Anal. Chem. 1987, 59, 2789-2794
COMPETITIVE 1.30
ASSAY
F O R HUMAN
falls off at higher concentrations as expected. The accuracy of the measurement is in the *2% region. The assays have been repeated for over 2 weeks a t the rate of 30 assays per day (600total)and the decrease in the immunological activity is less than 5%. The immunosorbent has been stored in the refrigerator for 2 years at 4 OC and the decrease in immunological activity is negligible. In summary the FIA system coupled to an immunoreactor with a thin-layer electrochemical detector forms a basis for carrying out immunoassays rapidly with a high degree of accuracy and precision.
IGG
I1
I
n 1s I. I -
0.00
2. 50
5 . 00
7. so
12.50
10.00
2789
Plcomoler HlgQ Added
Flgure 5. Calibration curve for the human IgG competitive assay.
Table 11. Recovery of Human 1%: from Control Serum amt taken, pmol
amt detected,” pmol
amt taken, pmol
amt detected: pmol
0.5 2.0
0.525 (*1%) 2.1 (&2%)
5.0 10.0
5.11 (&3%) 10.5 (&5%)
a Unknown samples were prepared by spiking control serum with known amounts of human gamma globulin.
accuracy due to the flattening of the curve. A calibration curve for this assay is shown in Figure 5. A dynamic range of a factor of 20 is obtained. The competitive assay has an inherently much shorter range than the sandwich assay and is much more tedious to optimize. The results of a well-optimized assay are shown in Table 11. The precision of the assay
ACKNOWLEDGMENT We thank American Qualex International, Inc., for generous donations of materials. LITERATURE CITED Shekarchi, I . C.; Sever, J. L.; Lee, Y. J.; Castellano, 0.;Madden, D.L. J . Clin. Microbiol. 1984, .79(2), 89-96. Cantarero, L. A., Butler, J. E.; Osborne, J. W. Anal. Blochem. 1980. 105, 375-382. Kenny, G. E.; Dunsmoor, C. L. J . Clin. Microbiol. 1983, 77(4), 655-665. De Alwls, U.; Wilson, G. S.Anal. Chem. 1985, 57, 2754-2756. Nllsson, K.; Mosbach, K. Methods Enzymol. 1984, 704, 56-69. De Alwls, U.; Hill, 6. S.; Meiklejohn, B. 1.; Wilson 0. S. Anal. Chem. 1987, 59, 2688-2691. Ternynck T.; Avrameas, S. Ann. Immunol. (Paris) 1976, 727C, 197-208. Miron, T.; Wilchek, M. Appl. Biochem. Biofechnol. 1985, 1 1 , 445-456. Sportsman, R. J.; Wllson, G. S.Anal. Chem. 1980, 52, 2013-2017. Weibel, M. K.; Bright, H. J. J . Biol. Chem. 1971, 249(9), 2734-3739.
RECEIVED for review March 23,1987. Accepted July 15,1987. We thank the National Institutes of Health (Grant DK 30718) for financial support.
Magnesium Excitation Mechanisms and Electronic-State Populations in an Argon Inductively Coupled Plasma Tetsuya Hasegawal and Hiroki Haraguchi* Department of Chemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan The excitation mechanlsms of magnesium in an argon inductlvely coupled plasma have been investigated by using a coiildonakadlative process theory, which includes the Penning lonlzatkn and charge transfer reactlaw in addltion to the electron impact and radiative processes. The calculated population denslty distributions in the electronic states of atoms and ions showed markedly large deviations from local thennodynamk equlllbrlum, Le., overpopulations of ail atomic levels and lower lonlc levels. The overpopulatlons of these levels are interpreted by 8IgnWlcant spontaneous emisslon and negllgble radlathre absorption In the case of magnesium. The discrepancy between the present resuit and prevlous observations, Le., overpopuiatlon of ions, is attributed to the dlfference of temperatures used for the LTE calculation. From the comparison of the reaction rates for the ionization processes, R has been concluded that the electron impact processes are predominant for magnesium exclation/ionlzation rather than the Penning lonlzation and charge transfer reactions. Present address: Department of Industrial Chemistry, Faculty of Engineering, University of Tokyo, Bunkyo-ku, Tokyo 113,Japan. 0003-2700/87/0359-2789$01.50/0
In recent years, an inductively coupled plasma (ICP) has been extensively used in analytical atomic spectroscopy as atomization (I), excitation (21, and ionization (3) sources. In particular, the ICP is an efficient excitation source in attomic emission spectrometry which provides the excellent analytical feasibilities such as good sensitivity, good stability and precision, less interelement interferences, wide dynamic ranges, multielement detection capability, and so forth (4-6). It is also well-known from the spectroscopic studies that ionic lines for most of analyks generally provide much better sensitivities than their atomic lines. According to the previous investigations (7), it has been considered that such spectroscopic characteristics of the emission lines of analytes in the argon ICP result in deviation from LTE (local thermodynamic equilibrium) and may be appreciated as the overpopulations of ionic species in the ICP, which are often called “suprathermal ionization”. Actually the non-LTE properties of the argon ICP, on the other hand, give analytical advantages of ICP-AES (inductively coupled plasma atomic emission spectrometry) mentioned above. In order to interpret the non-LTE phenomena of the ICP, various models for excitation and ionization mechanisms have been proposed by many research groups (8-19). 0 1987 American Chemlcal Soclety
2790
ANALYTICAL CHEMISTRY, VOL. 59, NO. 23, DECEMBER 1, 1987
Mermet (8)f i i t postulated the following Penning ionization reaction as a dominant process for analyte metals (X) (9):
-
+ X Ar + X+ + eArm + X -,Ar + X+* + eArm
(1)
(2) where Ar and Arm are the ground-state and metastable argon atoms and X+ and X+*the ground-state and excited-state ions, respectively. This assumption was based on the assumption that the metastable argon atoms might be significantly overpopulated in the ICP and efficiently produce the analyte ions, since the reverse processes of eq 1 and 2 could be neglected. However, the number density of Armwas found to be on the order of lo1’ (10, I I ) , which is not so overpopulated compared with the LTE value. Recently, the following charge transfer reaction has been considered to be a more possible process because of the larger population of argon ion (on the order of 1015~ m - ~ ) :
Ar+ + X
+
-
+ X+ + eAr + X+* + e-
-,Ar
the deviation from LTE for argon was ascribed mainly to the spontaneous emission and motions of electrons and argon ions. In the present paper, the excitation mechanism of magnesium as a representative analyte will be discussed by the collisional-radiative process theory which includes the Penning ionization and charge transfer reactions. By comparison of the reaction rates, it is concluded that the electron impact ionization is dominant and the Penning ionization and charge transfer are less significant in the case of magnesium.
THEORETICAL CONSIDERATION Rate Processes and Steady-State Approximation for a Collisional-Radiative Model. In the collisional-radiative model, the following rate processes for both analyte atom (X) and ion (X’) are taken into consideration, similar to the case of argon (20-22): (i) electron impact excitation and deexcitation
(3)
Ar+ X (4) Boumans (12) introduced the concept of ”recombining plasma” as a possible plasma model, in which the number density of analyte ion was superior to that of neutral analyte atom. The recombining plasma is established by the nonlinear reactions in eq 1-4 or the transport phenomena of species, where the reaction rates of the nonlinear processes should far exceed those of the electron impact ionization and recombination processes. Boumans proposed the Penning ionization processes as the main cause of recombining plasma, but the further investigations considering the mass and energy balances of the plasma species (13,14)suggested the significance of diffusion processes such as axial convection and lateral ambipolar diffusion of electrons. On the basis of the observation that the number densities of argon metastables were almost similar to those of the radiative (resonant) 4s levels of argon, Blades and Hieftje (15) offered the radiation trapping model. According to their model, the resonance radiation emitted from the hot surrounding zone is completely absorbed or trapped by cool argon atoms in the central channel zone, and, as a result, the population of resonant argon atoms is enhanced up to the number density of Arm. This idea was extended to the excitation model of analyte metals that the extremely overpopulated 4s levels due to the radiation trapping caused the significant Penning ionization (16).Recently de Galan (17)critically reviewed the excitation mechanisms mentioned above and emphasized the difficulty in elucidating the contributions of various processes occurring in the atmospheric pressure plasma. Rayson and Hieftje (18)evaluated semiquantitatively the ionization and excitation mechanism of calcium in the ICP under the steady-state approximation and suggested the importance of electron collision and radiative processes. Furthermore, Goldwasser and Mermet (19) investigated the contribution of the charge transfer process to the excitation of anal* in ICP-AES. Obviously, various models for excitation described above appear to interpret partly the excitation mechanisms and the overpopulations of ions. However they are still hypothetical and/or empirical, and in particular the discussion on kinetic processes is still insufficient. We have successively investigated the diagnostics of argon in the ICP by using a collisional-radiative model, which incorporated the electron impact and radiative reactions and transport of species as the rate processes occurring in the plasma (20-22). The spatial distribution of electron temperature (T,) and electron number density (ne),which were essential for the present model, were measured from the background continuum around 400 nm and Stark width of H, line, respectively (22),after Abel inversion. Consequently,
(ii) electron impact ionization and three-body recombination
+ & X+ (X2+)+ 2ekLP
X(p) (X+(p’)) e-
(6)
(iii) induced absorption and spontaneous emission PBW
’a,X(q) (X+(q’))
X b ) (X+(P’)) + b i n e
(7)
(iv) radiative recombination X+ (X2+)+ eX(p) (X+(p’)) + huCont (8) In addition, the nonlinear reactions are also considered as possible ionization processes for the analyte atom (v) Penning ionization
2
X(p) (X+(p’))
+ Ar*
X+ (X2+)+ Ar
mP
+ e-
(9)
(vi) charge transfer X(p)
+ Ar+
X+ +Ar
(10)
CP
where k , a,m, m f , c, and c f are the rate constant for each process, B,, the Einstein coefficient for transition from p to q, A, the transition probability of transition from q top, X(p) and X+(p’) the analyte atom and ion at levels p and p‘, respectively, and Ar* is, in general, the excited argon atoms, which are not necessarily metastable argon atoms. In the present calculation, however, only the 4s levels of Ar are considered Ar*, because the population densities of the radiative and metastable 4s levels were estimated to be about 70% of the total number densities of excited states (22). The rate equations for analyte atom in the electronic states can be described, as follows, in similar manner to those for argon (21, 22) dn(X(p))/dt =
C hqpnen(X(q)) 4
