of a sine bar. There are two basic modes provided by the manufacturer for wavelength tuning. One is the "track" mode which uses a photodetector to monitor the output power and allow feedback to control the crystal position, while the other is a "table" mode which employs a lookup table to locate a previously optimized crystal angle for each wavelength. In general, the "table" mode provides more short-term stability, as long as the laser has been aligned correctly. Two high-damage threshold 355 nm dichroics route the pump beam directly through the BBO crystal, then out into a beam dump. This design avoids any necessity to direct the pump beam through the cavity resonator optics which have a low-damage threshold due to

112

HOU, ZHOU, YANG, STCHUR, and MICHEL tuning

sealed OPO cavity

SHG

mirror [..................................................................i[ ............. ~

! :

t

~k

I ,

grating~i j

,

.

I

(

~ip

BBO

il "~,

:

/" I" " ....... .............................., ....................

pump retro-reflector /

half'halfw a v e plates

I

i

9

[,;

355 nm pump ;; ;

. ............................................. "

i .

delayed 355 nm

BBO

9

~

BBO

i

.!

A IX>,

i

i ;

iUV ~

r ,--v,,,

i

--

/

...... 3

-B

............................................. .: ~r ; sealed OPA cavity 355 nm to beam dump

!

'o?V] m

..... :

i 1

"r" LI~ } [

J'ii

o/2= O m m

[~TJ"

m

~ . . . . . . . . . . . . ....~... ......

~

_L.. I. /

prism

"9 '[

[ !!

t'

!

! i

: !

Figure 8. A schematic diagram of a Continuum Sunlite OPO laser system with a second harmonic generator (SHG). The laser system consists of an extraordinary resonance OPO and a single-pass OPA, with all crystals sealed in temperature stabilized enclosures. constraints imposed by the necessary broadband optical coating materials in the resonant cavity. A somewhat different commercial design is shown in Figure 8, and involves Type I phase matching, and an OPA pumped with a 10 Hz pulsed Nd:YAG laser. For this design, a typical pump energy at 355 nm is 320 mJ, of which 20% is directed to the OPO and the rest to the OPA. An attenuator and various telescopes are used to provide the oscillator with the proper pump beam divergence and energy density. Both the OPO cavity and OPA cavity are sealed in temperature-controlled enclosures to protect the hygroscopic BBO crystals. This minimizes the influence of ambient temperature, and stabilizes the crystals' phase-matching conditions. An interesting aspect of this laser system is its OPO cavity design (Figure 9) in which the polarization of the signal is rotated by 90 ~ with a half-wave plate, for dispersion by the grating in the extraordinary plane. This results in higher diffraction efficiency, and the small acceptance angle of both the grating and the crystal in their extraordinary planes combine to reduce the spectral linewidth of the signal. The tuning mirror retro-reflects the first-order diffraction of the signal onto the grating so that it returns into the cavity in the usual way. The cavity geometry is not designed to allow the idler to resonate in the cavity. After exiting the OPO cavity the signal is rotated back to the vertical polarization by another half-wave plate to seed the OPA. The active elements of the OPA are a pair of phase-matched Type I BBO crystals, the optical axes of which are a mirror image of each other. Therefore, when


113

Figure 9. The extraordinary resonance design of the OPO cavity of Figure 8, showing the BBO crystal dispersing light along its extraordinary plane, and its grating further dispersing the light in the same plane. The half waveplate rotates the polarization of light that strikes the grating, which increases its diffraction efficiency. (Reproduced with permission from Radunsky, M.B. (1995). Copyright 1995 Pennwell Publishing).

they counter rotate during a wavelength scan, the pointing direction of the laser beam at the output is kept constant. Compared to the resonant design described in Figure 7, the objective of the single-pass OPA design of Figure 8 is to produce less degradation of beam quality by the back-conversion that occurs during resonance.

C. Characteristics of Optical Parametric Oscillator Lasers Output Energies of the Laser Pulses To pump an OPO laser, the output energy of a YAG laser at 355 nm may vary from 320 mJ to more than 450 mJ, depending upon the design of the OPO laser and the spatial and temporal characteristics of the YAG pump beam. For an OPO laser system that was characterized in our laboratory, the YAG pump energy for a beam size of 9-10 mm was between 460 and 540 mJ per pulse over about 4 months of 40 hour weeks. It was found that when the energy of the YAG laser dropped below 400 to 450 rnJ, the OPO began to stop oscillating at a gradually increasing number of wavelengths. Although the oscillation could often be restarted, by tweaking the alignment of the BBO crystals and other optics at each wavelength of interest, this condition generally indicated that the flash lamps should be replaced and the OPO laser realigned. There was also a fear that the beam quality of the YAG laser might have deteriorated to the point that it might damage optical components, or that refocusing the 355 nm radiation more tightly into the BBO crystals to compensate for energy loss might also damage the optics. Figure 10 shows the average output

114

HOU, ZHOU, YANG, STCHUR, and MICHEL 120 100

80 60

(a) signal

40 "-3

E

>~

G) t--

w

0

20 0 400

500

600

700

60

45

idler

30

(b)

15

500

750

1000 1250 1500 1750 2000 Wavelength, nm

Figure 10. Average output energy of the OPO laser system of Figure 7 as a function of wavelength: signal (a) and idler (b). The error bars represent the long-term energy stability of the laser, and are the difference between the minimum and maximum energy of four scans obtained during the course of 6 days. The mean of the four scans was plotted. energy per pulse of four scans of the OPO laser system of Figure 7. The scans were obtained over a period of six days. The output energies per pulse were 35-90 mJ for the signal beam, and 1-40 mJ for the idler beam. The error bars in Figure 10 represent the extremes of the range of energies that were measured. The energy levels were stable at most wavelengths with variations in range between +2% and +10%, although the variations were up to +15% at the blue end, around 440 nm. The tuning range was extended by use of a second harmonic generation (SHG) section which had typical output energies in the range 1-11 mJ (Figure 11). The SHG wavelength range was 220-440 nm, with a gap around 355 nm due to the degeneracy of the OPO's fundamental signal and idler beams around 710 nm. For the laser of Figure 8, also characterized in our laboratory, the output of the idler beam had energy levels in the range of 1-40 mJ, while the signal beam had


115

12-

-:

E

crystal change

9-

>~

eIii

6-

=3 t'3

I degeneracygap O

3-

0 200

I

I

I

I

I

250

300

350

400

450

Wavelength, nm

Figure 11. Output energy of the frequency doubled radiation for the laser of Figure 7 in the wavelength range from 220 to 440 nm. Two crystals cut for 56 ~ and 36 ~ were used to cover the measured output range. The first part of the curve (e) was the frequency doubled output, obtained by use of the signal beam, while the second part (0) was obtained by use of the idler beam. Reproduced with permission from Zhou, J.X. et al. (1997). Copyright 1997 American Chemical Society.

energies in the 10-55 mJ range, and the YAG pump energy was about 320 mJ. The output energy of the SHG section was between 2 and 14 mJ in the wavelength range from 225 to 450 nm. This represented a high SHG conversion efficiency of about 20%. The stability of the output of the SHG section was 10-20% over a period of three days. The high energies that are obtained from these laser systems exceed those required for atomic spectroscopy, and it is useful to use an optical attenuator to control output energies. For laser ablation, these energies are the same or more than is required.

Pulse-to.Pulse Energy Stabifity The pulse-to-pulse stability can be obtained by measurement of the relative standard deviation (RSD) of the energy of the laser pulses. For commercial OPO lasers, pumped with 355 nm radiation from a Nd:YAG laser, there are several factors which affect the pulse-to-pulse stability of an OPO laser. These factors include the inherent pulse-to-pulse stabilities of the Nd:YAG laser, and the signal and idler laser beams. Other variables include the ambient temperature which must be maintained within about +2.5 ~ and the accuracy of the optical alignment of the OPO laser

116


system. Typical pulse-to-pulse RSDs of the 355 nm pump radiation are normally better than 2%. For the OPO part of the system, and through most of the wavelength range in the visible (signal) and infrared (idler), the RSD is 2-6%, but the RSDs degrade at the extreme wavelengths of operation to a level as high as 12-14%. Typical RSDs for the second harmonics of the signal and idler are around 3-12% for most of the wavelength range. Figure 12 shows the RSD of the second harmonic of the signal beam of the OPO of Figure 8. For the doubled radiation there appears to be no relationship between the RSD and wavelength, in contrast to the situation with the signal and idler beams which show increases in RSDs at the extremes of the wavelength range. A summary of approximate laser beam size, pulsed output energies, and pulseto-pulse RSDs for the lasers of Figures 7 and 8 appears in Table 1.

Signal Spectral Linewidth Figure 13 shows a typical set of interference fringes recorded with a linear charge coupled detector (CCD) array during a laser linewidth measurement at 595 nm with the laser of Figure 7. About three laser pulses were averaged to obtain the fringes, but significant multiple peaks in the interference fringes were observed when a larger averaging time was used at the CCD, which could indicate mode hopping or beam pointing variations between pulses. The spectral linewidth of the OPO laser of Figure 7 is typically between 0.1 and 0.25 cm -1 for the signal beam. Figure 14 16

l~" 1 2 oo 9

00

9

9 90

6 oo

9

a.

0

"

200

I

I

I

I

I

250

300

350

400

450

500

Wavelength, n m Figure 12. Pulse-to-pulserelative standard deviation (RSD)of the frequency doubled output of the signal beam from the OPO laser of Figure 8. The RSD was measured and calculated for 50 pulses at each wavelength.

117


Table 1. Comparison of Some Features of Two Designs of Nd:YAG Pumped Nanosecond OPO Laser

OPO Laser

Typical Beam Diameter (mm)

Laser Energy (mJ/pulse)

Typical Pulse-toPulseStability (RSD, %)

10

>450

2

8

35-90

5

idler SHG

7 8

1-40 1-11

5 5

YAG pump at 355 nm signal

8

320

2

7

10-55

5

idler SHG

5 6

1-40 2-14

8

Laser Beam

Spectra Physics MOPO (Figure 7)

Continuum sunlite (Figure 8)

YAG pump at 355 nm signal

5

1000 -

:j

800 -

c::

600 -

r

> :~

400

o) n~

200

I

I

I

I

I

I

200

400

600

800

1000

1200

CCD Pixel Figure 13. Typical interference fringes for the laser of Figure 7, recorded at 595 nm with a linear CCD array. The fringes were formed by an ~talon and imaged with a biconvex lens of 30 cm focal length onto a linear CCD array. Three consecutive interferograrns were averaged and recorded. The calculated laser spectral line width was about 0.2 cm -I.

118

HOU, ZHOU, YANG, STCHUR, and MICHEL 0.4

'

,7, 0.3E o

.=-o 0.2..... t.d .m

0.1-

.0

450

I

I

I

I

500

550

600

650

700

W a v e l e n g t h , nm

Figure 14. Linewidth versus wavelength for the signal beam from the laser of Figure

7. The interferograms were recorded at wavelength intervals of 5 nm from 460 to 690 nm for the signal beam. The integration time for the interference fringes was such that the average of two or three consecutive interferograms was recorded. The linewidth was calculated by use ofthe distances between the interference fringes and their width, together with the free spectral range of the ~talon.

shows a series of measurements of the same laser's linewidth as a function of wavelength. For the OPO laser system of Figure 8, the different cavity design leads to a narrower spectral linewidth by about a factor of 2.

Wavelength Calibration Accuracy The wavelength calibration accuracy of the output of an OPO laser can be measured by centering the laser's wavelength at known atomic lines and observing atomic fluorescence. Comparison of the known wavelength with the wavelength on the readout of the OPO laser system controller allows an estimate of the calibration accuracy of the laser. For the laser of Figure 8, the results of such an experiment are shown in Figure 15, where the deviation of the readout from the known atomic wavelength is within the laser linewidth. This degree of calibration accuracy allows an atomic line to be found, in the first instance, by a simple "go to" command on the laser controller.

Rapid ScanSpeed One of the most important advantages of an OPO laser is that it can be rapidly tuned from wavelength to wavelength over a broad range from 220 to 2000 nm. If the laser is slewed at high speed between wavelengths, typically at up to 2.5 nm


119

40 E 30,.,t" ~ t-LU

500" 250 0

7O0

|

750

!

800

Wavelength, nm

Figure 18. The output wavelength range and pulse energy of a 20 Hz pulsed Alexandrite laser at 50 ~ Reproduced with permission from Hecht, J. (1992). Copyright 1992 McGraw-Hill.

124


erties for lasing. Postgrowth annealing treatments have also been developed to further ensure high-quality, low-loss materials. Ti:sapphire lasers are available that range from CW, to pulsed, and to ultrafast, with output wavelengths between the UV and IR. A Ti:sapphire crystal has about 0.1% Ti3§ doped into the sapphire host, which replaces octahedrally coordinated A13§ ions in the crystalline lattice at sites with trigonal symmetry. The Ti 3§ ion has a single 3d electron outside the closed electronic shell of an argon core. Undoped A1203 crystals are very transparent in the nearinfrared and visible regions from approximately 2000 nm to 400 nm, and in the UV region from 400 nm to 200 nm, although there is somewhat increased absorption towards shorter wavelengths. Ti3§ A1203 crystals are pink, which results from the broad, double-humped absorption band that extends from about 400 nm to 600 nm in the blue-green region of the visible spectrum. This absorption band is due to photon-assisted excitation of the 3d electron of the Ti 3§ The various peak absorption bands at 266 nm, 216 nm, and 185 nm are of unknown origin (Sanchez et al., 1988). The free-space, five-fold degenerate, d-electron levels of Ti 3§ are split by the crystal field of the host A1203, which can be viewed as the sum of cubic and trigonal symmetry components. The cubic field dominates and splits the Ti 3§ energy levels into a triply degenerate 2T2 ground state and a doubly degenerate 2E excited state. The trigonal field splits the 2T2 ground state into two levels, of which the lower level is split further into two levels by the spin-orbit interaction. Figure 19 shows a schematic energy level diagram for Ti3§ in a Ti:sapphire crystal. Interactions between the Ti 3§ electronic energy levels and the vibrational energy levels of the crystal are responsible for the vibronically broadened 2E-2T2 transition (Figure 20). Although the laser emission is tunable from 660 to over 1100 nm, the maximum efficiency is in the wavelength range from 700 to 900 nm. The large tuning range is the result of the simple, 3d 1, energy-level structure, which avoids possible

2Eg

2E

~176176176~

i !

i

2D /

I

free ion

2A1

2T2g ,:....... 9 cubic field

2E

52

$~1 I trigonal ........... field

o~.........

spin-orbit

Figure 19. Schematic energy-level diagram for Ti 3+ ion in Ti:AI203.

125

New Types of Tunable Lasers 2E

relaxation

(9 C UJ

.o

.o

o

E

x

2~

relaxation

Figure 20. Interactions between the Ti 3+ electronic energy levels and the vibrational energy levels in the sapphire crystal produce a vibronically broadened 2E-2T2 transition, which is the origin of the tunability of a Ti:sapphire laser.

redirection of energy by reabsorption of the laser radiation. There are two key propertiesnthe concentration and the optical cross section of the Ti 3+ ionsmthat are responsible for the laser action. The absorption cross section at a wavelength ~. is given by cr= ~[Ti3+], where c~ is the absorption coefficient at g., while [Ti 3§ is the ionic concentration. Figure 21 shows the polarized absorption cross section for

~E 6.0 o 0

~" 4.0

o 2.0 L)

400

500 600 Wavelength, nm

700

Figure 21. Polarized absorption cross section for the 2T2---~2Etransition in Ti:AI203. The baseline was arbitrarily set to zero for both polarizations at 700 nm. Reproduced with permission from Moulton, P.F. (I 986). Copyright 1986 Optical Society of America.

126


the 2T2-2E transition in the Ti:sapphire crystal (Moulton, 1986), while a generalized absorption-fluorescence spectrum is shown in Figure 22. For rr polarization, which is the polarization that yields the strongest absorption, the absorption cross section is 6.5 x 10-20 cm 2 at 490 nm. The absorption band from 400 nm to 650 nm allows for convenient optical pumping by a number of blue-green sources including argon lasers, frequencydoubled Nd:YAG lasers, copper vapor lasers, and flashlamp pumped dye converter systems. The most commonly used pump sources are argon ion lasers and frequency-doubled Nd:YAG lasers because the temporal pulse width of the Nd:YAG laser is appropriate for the Ti:sapphire material's short fluorescence lifetime at room temperature, about 3.2 Its. Also, the output wavelength of both these pump lasers matches the narrow range for pumping at the peak wavelength near 500 nm (Figure 22). Efficient CW laser operation requires sufficiently high intensity within the gain medium to maximize the stimulated emission process. For the Ti:sapphire laser's transition, which peaks at 800 nm, the calculated saturation intensity is 2.6 x 105 W/cm 2. This high saturation intensity necessitates tight focusing within the crystal, especially at low pump power levels. Tight focusing requires the use of a physically short laser crystal with a high concentration of dopant. The concentration must not be too high, in order to minimize potential losses associated with scattering, concentration quenching, and parasitic absorption that overlaps with the emission region of the Ti:sapphire laser. The parasitic absorption is caused by Ti3§ 4+ defect pairs in the host material. After crystal growth, and under a reducing atmosphere of hydrogen gas, carefully controlled treatment during annealing at high tempera-

1.0 s

absorption

fluorescence

t"

r 0.6-

0.2-

400

600 800 Wavelength, nm

1000

Figure 22. Generalized absorption and fluorescence spectra of a Ti:sapphire crystal.


127

ture has been developed to minimize the number of Ti3+:Ti 4+ defect pairs. An absorption figure of merit (FOM), defined as the ratio of the absorption coefficients at the pump and emission wavelengths, has been used to assess the quality of Ti:sapphire crystals by Pinto et al. (1994). Figure 23 shows the dependence of laser performance on the crystal's FOM. FOM values for Ti:sapphire crystals decrease with increased Ti 3§ concentration. Since the Ti:sapphire medium has a relatively low gain, a compromise must be made between low-threshold operation and high-output efficiency. Laser rods with Ti 3+ ion concentration in the range of 0.1-0.15 wt% have been demonstrated to give superior CW laser performance. The product of the excited-state lifetime and the cross section for stimulated emission is approximately proportional to the inverse of the spectral bandwidth over which useful laser gain is obtained. A tunable solid-state laser, with its broad gain bandwidth, cannot match both the high cross section and long excited-state lifetime of a laser such as the Nd:YAG laser with its relatively narrow spectral output. In addition, the balance between lifetime and cross section affects the choice of pump laser. A Ti:sapphire laser has a cross section comparable to that of Nd:YAG, but its radiative lifetime is only 3.2 lets, and this is the reason that it must be pumped with short-pulsed, Q-switched, or CW lasers, or with short-pulsed flashlamps. Ti:sapphire lasers are usually designed for laser pumping and resemble tunable dye lasers in this regard. Both linear standing wave cavities and ring cavities similar to those for CW lasers are employed for CW Ti:sapphire lasers. Figure 24 shows a schematic diagram of a typical ring configuration for a CW Ti:sapphire laser

C

3 b 0

n 0

2

1

==

a

=,,,

0

2

4

6

8

10

Input Power, W

Figure 23. Laser performance data as a function of FOM, as defined in the text, for several Ti:AI203 rods. FOM = 90, 560, 1000, for a, b, and c, respectively. The wavelength of the argon ion laser pumped CW Ti:sapphire laser was at 800 nm. Reproduced with permission from Pinto, J.F. et al. (1994). Copyright 1994 IEEE.

128

HOU, ZHOU, YANG, STCHUR, and MICHEL output

birefringent crystal

lens

pump

Figure 24. Schematic diagram of a ring configuration for a CW Ti:sapphire laser cavity. Reproduced with permission from Cunningham, R. (1991). Copyright 1991 Gordon Publications.

(Cunningham, 1991). In this design, the Ti:sapphire rod is cut and polished at Brewster's angle. The broad-band folding mirrors are dielectrically coated for both high reflection at around 800 nm and high transmission in the pump wavelength range. A birefringent filter, which restricts the oscillation within a narrow wavelength range, is used for wavelength tuning, while an 6talon is employed for subsequent narrowing of the linewidth. The absorption band of Ti:sapphire is a maximum near 500 nm, which allows for argon laser pumping, or pumping with frequency-doubled Nd:YAG lasers, or copper vapor lasers. Gallium aluminum arsenide (GaA1As) diode lasers cannot pump Ti:sapphire lasers directly, but they can be used to pump a Nd:YAG laser that can be frequency-doubled to pump a Ti:sapphire lasers. Figure 25a shows the tuning curve of a Ti:sapphire laser pumped by an argon laser. The tuning range is from 665 nm to 1070 nm by use of four sets of cavity optics. For some commercial versions, five sets of mirrors are employed to maximize performance (Gray, 1989; Figure 25b). Higher pump powers from a CW argon laser produces higher output powers (Figure 26). Commercial Ti:sapphire lasers can generate CW powers of up to several watts of fundamental output. Ground-state absorption by Ti 3§ limits tunability at short wavelengths, and tunability at long wavelengths is limited by a lower gain cross section for stimulated emission. While dye lasers can be tuned across a broad range only by changing the dye, Ti:sapphire lasers can be tuned across the entire range by exchanges of cavity mirrors. Compared to a change of dye, switching mirrors in a Ti:sapphire laser is easier to realize because it can be computer-controlled.The gain curve also depends on the temperature because the vibrational sublevels of the ground electronic state of the lasing levels are thermally populated. Pulses produced by a Ti:sapphire laser pumped with a Q-switched Nd:YAG laser are eminently suitable for conversion to other wavelengths in order to extend the tuning range, due to the high beam quality and relatively short pulses. Extension of the tunable wavelength range is possible with harmonic generation or other


129

2.0

1.5

(a)

1.0

~

0.5

700

0

0

800

4.0

900

1000

~ **~

2.r

!

650

i I i, 750

850

Wavelength,

950

1100

(b)

1050

nm

Figure 25. Typical tuning curves for CW Ti:sapphire lasers with four mirror sets (a) and five mirror sets (b), at 5 W and 20 W pump power for (a) and (b), respectively. Reproduced with permission from Pinto, J.F. et al. (1994) and Gray, T. (1989). Copyright 1994 IEEE and 1991 Gordon Publications, respectively.

nonlinear wavelength conversion techniques. The use of Raman shifting and second and third harmonic generation is possible for extension of the tuning range to encompass 202 to 3180 nm (Funayame et al., 1993). From 260 nm, over 3 mJ of pulse energy is obtained for most of the spectral range, but the pulse energy drops rapidly in the deep UV, ranging from less than 1 mJ at 250 nm to 1 l.tJ at 202 nm (Figure 27a). Including Raman shifting, many stages of frequency conversion (Figure 27a) are required to cover the wavelength range from 202 nm to about 700 nm. In contrast, only two SHG crystals are needed to extend the tuning range of an OPO laser over the wavelength range of 220 nm to around 2 l.tm, and higher output energy levels are available for most of this wavelength range (Figure 27b). Also, it is worth noting that the tuning range of OPO lasers can be up to 2000 nm in the red, while it is difficult for Ti:sapphire lasers to produce wavelengths longer than 1 l.tm. The broad-gain bandwidth of Ti:sapphire laser has been used to generate ultrashort pulses directly. Figure 28 shows a block diagram of a commercial 200 fs

130

HOU, ZHOU, YANG, STCHUR, and MICHEL 2.0

_ 1.5

0

n

1.0

0

0.5

a

700

800

900

1000

Wavelength, nm Figure 26. Higher CW argon-ion pump powers produce higher Ti:sapphire output powers, but they vary as a function of wavelength. Three sets of mirrors were used. Pump powers were, 3, 5, and 7 W for a, b, and c, respectively. Reproduced with permission from Hecht, J. (1992). Copyright 1992 McGraw-Hill.

pulse 76 MHz repetition rate mode-locked Ti:sapphire laser (Fisher et al., 1997). Mode locking is used to produce ultrashort pulses in Ti:sapphire lasers. With mode locking, a phase relationship is created such that completely constructive interference between all the modes is realized at just one point, with destructive interference everywhere else. By use of pulse compression techniques, together with mode locking, Ti:sapphire lasers can produce ultrashort pulses down to the femtosecond level. In order to amplify short pulses, three main requirements have to be satisfied by the amplification medium. First, the bandwidth of the amplifier must be large enough to accommodate the full spectrum of the short pulse. Second, the fluence of the pulse has to be near the saturation fluence of the medium. Finally, the intensity within the amplifier has to stay below a critical level at which nonlinear effects can distort the spatial and temporal profiles of the pulse (Maine et al., 1988). A new technique, chirped pulse amplification (CPA), has been used to amplify short pulses to saturation energies while maintaining low power levels in the amplifier (Maine and Strickland, 1988). In CPA, a short optical pulse is first temporally stretched, thus allowing it to be amplified to saturation while maintaining relatively low peak power. Then the original short pulse is restored by use of an optical compressor, which produces a short pulse at its Fourier-transform limit, and at high energy. CPA instrumentation includes four basic components: a short pulse oscillator, a pulse stretcher, an amplifier, and a pulse compressor. The CPA technique is schematically shown in Figure 29. The stretcher can be a single-mode optical fiber, a prism pair, or an antiparallel pair of gratings. The compressor can

131


~~ lo'1

"

ShG

THG

"

/ " ~ S,

S, (

~"~ ~\

100

(a)

10"2~ [ 1

AS1

1 0 3 . IAS2 >~ 200 (l) eLU

10 3

Q.

102

O

101

.

doubled signal

, 400

~

. . . . . 600 800 1000

signal

idler

(b)

100 10-1

doubled idler

10.= 10.3

200

=

500 Wavelength,

=

1000

2000

nm

Figure 27. (a) Several stages of frequency conversion are needed for extension of the wavelength tuning range of a Ti:sapphire laser to 202-1 txm. Reproduced with permission from Funayama, M. et al. (1993). Copyright 1993 Elsevier Science Publisher B. V. (b) Only one stage of SHG frequency conversion, with two BBO crystals, are involved for extension of the wavelength tuning range of an OPO laser to cover 220-2 t.tm.

be a parallel pair of gratings, or a glass-block compressor. The CPA method greatly reduces the peak power of the optical pulse during amplification by avoiding any nonlinear interaction with the gain medium which can result in catastrophic damage. The tuning range of ultrafast Ti:sapphire lasers has been extended to the deep UV by various optical methods. For a 1 kHz femtosecond Ti:sapphire laser, a spectral range from 173 nm to 1.5 txm has been demonstrated (Petrov et al., 1994) which involved the use of cascaded second-order nonlinear frequency conversion processes. Extension of the wavelength tuning range of ultrashort pulses generated by Ti:sapphire lasers Can also be obtained by use of an optical parametric oscillator where the nonlinear crystal of such an arrangement is usually in a fixed position. The tunable OPO output wavelengths are achieved by tuning the Ti:sapphire pump wavelength rather than angle tuning the OPO. By tuning a Ti:sapphire laser in the range 720 to 853 nm, a synchronously pumped near-infrared OPO can produce a

132


M8 /1 . . . . . . . . .alignment cavity ........... P1

M7 pump beam

~

riM9

L

M5

P2

M6

BRF

M1

M3L/slit

Figure 28. Optical schematic diagram ofthe Coherent Model 900-F Ti:sapphire laser. M1, output coupler; M2, M3, M6, and MT, high reflecting mirrors; M4, focusing mirror coated to pass the pump beam and reflect the intracavity beam; M5, focusing, high-reflector mirror; XTL, Ti:sapphire crystal; P1, and P2, prism pair for introducing negative GVD; L, lens. Reproduced with permission from Fisher, W.G. et al. (1997). Copyright 1997 Society for Applied Spectroscopy.

signal wave from 1.05 to 1.2 l.tm and an idler wave from 2.28 to 2.87 l.tm with a maximum average power of about 700 mW (Nebel et al., 1993). In an arrangement comprised of a 250 kHz Ti:sapphire regenerative amplifier and an OPA, it is possible to achieve (Reed et al., 1994) tunable outputs in the signal beam of 460 to 700 nm and in the idler beam from 2.4 to 0.9 I.tm with a peak pulse energy of about 150 nJ. In order to improve the tuning speed of a pulsed Ti:sapphire laser, an acousto-optical tunable filter (AOTF) can be incorporated into an OPO cavity (Chang, 1981) which enables rapid and randomly accessible wavelength tuning, with signal and idler tuning ranges typically from 1.06 to 1.31 and 2.97 to 2.27 ~m, respectively, and very rapid tuning speeds as high as 4 kHz (Akagawa et al., 1997).

Nd:YAG"'

argonlaser

Ti:sapphirelaser

I

"1 amplifier I

oompressor l .

Figure 29. Block diagram of the chirped pulsed amplification technique.


133

Other Vibronic Lasers Other promising materials suitable for vibronic lasers include cobalt-doped magnesium fluoride (Co:MgF 2, tunable from 1750 to 2400 nm) (Welford and Moulton, 1988), chromium-doped crystals such as chromium-doped LiSrA1F6 (tunable from 750 to 1000 nm) (Payne, 1989), and chromium-doped LiCaA1F6 (tunable from 700 to 900 nm). The Co:MgF 2 laser was first commercialized in 1989 with a pulse energy of 75 mJ at its peak wavelength. Many other hosts have also been investigated. Practical problems that are encountered with some of the crystals include poor mechanical, thermal, or optical properties. For example, in some host materials absorption by excited states is found, which reduces laser efficiency.

IV.

D I O D E LASERS

Some significant advances in diode laser technology have been made in recent years. In many ways, diode lasers are the most promising lasers for commercial laser atomic spectrometric instruments because of their small size and potentially low cost. Presently, the main limitation is the lack of reliable lasers that emit in the blue part of the spectrum.

A. Basicsof Diode Lasers A simplified schematic diagram of a semiconductor laser is shown in Figure 30a in which the typical small size, 300 x 250 x 150 ktm, is indicated. The active laser region is a spatially confined layer. When an injection current is sent through the active pn junction region of the diode, which is between the n- and p-type cladding layers, electrons and holes move to the pn junction where they recombine and emit photons. If the densities of electrons and holes are large enough, this radiation can stimulate the recombination of electrons and holes, and laser action can be realized if the amplification of radiation exceeds the loss. The ends of the substrate act as resonator mirrors. The spatial mode of the laser can be defined either by varying the spatial injection current density through the active region, which is called gain guided, or by changing the semiconductor material to change the spatial distribution of the index of refraction, which is called index-guided. The beam divergence directly from the laser diode is always large because the light is emitted from a small rectangular region, in the order of 0.1 lxm by 0.3 lxm. The typical divergence angle is 30 ~ in the direction perpendicular to the junction and 10 ~ in the parallel direction. Therefore, a lens with a smallf number is used at the exit of the laser diode to collimate the laser beam. In addition, the output beams of most diode lasers are also astigmatic. However, a laser wavefront with a good Gaussian profile can be obtained by use of relatively inexpensive lenses and spatial filtering.

134


current blocking

layer

250 pm

active layer

(pn junction)

r

0

p-type material

2

laser output

\

n-type material polished end

polished end

(a) electron flow

J

"10 t-. c~

T

Eg=band gap

I

conduction bands

VV~~

p-type

~I~

laser emission

n-type

(b) Figure 30. (a) Simplified diagram of a semiconductor laser. The active layer (pn junction) is formed when heavily doped p and n materials are joined. The thickness of the layer is about 1 l~m. When an injection current is applied to the laser diode, holes in p type material and electrons in n type material will migrate to the pn junction to recombine and produce laser radiation. The laser radiation is emitted from a well-defined region in the active layer because of the stripe design. (b) Schematic diagram for the energy levels of the p and n-type semiconductor. The laser transition occurs between the conduction band of the n-type semiconductor and the valence band of the p-type semiconductor.

New Typesof Tunable Lasers

135

The semiconductor material determines the wavelength range of the laser. For example, A1GaAs devices provide radiation in the near-IR spectral range, 770-810 nm, with a typical output power of 3 mW; InGaAsP lasers operate at around 670 nm with a power of 3 mW, while InGaN lasers can provide radiation at 420 nm, but these lasers are still under development to improve the lifetime. Among diode lasers that are available in the market, typical output powers are in the range from 2 to 200 mW. At a current that is higher than a threshold value, the output power increases abruptly with increased injection current at a fixed temperature. On the other hand, if the injection current is fixed, the output power increases rapidly when the temperature drops.

B. Characteristics of Diode Lasers Diode lasers have excellent practical and spectroscopic characteristics, including compactness, narrow bandwidth, wavelength tunability, and facile wavelength modulation. In addition, they are inexpensive compared to other lasers, but they have low peak power and relatively limited tuning range. Also, they are temperature-sensitive, as they require a temperature stability of a few mK, and they have a somewhat limited lifetime especially for blue wavelength diode lasers (Niemax et al., 1996).

Compactness The small size and low cost of diode lasers make it possible to install and operate several units simultaneously in a single spectrometer to realize multielement determination. Up to six laser diodes have been used simultaneously, and independently tuned by temperature and locked to the wavelength of several analytes of interest (Niemax et al., 1993). A limiting factor in the simultaneous use of a number of laser diodes is the number of independently operated power supplies that is required.

Narrow Linewidth The linewidth of a diode laser is much narrower than other lasers such as a tunable dye laser or an OPO laser. Typically, at constant temperature and injection current, the spectral linewidth of a diode laser is 30 fm. Whether the characteristic of the narrower linewidth is an advantage or disadvantage in elemental analysis depends on the pressure conditions in the sample atomizer. For example, for laser-excited atomic fluorescence in a graphite furnace, LEAFS, the linewidth of a pulsed dye laser is usually wider than the linewidth of the homogeneously broadened atomic transition. The unabsorbable radiation in the wider bandwidth increases the stray light and degrades the detection power of the technique. The narrower linewidth of a laser diode may be an advantage in this respect. For the acquisition of qualitative spectroscopic information, the biggest advantage of a narrow linewidth is that it

136


can be used to allow higher resolution of atomic and molecular spectral features in the gas phase. For example, one important application is isotope detection by Doppler-free laser spectroscopy (Lawrenz et al., 1987).

Wavelength Tunability The wavelength of a laser diode is determined primarily by the band gap of the semiconductor material, while the tunable range is determined by the width of both the conduction and valence bands, and by the injection current and pn junction temperature (Figure 30b). Generally, this means that the wavelength of a laser diode can be tuned over an interval of about 20 nm. Tuning by variation of the temperature is much slower than by variation of the injection current because it takes time for the heat to transfer to the semiconductor chip. At constant current, the behavior of temperature tuning is illustrated in Figure 3 l a. The temperature tuning curve is a staircase with sloping steps in which the smooth tuning section spans about 0.25 to 0.4 nm, and is due to the continuous change of the optical path length of the cavity (0.06 nrn/K) with temperature. The jump between steps corresponds to a hop from one longitudinal mode to another, which occurs because the slope of the laser gain curve is steeper than the change in cavity length caused by the change in temperature. The spectral gaps that result are a drawback in the application of diode lasers to atomic spectrometry, although a technique that involves external optical feedback can be used to reduce the problem. Changes in the injection current affect both the diode temperature and the index of refraction, both of which affect the wavelength. At a stable temperature, the injection current can be modulated in such a way that the variation of the laser wavelength is within the smooth tuning section, and the wavelength is modulated around a center wavelength (Figure 31 b). The depth of the wavelength modulation depends on the modulation frequency. For example, the

E t-

J

.c"

J

t(!,1

Case Temperature,~

(a)

Injection Current, mA

(b)

Figure 31. (a) Temperature tuning of a diode laser (see text). The typical total tunable wavelength range is 20 nm, while the continuously tunable range at each segment is about 0.25 to 0.4 nm. (b) Injection current tuning of a diode laser. Reproduced with permission from Niemax, K. et al. (1996). Copyright 1996 American Chemical Society.


13 7

wavelength of a typical A1GaAs diode laser can be changed at a rate of 1 prn/mA for modulation frequencies below 1 MHz, then it drops to 100 fm/mA for frequencies from 1 to 3000 MHz. The modulation frequency can rise as high as 3 GHz, after which point the wavelength barely changes as the modulation frequency is raised (Telle, 1993).

Extension of Wavelength TuningRange Laser diodes are now commercially available to cover the spectral range from 625 to 1600 nm, although each laser diode has a limited tuning range of about 20 nm. Both external and internal cavity SHG techniques have been demonstrated for the wavelength extension of diode lasers. Internal methods are advantageous because the SHG beam is automatically created with the same narrow linewidth as the diode fundamental wavelength, and can be directly used for spectroscopic applications, or for pumping another frequency doubler to produce UV radiation. By use of SHG techniques, near-infrared diode lasers with an output wavelength around 780 nm can be frequency-doubled to 390 nm with a potassium dihydrogen phosphate (KDP) crystal (Okazaki et al., 1988). The power achieved is around 50 nW for a 20 mW diode laser, which is a conversion efficiency of 2.5 • 10-6. The conversion efficiency depends on the angle and temperature of the crystal because phase matching is necessary. The conversion efficiency is not very sensitive to the focal length of the lens and optical configuration of the SHG system. Most frequently, potassium niobate (KNbO3) has been used as the SHG crystal for frequency-doubling of diode lasers. It possesses the highest nonlinearity among the commercially available crystals, but high conversion efficiency is not usually realized with an external SHG cavity (Lodahl et al., 1997). One of the main reasons for this is the process of blue light induced infrared absorption (BLIIRA). After accounting for BLIIRA, the conversion efficiency, as measured, is in good agreement with calculated values (Lodahl et al., 1997; Figure 32). Generally, it is easier to obtain high conversion efficiency at higher input power due to the nonlinear character of the process. The data in Figure 32, which were obtained with a 20 mW laser diode, are encouraging because 10 mW of coherent tunable blue light can be generated by using only 20 mW of diode laser radiation. At higher powers, the conversion efficiency reaches 60% and then levels off, which can be explained by the effect of BLIIRA. To take advantage of the compactness of diode lasers, the frequency conversion system should be as small as possible. For this purpose, a technique based on internal cavity SHG has been used (Imasaka et al., 1989) with gallium arsenide diode lasers. The second harmonic can be generated even under phase-mismatched conditions because of the high radiation field inherent in an internal cavity design. This approach allows SHG from a diode laser without the use of extracavity optics and a nonlinear crystal, so the advantages of compactness and convenience of diode laser are retained. The main problem is low conversion efficiency, which can be as low as 10-11 for a CW diode laser. By use of a 1000 Hz

138


80

60

40

g

O

20

'2b

4b

6'0

8'o 160

Input Power, mW Figure 32. The measured conversion efficiency as a function ofthe input fundamental power (N) for a laser diode. Also, the conversion efficiencies were calculated with BLIIRA taken into account (A) (see text). The solid curve was calculated in the absence of BLIIRA. Reproduced with permission from Figure 4 of Lodahl, P. et al. (1997). Copyright 1997 Springer-Verlag.

pulsed laser, operated at a peak power of 10 W, the output is about 0.4 mW at 452 nm, which is a conversion efficiency of about 10-5. The lowest output wavelength, 625 nm, from a commercial diode laser can be frequency-doubled down to 313 nm. With the fundamental input at a level of 50 mW, typical frequency-doubled output power is 50 nW, with a maximum of 3 l.tW (Niemax, 1997). While the wavelength tuning range can be extended by SHG methods, generally, the conversion efficiency is low, especially when converted to blue radiation. Therefore, efforts have been made to extend the short wavelength coverage by development of blue diode lasers. New Type II-VI diodes (ZnSe) with a lasing wavelength in the blue-green range (470-515 nm at room temperature) have been developed in research laboratories. Blue diode lasers have been made that emit at wavelengths as low as 416 nm with an output power of 50 mW, but the lifetime of 300 h is too short. Wavelengths produced by diode lasers have been obtained as low as 400 nm, but with a lifetime of only 0.5 h at room temperature. This type of diode


139

laser is made of InGaN with a multiquantum well structure which consists of lasers of various types of n- and p-doped semiconductor materials, grown by a two-flow organometallic chemical vapor deposition method. Further improvement of the lifetime may be achieved in the future by reducing the threshold current and voltage (Nakamura et al., 1997).

Wavelength Modulation and Wavelength Stabilization The wavelength of a diode laser can be easily and rapidly modulated by variation of the injection current. For common diode lasers, the modulation can be as high as a few gigahertz, while the depth of modulation can be greater than a few picometers. Wavelength modulation allows for discrimination against 1/f noise, which enables measurements to be made near the shot noise limit. Also, for atomic spectrometry, this rapid tunability allows the laser wavelength to alternate between resonance with an atomic line and off-resonance, which can be used for rapid background corrected measurements. Modulation of the injection current also changes the amplitude of the output, but the change is much weaker than that caused by the wavelength change. For many applications of atomic spectrometry, the change in output amplitude can be ignored during the wavelength modulation process. Optical and electronic devices can also be used to improve the spectral quality of diode lasers. By use of a small mirror or a glass plate to provide optical feedback, a multimode laser can be forced to oscillate in a single mode, which reduces the spectral linewidth (Andrews, 1985). For electronic feedback, a small portion of the injection current is injected back to control the laser wavelength. If a very fast electronic feedback is applied, the spectral linewidth can be improved to 100 kHz (Saito et al., 1985). However, these simple feedback devices cannot be applied generally to all types of diode lasers. More sophisticated devices such as external cavity lasers (Fleming and Mooradian, 1981), pseudo-external cavity lasers (Wieman and Hollberg, 1991), and feedback from a high-Q optical cavity (Dahmani et al., 1987) have been developed. It is essential that the output wavelength remains constant for spectroscopic applications, but the wavelength of a distributed feedback diode laser drifts with temperature at 0.1 nm/K. Accordingly, the temperature of these lasers must be stabilized carefully. This is usually done by mounting the laser on a thermoelectric cooler, and by provision of a temperature-sensing feedback loop. Although this approach works, the coolers are bulky, consume extra power, and increase the cost of the diode laser. Stress from differential thermal expansion can be used (Cohen et al., 1996), to counteract the effects of temperature. The temperature sensitivity of the wavelength of a 1.55 mm GalnAsP/InP laser can be reduced by 50%, through use of differential thermal expansion between various fluids incorporated into the laser package. This method automatically generates a temperature-dependent pressure.

Table 3. Summary of the Output Characteristics of Alexandrite, Ti:Sapphire, OPO, and Semiconductor Lasers Laser Type

Alexandrite

Typical pump

flashlamps

Repetition rate, Hz

10-250 (Sam et al., 1988)

Pulse duration

ps-CW (Hecht, 1992)

Fundamental tunable range, 701-826 (Hecht, 1 9 9 2 ) nm

Titanium:Sapphire Nd:YAG or CVL (copper vapor laser) 10-250K and around 100 MHz (Petrov et al., 1994) fs-CW (Padgett and Dunn, 1994) 680-930 (Funayama et al., 1993) to 202 (Funayama et al., 1993)

Wavelength extension, nm, incl. Raman shift, SHG, THG Typical av CW power, W

to 248 (Hobbs, 1993)

Typical av pulsed power, W

up to 5.5 (Knowles and 10-150 (Walling et al., Webb, 1993) 1985; Hecht, 1992) 10-2500 (Sam et al., 1988; 4-100 (Funayama et al., 1993) Walling et al., 1985) 10-s-4 (Padgett and Dunn, 0.003-30 (Bruneau et al., 1994: Knowles and 1994; Volker et al., 1991) Webb, 1993) wide tunable range with high output power, but several stages of relatively narrow tuning frequency conversion; range several sets of mirrors needed for tuning

Typical pulse energy, mJ Linewidth, cm -1 Comments

2-60 (Samelson et al., 1988; 1-43 (Erbert et al., 1991) Walling et al., 1985)

OPO Nd:YAG

Semiconductor Diode electrical

10-250 K (Reed et al., 1994) can be modulated fs-CW (Padgett and Dunn, 1994) 400-2500 with degeneracy gap (Zhang et al., 1 9 9 3 ) to 220 (Zhou et al., 1997) 0.01-0.8 (Padgett and Dunn, 1994; Gerstenberger and Wallace, 1993) 0.01-0.4 (Padgett and Dunn, 1994) 1-100 (Zhou et al., 1997)

continuous wave (CW), usually 625-1600 many lasers/wavelength gaps (Niemax et al., 1996) to 313 nm (Niemax, 1997) 1-50 mW (Wieman and Hollberg, 1992) (up to several W for diode arrays) N/A N/A

0.02-300 (Radunsky, 1995)

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