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Cyanogen flame emission and long pathlength atomic absorption spectrometry
05&2-8547/80/1101-0657$02.00/0
p&x&mica Acta. Vol.3SB.m 657to661 i PcrgmmnFY~ Ltd.1980. F?intcdin Great Britain
Cyanogen flame emission and long pathlength atomic absorption spectrometry K. FUWA Department of Chemistry, University of Tokyo, Bunkyo-ku, Tokyo, Japan
and B. L.
VALLEE
BiophysicsResearch Laboratory, Harvard Medical School and P. B. Brigham Hospital, Boston, MA, U.S.A. (Received 20 June 1980) IN THE 1940s the Spectroscopy Laboratory of Massachusetts Institute of Technology was under the direction of three scientists, G. R. HARRISON, Professor in Physics, R. C. LORD, Professor in Chemistry, and J. R. LOOFBOUROW, Professor in Biophysics. After the untimely death of Prof. LOOFBOUROW in 1951, Dr. B. VALLEE, one of the members of the biophysics laboratory moved to Harvard Medical School and started the Biophysics Research Laboratory within the building of Peter Bent Brigham Hospital, one of the Hospitals afhliated with the university. The activity of the new laboratory concerned the composition, structure and function of trace metals in biological systems. One of the major activities of the laboratory was spectrochemical analyses for trace metals in various biological materials. 1955 was the inaugural year for atomic absorption spectrometry due to A. WALSH’S publication, and at that time, two particular scientists who belonged to the spectroscopy group of the Biophysics Research Lab., that is, Dr. R. E. THIERS previously from the University of Virginia, and Dr. M. MARGOSHES from Iowa State University should be mentioned. They had been developing a cyanogen emission flame and a four-channel direct-reading flame photometer together with Dr. VALLEE. At that time, Dr. K. FUWA was instructor in the laboratory of Prof. E. MINAMI, the Department of Chemistry, University of Tokyo, and was completing a Ph.D. thesis under the title “Spectrochemical Analysis of Fluorine by Emission Spectra of Alkaline Earths Bifluorides and Its Application to Geochemistry”. Prof. MINAMI had studied geochemistry at the school of Prof. V. GOLDSCHMIDT of Gottingen University, Germany, in the 1930s. Colleagues at Giittingen included Drs. R. MANNKOPF, C. PETERS and L. W. STROCK.* The technique of cathode layer excitation was developed by these scientists and became a well-known technique of high sensitivity in the field of DC arc emission spectroscopy. When FUWA was selected as a Fulbright Exchange Scholar to the U.S. from Japan in 1955, Dr. MINAMI wrote to Dr. STROCK asking if he could work in his laboratory in the U.S.A. Dr. STROCK was then a member of the research laboratory of Sylvania Electric Co. at Long Island and suggested that he should join Dr. VALLEE'S laboratory as activities at Sylvania Co. were not confined to spectrochemical analysis. Consequently, FUWA joined the spectroscopy group of the Biophysics Research Laboratory in Boston in the spring of 1956. This was already a year after atomic absorption was suggested by Dr A. WALSH of Australia, and Dr. C. T. J. ALKEMADE in the Netherlands Cl], but we did not immediately investigate the method but continued to work in the field of flame emission spectroscopy. Flame emission work was the general trend of research in the 1950s in the U.S.A.
*Dr. Strock was from Philadelphia and was also studying geochemistry as well as spectroscopy in Goldschmidt’s laboratory. [I] A. WALSH, Spechochim. Acta. 7, 108 (1955); C. T. J. ALKEMADE 45, 583 (1955). 657
and J. M. W. MILATZ, J. Opt. Sot. Am.
658
K. FUWA
and
B. L.
VALLEE
METALLOENZYME AND CYANOGEN FLAME As was briefly described above, the Biophysics Research Laboratory was established to study the role of “trace metals” in biology, particularly enzymes. Carboxypeptidase from bovine pancreas, alcohol dehydrogenase from yeast and horse liver, and alkaline phosphatase from E. coli were the first of now more than 120 enzymes which Dr. VALEE and his colleagues quickly established to be zinc containing and dependent enzymes. In order to investigate these metalloenzymes, sensitive, accurate and yet simple detection techniques for zinc were required as only one or a few metal atoms were contained in a large protein molecule of the enzyme. The second biological material which was under investigation in the laboratory was a cadmium containing protein. Cadmium was chosen as the second metal because of its close chemical similarity to zinc. The cadmium-containing protein is concentrated in the cortex of the mammalian kidney, particularly that of the horse. This most interesting protein was named metallothionein, and attracted the interests of many people working in the toxicological and environmental fields. Dr. MARGOSHESstarted work on this cadmium protein and had to use the dithizone calorimetric method as sensitive flame emission techniques were not available. Thus, we needed also a sensitive, accurate and simple method for cadmium. The third metal of interest in the laboratory was magnesium. An accurate method was not available and the function of magnesium in human blood was unknown. Dr. W. E. C. WACKERwas investigating this field and here again a good method was required for magnesium. For decades before atomic absorption spectrometry, analytical spectroscopists were making various modifications of the three emission light sources, i.e. d.c. arc, spark and flames which had been used for nearly a hundred years since the age of KIRCHHOFFand BUNSEN. In the Biophysics Research Laboratory, the arc-like spark source with a porous cup electrode was used for both qualitative and quantitative analyses. A photographic method using a Jarrell-Ash 3.4-m Wadsworth mounting grating spectrograph together with densitometry of the lines for the internal standard process were adapted. The first survey of the sample was carried out by photography and then individual elements were determined by either direct-reading flame photometry or calorimetry. A four-channel flame photometer with automatic background correction was under development for sodium, potassium, calcium and magnesium by MARGOSHES [2]. The flame used was mainly the oxy-hydrogen flame supported on a Beckman burner. In order to determine heavy metals other than the alkali metals and alkaline earths but including zinc and cadmium, a higher temperature flame was needed. It was known then that both the cyanogen-oxygen flame and the hydrogen-fluorine flame reach approximately 5000 K when they react in the following manner C,N, + 202 = 2C02 + Na, H2 + F, = 2HF. Dr. VALLEE and his colleagues decided to investigate the cyanogen-oxygen flame [3]. The design of the burner for the system of cyanogen and oxygen was the first project FUWAwas given when he joined the laboratory in April of 1956. The principal prerequisite of the burner for a total consumption of the analytical Aame is to make a stable flame of correct size and from a mixture of the fuel and oxidizing gases (in approximately chemical equilibrium amounts) and to nebulize the sample solution pneumatically into the flame. In order to fulfil these prerequisites the flame speed or the burning velocity of the system ought to be high as pneumatic nebulization require(s) also a certain high pressure. The burning velocities of oxyhydrogen and oxyacetylene flames are 3680 cm/s and 2480 cm/s respectively, but that of oxycyanogen is only 270 cm/s. [2] B.
L. VALLEE and M. MARGOSHES, Anal.
Chem.
28, 17.5
(1956).
r33 K. FUWA, R. E. THIERS, B. L. VALLEE and M. R. BAKER, Anal. Chem. 31, 1419 and
2039 (1959).
Cyanogen flame emission and long pathlength atomic absorption spectrometry
659
ctN,e02
to main flame J Fig. 1. Burner for oxycyanogen flame [3]. It was made at the machine shop of P. B. Brigham Hospital in Boston. Two syringe needles were used for the pilot flame, which supports the reaction of the main flame.
After a series of trials and errors, we came to the conclusion that a pilot flame, burning independently from the central main flame, was needed. The schematic diagram of oxycyanogen burner which was first made at the machine shop of the Biophysics Research Laboratory is shown in Fig. 1. Various investigations were carried out with this burner to evaluate the nature of the oxycyanogen flame as a source for emission flame photometry of metals. Interesting results were obtained such as a very low sample flow rate for optimal intensity by this flame [3]. Sensitivities for zinc and cadmium, however, were by no means satisfactory. LONG PATHLENGTH ATOMIC ABSORPTION To our disappointment, the oxycyanogen flame failed to give the required sensitivity for elements such as zinc and cadmium, the elements for which sensitive methods were needed most in our laboratory to study metalloproteins of these two elements. Although we had followed up on the early publications from the laboratory of Dr. A. WALSH, who suggested the use of atomic absorption, we now had to investigate it because the reported sensitivity for zinc, 0.3 ppm, was far better than that obtained by oxycyanogen emission. The year was now 1960, several years after the first publication of Dr. WALSH, but, as yet, suitable apparatus for atomic absorption was not available in the U.S.A. Therefore, we purchased an atomic absorption of Hilger and Watts in the U.K. The atomic absorption burner attachment of Hilger and Watts was a premixed type burner using air-acetylene. A concentric nebulizer transported sample solution into a
660
K. FUWAand B. L. VALLEE
chamber, within which the fuel gas and air with sample mist are premixed, the mixed gas penetrates through a series of small holes at the surface of the burner head and upon ignition the flame is generated. The lateral length of the flame was about 10 cm. The principle was similar to that of present-day regular atomic absorption burner, but the efficiency was quite poor, perhaps due to the design of the burner head, which had two lines of ten small holes instead of the present-day single slit. The loss of sample was over 90% of that nebulized into the chamber. Nevertheless, we obtained sensitivities of 0.3 ppm for zinc as was expected. Zinc enzymes were the substance under investigation and minute amounts of zinc had to be measured in micro amounts of purified samples. Hence, we next considered the improvement of both relative and absolute sensitivity. The total consumption burner of the oxyhydrogen emission flame was employed for zinc absorption. The concentrational sensitivity was approximately the same as that obtained from the Hilger and Watts attachment. As the sample volume required was one tenth, we had already gained an order of magnitude in absolute sensitivity. Air instead of oxygen was used as the flame temperature was not critical. The gain from the increase of the light path for atomic absorption was addressed next. The air-hydrogen flame from the Beckman burner was inserted straight up into a Vycor T-tube and the absorption was measured at the central part of the upper horizontal tube, the length of which was 20 cm. The sensitivity was again increased about ten fold. However, when the flame is inserted into the T-shape tube, the direction of the burning gas flow was altered at right angles by hitting the middle portion of the internal surface of the wall of the horizontal Vycor tube. To avoid this unnatural sudden change of the stream of the flame, the burner was tilted at a right angle and the flame was directly inserted into the horizontally held Vycor tube. The flame, being thus directed into the cell and separated from the surrounding atmosphere, was elongated inside the tube up to and beyond one meter but without too much of a temperature loss, as was readily observed by nebulizing a sodium salt solution. Since the beam from the light source of the hollow cathode could be introduced into the absorption cell by tilting the burner a little from the parallel position to the tube without changing much of the shape of the inside flame, a long cell absorption system was arranged, as is shown in Fig. 2. The Vycor absorption cell was kept inside a horizontal chimney and tested by substitution of various types of tube, with different geometries, i.e. length, inside diameter, tapered shape and so forth. A result of such tests for zinc is reproduced in Table 1. Both concentrational and absolute sensitivities were increased about a thousand fold or more [4]. The system described above, a long pathlength atomic absorption cell, proved to be useful for the trace element research in the Biophysics Laboratory. It was rather an interesting coincidence that the elements which were selected for biological study by Dr. VALLEE and his colleagues were also those which gave the best sensitivity by atomic absorption. They were zinc, cadmium and magnesium. Further studies of the long cell absorption method showed a different elongation factor inside the cell for individual elements. But here again zinc and cadmium were the elements whose determination benefited most from the elongation. The matrix effects for the method are of course a problem. As the flame which carries the sample mists must make contact with the surface of the Vycor tube, the method is sensitive to any concomitant salt. Water solutions of metalloenzymes, however, did not give rise to interference of this kind, as the major matrices in the solution or the protein, were oxidized immediately after nebulization. [4] K. FUWA and B. L. VALLEE, Anal. Chem. 35, 942 (1963); K. FUWA, Flame Absorption Spectrometry, in: Spectrochemical Methods of Analysis (edited by J. D. WINEFORDNER).Wiley, New York (1971).
Cyanogen
flame emission
Hollow Cathode
and long pathlength
atomic
absorption
spectrometry
661
Lamp
of Optical Systern APPARATUS FOR ATOMIC AFJSORPT~ON Fig. 2. Long pathlength covered with magnesium
absorption cell assembly for atomic oxide sheath and 3 mm exit orifice, shown in the insert.
absorption spectrometer [4]. Cell which gives the best sensitivity, is
As mentioned earlier, in this type of work samples are always precious and limited, and analyses have to be performed with limited amounts of material. The long pathlength atomic absorption technique, a combination of a total consumption burner with a long pathlength cell was needed for the research. The method was first announced in June 1962,, at the Xth Colloquium Spectroscopicum Internationale held at University of Maryland. When FUWA read the paper, both Dr. L. STRUCKand Dr. A. WALSH and his colleagues were in the audience. Dr. WALSH told us later that year, that since the conference at Maryland, the atomic absorption technique had become increasingly appreciated by many people and had begun to be popular. The history of spectroscopy began in 1666 by the discovery of the sun’s emission spectrum by Sir ISAACNEWTON.Knowledge has been accumulated since through the efforts of many including J. FRAUNHOFER,G. KIRCHHOFFand R. BUNSEN in both absorption and emission, to which Sir ALAN WALSH added importantly in 1955. The emphasis on absorption phenomena seems to be altering again with the rise of emission approaches recently emphasized in plasma spectrometry. Table
1. Enhancement
of sensitivity
Condition 1. Elongated burner* 2. Beckman burner a. Vertical flame b. Horizontal flame c. Asbestos cell* Diameter 1 cm d. Vycor cellt Diameter 2.6 cm e. Vycor cellt Diameter 1 cm f. Vycor cell? Diameter 1 cm MgO sheath g. Vycor cellt Diameter 1 cm MgO sheath Higher pressure * Hilger-Watts, Ltd., attachments t Length of cells is 91 cm.
for zinc analysis
by absorption
Concentration for 1% absorption, pg Zn ml-’
Zn detectable (g)
0.3
3 x lo-’
0.3 0.04 0.01
3x 1OP 4 x 1OP 1x10-9
0.006
6x 10-r’
0.0006
6x lo-”
0.0004
4 x lo-”
0.0002
2 x 10-l’
No. H909
and H1090.
cell
p&x&mica Acta. Vol.3SB.m 657to661 i PcrgmmnFY~ Ltd.1980. F?intcdin Great Britain
Cyanogen flame emission and long pathlength atomic absorption spectrometry K. FUWA Department of Chemistry, University of Tokyo, Bunkyo-ku, Tokyo, Japan
and B. L.
VALLEE
BiophysicsResearch Laboratory, Harvard Medical School and P. B. Brigham Hospital, Boston, MA, U.S.A. (Received 20 June 1980) IN THE 1940s the Spectroscopy Laboratory of Massachusetts Institute of Technology was under the direction of three scientists, G. R. HARRISON, Professor in Physics, R. C. LORD, Professor in Chemistry, and J. R. LOOFBOUROW, Professor in Biophysics. After the untimely death of Prof. LOOFBOUROW in 1951, Dr. B. VALLEE, one of the members of the biophysics laboratory moved to Harvard Medical School and started the Biophysics Research Laboratory within the building of Peter Bent Brigham Hospital, one of the Hospitals afhliated with the university. The activity of the new laboratory concerned the composition, structure and function of trace metals in biological systems. One of the major activities of the laboratory was spectrochemical analyses for trace metals in various biological materials. 1955 was the inaugural year for atomic absorption spectrometry due to A. WALSH’S publication, and at that time, two particular scientists who belonged to the spectroscopy group of the Biophysics Research Lab., that is, Dr. R. E. THIERS previously from the University of Virginia, and Dr. M. MARGOSHES from Iowa State University should be mentioned. They had been developing a cyanogen emission flame and a four-channel direct-reading flame photometer together with Dr. VALLEE. At that time, Dr. K. FUWA was instructor in the laboratory of Prof. E. MINAMI, the Department of Chemistry, University of Tokyo, and was completing a Ph.D. thesis under the title “Spectrochemical Analysis of Fluorine by Emission Spectra of Alkaline Earths Bifluorides and Its Application to Geochemistry”. Prof. MINAMI had studied geochemistry at the school of Prof. V. GOLDSCHMIDT of Gottingen University, Germany, in the 1930s. Colleagues at Giittingen included Drs. R. MANNKOPF, C. PETERS and L. W. STROCK.* The technique of cathode layer excitation was developed by these scientists and became a well-known technique of high sensitivity in the field of DC arc emission spectroscopy. When FUWA was selected as a Fulbright Exchange Scholar to the U.S. from Japan in 1955, Dr. MINAMI wrote to Dr. STROCK asking if he could work in his laboratory in the U.S.A. Dr. STROCK was then a member of the research laboratory of Sylvania Electric Co. at Long Island and suggested that he should join Dr. VALLEE'S laboratory as activities at Sylvania Co. were not confined to spectrochemical analysis. Consequently, FUWA joined the spectroscopy group of the Biophysics Research Laboratory in Boston in the spring of 1956. This was already a year after atomic absorption was suggested by Dr A. WALSH of Australia, and Dr. C. T. J. ALKEMADE in the Netherlands Cl], but we did not immediately investigate the method but continued to work in the field of flame emission spectroscopy. Flame emission work was the general trend of research in the 1950s in the U.S.A.
*Dr. Strock was from Philadelphia and was also studying geochemistry as well as spectroscopy in Goldschmidt’s laboratory. [I] A. WALSH, Spechochim. Acta. 7, 108 (1955); C. T. J. ALKEMADE 45, 583 (1955). 657
and J. M. W. MILATZ, J. Opt. Sot. Am.
658
K. FUWA
and
B. L.
VALLEE
METALLOENZYME AND CYANOGEN FLAME As was briefly described above, the Biophysics Research Laboratory was established to study the role of “trace metals” in biology, particularly enzymes. Carboxypeptidase from bovine pancreas, alcohol dehydrogenase from yeast and horse liver, and alkaline phosphatase from E. coli were the first of now more than 120 enzymes which Dr. VALEE and his colleagues quickly established to be zinc containing and dependent enzymes. In order to investigate these metalloenzymes, sensitive, accurate and yet simple detection techniques for zinc were required as only one or a few metal atoms were contained in a large protein molecule of the enzyme. The second biological material which was under investigation in the laboratory was a cadmium containing protein. Cadmium was chosen as the second metal because of its close chemical similarity to zinc. The cadmium-containing protein is concentrated in the cortex of the mammalian kidney, particularly that of the horse. This most interesting protein was named metallothionein, and attracted the interests of many people working in the toxicological and environmental fields. Dr. MARGOSHESstarted work on this cadmium protein and had to use the dithizone calorimetric method as sensitive flame emission techniques were not available. Thus, we needed also a sensitive, accurate and simple method for cadmium. The third metal of interest in the laboratory was magnesium. An accurate method was not available and the function of magnesium in human blood was unknown. Dr. W. E. C. WACKERwas investigating this field and here again a good method was required for magnesium. For decades before atomic absorption spectrometry, analytical spectroscopists were making various modifications of the three emission light sources, i.e. d.c. arc, spark and flames which had been used for nearly a hundred years since the age of KIRCHHOFFand BUNSEN. In the Biophysics Research Laboratory, the arc-like spark source with a porous cup electrode was used for both qualitative and quantitative analyses. A photographic method using a Jarrell-Ash 3.4-m Wadsworth mounting grating spectrograph together with densitometry of the lines for the internal standard process were adapted. The first survey of the sample was carried out by photography and then individual elements were determined by either direct-reading flame photometry or calorimetry. A four-channel flame photometer with automatic background correction was under development for sodium, potassium, calcium and magnesium by MARGOSHES [2]. The flame used was mainly the oxy-hydrogen flame supported on a Beckman burner. In order to determine heavy metals other than the alkali metals and alkaline earths but including zinc and cadmium, a higher temperature flame was needed. It was known then that both the cyanogen-oxygen flame and the hydrogen-fluorine flame reach approximately 5000 K when they react in the following manner C,N, + 202 = 2C02 + Na, H2 + F, = 2HF. Dr. VALLEE and his colleagues decided to investigate the cyanogen-oxygen flame [3]. The design of the burner for the system of cyanogen and oxygen was the first project FUWAwas given when he joined the laboratory in April of 1956. The principal prerequisite of the burner for a total consumption of the analytical Aame is to make a stable flame of correct size and from a mixture of the fuel and oxidizing gases (in approximately chemical equilibrium amounts) and to nebulize the sample solution pneumatically into the flame. In order to fulfil these prerequisites the flame speed or the burning velocity of the system ought to be high as pneumatic nebulization require(s) also a certain high pressure. The burning velocities of oxyhydrogen and oxyacetylene flames are 3680 cm/s and 2480 cm/s respectively, but that of oxycyanogen is only 270 cm/s. [2] B.
L. VALLEE and M. MARGOSHES, Anal.
Chem.
28, 17.5
(1956).
r33 K. FUWA, R. E. THIERS, B. L. VALLEE and M. R. BAKER, Anal. Chem. 31, 1419 and
2039 (1959).
Cyanogen flame emission and long pathlength atomic absorption spectrometry
659
ctN,e02
to main flame J Fig. 1. Burner for oxycyanogen flame [3]. It was made at the machine shop of P. B. Brigham Hospital in Boston. Two syringe needles were used for the pilot flame, which supports the reaction of the main flame.
After a series of trials and errors, we came to the conclusion that a pilot flame, burning independently from the central main flame, was needed. The schematic diagram of oxycyanogen burner which was first made at the machine shop of the Biophysics Research Laboratory is shown in Fig. 1. Various investigations were carried out with this burner to evaluate the nature of the oxycyanogen flame as a source for emission flame photometry of metals. Interesting results were obtained such as a very low sample flow rate for optimal intensity by this flame [3]. Sensitivities for zinc and cadmium, however, were by no means satisfactory. LONG PATHLENGTH ATOMIC ABSORPTION To our disappointment, the oxycyanogen flame failed to give the required sensitivity for elements such as zinc and cadmium, the elements for which sensitive methods were needed most in our laboratory to study metalloproteins of these two elements. Although we had followed up on the early publications from the laboratory of Dr. A. WALSH, who suggested the use of atomic absorption, we now had to investigate it because the reported sensitivity for zinc, 0.3 ppm, was far better than that obtained by oxycyanogen emission. The year was now 1960, several years after the first publication of Dr. WALSH, but, as yet, suitable apparatus for atomic absorption was not available in the U.S.A. Therefore, we purchased an atomic absorption of Hilger and Watts in the U.K. The atomic absorption burner attachment of Hilger and Watts was a premixed type burner using air-acetylene. A concentric nebulizer transported sample solution into a
660
K. FUWAand B. L. VALLEE
chamber, within which the fuel gas and air with sample mist are premixed, the mixed gas penetrates through a series of small holes at the surface of the burner head and upon ignition the flame is generated. The lateral length of the flame was about 10 cm. The principle was similar to that of present-day regular atomic absorption burner, but the efficiency was quite poor, perhaps due to the design of the burner head, which had two lines of ten small holes instead of the present-day single slit. The loss of sample was over 90% of that nebulized into the chamber. Nevertheless, we obtained sensitivities of 0.3 ppm for zinc as was expected. Zinc enzymes were the substance under investigation and minute amounts of zinc had to be measured in micro amounts of purified samples. Hence, we next considered the improvement of both relative and absolute sensitivity. The total consumption burner of the oxyhydrogen emission flame was employed for zinc absorption. The concentrational sensitivity was approximately the same as that obtained from the Hilger and Watts attachment. As the sample volume required was one tenth, we had already gained an order of magnitude in absolute sensitivity. Air instead of oxygen was used as the flame temperature was not critical. The gain from the increase of the light path for atomic absorption was addressed next. The air-hydrogen flame from the Beckman burner was inserted straight up into a Vycor T-tube and the absorption was measured at the central part of the upper horizontal tube, the length of which was 20 cm. The sensitivity was again increased about ten fold. However, when the flame is inserted into the T-shape tube, the direction of the burning gas flow was altered at right angles by hitting the middle portion of the internal surface of the wall of the horizontal Vycor tube. To avoid this unnatural sudden change of the stream of the flame, the burner was tilted at a right angle and the flame was directly inserted into the horizontally held Vycor tube. The flame, being thus directed into the cell and separated from the surrounding atmosphere, was elongated inside the tube up to and beyond one meter but without too much of a temperature loss, as was readily observed by nebulizing a sodium salt solution. Since the beam from the light source of the hollow cathode could be introduced into the absorption cell by tilting the burner a little from the parallel position to the tube without changing much of the shape of the inside flame, a long cell absorption system was arranged, as is shown in Fig. 2. The Vycor absorption cell was kept inside a horizontal chimney and tested by substitution of various types of tube, with different geometries, i.e. length, inside diameter, tapered shape and so forth. A result of such tests for zinc is reproduced in Table 1. Both concentrational and absolute sensitivities were increased about a thousand fold or more [4]. The system described above, a long pathlength atomic absorption cell, proved to be useful for the trace element research in the Biophysics Laboratory. It was rather an interesting coincidence that the elements which were selected for biological study by Dr. VALLEE and his colleagues were also those which gave the best sensitivity by atomic absorption. They were zinc, cadmium and magnesium. Further studies of the long cell absorption method showed a different elongation factor inside the cell for individual elements. But here again zinc and cadmium were the elements whose determination benefited most from the elongation. The matrix effects for the method are of course a problem. As the flame which carries the sample mists must make contact with the surface of the Vycor tube, the method is sensitive to any concomitant salt. Water solutions of metalloenzymes, however, did not give rise to interference of this kind, as the major matrices in the solution or the protein, were oxidized immediately after nebulization. [4] K. FUWA and B. L. VALLEE, Anal. Chem. 35, 942 (1963); K. FUWA, Flame Absorption Spectrometry, in: Spectrochemical Methods of Analysis (edited by J. D. WINEFORDNER).Wiley, New York (1971).
Cyanogen
flame emission
Hollow Cathode
and long pathlength
atomic
absorption
spectrometry
661
Lamp
of Optical Systern APPARATUS FOR ATOMIC AFJSORPT~ON Fig. 2. Long pathlength covered with magnesium
absorption cell assembly for atomic oxide sheath and 3 mm exit orifice, shown in the insert.
absorption spectrometer [4]. Cell which gives the best sensitivity, is
As mentioned earlier, in this type of work samples are always precious and limited, and analyses have to be performed with limited amounts of material. The long pathlength atomic absorption technique, a combination of a total consumption burner with a long pathlength cell was needed for the research. The method was first announced in June 1962,, at the Xth Colloquium Spectroscopicum Internationale held at University of Maryland. When FUWA read the paper, both Dr. L. STRUCKand Dr. A. WALSH and his colleagues were in the audience. Dr. WALSH told us later that year, that since the conference at Maryland, the atomic absorption technique had become increasingly appreciated by many people and had begun to be popular. The history of spectroscopy began in 1666 by the discovery of the sun’s emission spectrum by Sir ISAACNEWTON.Knowledge has been accumulated since through the efforts of many including J. FRAUNHOFER,G. KIRCHHOFFand R. BUNSEN in both absorption and emission, to which Sir ALAN WALSH added importantly in 1955. The emphasis on absorption phenomena seems to be altering again with the rise of emission approaches recently emphasized in plasma spectrometry. Table
1. Enhancement
of sensitivity
Condition 1. Elongated burner* 2. Beckman burner a. Vertical flame b. Horizontal flame c. Asbestos cell* Diameter 1 cm d. Vycor cellt Diameter 2.6 cm e. Vycor cellt Diameter 1 cm f. Vycor cell? Diameter 1 cm MgO sheath g. Vycor cellt Diameter 1 cm MgO sheath Higher pressure * Hilger-Watts, Ltd., attachments t Length of cells is 91 cm.
for zinc analysis
by absorption
Concentration for 1% absorption, pg Zn ml-’
Zn detectable (g)
0.3
3 x lo-’
0.3 0.04 0.01
3x 1OP 4 x 1OP 1x10-9
0.006
6x 10-r’
0.0006
6x lo-”
0.0004
4 x lo-”
0.0002
2 x 10-l’
No. H909
and H1090.
cell