proposal for standardisation of methods of reporting fluorescence emission spectra

1963 431

Proposal for Standardisation of Methods of Reporting Fluorescence Emission Spectra

(Eingegange~ am 1. MSrz 1963J

In view of the increasing use of fluorescence spectroscopy in analytical and research work, it would be desirable to get some general agreement on the method of reporting fluorescence spectra. This question has already been discussed in publications by some of us 1-3 in which both the fluorescence excitation and fluorescence emission spectra were con- sidered. At the present time fluorescence emission spectra are more widely used than excitation spectra and the corrected spectra can be presented in a variety of ways. The present proposals therefore refer only to fluorescence emission spectra.

We have borne in mind that there arc two distinct kinds of informa- tion, one qualitative and one quantitative and that for some purposes only the qualitative information is required. This qualitative information simply involves the determination of the true shape of the fluorescence band, reported in relative intensity units and without reference to absolute fluorescence intensities. The quantitative information involves some statement of absolute fluorescence intensity, either by statement of an absolute fluorescence efficiency alongside the shape of the fluo- rescence band or by the quotation of the spectrum of a standard fluorescent solution measured under the same conditions. Production of data of the latter type requires more careful control of conditions, particularly in regard to the puri ty of the exciting light and its degree of absorption by the solution, but in view of the greatly increased value of such data it is suggested that this method of reporting results should be adopted whenever possible. We therefore put forward the following proposals:

1. The spectrum should be corrected for variations in photomultiplier sensitivity, spectrometer dispersion and light losses. This requires the preparation of an instrument calibration curve which may be ob- tained by:

a) measurements with a calibrated tungsten lamp (for the visible region), the intensity of which is lowered by a neutral method (e.g. see ref.1), or

b) by measurement of reference fluorescent solutions whose true fluorescence spectra have been determined and agreed, or

c) by other methods, e.g. measurements with a xenon lamp calibrated by the use of fluorescent solutions used as quantum counters.

I f the correction of spectra is not vital to the particular work being reported, reference should be made to the correction curve of the in-

432 Stand~rdis~tion of fluorescence emission spectra Bd. 197

strument concerned so that other workers may apply the correction themselves ff desired.

2. The corrected spectrum should be plotted as relative quanta per unit fregaency interval on the vertical scale versus wavennmber in reciprocal microns (#-1), or em -1, on the horizontal scale. I f possible, the absolute fluorescence efficiency should be quoted or quantitative comparison with a suitable standard solution (e.g. quinine sulphate in 0.1 N sulphurie acid at a stated concentration and optical density per cm) should be given.

3. The spectra should be corrected for "background" (fluorescence from solvent or cnvette, Raman emission, scattered exciting light etc.) or alternatively, the total "background" (ff appreciable) should be recorded on the graph by taking measurements on the pure solvent.

4. The following experimental details should be quoted: a) The spectrum of the exciting light; ff a monochromator is used,

the ~ght source and the band width; ff filters are used, their transmission curves and the nature of the light source (or better, the spectral distri- bution of the exciting light should be plotted to avoid any ambiguity with regard to filter transmissions etc.).

b) The geometrical arrangement of the cuvette width respect to the light beams. The path lengths of exciting and fluorescence light through the liquid from the point from which the fluorescence is viewed. The optical density per cm of the solution for the frequency of the exciting light should be stated and an estimate given of the distortion of the fluorescence spectrum by self-absorption (if this is appreciable).

c) Puri ty and concentration of solute and nature of the solvent. d) Temperature of solution. e) Whether the solution is aerated or de-aerated. f) Type of analysing monochromator and the band widh at an ap-

propriate frequency. We appreciate tha t adoption of these proposals will involve the

quotation of considerably more detail than has been usual in publications of fluorescence spectra, but the information is most desirable if the published spectra are to be of maximum and lasting value to other workers. Since many of the parameters will often remain constant for a large number of experiments, some of them can be stated by simply quoting a reference to a previous publication.

We have deliberately avoided making proposals regarding nomen- clature because this does not affect the value of the published data, and is likely to prove more controversial (e.g. the choice between "spectro- fluorimeter" and "spectrofluorophotometer", or between "excitation spectrum" and "activation spectrum").

1 9 6 3 Bericht: Analyse organischer Stoffe. 1. Elementaranalyse 433

References 1 LIPPERT, E., W. NAGELE, I. SEIBOLD-BLAIqKENSTEIN, W. STAXGER U.

W. VOSS: Z. analyt. Chem. 170, 1 (1959). -- ~ M]~L~S~, W. H.: J. Phys. Chem. 64, 762 (1960). -- a P ~ : ] ~ , C . A . , and W.T.R~Es: Analyst 85, 587 (1960); cf. Z. analyt. Chem. 182, 198 (1961).

J. H. C~A~A~, Glaxo Laboratories Ltd., Greenford, Middlesex, England

TE. F S ~ s ~ , Laborat0rium ffir physik. Chemie der Technischen Hochsehule, 7 Stuttgart

G. Ko~Tti~, Institut ffir physik. Chemie der Universit~t, 74 Tfibingen

E. L ~ e ~ T , Laboratorium fiir physik. Chemie der Teehnischen I~Ioehsehule, 7 Stuttgart,

W. It. ME~Ws~, University of California, Los Angeles, U.S.A.

G. N ~ B ~ , Istituto di Merceologia, Universit& 4egli Studi, Bari, Italy

C. A. P~RKn~, 1% oyal Naval Scientific Service, Admiralty Materials Laboratory, Holton I-Ieath, Poole, Dorset, England

Bericht fiber die Fortschritte der analytischen Chemie

IH. Analyse organischer Stoffe

1. E l e m e n t a r ~ n a l y s e

Den Wasserstollgehalt in organisehen Verbindungen bestimm~ S. MLI~:5 ~ jodometrisch mit einer Genauigkei~ yon ~ 0,04~ . Die Probe wird w~hrend 5 bis 6 rain im Sauerstoffstrom mit einer StrSmungsgesehwindigkeit yon 12 ml/min verbr~nnt. Das d~bei entstehende Wasser wir4 ausgefroren und mit Sehwefel- kohlenstoff fiber einem Korundkatalysator zu Schwefelwasserstoff und Koh]en- dioxid umgesetzt. Man absorbiert den Schwefelwasserstoff in einer Misehung aus ZinksulfatlSsung, NatriumacetatlSsung und Eisessig. Man versetzt die LSsung mit einem ~berschu[~ 0,02 n JodlSsung und titriert den UberschuB mit 0,02 n Natrium- thiosulfatlSsung gegen Sti~rke als Indicator zurfick. -- Die Versuehsapparatur und die Arbeitsweise werden in der Originalarbeit genau beschrieben.

1 Mikrochim. Acta (Wien) 1962, 638--649. Zentralforsehungsinst. f. Chemie, Ungar. Akad. Wins., Budapest (Ungarn). A ~ A n ~ t-IOLLST~I~

Die Oxydationswirkung verschiedener Yerbrennungskatalysatoren, die bei der ]~lementaranalyse fiir Kohlenstoff- und Wasserstoffverbindungen benutzt werden, prfiften J. HORXS]~K, V. P E c ~ v , c und J. KS~L 1. Kohlenmonoxid, n-I-Iept~n und Benzol wurden als Testsubstanze~ verwendet und die nieclrigste Temperatur festgestellt, bei tier die Verbrennung zu I(ohlendioxid und Wasser gerade noeh quantitativ verl~uft. Naeh Ausffihrung aller Teste erwiesen sich als wirksame Katalysatoren folgende Substanzen: MnO~ (gefi~llt), ~_aO 2 (durch Gliihen

z. analyt. Chem., Bd. 197 29