Determination of Lead, Cadmium, and Zinc Using the Zeeman Effect in Atomic Absorption Spectrometry Hideaki Kolzumi" and Kazuo Yasuda Naka Works Hitachi Ltd., Katsuta, Ibaraki, Japan
A new method of atomic absorption spectrometry using the Zeeman effect was applied to the analyses of lead, cadmium, and zinc. The polarized lights of the Zeeman splitting emission line, i.e., the x and the u components, were used for a measuring and for a reference light, respectively, to correct the background absorptlon. The error due to the background absorption is completely elimlnated in the sample of background absorbance less than 1.5. Lead in blood and liver, cadmlum In urlne were determined within 30 s by directly Introducing the samples into a carbon tube atomizer without any chemical treatment, drying, or ashing processes.
dicular to the field when they are observed transverse to the magnetic field. Since the emission line from the light source in the magnetic field can be separated into each Zeeman component such as a and cr by a rotating linear polarizer, it is possible to use the 7r component as a measuring beam and the (r component as a reference beam to correct the molecular absorption and scatterings, as shown in Figure 2. An absorption line of an atom in a carbon tube atomizer is broadened by the Doppler and the Lorentz effects. The half intensity widths of the Doppler and the Lorentz broadening are shown by the following equations, respectively ( 4 ) .
AVD= Accurate and rapid determination of trace elements in food or human organs is acquiring more importance day by day for the detection of environmental pollution. It is highly desirable to develop a new method which gives accurate and rapid determination of these elements easily. Although the sensitivity of the flameless sampling method in atomic absorption spectrometry is high, chemical pretreatment is a necessity. As a result, the process of analysis becomes very long and tedious, and considerable error could be introduced from various sources of contamination and loss. In the previous papers (1,2),we reported a new method of atomic absorption spectrometry of mercury using the Zeeman effect. The method is useful for the determination of trace elements in biological and botanical samples, sea water, and soil. The measurement can be performed without chemical pretreatment in this method. In the present article, the Zeeman technique of atomic absorption spectrometry applied to lead, cadmium, and zinc is described and the results of analyses of biological samples are reported. Figure 1shows the Zeeman patterns of the resonance lines of lead, cadmium, zinc, and mercury. The shift in atomic energy level is given as follows ( 3 ) . - A T M = MgLH Here, A T M is the energy in cm-l; H , magnetic induction in gauss; M , the magnetic quantum number; L and g are the Lorentz unit and Land6 factor, respectively, defined by the following equations.
L=- e g=l+
J(J +
47rmc2 1) S(S 1)- L ( L 2J(J+ 1)
+
+
(2)
+ 1)
(3)
Here, e is the electronic charge; m , electronic mass; c, velocity of light, S, L, and J are the quantum numbers of spin, orbital, and total angular momentum, respectively. The lines due to the transitions of AM = 0 and AM = f l are called a and crh components, respectively. The sum of the intensities of the TC components is equal to that of the u components. The pattern A in Figure 1 shows the normal Zeeman triplet. The patterns B and C also consist of three lines, but the magnitude of the shift is different from that of the normal Zeeman effect. The a component is linearly polarized parallel to the magnetic field, and the cr components are linearly polarized perpen1178
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
2v3R-G-2 C
(4)
AUL= 1.95 x 1019 q(rL2
Here, R is the gas constant, VO, frequency at line center; M , atomic weight; T , absolute temperature; q , partial pressure of perturbers, c r ~ effective ~ , cross section of Lorentz broadening; Mp, atomic or molecular weight of perturbers. The total half intensity width is ( 5 )
The strength of the magnetic field applied to the light source must be large enough to deviate the cr components from the broadened absorption line.
EXPERIMENTAL Apparatus. The schematic diagram of the experimental arrangement is shown in Figure 3. Electrodeless discharge lamps of cadmium, zinc, and lead constructed in our laboratory were operated at high frequency power of 2 W a t 100 MHz for use as light sources. The cadmium, zinc, and lead lamps were heated by a small oven at 230, 250, and 280 "C, respectively, to keep the vapor pressure of the elements a t about Torr. Argon gas was filled a t 2 Torr in the tube as buffer gas. These light sources including the oven were placed between the pole pieces of a small permanent magnet which could produce a magnetic field of 10 kgauss. The emission lines corresponding to the r a n d the u components were picked up by the rotating linear polarizer (Polacoat 105 UVW) fixed in the shaft of a synchronous motor, and were detected alternately. The sampling interval of these measuring and reference lights was 10 ms. The conventional atomic absorption spectrophotometer (Hitachi Model 308) was used to select an absorption line. The resonance line of each element used for the measurement was 2833 %, for lead, 2288 8, for cadmium, and 2138 %, for zinc. The signal of the photomultiplier (Hamamatsu TV-R106) was transformed into a logarithm, and the difference between the signal of the measuring and the reference lights was amplified by a lock-in amplifier. The high voltage applied to this photomultiplier was automatically controlled so that the signal of the reference light was kept at a constant level. The output signal, in which the background absorption was corrected, was recorded by a recorder with response time of 0.5 s/full scale. Each absorption of the measuring and the reference light was also observed alternately by a high speed recorder with response time of 300 ps/full scale. The high speed recorder was directly connected with the photomultiplier by which the signal intensities of 7 and u components were observed alternately. A modified Massmann type carbon tube atomizer was used for the determination of lead, cadmium, and zinc; and the shape of the carbon tube was slightly changed so as to give efficient atomization. Its central part, 35 mm (full length 60 mm), was kept a t a constant temperature
Element
Wavelength [
2022.02
Pb
I
Term
t
Magnet ( 1 0 kgauss)
Zeeman pattern
Monochromator Lamp compensater (Pb,Cd,Zn)
Rotating linear polarizer
Photomultiplier
3Po-1P1
2053.27
3P0..3Pl
2169.99
3P0-3D1
2833.06
3Po-3P1 1S0-lP1 1 s -3P 0 1 1S0-lP1
ls -3P 0 1 ls -1P 0 1 1So-3P1
Figure 3. Schematic diagram of the experimental arrangement
Figure 1. Zeeman patterns of the resonance lines
'08Pb 52.3 %
Zeernan shifted
emission line Atomic absorption line
Lamp 206Pb
-
23.6
207Pb [a) 15.1 %
I
-10.35
% 207Pb (b)
'04Pb
7.5 %
1.5 % T
-4.59
-2.43
0
T 2.94
Frequency [ GHz )
linear polarizer
Figure 4. Hyperfine structure of the resonance line of lead at 2833 A (3P0-3P1)
Figure 2. Principle of Zeeman atomic absorption spectrometry
of about 1800 OC by cutting both ends thin. Owing to large resistances, these cut ditches show higher temperaturesthan the central part and the temperature of the central part is almost constant, unlike the case of the conventional Massmann furnace ( 6 ) .A sample holder was used to make accurate positioning of milligram-levelsolid sample in the carbon tube. Eppendorff's micropipets were used for loading the liquid samples. Argon gas flowed through the tube at the rate of 1 l./min. As the carbon tube had a considerableheat capacity,its temperature rose gradually with rise time of about 5 s when an electric current was supplied to it. In the present experiment,no drying or ashing process was used, except for the case where conventional stepwise heating was made to compare with the present method. Reagents. Standard solutions of lead, cadmium,and zinc in 0.1 N "03 were prepared just before measurementsfrom stock solutions of 1000 ppm in 1 N " 0 3 . Sea water containing 3.4%of salts was obtained at Ohoarai beach. Blood and urine were taken from one of the authors, and sodium citrate was added to the blood at 2 mg/ml as anticoagulant.Special grade reagents were employed throughout this experiment, and lead and cadmium impurities in the reagents were checked before the analysis. NBS Standard SRM-1577 (Bovine Liver) was dried for 48 h in a desiccator and was weighed. RESULTS AND DISCUSSION
T o choose a suitable magnetic field strength for the light source, the emission line profile a t a certain magnetic field and the absorption line profile were calculated. In the calculation, the following conditions were assumed: the gas temperature in the absorption cell is 2100 K, and the effective cross section of the Lorentz broadening is 60 X The half-intensity widths AUT obtained by Equations 1-3 were 5.5 GHz for the cadmium line a t 2288 8, and 3.9 GHz for the lead line a t 2833 8,.The damping constant in the Voigt function ( 4 ) is 0.54 for the cadmium line and 0.84 for the lead line. The Doppler widths of emission lines were 1.33 GHz for cadmium and 0.98 GHz for lead, and the Lorentz widths of the emission lines were negligibly small in the argon gas a t 2 Torr. Then, the absorption sensitivity is reduced to about 50of its peak value when the frequency of an absorbing light shifts by 1.5 AUT from the center of the absorption line. The shift of the u components are calculated from Equation 4 to be 1.4 H GHz for cadmium and 2.0 H GHz for lead, respectively, when the magnetic field H is expressed in kgauss. Then a shift of 1.5 AUT
GHz for the u components of the cadmium line obtained at the field strength of 5.9 kgauss. The hyperfine structure of the cadmium line a t 2288 8,is negligibly small (7), but that of the lead line a t 2833 8, is considerably large as shown in Figure 4 (8).The over-all absorption line profile consists of two lines, i.e., the 207Pb(a)line with half intensity width of 3.9 GHz and the line of the other isotopic components with half intensity width of 4.8 GHz. The frequency difference between these lines is about 10 GHz. Then, the applied magnetic field to the light source must be larger than about 7 kgauss to separate the u components from the absorption line. We applied a magnetic field of 10 kgauss to the cadmium and lead light sources, because a field strength of 10 kgauss is easily obtainable from a small permanent magnet, and it is large enough to shift the u components out of the absorption line. Figure 5 shows the absorption signals of P and u components alternately recorded with the rotation of the linear polarizer. The upper and the lower points of track correspond to the absorption of a and u, respectively. Figure 5(A) shows that the absorptions of a and u of the lead resonance line a t 2833 8, changes with time when the 0.1 N "03 solution containing 5 ng of lead was atomized in the carbon tube. The absorption signals of a and u of the cadmium resonance line a t 2288 8, obtained by the 0.1 N "03 solution containing 150 pg of cadmium were also shown in Figure 5(B). The atomic vapor absorbed only the a component, because its wavelength does not shift by the magnetic field and is coincident with the absorption line. The scattering and molecular absorptions, which is called the background absorption, decrease the intensity of both the a and the u components equally, since the molecular absorption spectra are much broader than the absorption line of the atom. The weak absorption of the u components in Figure 5 is partly due to the background absorption, and partly due to the wings of the broadened absorption line of cadmium or lead. The signals of a and u are transformed into logarithms and the difference of these signals, which corresponds to true atomic absorption, is obtained. The reduction in the sensitivity of analysis caused by the wing absorption is negligible as described in our previous paper ( 2 ) . ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
1179
I
.
9 I
sample : sea water Wavelength : 2833 A
+
0.2
ppm
+
Pb
lopL
0.1 ppm Direct recording
Pb
m
p
0.
u
M B
B
-
0.
0
e2 0.
l
Corrected signal 10
5
Time ( min )
0
Figure 6. Determination of lead added to the sea water (The upper and the lower tracks show uncorrectedand corrected signals, respectively.)
Flgure 5. Time dependence of the absorption of lead and cadmium in 0.1 N HN03 observed by the T and the u components of the emission line. (A) lead, (e) cadmium
The accuracy of the background correction by this method was compared with the conventional method using a Dz continuum source. In the case of the Dz lamp correction method, it is very important to superpose precisely the two light beams from two different light sources. If the alignment is not perfect, over- or underestimation of the background absorption is made when the decomposed materials are distributed inhomogeneously in the absorption cell. In the analysis using a carbon tube or metal filament atomizer, it gives a serious error sometimes because of the temporal and spatial inhomogeneities of atomic densities in the cell. Furthermore, the intensities of lights from the different kinds of lamps are not all the same on each point of the cross section of the light beams. In our experiment of D2 lamp correction with a Massmann type carbon tube atomizer, a background of absorbance 1.0 was corrected to 0.05 of absorbance when the alignment was carefully fixed and the optimum electronics condition was used. However, when the alignment was slightly off, the correction became incomplete and 10 to 20% of the background absorption remained in the corrected signal. On the other hand, since only a single light source was used in the present method, optical alignment poses no problem. Even if a background absorption reached 1.7 of absorbance unit (2% of transmittance), our corrected signal had only the error less than 0.002 in absorbance unit. This error was attributed to the electronics. No absorption signal was detected above the noise level in the corrected signal when a background absorption was less than 1.0 of absorbance. Figure 6 shows an example 1180
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
of the background correction by the present method. The upper track shows an uncorrected signal in a transmittance scale and the lower track shows the corrected signal in an absorbance scale, when sea water of 10 11.1containing additive lead was introduced into the carbon tube atomizer and atomized directly without drying and ashing processes. The corrected absorption signal of lead added in the sea water could be observed without any interference of the strong background absorption, although it was impossible to recognize the resonance absorption peak of lead in the uncorrected signals. The detection limits were 0.1 ppb ( 5 pg) for cadmium at 2288 A, and 2 ppb (100 pg) for lead at 2833 A. The detection limit was defined as the concentration of the element in aqueous solution of 50 11.1which gave a signal intensity as large as twice the standard deviation in signal near the lower limit of the analytical curve. Almost the same detection limit in pg could be obtained in the analysis of actual samples, such as milk, urine, serum, etc., under the condition where 10 mg of samples were used for one analysis. After urine was introduced into the atomizer, time dependence of the absorption of T and u components of the cadmium line at 2288 A was observed as shown in Figure 7(A). At first, volatile materials such as organic compounds give rise to strong background absorption and, after that, the resonance absorption due to the cadmium, less volatile material, added to the urine is observed with the rise of temperature in the carbon tube. The absorption of cadmium overlaps with the second strong background absorption which is attributed to nonvolatile materials such as salts. Therefore, it is impossible to analyze cadmium in urine by the conventional ashing method accurately (91, because the cadmium signal cannot be separated from the background absorption. The selective atomization method, which is performed to separate the atomic absorption from background absorption by using fractional distillation, is not applicable to the cadmium determination. The same is true for the determination of comparatively volatile elements such as lead, zinc, and mercury in urine. Figure 7(B) shows a temporal behavior of absorption of the T and the u components of the lead line at 2833 8, when blood was introduced into the carbon tube atomizer, demonstrating the difficulty in separation of the lead resonance absorption from its background. We could not accurately determine the
Figure 7. (A). Time dependence of absorption of cadmium added to urine
Figure 7 (B). Time depencence of absorption of lead added to blood
Figure 8. Determinationof cadmium in urine by Zeeman AAS
The upper and the lower points of tracks correspond to the absorption of ?r and d components of the emission line, respectively
lead in blood by the conventional ashing method with a Dz lamp correction, because when the ashing temperature was slightly low, enormous background absorption made it impossible to perform any accurate correction. When the ashing temperature was slightly increased to eliminate the coexistent materials, a considerable amount of lead was easily lost in the ashing process. However, by the present method of Zeeman atomic absorption, we could directly determine cadmium in urine and lead in blood without any ashing process. Quantitative analysis for cadmium in urine was performed by standard addition method. The corrected absorption signals and the analytical curve are shown in Figure 8. We obtained a result of 1.85 ppb cadmium content in the urine. Figure 9 shows the result of lead determination in blood. Sodium citrate was added to the blood by 2 mg/ml as anticoagulant, and the samples of blood were diluted into 20% solution with standard solutions of lead. Lead in this solution was determined directly in the same way as the analysis for cadmium in urine. Although the lead background of the sodium citrate was 0.20 ppm, it was found that the blood sample contained 0.10 ppm of lead.
Figure 9. Determination of lead in blood by Zeeman AAS ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
1181
of SRM-1577 are N, 10.6%;K, 0.97%; Na, 0.243%; and the main trace constituents are Fe, 270 ppm; Cu, 193 ppm; and Zn, 130 ppm. When this sample was analyzed by a working curve which is made by a lead solution of 0.1 N “03, the observed result was 0.342 f 0.07 ppm. The result agreed precisely with the NBS value of 0.34 & 0.14 ppm. Thus, it shows that accurate results can be obtained by the present method even if the standard additions method is not applied. The heated area of the carbon tube must be sufficiently large to prolong the residence time so as to decompose the sample completely. If only a small portion of the central part of the tube was heated to high temperature, the good reproducibility and the high sensitivity were not obtained in the case of a solid sample. The temperature distribution of the carbon tube was important to perform an accurate analysis. We did not observe any noticeable chemical interference in all the samples analyzed. The determination of zinc by the present method was also performed, and good results as well as those of cadmium and lead were obtained under the same conditions as in the case of cadmium. Figure 10. Determination of lead in the standard reference material of NBS (SRM-1577, Bovine Liver) by Zeeman AAS (a) Weight, mg Pb, ppm (1) 1.42 (2)2.12 (3)1.65 (4)1.73 (5)1.85
0.370 0.307 0.340 0.356 0.341
(b) Result: 0.343f 0.023ppm. Certified value of NBS: 0.34f 0.08 ppm
The biological standard reference material of NBS was analyzed to confirm the accuracy of this method. Figure 10 shows the results and the procedure when the lead in the SRM-1577 Bovine Liver was determined. Dried and weighed samples of 1to 3 mg were positioned exactly in the center of the carbon tube by the sample holder. The major constituents
ACKNOWLEDGMENT
We thank T. Hadeiahi of the University of California and M. Katayama of the University of Tokyo for their useful suggestions. Thanks are also due to K. Ohishi and K. Uchino of Hitachi Ltd. for their great help. LITERATURE CITED (1) H. Koizumi and K. Uchino, HitachiHyoron, 56, 1037 (1974). (2) H. Koizumi and K. Yasuda, Anal. Chem., 47, 1679 (1975). (3) H. E. White, “Introduction to Atomic Spectra”, McGraw-Hill, New York, 1934. (4) A. C. G. Mitchell and M. W. Zemansky, “Resonance Radiation and Excited
Atoms”, Cambridge, 1934. (5) E. E. Whiting, J. Quant. Spectrosc. Radiat. Transfer, 8, 1379 (1968). (6) H. Massmann, Spectrochim. Acta, Parts, 23, 215 (1968). (7) A. G. Shenstone, Phys. Rev., 47, 317 (1935). (8) T. E. Manning, Phys. Rev., 78, 417 (1950). (9) J. P. Matousek, Am. Lab., No. 6, 45 (1971).
RECEIVEDfor review January 19,1976. Accepted March 15, 1976.
FIuorometric Determination of Chlordiazepoxide in Dosage Forms and Biological Fluids with Fluorescamine James T. Stewart” and Jonathan L. Williamson School of Pharmacy, University of Georgia, Athens, Ga. 30602
A fluorometrlc procedure for chlordiazepoxide is reported based upon a fluorophor formed with fluorescamine after acid hydrolysis of the drug. A study of optlmum pH, hydrolysis time, and fluorescamineconcentrationis presented. Fluorescence is linear over the range 0.25-6 pg chlordlazepoxlde hydrochloride per ml of sample. The method is free from interference from chlordiazepoxide’s major metabolites as well as other drugs such as clldinlum bromide, oxazepam, and dextropropoxyphene hydrochlorlde. The procedure Is subject to interference from other amlne-containingdrugs such as amphetamines that might be present In analytical samples. Applicatlon of the procedure to the analysis of chlordiazepoxide in dosage forms, urlne, and plasma gave an accuracy of 1-3 %. 1182
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
Chlordiazepoxide (I), a 1,4-benzodiazepine derivative, has been widely used therapeutically because of its muscle relaxant, taming, sedative, anti-anxiety and anticonvulsant properties (1-3).
NHCH,
I
A new method of atomic absorption spectrometry using the Zeeman effect was applied to the analyses of lead, cadmium, and zinc. The polarized lights of the Zeeman splitting emission line, i.e., the x and the u components, were used for a measuring and for a reference light, respectively, to correct the background absorptlon. The error due to the background absorption is completely elimlnated in the sample of background absorbance less than 1.5. Lead in blood and liver, cadmlum In urlne were determined within 30 s by directly Introducing the samples into a carbon tube atomizer without any chemical treatment, drying, or ashing processes.
dicular to the field when they are observed transverse to the magnetic field. Since the emission line from the light source in the magnetic field can be separated into each Zeeman component such as a and cr by a rotating linear polarizer, it is possible to use the 7r component as a measuring beam and the (r component as a reference beam to correct the molecular absorption and scatterings, as shown in Figure 2. An absorption line of an atom in a carbon tube atomizer is broadened by the Doppler and the Lorentz effects. The half intensity widths of the Doppler and the Lorentz broadening are shown by the following equations, respectively ( 4 ) .
AVD= Accurate and rapid determination of trace elements in food or human organs is acquiring more importance day by day for the detection of environmental pollution. It is highly desirable to develop a new method which gives accurate and rapid determination of these elements easily. Although the sensitivity of the flameless sampling method in atomic absorption spectrometry is high, chemical pretreatment is a necessity. As a result, the process of analysis becomes very long and tedious, and considerable error could be introduced from various sources of contamination and loss. In the previous papers (1,2),we reported a new method of atomic absorption spectrometry of mercury using the Zeeman effect. The method is useful for the determination of trace elements in biological and botanical samples, sea water, and soil. The measurement can be performed without chemical pretreatment in this method. In the present article, the Zeeman technique of atomic absorption spectrometry applied to lead, cadmium, and zinc is described and the results of analyses of biological samples are reported. Figure 1shows the Zeeman patterns of the resonance lines of lead, cadmium, zinc, and mercury. The shift in atomic energy level is given as follows ( 3 ) . - A T M = MgLH Here, A T M is the energy in cm-l; H , magnetic induction in gauss; M , the magnetic quantum number; L and g are the Lorentz unit and Land6 factor, respectively, defined by the following equations.
L=- e g=l+
J(J +
47rmc2 1) S(S 1)- L ( L 2J(J+ 1)
+
+
(2)
+ 1)
(3)
Here, e is the electronic charge; m , electronic mass; c, velocity of light, S, L, and J are the quantum numbers of spin, orbital, and total angular momentum, respectively. The lines due to the transitions of AM = 0 and AM = f l are called a and crh components, respectively. The sum of the intensities of the TC components is equal to that of the u components. The pattern A in Figure 1 shows the normal Zeeman triplet. The patterns B and C also consist of three lines, but the magnitude of the shift is different from that of the normal Zeeman effect. The a component is linearly polarized parallel to the magnetic field, and the cr components are linearly polarized perpen1178
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
2v3R-G-2 C
(4)
AUL= 1.95 x 1019 q(rL2
Here, R is the gas constant, VO, frequency at line center; M , atomic weight; T , absolute temperature; q , partial pressure of perturbers, c r ~ effective ~ , cross section of Lorentz broadening; Mp, atomic or molecular weight of perturbers. The total half intensity width is ( 5 )
The strength of the magnetic field applied to the light source must be large enough to deviate the cr components from the broadened absorption line.
EXPERIMENTAL Apparatus. The schematic diagram of the experimental arrangement is shown in Figure 3. Electrodeless discharge lamps of cadmium, zinc, and lead constructed in our laboratory were operated at high frequency power of 2 W a t 100 MHz for use as light sources. The cadmium, zinc, and lead lamps were heated by a small oven at 230, 250, and 280 "C, respectively, to keep the vapor pressure of the elements a t about Torr. Argon gas was filled a t 2 Torr in the tube as buffer gas. These light sources including the oven were placed between the pole pieces of a small permanent magnet which could produce a magnetic field of 10 kgauss. The emission lines corresponding to the r a n d the u components were picked up by the rotating linear polarizer (Polacoat 105 UVW) fixed in the shaft of a synchronous motor, and were detected alternately. The sampling interval of these measuring and reference lights was 10 ms. The conventional atomic absorption spectrophotometer (Hitachi Model 308) was used to select an absorption line. The resonance line of each element used for the measurement was 2833 %, for lead, 2288 8, for cadmium, and 2138 %, for zinc. The signal of the photomultiplier (Hamamatsu TV-R106) was transformed into a logarithm, and the difference between the signal of the measuring and the reference lights was amplified by a lock-in amplifier. The high voltage applied to this photomultiplier was automatically controlled so that the signal of the reference light was kept at a constant level. The output signal, in which the background absorption was corrected, was recorded by a recorder with response time of 0.5 s/full scale. Each absorption of the measuring and the reference light was also observed alternately by a high speed recorder with response time of 300 ps/full scale. The high speed recorder was directly connected with the photomultiplier by which the signal intensities of 7 and u components were observed alternately. A modified Massmann type carbon tube atomizer was used for the determination of lead, cadmium, and zinc; and the shape of the carbon tube was slightly changed so as to give efficient atomization. Its central part, 35 mm (full length 60 mm), was kept a t a constant temperature
Element
Wavelength [
2022.02
Pb
I
Term
t
Magnet ( 1 0 kgauss)
Zeeman pattern
Monochromator Lamp compensater (Pb,Cd,Zn)
Rotating linear polarizer
Photomultiplier
3Po-1P1
2053.27
3P0..3Pl
2169.99
3P0-3D1
2833.06
3Po-3P1 1S0-lP1 1 s -3P 0 1 1S0-lP1
ls -3P 0 1 ls -1P 0 1 1So-3P1
Figure 3. Schematic diagram of the experimental arrangement
Figure 1. Zeeman patterns of the resonance lines
'08Pb 52.3 %
Zeernan shifted
emission line Atomic absorption line
Lamp 206Pb
-
23.6
207Pb [a) 15.1 %
I
-10.35
% 207Pb (b)
'04Pb
7.5 %
1.5 % T
-4.59
-2.43
0
T 2.94
Frequency [ GHz )
linear polarizer
Figure 4. Hyperfine structure of the resonance line of lead at 2833 A (3P0-3P1)
Figure 2. Principle of Zeeman atomic absorption spectrometry
of about 1800 OC by cutting both ends thin. Owing to large resistances, these cut ditches show higher temperaturesthan the central part and the temperature of the central part is almost constant, unlike the case of the conventional Massmann furnace ( 6 ) .A sample holder was used to make accurate positioning of milligram-levelsolid sample in the carbon tube. Eppendorff's micropipets were used for loading the liquid samples. Argon gas flowed through the tube at the rate of 1 l./min. As the carbon tube had a considerableheat capacity,its temperature rose gradually with rise time of about 5 s when an electric current was supplied to it. In the present experiment,no drying or ashing process was used, except for the case where conventional stepwise heating was made to compare with the present method. Reagents. Standard solutions of lead, cadmium,and zinc in 0.1 N "03 were prepared just before measurementsfrom stock solutions of 1000 ppm in 1 N " 0 3 . Sea water containing 3.4%of salts was obtained at Ohoarai beach. Blood and urine were taken from one of the authors, and sodium citrate was added to the blood at 2 mg/ml as anticoagulant.Special grade reagents were employed throughout this experiment, and lead and cadmium impurities in the reagents were checked before the analysis. NBS Standard SRM-1577 (Bovine Liver) was dried for 48 h in a desiccator and was weighed. RESULTS AND DISCUSSION
T o choose a suitable magnetic field strength for the light source, the emission line profile a t a certain magnetic field and the absorption line profile were calculated. In the calculation, the following conditions were assumed: the gas temperature in the absorption cell is 2100 K, and the effective cross section of the Lorentz broadening is 60 X The half-intensity widths AUT obtained by Equations 1-3 were 5.5 GHz for the cadmium line a t 2288 8, and 3.9 GHz for the lead line a t 2833 8,.The damping constant in the Voigt function ( 4 ) is 0.54 for the cadmium line and 0.84 for the lead line. The Doppler widths of emission lines were 1.33 GHz for cadmium and 0.98 GHz for lead, and the Lorentz widths of the emission lines were negligibly small in the argon gas a t 2 Torr. Then, the absorption sensitivity is reduced to about 50of its peak value when the frequency of an absorbing light shifts by 1.5 AUT from the center of the absorption line. The shift of the u components are calculated from Equation 4 to be 1.4 H GHz for cadmium and 2.0 H GHz for lead, respectively, when the magnetic field H is expressed in kgauss. Then a shift of 1.5 AUT
GHz for the u components of the cadmium line obtained at the field strength of 5.9 kgauss. The hyperfine structure of the cadmium line a t 2288 8,is negligibly small (7), but that of the lead line a t 2833 8, is considerably large as shown in Figure 4 (8).The over-all absorption line profile consists of two lines, i.e., the 207Pb(a)line with half intensity width of 3.9 GHz and the line of the other isotopic components with half intensity width of 4.8 GHz. The frequency difference between these lines is about 10 GHz. Then, the applied magnetic field to the light source must be larger than about 7 kgauss to separate the u components from the absorption line. We applied a magnetic field of 10 kgauss to the cadmium and lead light sources, because a field strength of 10 kgauss is easily obtainable from a small permanent magnet, and it is large enough to shift the u components out of the absorption line. Figure 5 shows the absorption signals of P and u components alternately recorded with the rotation of the linear polarizer. The upper and the lower points of track correspond to the absorption of a and u, respectively. Figure 5(A) shows that the absorptions of a and u of the lead resonance line a t 2833 8, changes with time when the 0.1 N "03 solution containing 5 ng of lead was atomized in the carbon tube. The absorption signals of a and u of the cadmium resonance line a t 2288 8, obtained by the 0.1 N "03 solution containing 150 pg of cadmium were also shown in Figure 5(B). The atomic vapor absorbed only the a component, because its wavelength does not shift by the magnetic field and is coincident with the absorption line. The scattering and molecular absorptions, which is called the background absorption, decrease the intensity of both the a and the u components equally, since the molecular absorption spectra are much broader than the absorption line of the atom. The weak absorption of the u components in Figure 5 is partly due to the background absorption, and partly due to the wings of the broadened absorption line of cadmium or lead. The signals of a and u are transformed into logarithms and the difference of these signals, which corresponds to true atomic absorption, is obtained. The reduction in the sensitivity of analysis caused by the wing absorption is negligible as described in our previous paper ( 2 ) . ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
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I
.
9 I
sample : sea water Wavelength : 2833 A
+
0.2
ppm
+
Pb
lopL
0.1 ppm Direct recording
Pb
m
p
0.
u
M B
B
-
0.
0
e2 0.
l
Corrected signal 10
5
Time ( min )
0
Figure 6. Determination of lead added to the sea water (The upper and the lower tracks show uncorrectedand corrected signals, respectively.)
Flgure 5. Time dependence of the absorption of lead and cadmium in 0.1 N HN03 observed by the T and the u components of the emission line. (A) lead, (e) cadmium
The accuracy of the background correction by this method was compared with the conventional method using a Dz continuum source. In the case of the Dz lamp correction method, it is very important to superpose precisely the two light beams from two different light sources. If the alignment is not perfect, over- or underestimation of the background absorption is made when the decomposed materials are distributed inhomogeneously in the absorption cell. In the analysis using a carbon tube or metal filament atomizer, it gives a serious error sometimes because of the temporal and spatial inhomogeneities of atomic densities in the cell. Furthermore, the intensities of lights from the different kinds of lamps are not all the same on each point of the cross section of the light beams. In our experiment of D2 lamp correction with a Massmann type carbon tube atomizer, a background of absorbance 1.0 was corrected to 0.05 of absorbance when the alignment was carefully fixed and the optimum electronics condition was used. However, when the alignment was slightly off, the correction became incomplete and 10 to 20% of the background absorption remained in the corrected signal. On the other hand, since only a single light source was used in the present method, optical alignment poses no problem. Even if a background absorption reached 1.7 of absorbance unit (2% of transmittance), our corrected signal had only the error less than 0.002 in absorbance unit. This error was attributed to the electronics. No absorption signal was detected above the noise level in the corrected signal when a background absorption was less than 1.0 of absorbance. Figure 6 shows an example 1180
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
of the background correction by the present method. The upper track shows an uncorrected signal in a transmittance scale and the lower track shows the corrected signal in an absorbance scale, when sea water of 10 11.1containing additive lead was introduced into the carbon tube atomizer and atomized directly without drying and ashing processes. The corrected absorption signal of lead added in the sea water could be observed without any interference of the strong background absorption, although it was impossible to recognize the resonance absorption peak of lead in the uncorrected signals. The detection limits were 0.1 ppb ( 5 pg) for cadmium at 2288 A, and 2 ppb (100 pg) for lead at 2833 A. The detection limit was defined as the concentration of the element in aqueous solution of 50 11.1which gave a signal intensity as large as twice the standard deviation in signal near the lower limit of the analytical curve. Almost the same detection limit in pg could be obtained in the analysis of actual samples, such as milk, urine, serum, etc., under the condition where 10 mg of samples were used for one analysis. After urine was introduced into the atomizer, time dependence of the absorption of T and u components of the cadmium line at 2288 A was observed as shown in Figure 7(A). At first, volatile materials such as organic compounds give rise to strong background absorption and, after that, the resonance absorption due to the cadmium, less volatile material, added to the urine is observed with the rise of temperature in the carbon tube. The absorption of cadmium overlaps with the second strong background absorption which is attributed to nonvolatile materials such as salts. Therefore, it is impossible to analyze cadmium in urine by the conventional ashing method accurately (91, because the cadmium signal cannot be separated from the background absorption. The selective atomization method, which is performed to separate the atomic absorption from background absorption by using fractional distillation, is not applicable to the cadmium determination. The same is true for the determination of comparatively volatile elements such as lead, zinc, and mercury in urine. Figure 7(B) shows a temporal behavior of absorption of the T and the u components of the lead line at 2833 8, when blood was introduced into the carbon tube atomizer, demonstrating the difficulty in separation of the lead resonance absorption from its background. We could not accurately determine the
Figure 7. (A). Time dependence of absorption of cadmium added to urine
Figure 7 (B). Time depencence of absorption of lead added to blood
Figure 8. Determinationof cadmium in urine by Zeeman AAS
The upper and the lower points of tracks correspond to the absorption of ?r and d components of the emission line, respectively
lead in blood by the conventional ashing method with a Dz lamp correction, because when the ashing temperature was slightly low, enormous background absorption made it impossible to perform any accurate correction. When the ashing temperature was slightly increased to eliminate the coexistent materials, a considerable amount of lead was easily lost in the ashing process. However, by the present method of Zeeman atomic absorption, we could directly determine cadmium in urine and lead in blood without any ashing process. Quantitative analysis for cadmium in urine was performed by standard addition method. The corrected absorption signals and the analytical curve are shown in Figure 8. We obtained a result of 1.85 ppb cadmium content in the urine. Figure 9 shows the result of lead determination in blood. Sodium citrate was added to the blood by 2 mg/ml as anticoagulant, and the samples of blood were diluted into 20% solution with standard solutions of lead. Lead in this solution was determined directly in the same way as the analysis for cadmium in urine. Although the lead background of the sodium citrate was 0.20 ppm, it was found that the blood sample contained 0.10 ppm of lead.
Figure 9. Determination of lead in blood by Zeeman AAS ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
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of SRM-1577 are N, 10.6%;K, 0.97%; Na, 0.243%; and the main trace constituents are Fe, 270 ppm; Cu, 193 ppm; and Zn, 130 ppm. When this sample was analyzed by a working curve which is made by a lead solution of 0.1 N “03, the observed result was 0.342 f 0.07 ppm. The result agreed precisely with the NBS value of 0.34 & 0.14 ppm. Thus, it shows that accurate results can be obtained by the present method even if the standard additions method is not applied. The heated area of the carbon tube must be sufficiently large to prolong the residence time so as to decompose the sample completely. If only a small portion of the central part of the tube was heated to high temperature, the good reproducibility and the high sensitivity were not obtained in the case of a solid sample. The temperature distribution of the carbon tube was important to perform an accurate analysis. We did not observe any noticeable chemical interference in all the samples analyzed. The determination of zinc by the present method was also performed, and good results as well as those of cadmium and lead were obtained under the same conditions as in the case of cadmium. Figure 10. Determination of lead in the standard reference material of NBS (SRM-1577, Bovine Liver) by Zeeman AAS (a) Weight, mg Pb, ppm (1) 1.42 (2)2.12 (3)1.65 (4)1.73 (5)1.85
0.370 0.307 0.340 0.356 0.341
(b) Result: 0.343f 0.023ppm. Certified value of NBS: 0.34f 0.08 ppm
The biological standard reference material of NBS was analyzed to confirm the accuracy of this method. Figure 10 shows the results and the procedure when the lead in the SRM-1577 Bovine Liver was determined. Dried and weighed samples of 1to 3 mg were positioned exactly in the center of the carbon tube by the sample holder. The major constituents
ACKNOWLEDGMENT
We thank T. Hadeiahi of the University of California and M. Katayama of the University of Tokyo for their useful suggestions. Thanks are also due to K. Ohishi and K. Uchino of Hitachi Ltd. for their great help. LITERATURE CITED (1) H. Koizumi and K. Uchino, HitachiHyoron, 56, 1037 (1974). (2) H. Koizumi and K. Yasuda, Anal. Chem., 47, 1679 (1975). (3) H. E. White, “Introduction to Atomic Spectra”, McGraw-Hill, New York, 1934. (4) A. C. G. Mitchell and M. W. Zemansky, “Resonance Radiation and Excited
Atoms”, Cambridge, 1934. (5) E. E. Whiting, J. Quant. Spectrosc. Radiat. Transfer, 8, 1379 (1968). (6) H. Massmann, Spectrochim. Acta, Parts, 23, 215 (1968). (7) A. G. Shenstone, Phys. Rev., 47, 317 (1935). (8) T. E. Manning, Phys. Rev., 78, 417 (1950). (9) J. P. Matousek, Am. Lab., No. 6, 45 (1971).
RECEIVEDfor review January 19,1976. Accepted March 15, 1976.
FIuorometric Determination of Chlordiazepoxide in Dosage Forms and Biological Fluids with Fluorescamine James T. Stewart” and Jonathan L. Williamson School of Pharmacy, University of Georgia, Athens, Ga. 30602
A fluorometrlc procedure for chlordiazepoxide is reported based upon a fluorophor formed with fluorescamine after acid hydrolysis of the drug. A study of optlmum pH, hydrolysis time, and fluorescamineconcentrationis presented. Fluorescence is linear over the range 0.25-6 pg chlordlazepoxlde hydrochloride per ml of sample. The method is free from interference from chlordiazepoxide’s major metabolites as well as other drugs such as clldinlum bromide, oxazepam, and dextropropoxyphene hydrochlorlde. The procedure Is subject to interference from other amlne-containingdrugs such as amphetamines that might be present In analytical samples. Applicatlon of the procedure to the analysis of chlordiazepoxide in dosage forms, urlne, and plasma gave an accuracy of 1-3 %. 1182
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
Chlordiazepoxide (I), a 1,4-benzodiazepine derivative, has been widely used therapeutically because of its muscle relaxant, taming, sedative, anti-anxiety and anticonvulsant properties (1-3).
NHCH,
I
