Some observations on fluorometric determinations

:ANALYTICAL

BIOCHEMISTRY

Some

1,

*Observations HANNELORE

From

the

443-451 (1960)

on Fluorometric BRAUNSBERG

AND

Determinations V. H. T. JAMES

Department of Chemical Pathology, St. Mary’s Hospital Medical School, London, England Received August 12, 1960 INTRODUCTION

The use of fluorometry as a qualitative and quantitative technique offers many advantages. First, advances in instrumentation now permit the measurement of fluorescence with a high degree of sensitivity, and therefore fluorometric methods are attractive in cases where the compounds to be determined or the mat,erial from which they are derived are not available in large quantities. Even when larger amounts are accessible, it may be an advantage to use smaller quantities of material. Furthermore, the fact that two characteristics of a compound are utilized (absorption of light of one wavelength and emission at another) rather than one only, as in absorptiometric determinations, may contribute to the specificity of such a measurement. The availability of spectrophotofluorometers during recent years has facilitated the application of fluorometric methods, and the present paper reports a detailed examination of the Aminco-Bowman instrument. This was undertaken with the aim of using it for the determination of steroids in human blood (1). METHODS

The Aminco-Bowman spectrophotofluorometer (American Instrument Co.) was used with a lP21 photomultiplier (Radio Corporation of America). The quartz cells (external dimensions: 1.2 cm square X 4.8 cm high; internal volume: 4.8 ml) were supplied with the instrument. The apparatus has high sensitivity which can be varied by a factor of 1000. The full-scale meter deflection is 100 divisions, but readings above 40 divisions are unstable and can be avoided by using a less sensitive range. The slit system consists of three metal strips with slits of various widths, which are placed in both the primary and secondary light paths. The center slits determine the band widths, and therefore the resolution of the incident and fluorescent light beams; slightly wider slits placed either side of these reduce stray light. The increase in sensitivity obtained with wider slits is accompanied by decreased resolution and more scatter. The 443

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system is rather crude, and a set of easily and finely adjustable slits would offer considerable advantage. A further slit, placed in front of the photomultiplier also affects resolution and sensit.ivity. The secondary slit system determines t.he size of the image falling on the secondary grating, while the photomultiplier slit affects the band width reaching the detector. To measure the variation of light intensity reaching the test solution in the Aminco instrument, a selenium photocell (14 mm diameter, type B, metal mount with terminals at the back, from Megatron Ltd., London) of known wavelength response was placed in the cell compartment and connected to suitable resistances and a galvanometer. The fluorescence monochromator was calibrated as recommended by the manufacturers, using a Pen-ray mercury lamp (Ultra-violet Products Inc., San Gabriel, California). It has been pointed out by Hercules (2), that certain instrumental factors can affect fluorescence spectra. In the present work, spectra were determined photometrically to avoid errors due to recorder-pen response; those due to reabsorption of emitted light were not likely to occur, since adequately purified material was used at low concentrations and the fluorescence efficiencies were relatively high. The methods used for obtaining fluorescent solutions are described in another paper (3). RESULTS Effect of Slit Size on Spectral Resolution

To study the effect of slit size on the resolution of the instrument, the Pen-ray lamp was placed in the cell compartment and the spectrum of the 365-rnp mercury line was recorded. Figure 1 shows the results obtained by varying the secondary slit width. The’effect of the photomultiplier slit was studied at three wavelengths: 313, 365, and 577 mp. These spectra are shown in Fig. 2; the results are essentially the same at these three wavelengths, and slits wider than l/32 in. cause a decrease in resolution and some distortion. Results with a l/64-in. slit (not shown in Fig. 2) were almost identical with those for the l/32-in. slit. Figure 3 shows the effect of t,he photomultiplier slit size on the resolution of the fluorescence spectrum of corticosterone in ethanolic sulfuric acid. The primary and secondary slits were l/32-in. (0.795 mm) wide, and l/16-in. (1.59 mm) slits were placed on each side of these. While affecting the sensitivity of these measurements and the scatter recorded at the shorter wavelengths, the width of the photomultiplier slit did not alter the spectrum obtained. This indicates that in this case the spectrum is wide and no loss in resolution occurs even with the 3/16-in. photomultiplier slit.

FLUOROlMETRIC

Fluoresceoce

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DETERMINATIONS

reading

(0.01

x

2s stole)

2E

1

Wavelength Calibrations

Caiibration of the secondary monochromator with the Pen-ray lamp revealed errors on the wavelength disk of O-8 ml* in the range 235-730 mp. To test the monochromator calibratians with respect to one another, the scatter from a quartz cell was used. The primary wavelength WFG

(a) I40I

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AND

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FIG. 2. Effect of width of photomultiplier slit on the spectral resolution of mercury lines at (a) 313 mp, (b) 365 mp, and (c) 577 mp. A secondary slit of l/64 in. was used. fixed at the desired value, and the secondary grating was moved until a maximum galvanometer reading was obtained. Errors of up to 10 my were found in the range 250-550 mp, and attempts to adjust the monochromators were not successful. Correction of some spectra was, therefore, necessary [cf. (2)]. The calibration experiments also revealed considerable variation of the complete optical system of the instrument with wavelength. Figure 4 shows the variation of maximum scatter readings with wavelength. Three factors are likely to contribute to this: (a) variation in lamp output with

447

I

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500

600 Wavelength

3. Fluorescence spectra of corticosterone recorded with different photomultiplier slits. I. l&bin. slit. 0.63 cg corticosterone/ml. II. l/32&. slit. 0.32 pg corticosterone/ml. III. 3/16-k. slit. 0.08 pg corlicosterone/ml. FIQ.

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4. Variation

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in 70% (v/v)

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of scatter readings with wavelength.

sulfuric acid

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wavelength; (b) variation in the recording system with wavelength; and (c) possible fault.5 in t,he gratings. An attempt) was therefore made to investigate this further. Experiments

to Investigate

Instrumental Vnriation with Wavelength

It was apparent from scatter readings (Fig. 4) that the over-all sensitivity of the instrument varied considerably with wavelength. The lamp output (through the primary grating) over the range of wavelengths generally used in the present work was measured with a selenium cell as described under Methods. The galvanometer readings obtained were corrected for variations in the photocell response with wavelength, and the results are plot,ted in Curve I (Fig. 5). The lamp output thus appeared to vary slightly with wavelength in the range investigated.

FIG. 5. Variations in the Amine0 spectrofluorometer with wavelength. I. Variation of light intensity reaching the solution, as recorded by a selenium cell. II. Variation of sensitivity of fluorescence measurements with wavelength, not caorrected for fluctuations in the primary light beam.

To examine the over-all instrumental response, a front-silvered plane mirror was placed in t,he cell compartment at 45O to the incident light beam. To cut down the light reaching the photomultiplier, l/64-in. slits mere placed into the primary and reflected light beams. With the primary wavelength setting at a given value, the secondary grating was adjusted to give a maximum reading (this was necessary because the two gratings were slightly out of alignment), and t,he readings are plotted in Curve II (Fig. 5). In the range studied, there is a sharp increase in sensitivity with

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a peak at 470 my, falling off rapidly wit.h increasing wavelength. These experiments appeared to indicate that the instrumental variation was confined to t,he secondary grating or the recorder system, or both. However, at this time, White, Ho, and Weimer (4) published the results of a similar investigation, and since their data conflicted with those obtained here, the primary system w-as further examined by an indirect method. Since the intensity of fluorescence is proportional to the number of absorbed light quanta, and the ratio Ef/E,fluorescent light energy to energy absorbed-is practically independent of exciting wavelength from 300 to 480 in,,, for fluorescein [cf. (5) 1, a quantity proport.ional to this ratio was caIculated from the measured absorption and fluorescence activation spectra for this dye and plotted against wavelength. It was found that there was a marked peak for this ratio in the region 460470 m+ Similar peaks ivere found for the fluorescent solutions of steroids in sulfuric acid. These findings are considered in the Dismrssion. r

Stability

The instrumehtal stability was tested by taking sets of 20 replicate readings on standard solutions of cortisol in sulfuric acid. For the three most sensitive settings of the recording equipment, readings between 10 and 40: divisions ,gave standard deviations of 2% of t,he mean readings. Thi& a wider variation than that reported by Goldzieher et ~1. (6), but the&workers did not’ st,ate at which sensitivity they examined the precitijon. DISCUSSIOK

A n&&i ,.pf : satisfactory fluorometers have been described in the literature f6-19) for [email protected] of fluorescence in solution, and that of Ayres, Simpson, a&d Tait (10) can also be used for sections cut from paper [email protected]_a,ms. More recently, instruments have become available commerciaJly,l and, for those employing optical filters’ the best combinn1 Filter instruments are’manufactured by Farrand Optical Co., New York; Netelel and Hinz, Hamburg, Germaqy ; ,Kipp and Zonen, Delft, Holland; Fisher Scientific Co., Pittsbu$$, Pa.; Locarte: Co.; London; Hilger and Watts, London; Central Scientific Co., Chicago, Ill.; Photovolt Corp., New York; Klett Manufacturing Co. New York. Spectrofluorometers are available from Farrand Optical Co., Nem York American Instrument Co., Silver Spring, Maryland : Perkin-Elmer Corp., ?iorwallc Conn.; Carl Zeiss, Oberkochen, Wiirttbg., Germany. 2 Glass filters are available from Corning Glass Works, Corning, N. I’.; Chnncc+ Pilkington Optical Works, St. Asaph, F&shire, England; and Schott, Mninz, Germany; gelatin filters from Kodak, England and United States; and Ilford Lid.. Ilford England; interference filters from Geraetebau-Anstalt, Balzers, Liechtrnsicin Farrand Optical Co., New York; Schott, Mainz, Germany: Bausch ant1 Loml~ Optical Co., Rorhester, N. T., nnd Barr antI Stroud. T,td., Glnsgon., Scotl:rntl

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tion often has to be found empirically. The interference filters with high transmissions and narrow band widths are perhaps preferable to other types, but their higher order transmissions may result in high “blanks” if scatter from cells or reagent cannot be eliminated. More versatility is offered by spectrophotofluorometers, which ultilize gratings or prisms in incident and fluorescent light paths, and the establishment of optimum conditions for fluorescence production and measurement of a given compound is greatly facilitated. The Aminco instrument is of this type, and employs a xenon source and two gratings, together with a simple slit system. The resolution of the instrument depends upon the slit system employed, and the effect of variations in the arrangement of slits is shown in Figs. 1 and 2, which record spectra of certain mercury lines. It can be seen that the optimum resolution is not high, as was observed by Goldzieher et al. (6), but, on the other hand, the high sensitivity available would almost certainly be decreased by improved resolution. It is also apparent that the use of larger slits introduces considerable spectral distortion, and care should thus be exercised in the choice of slits if information is required on spectral characteristics of compounds under study. Spectral distortion may also be due to variation in the energy output of the light source with wavelength, and attempts were made to investigate this. Direct measurement of light reaching the cell compartment, using a selenium cell, indicated only a small variation with wavelength (Fig. 5) ; when measurements were made through the whole optical system using a mirror or scatter (Figs. 4 and 5), a peak was found at about 470 my. It would thus appear that the increased sensitivity in this region was due to variations in the secondary grating or the recording system. However, White et al. (4) had also investigated the variation of the primary system by using a photomultiplier tube of known characteristics, and observed an increased light intensity in the 470-rnp region. Their method is basically similar to the one employed here with a selenium photocell, and in view of the discrepancy between the results, an attempt was made to investigate further the primary system by an indirect method. This gave the variation of E,/E, ratios with wavelength, and since these ratios were obt.ained from the activation spectrum with no alteration in the secondary or recording systems, the increase observed at about 460-470 rn,p must be due to variations in the primary system. This finding would substantiate that reported by White et al. (4) for their instrument, but is not in agreement with the observations made directly using a selenium cell in the cell compartment. It is possible that the data supplied by the makers for the spectral response of the cell were inaccurate, and hence the corrections for its variation which were applied to

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the experimental data may have been in error. Further work is required to elucidate the cause of the variation of response with wavelength in the Aminco spectrofluorometer. Results on the variation of incident radiation and sensitivity of the recording unit with wavelength, and on the stability, sensitivity, and resolution of other instruments, such as the Farrand, would be a welcome addition to the literature on fluorometric analysis. Indeed, publication of activation and fluorescence spectra without such data limits their usefulness. It appears that further work on instrumentation is also desirable if fluorometric techniques are to be firmly established in analysis. SUMMARY

A detailed study has been made of the Aminco-Bowman spectrophotofluorometer with reference to its variation of sensitivity with wavelength, spectral resolution, and stability. ACKNOWLEDGMENTS We are grateful to Dr. A. Rosen for calibrating the Aminco instrument, and to Dr. Vezzi of Megatron Limited, who very kindly made available the selenium cell. It is a pleasure to acknowledge the advice and loan of equipment from Dr. S. Rowlands. One of us (HR.) is indebted to St. Mary’s Hospital and Medical School for grants. We should like to thank Professor A. Neuberger for his interest and help. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

BRAUNSBERG,H., AND JAMES, V. H. T., J. Endocrinol. 21.333 (19601. HERCULES, D. M., Science 125, 1242 (1957). BRAUNSBERQ,H., AND JAMES, V. H. T., Anal. Biochem. 1,452 (1960). WHITE, C. E., Ho, M., AND WEIMER, E. Q., Anal. Chem. 32,438 (1960). PRINCSHEIM, P., in “Fluorescence and Phosphorescence,” p. 307. Interscience, New York and London, 1949. GOLDZIERER, J. W., BAULD, W. S., ENGEL, L. L., AND GIVNER, M. L., Can. J. B&hem. 38,233 (1966). MCANALLY, J. S., Anal. Chem. 26.1526 (1954). BRAUNSBERG,H., OSBORN, S. B., AND STERN, M. I., J. Endocrinol. 11, 177 (1954). FREEMAN, D. C., PhD. Thesis, University of Maryland, June, 1955. AYRES, P. J., SIMPSON, S. A., AND TAIT, J. F., Biochem. J. 65, 647 (1957).