《材料测试技术及方法》课程教学资源（书籍文献）分子光谱之紫外可见光谱 UV-VIS Spectroscopy and Its Applications，Translated by H. Charlotte Grinter and Dr. T. L. Threlfall

Heinz-Helmut Perkampus UVVIS Spectroscopy and Its Applications Translated by H.Charlotte Grinter and Dr.T.L.Threlfall With 78 Figures and 21 Tables Springer-Verlag Berlin Heidelberg New York London Paris Tokyo Hong Kong Barcelona Budapest

Heinz-Helmut Perkampus UV-VIS Spectroscopy and Its Applications Translated by H. Charlotte Grinter and Dr. T. L. Threlfall With 78 Figures and 21 Tables Springer -Ver lag Berlin Heidelberg New York London Paris Tokyo Hong Kong Barcelona Budapest

Preface UV-VIS spectroscopy is one of the oldest methods in molecular spectroscopy.The definitive formulation of the Bouguer-La Beer law in 1852 created the basis for the quantitative evaluation of absorption measurements at an early date.This led firstly to colorimetry,then to photometry and finally to spectrophotometry. This evolution ran parallel with the development of detectors for measuring light intensities,i.e.from the human eye via the photo- element and photocell,to the photomultiplier and from the photo- mln me mn of eomlee With the development of quantum chemistry,increasing atten- s paid to the en light abs tion a and the structur the lt that ent de t dis ssions of the theory of des a numbe (UV-VIS and c spectr spectroscopy)have been publishe Consequently,this extremely interesting as ect o mo r spec troscopy has dominated the teaching of the subject both in my own lectures and those of others.However,it is often overlooked that,in addition to the theory,applications of spectroscopic methods are of particular interest to scientists.For this reason,a lecture series about electronic spectroscopy given in the Institute for Physical Chemistry at the Heinrich-Heine-University in Dusseldorf was supplemented by one about "UV-VIS spectroscopy and its applications".This formed the basis of the present book. UV-VIS spectro opy owes its importance not least to its varied in This book to show W UV-VIS 0P2 be applied an to the kine tics of chemical reac ctions,incluc ng phot theoretical section has been kept to a minimum since,as men tioned above,excellent discussions of such matters are ava lable in the literature.The details of the equipment are also described very briefly because G.Kortum gave an outstanding discussion of this subject in volume II of the series "Anleitung fur die chemische Laboratoriumspraxis";and its basic details still apply today. In addition to the applications,a number of UV-VIS spec- troscopic techniques are discussed.However,in this case the selec- tion has been influenced by the author's own interests.In order to expe ime tal mples ous me ents ha e beer

Preface UV-VIS spectroscopy is one of the oldest methods in molecular spectroscopy. The definitive formulation of the Bouguer-Lambert￾Beer law in 1852 created the basis for the quantitative evaluation of absorption measurements at an early date. This led firstly to colorimetry, then to photometry and finally to spectrophotometry. This evolution ran parallel with the development of detectors for measuring light intensities, i.e. from the human eye via the photo￾element and photocell, to the photomultiplier and from the photo￾graphic plate to the present silicon-diode detector both of which allow simultaneous measurement of the complete spectrum. With the development of quantum chemistry, increasing atten￾tion was paid to the correlation between light absorption and the structure of matter with the result that in recent decades a number of excellent discussions of the theory of electronic spectroscopy (UV-VIS and luminescence sp,~ctroscopy) have been published. Consequently, this extremely ivteresting aspect of molecular spec￾troscopy has dominated the teaching of the subject both in my own lectures and those of others. However, it is often overlooked that, in addition to the theory, applications of spectroscopic methods are of particular interest to scientists. For this reason, a lecture series about electronic spectroscopy given in the Institute for Physical Chemistry at the Heinrich-Heine-University in Dusseldorf was supplemented by one about "UV-VIS spectroscopy and its applications". This formed the basis of the present book. UV-VIS spectroscopy owes its importance not least to its varied applications in chemistry, physics and biochemistry. This book aims to show how UV-VIS spectroscopy can be applied to analytical problems, to the investigation of chemical equilibria and to the kinetics of chemical reactions, including photokinetics. The theoretical section has been kept to a minimum since, as men￾tioned above, excellent discussions of such matters are available in the literature. The details of the equipment are also described very briefly because G. KortUm gave an outstanding discussion of this subject in volume II of the series "Anleitung fUr die chemische Laboratoriumspraxis"; and its basic details still apply today. In addition to the applications, a number of UV-VIS spec￾troscopic techniques are discussed. However, in this case the selec￾tion has been influenced by the author's own interests. In order to obtain experimental examples, numerous measurements have been

VI Preface made which might also be set as practical work for students of ad- chemis like my colleagues for making these measure. ments,and rawing grams The English translation of the s onddition of this volume is due to the stimulating interest of the "Ultra iolet Spectrometry Group",London,to whom I am very gratefu Mrs.Charlotte Grinter undertook the translation with great in terest and engagement,professionally supported by Dr.T.L. Threlfall and Dr.Grinter.I would like to express my sincere thanks to Mrs.Grinter and the colleagues mentioned above for their hard labors. A few additions have been made to the first edition,i.e.the brief section "Chemometrics"was added by the English colleagues, me have e been changed,others are new and the cited ated w y.Here also the English ed to be very helpful for ich vould like to ex press my thank Thanks are also due to Dr.Enders of the Springer -Verlag interest and support of the publication of the English Dusseldorf,June 1992 HEINZ-HELMUT PERKAMPUS

VI Preface made which might also be set as practical work for students of ad￾vanced physical chemistry. I would like to thank my colleagues for making these measure￾ments, and drawing the diagrams. The English translation of the second edition of this volume is due to the stimulating interest of the "Ultraviolet Spectrometry Group", London, to whom I am very grateful. Mrs. Charlotte Grinter undertook the translation with great in￾terest and engagement, professionally supported by Dr. T. L. Threlfall and Dr. Grinter. I would like to express my sincere thanks to Mrs. Grinter and the colleagues mentioned above for their hard labors. A few additions have been made to the first edition, i.e. the brief section "Chemometrics" was added by the English colleagues, some figures have been changed, others are new and the cited literature has been updated where necessary. Here also the English colleagues proved to be very helpful for which I would like to ex￾press my thanks. Thanks are also due to Dr. Enders of the Springer-Verlag for his interest and support of the publication of the English edition. Dusseldorf, June 1992 HEINZ-HELMUT PERKAMPUS

Contents 1 Introduction 1 Principles .。4”4。4 2.2 Primary Photophysical Processes . 5 2.3 Vibrational Structure of Electronic Spectra. 6 2.4 Electronic Spectra and Molecular Structure . 8 References. Photometers and Spectrophotometers. o 3.1 Photometers. 11 3.2 Spectrophotometers. 12 3.3 The Stray Light Error 17 3.3.1 General Observations 3.3.2 The Stray Light Error of Transmission and Absorbance and Its Measurement 19 3.4 Light Sources for UV-VIS Spectroscopy . 21 References 24 Analytical Applications of UV-VIS Spectroscopy 26 4.1 Photometric Determination of a Single Substance 26 4.1.1 Photometric Det mination of Ei lements by Means of Co omp Ag 29 4.1.2 metric Determ in f Anions and Amm 4.1.3 Photometric Water Analyses 4.1.4 Photometric Determination of Organic Compounds. 。,。,。 4.1.5 Enzymatic Analysis and Enzyme Kinetics. 4.2 2. Multicomponent Analysis +。 Basic Equa tions 42 An Example of a Multicomponent Analysis . 886 43 Identification and Structure Determination. 68

Contents 1 2 2.1 2.2 2.3 2.4 Introduction Principles . . The Bouguer-Lambert-Beer Law and Its Practical Application . . Primary Photophysical Processes . . Vibrational Structure of Electronic Spectra Electronic Spectra and Molecular Structure . . 1 3 3 5 6 8 References . 9 3 3.1 3.2 3.3 3.3.1 3.3.2 3.4 Photometers and Spectrophotometers . . Photometers . . Spectrophotometers . . The Stray Light Error . . General Observations . . The Stray Light Error of Transmission and Absorbance and Its Measurement . . Light Sources for UV-VIS Spectroscopy . . 10 11 12 17 17 19 21 References . 24 4 4.1 4.1.1 4.1.2 4.1.3 4.1.4 4.1.5 4.2 4.2.1 4.2.2 4.3 Analytical Applications of UV-VIS Spectroscopy Photometric Determination of a Single Substance Photometric Determination of Elements by Means of Complexing Agents . . Photometric Determination of Anions and Ammonia . . Photometric Water Analyses . . Photometric Determination of Organic Compounds . . Enzymatic Analysis and Enzyme Kinetics . . Multicomponent Analysis . . Basic Equations . . An Example of a Multicomponent Analysis . . Identification and Structure Determination . . 26 26 29 38 43 44 49 58 58 65 68

VIII Contents 4.4 Chemometrics 六 References . 76 5 Recent Developments in UV-VIS Spectroscopy. 8 5.1 Dual-Wavelength Spectroscopy 。+，。+ 81 5.2 Derivative Spectroscopy. 5.3 Reflectance Spectroscopy 9 5.4 Photoacoustic Spectroscopy 。,。4.4。年。4。，。g: 5.4.1 Principles of PAS . 181 5.4.2 PAS Applications. 110 5.5 Luminescence-Excitation Spectroscopy ·。” 120 References . . 128 6 Investigation of Equilibria 3 61 General. 131 6.2 Protolytic Equilibria;pK-Values ，。，。，。÷。4.4944。 132 6.3 Complex-Forming Equilibria 142 63, As ation. 143 6.3 EDA 149 +”4。年”年”年”中年车”卡”中”·。”。 6.3.3 Metal Complexes 年年。+。+4++4年44。”。”*”。卡” 158 References 162 Investigation of the Kinetics of Chemical Reactions. 165 7. Fundamental Equations of Kinetics 165 11.1 Introduction of Absorbance a meas 165 7.1.2 sific of Other Types of Reaction 167 7.1.2.1 2nd o 167 年。卡年无，8”卡”卡”卡”中”中”卡” 7.1.2.2 3rd Order 169 00年卡年卡+”年9年。，。年。” 7.1.2. 3 Pseudo 1st Order Reactions ▣0。t0t。t。t”年”+”4”4 171 7.1.2.4 Consecutive Reactions . 172 7.1.2.5 Parallel Reactions., 173 7.2 The Number of Linearly Independent Partial Reactions. 175 7.3 Evaluation of Kinetic Measurements .179 7.4 Examples,.,。.。,.,。.。,。,.,。 183 Fast Reactions 190 Flow Method opped-Flov 190 Spectroscopic Relaxation Techniques. 193

VIII .Contents 4.4 Chemometrics 75 References . 76 5 5.1 5.2 5.3 5.4 5.4.1 5.4.2 5.5 Recent Developments in UV-VIS Spectroscopy . . Dual-Wavelength Spectroscopy . . Derivative Spectroscopy . . Reflectance Spectroscopy . . Photoacoustic Spectroscopy . . Principles of PAS . . PAS Applications . . Luminescence-Excitation Spectroscopy . . 81 81 88 95 101 101 110 120 References . 128 6 Investigation of Equilibria . 131 6.1 General. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 6.2 Protolytic Equilibria; pK-Values . . 6.3 Complex-Forming Equilibria . . 6.3.1 H-Bond Association . . 6.3.2 EDA Complexes . . 6.3.3 Metal Complexes . . References . . 7 Investigation of the Kinetics 132 142 143 149 158 162 of Chemical Reactions . 165 7.1 Fundamental Equations of Kinetics. . . . . . . . . 165 7.1.1 Introduction of Absorbance as a Measurement Parameter . 165 7.1.2 Classification of Other 'JYpes of Reaction . 167 7.1.2.1 2nd Order Reactions . 167 7.1.2.2 3rd Order Reactions . 169 7.1.2.3 Pseudo 1 st Order Reactions . 171 7.1.2.4 Consecutive Reactions . 172 7.1.2.5 Parallel Reactions . 173 7.2 The Number of Linearly Independent Partial Reactions . 175 7.3 7.4 7.5 7.5.1 7.5.2 Evaluation of Kinetic Measurements . . Examples . . Fast Reactions . . Flow Methods: The Stopped-Flow Technique . . Spectroscopic Relaxation Techniques . . 179 183 190 190 193

Contents IX 7.6 Photoreactions . 197 77 . 203 771 pectrometers· 204 Spectrometers.,.,.,.,.,.。.,.,。.。. 204 Diode Array Spectrometers. 205 7.8 Determination of the Spectra of Intermediates. 207 References . 210 8 Evaluation of UV-VIS Spectral Bands . 215 8.1 Oscillator Strength and Transition Moment. 215 8.2 Band Analysis. .。.。.。.。 220 8.2.1 Gaussian and Lorentzian Functions. 220 8.2.2 Application of Derivative Spectra. 223 8.3 Vibrational Structure. 228 References. 233 Index of Ilustrated Absorption Spectra . 235 Subject Index 237

Contents IX 7.6 7.7 7.7.1 7.7.2 7.7.3 . 7.8 Photo reactions . . Spectrometers for Kinetic Measurements . . Rapid Spectrometers . . FT-UV Spectrometers . . Diode Array Spectrometers . . Determination of the Spectra of Intermediates . . 197 203 204 204 205 207 References . 210 8 Evaluation of UV-VIS Spectral Bands . 215 8.1 Oscillator Strength and Transition Moment . 215 8.2 Band Analysis . 220 8.2.1 Gaussian and Lorentzian Functions . 220 8.2.2 Application of Derivative Spectra . 223 8.3 Vibrational Structure . . References . . Index of llIustrated Absorption Spectra . . Subject Index . . 228 233 235 237

Introduction Optical spectroscopy is based on the Bohr-Einstein frequency relationship 4E=E2-E=hy. (1) This relationship links the discrete atomicr states E with the The proportionality ergs).In spectroscopy,it is appropriate to use the wavenumber instead of frequency v.Equation (1)then takes the form: E=E2-E=he where v=c/i=c (2) Absorbed or emitted radiation of frequency v or wavenumber y can thus be assigned to specific energy differ ces or,applying the definition of the 'term value'(energy level),to specific energy-level dif =4E/he E2/hc-Ej/hc=T2-T1 (3) the term value From this definition it follows that it has in the SI-system.However,it is still commonly given as msice the munberyemthe lrature will also be used in this book (1 cm100m). For absorption spectroscopy in the ultraviolet (UV)and visible (VIS) region,this range can be characterized by the information in Fig.1. Within the overall range of electromagnetic radiation which is of interest to chemists,UV-and VIS-absorption spectroscopy occupies only a very narrow frequency or wavenumber rthe ss.this ng is of treme imr rtance,since the en electronic sta tes of atoms rrespon of th cep spectroscopy hermore,in th n the interaction matt and electromagnetic radi ation manifest themselves as color. This led th early investigators to methods of measurement,the basic prin- ciples of which still apply today. The limits given in Fig.1 are not fixed limits because molecules exhibit absorption below 200nm ie.above 50000 cm-.However,this spectral region is not accessible to routine measuring techniques.The short-wave- H.H.Perkampus,UV-VS Springer-Verlag Berlin Heidelberg 1992

1 Introduction Optical spectroscopy is based on the Bohr-Einstein frequency relationship (1) This relationship links the discrete atomic or molecular energy states Ej with the frequency v of the electromagnetic radiation. The proportionality constant h is Planck's constant (6.626 x 10-34 J s or 6.626 x 10- 27 erg s). In spectroscopy, it is appropriate to use the wavenumber v instead of frequency v. Equation (1) then takes the form: LIE = E2 -Ej = hcv where V=CIA=CV (2) Absorbed or emitted radiation of frequency v or wavenumber v can thus be assigned to specific energy differences or, applying the definition of the 'term value' (energy level), to specific energy-level differences: (3) Tj = E/h c is the term value. From this definition it follows that it has dimension m -j in the SI -system. However, it is still commonly given as cm-I; thus, wavenumber v as a term difference may be given in m-I or cm -I. Since the wavenumber is always given in cm -I in the literature, it will also be used in this book (1 cm-I~100m-I). For absorption spectroscopy in the ultraviolet (UV) and visible (VIS) region, this range can be characterized by the information in Fig. 1. Within the overall range of electromagnetic radiation which is of interest to chemists, UV- and VIS-absorption spectroscopy occupies only a very narrow frequency or wavenumber region. Nevertheless, this range is of ex￾treme importance, since the energy differences correspond to those of the electronic states of atoms and molecules; hence the concept of "electronic spectroscopy". Furthermore, in the visible spectral region the interactions between matter and electromagnetic radiation manifest themselves as color. This led the early investigators to methods of measurement, the basic prin￾ciples of which still apply today. The limits given in Fig. 1 are not fixed limits because molecules exhibit absorption below 200 nm ie. above 50000 cm -I. However, this spectral region is not accessible to routine measuring techniques. The short-wave￾H.-H. Perkampus, UV-VIS Spectroscopy and Its Applications © Springer-Verlag Berlin Heidelberg 1992

2 Introduction 175200 250 33400500 m80 057.102cm150 40一 302520 12510 Near IR Short-wavelength iig-wavelength limit Monochromators Photomultiplier Solvent Empirical:defintion of the VIS-region 02-absorptior Extension1cm Extension ot range→near IR Photocells.PbS-cells N2-flushing >57×103cm Grating spectrometer Vacuum UV Fig.1.The ranges of electronic spectra and their limits ongdpparats and bypetalch dep nds less on considerations of apparatus because, apart from a few comp nds exhibit no absorption traceable to ele There a exceptions:for ex ample polymethine dyes.used as photographic and so organic complexes which have absorption bands that can be observed upto 2μm±5×103m-1(5000cm-)

2 Introduction A- m ~ ~ ill ~ ~ ~~ I I I I I I I I I 60 57 '103cm-1 50 40 30 25 20 12.5 10 -v vacu~m UV I_ Nz- UV -1°11-.- UV -.If.o-~- VIS • liNear IR￾I Short-wavelength , limit Monochromators Solvent 02-absorption Extension of range:-+ - 57 x 103cm-1 { CaF2-prisms N2-f1ushing > 57 x 103cm-1 Grating spectrometer VacuilmUV Fig. 1. The ranges of electronic spectra and their limits Long-wavelength I limit , Photomultiplier Empirical; definition of the VIS-region Extension of range -+ near IR Photocells, PbS-celis Less problematical than at short-wavelength limit length limit is restricted by apparatus and by experimental techniques. The long-wavelength limit depends less on considerations of apparatus because, apart from a few exceptions, most compounds exhibit no absorption traceable to electronic excitation in this region. There are exceptions; for ex￾ample polymethine dyes, used as photographic sensitizers, and some in￾organic complexes which have absorption bands that can be observed up to 2~m~5xl05m-l (5000cm- 1)

2 Principles 2.1 The Bouguer-Lambert-Beer Law and Its Practical Application The Bouguer-Lambert-Beer law forms the mathematical-physical basis of light-absorption measurements on gases and solutions in the UV-VIS and IR-region [1]: (4④) where is the absorbance, 五-=名o0n%the nramanc, is the molar decadic extinction coefficient. Io is the intensity of the monochromatic light entering the sample and I is the intensity of this light emerging from the sample;c is the concentration of the light-absorbing substance and d is the pathlength of the sample in cm. Equation(4)then gives: 64 c.d with dimensions for e of: Imol-1cm-1 for "e"in mol 1-1 or 1000cm2mol1for“c”in mol10-3cm-3 The molar decadic extinction coefficient,is a quantity characteristic of the substance which also depends on wavenumber(cm)or on wavelength A(nm). H.H.Perkampus,UV-VS Springer-Verlag Berlin Heidelberg 1992

2 Principles :-:.:. -·r ~ _ . . .; . . '.'.', . • I ~ • ,; . . . . . . 1_'" . ~ . • _ . The Bouguer-Lambert-Beer law forms the mathematical-physical basis of light-absorption measurements on gases and solutions in the UV-VIS and IR-region [1]: ( 10) ( 100 ) _ Ig - = Ig - =Av = ev·c·d , 1 v T(OJo) v where Av= Ig (~)v is the absorbance, Tv = .!. tOO in 070 is the transmittance, 10 ev is the molar decadic extinction coefficient. (4) 10 is the intensity of the monochromatic light entering the sample and 1 is the intensity of this light emerging from the sample; c is the concentration of the light-absorbing substance and d is the pathlength of the sample in cm. Equation (4) then gives: Av ev=- c·d with dimensions for ev of: 1 mol- 1 cm - 1 for "c" in mol 1- 1 or 1000cm2 mol- 1 for "c" in moltO- 3 cm- 3 The molar decadic extinction coefficient, ev, is a quantity characteristic of the substance which also depends on wavenumber ii (cm- 1) or on wavelength A (nm). H.-H. Perkampus, UV-VIS Spectroscopy and Its Applications © Springer-Verlag Berlin Heidelberg 1992

4 Principles "absorption spectrum"of a compound vary by several orders of magnitude within the absorption spectrum of a single inorganic or organic compound,the logarithmic value lg e=f()can be used instead of e=f()to plot an absorption spectrum [2]. The Bouguer-Lambert-Beer law is a limiting law for dilute solutions,i.e. the assertion that the extinction coefficient e is independent of the concen- tration of a substance at the given wavenumber y (wavelength a)applies on to dilute solutions.e is no longer constant for concentrated solutions but the of the solution i1l.at concentrations up to 102n ive index and lies 1 or 2 powers of ten below the usual photometri accu owed with precise easurements on aqueous solutions of K [Fe(CN[3]. According to Eq.(4),the applicati of the Bouguer-Lambert-Beer law of the relationship between theight int Iand However,when measuring in quartz cuvettesUVV sitic cuvettes made of special optical glass(VIS region),part of the light is los through reflection at the cuvette surfaces.In order to eliminate thi s source of error,a reference measurement is made in a cuvette with the same pathlength but not containing the substance to be measured.Since most UV-VIS spectroscopy is carried out with solutions,the standard cuvette con- tains the pure solvent,which ideally should not absorb in the spectral region under c nsideration. after the light has traversed the standard or the sample has tra versed the cuvette containing ing on the otmiona mode of n as either analog or digital form.This result is independen )or eration of the of T losses due to reflection and the influence of the solvent. This still presupposes that the two cuvettes used for the meas have the same pathlength and have been matched prior to making the mea surements.Most manufacturers keep the accuracy of the pathlengths of a matched cuvette set within a few ppm.However,the continued matching of a previously used pair of cuvettes depends entirely on the care taken by the individual user of an UV-VIS spectrophotometer.In many applications, standard cuvettes are suitable;they are available in pathlengths of 1,2,5, 102050 nd 100 m and.depe nding on the spectral region,are produced ther fron or S artz glass or special optical glass.Fur- thermo r special methods of measure ment [4]. The choice of solvent depends on an adequate solubility y of the substance to be measured.For example,n-heptane water and triflu 0 ethanol r hexafluoroisopropanol may be considered as gooc scopic so bcu th r paret o th UVVI ever,below 200 nm the pathlength must be reduced to 1 mm and in this region the spectrophotometer must be flushed with pure nitrogen in order

4 Principles The functional correlation between eji and wavenumber v is called the "absorption spectrum" of a compound. Since the extinction coefficient can vary by several orders of magnitude within the absorption spectrum of a single inorganic or organic compound, the logarithmic value 19 e = f(v) can be used instead of e = f(v) to plot an absorption spectrum [2]. The Bouguer-Lambert-Beer law is a limiting law for dilute solutions, i.e. the assertion that the extinction coefficient e is independent of the concen￾tration of a substance at the given wavenumber v (wavelength A) applies on￾ly to dilute solutions. e is no longer constant for concentrated solutions but depends on the refractive index of the solution [1]. At concentrations up to c ~ 10 - 2 moll-I, the effect is slight and lies 1 or 2 powers of ten below the usual photometric accuracy, as Kortiim showed with precise measurements on aqueous solutions of K3 [Fe(CN)6] [3]. According to Eq. (4), the application of the Bouguer-Lambert-Beer law presupposes a measurement of the relationship between the light intensities I and 10, However, when measuring in quartz cuvettes (UV-VIS region) or cuvettes made of special optical glass (VIS region), part of the light is lost through reflection at the cuvette surfaces. In order to eliminate this source of error, a reference measurement is made in a cuvette with the same pathlength but not containing the substance to be measured. Since most UV-VIS spectroscopy is carried out with solutions, the standard cuvette con￾tains the pure solvent, which ideally should not absorb in the spectral region under consideration. Thus, 10 is measured after the light has traversed the standard or reference cuvette and I after the light has traversed the cuvette containing the sample. Depending on the construction and mode of operation of the equipment, the relationship I1Io is shown as a value of T ji (070) or Aji in either analog or digital form. This result is independent of losses due to reflection and the influence of the solvent. This still presupposes that the two cuvettes used for the measurement have the same pathlength and have been matched prior to making the mea￾surements. Most manufacturers keep the accuracy of the pathlengths of a matched cuvette set within a few ppm. However, the continued matching of a previously used pair of cuvettes depends entirely on the care taken by the individual user of an UV-VIS spectrophotometer. In many applications, standard cuvettes are suitable; they are available in pathlengths of 1, 2, 5, 10,20,50 and 100 mm and, depending on the spectral region, are produced either from Suprasil or Spectrosil quartz glass or special optical glass. Fur￾thermore, there is a wide range of cuvettes for special methods of measure￾ment [4]. The choice of solvent depends on an adequate solubility of the substance to be measured. For example, n-heptane, water and trifluoroethanol or hexafluoroisopropanol may be considered as good spectroscopic solvents because they are transparent from ca. 180 nm in the UV-VIS region. How￾ever, below 200 nm the pathlength must be reduced to 1 mm and in this region the spectrophotometer must be flushed with pure nitrogen in order