Internal-Standardization in Flame Analyses Using a Vidicon Spectrometer N. G. Howell, J. D. Ganjei, and G. H. Morrison* Department of Chemistry, Cornell University, Ithaca, N. Y. 14853
Routine application of internal standard measurements to flame analyses is facilitated by the use of a vldlcon spectrometer. The system is readily adaptable to ratio method procedures. The analyses performed display the technique’s capability to compensate for a variety of common matrix handling problems. A matrix interference of about 10% is reduced to a 1% devlation through the incorporation of an internal standard in the determination. Flame emission analyses of Na, K, and Ca simultaneously using a Li internal standard in serum, and Mg using a Mn internal standard for both serum and bovine liver samples are presented. While the vidicon tube has previously been used as a detector for multlelement determinations, it also provides a rapid, accurate, and efficient means of performing internal standard analyses.
T h e method of internal standardization has been utilized in emission spectrographic analyses for a t least a half-century ( 1 ) . Its application to flame methods was described over 30 years ago where t h e benefits of improved accuracy, precision, and matrix interference compensation were shown (2). Subsequent flame spectrometric studies have revealed a number of specific analytical problems for which t h e method compensates, Le., fuel-to-oxidant ratio shifts, aspiration rate variations, sample viscosity a n d surface tension differences, a n d errors caused by improper sample manipulations (3-6). Barnett e t al. have provided theoretical a n d experimental insight on t h e criteria which are essential for t h e effective use of t h e ratio method in spectrochemical analyses ( 7 , 8 ) . Conventional flame spectrometers are single channel instruments which limit internal standard measurements t o t h e sequential or scanning mode ( 3 ) . Dual spectrometer a n d multiple filter instruments have recently become available to incorporate internal standard methods (5, 6). With t h e advent of simultaneous multichannel instruments, it is now possible to take full advantage of the internal standard method’s inherent accuracy and flexibility (9-13). This report describes t h e application of a vidicon flame spectrometer, developed in this laboratory (9, 14), to a n evaluation of the internal standard method’s matrix compensation capability. T h e spectrometer system utilizes a sensitive television camera tube and an Optical Multichannel Analyzer (OMA). T h e OMA contains t h e circuitry which digitizes 500 electronic channels across t h e tube’s horizontal face. T h e integrating nature of t h e silicon diode tube target provides a spatial detector (15) for making simultaneous internal standard measurements on transient signals from microsamples over a wide wavelength region. T h e matrix interference compensation feature of t h e internal standard method is illustrated by t h e use of well characterized bovine serum and bovine liver samples. I n particular, t h e serum matrix is utilized to show how a previously encountered matrix effect (14) is easily compensated through t h e incorporation of a well matched internal standard spectral line into t h e observed wavelength region.
T h e additional capacity of t h e ratio method t o overcome significant sample manipulation errors is demonstrated by t h e bovine liver analysis.
EXPERIMENTAL Apparatus. The experimental facilities used are listed in Table I. Two spectrometers, a 0.5-m Ebert mount monochromator, and a 0.3-m modified Ebert mount polychromator, are focused on the same flame source. A block diagram of the system layout is shown in Figure 1. T h e 0.5-m Spectrometer S y s t e m . The apparatus used with this system has previously been described (14). The silicon intensified target (SIT) vidicon tube monitored a 40-nm window of spectral information simultaneously. The data obtained off the 12.5-mm active face of the tube was directed into the OMA for real-time data observation, or spectrum storage and manipulation. The 500 digital data points obtained with each 32.8 ms frame scan of the tube was either displayed on the oscilloscope in real-time, or successive scans were totaled and stored in either of two memories labeled A or B. Channel by channel subtraction of memory B from memory A via the arithmetic unit within the OMA yielded blank corrected sample spectra. The detailed operational description of the SIT tube (14) and OMA (9) were presented in earlier articles. The flame source for all experiments with both spectrometers was a nitrous oxide-acetylene flame. T h e 0.3-rn Spectrometer S y s t e m A 0.3-m polychromator supplied by Princeton Applied Research was fashioned from a JarrellAsh 0.25-m Ebert mount monochromator. The spectrometer included the mounting hardware required to position any of the manufacturer’s vidicon tube assemblies near the polychromator focal plane. The final focusing of the spectral image onto the tube target was facilitated by a sliding sleeve entrance slit assembly. The 25-pm fixed entrance slit supplied with the spectrometer was replaced by a 0-2000 pm variable slit. The bulkier 0.5-m bayonet mount design required the slit holder assembly on the polychromator to be rebuilt. The variable slit provided an easy method of optimizing the signal to prevent tube and electronics saturation while maintaining maximum sensitivity. The modification specifications can be furnished upon request. The flame was focused on the entrance slit by a 5-cm diameter (stopped down to 3.0 cm diameter) 10-cm focal length plano-convex supracil lens. The lens was stopped to prohibit the overfill of the collimator mirror. A 590-grooves-per-millimeter grating resulted in a 5.6 nm mm-’ linear dispersion and an observed 70-nm spectral window displayed across the vidicon tube. The ultraviolet sensitized silicon vidicon described previously ( 9 ) , transfers the spectral information to the OMA for subsequent data manipulation. Absorption Filters. Two sets of absorption filters were employed in this study to reduce the dynamic range of the radiation emitted by the flame. These filters partially absorbed the stronger lines preventing them from saturating the detector system at slit width settings sufficient to easily observe the weaker, unfiltered potassium or magnesium lines. One-centimeter path length quartz cells placed on a platform directly in front of the entrance slit held the filter solutions. The sodium, potassium, and calcium analyses used the same filters as previously described (14). The copper sulfate solution was unchanged in strength, while the absorbance of the iodine-carbon tetrachloride filter was reduced to 1.05 with respect to water at the 422.7-nm calcium line. The serum magnesium determination required the simultaneous monitoring of the magnesium 285.2-nm line and the sodium 330.2and 330.3-nm doublet. An aqueous nickel chloride solution was found to absorb at the strong sodium lines while passing the weaker magnesium line. Four grams of reagent grade NiCIy6H20 were ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976
319
PECTQOMETER
IC~RONATOR
LENS
DUCTION SYSTEM BURNER
Figure 1. Block diagram of experimental facilities
dissolved in 10 ml of water and diluted to the extent that an absorbance of 0.3 was obtained with respect to water at the unresolved 330.2, 330.3-nm sodium lines. Solutions and Samples. The stock solutions were prepared from high purity metals and salts using the methods described by Dean and Rains (16). Since the concentration requirements for sodium, potassium, lithium, and cesium were high, these stock solutions were prepared at 10000 rug ml-’ levels. The lithium stock solution was prepared from 99.999% lithium carbonate obtained from Spex Industries. The sodium solution, also utilized for spectral stripping, was prepared from ultra pure sodium carbonate obtained from Alfa Products Division of the Ventron Corporation. For the internal standard analyses, the samples and standards were diluted with an appropriate internal standard-ionization buffer solution. A dilution of one part sample or standard to two parts of the buffering solution was used. Thus, serum samples were compared with aqueous standards containing only the species to be analyzed. The solutions were diluted just prior to analysis, as described above, to facilitate the precise introduction of the internal standards. The actual amounts of samples and solutions used for each analysis are listed in Table 11. A 1500 pg ml-l lithium internal standard-10500 pg ml-’ cesium ionization buffer solution was used to dilute the serum samples and aqueous standards for the sodium, potassium, and calcium analysis. Manganese at 60 @gml-’ in a 1500 pg ml-’ cesium solution was used as the internal standard-ionization buffer solution for the serum magnesium determinations. The serum samples used in this study are experimental reference sera supplied by the Center for Disease Control (CDC), Atlanta, Ga. The bovine calf sera had previously been treated with streptomycin, penicillin, and fungizone to inhibit bacterial action. The elemental concentrations tabulated as CDC values were provided by the Center as their values. The bovine liver analysis was performed on NBS standard reference material SRM 1577. The material was dried for 12 hours at 80 “C and stored in a desiccator. Dried samples of 200 to 300 mg were placed in Teflon-lined bombs. To each of the two samples, 1.0 ml of a 1312 fig ml-I Mn internal standard solution, 1.5 ml of concentrated nitric acid, and 1.5 ml of concentrated sulfuric acid were added. The two bombs were then placed in an oven at 107 OC for 5 h. After cooling, each of the samples was transferred to a 25-ml volumetric flask along with 5.0 ml of the cesium ionization buffer solution. Sample No. 1 was carefully transferred and diluted as the control sample. Sample 2 was purposely diluted to some unknown volume after an incomplete sample transferral. Both samples were analyzed without further dilution. The value for magnesium was supplied by NBS as an “information value”. Procedures. A listing of the analytical conditions used in the analyses performed is given in Table 11. The individual procedures are detailed below. Determination of N a , K , and Ca i n S e r u m with a L i Internal Standard. DIRECTASPIRATION MODE.The 0.5-m spectrometer was used for this analysis with the grating positioned such that first order radiation at 837 nm would strike the tube center. At this setting, the analytical lines found in Table I11 for lithium, sodium, potassium, and calcium were simultaneously monitored on the tube face. The potassium and calcium lines were observed in the 320
second order. Iodine and copper sulfate filters reduced the calcium and sodium line intensities. The diluted sample and standard solutions were directly aspirated into the slightly fuel rich nitrous oxide-acetylene flame, and spectral data was obtained in the same manner for all. Once a steady-state aspiration was attained the accumulation button for memory A on the OMA was enabled for a preset of 150 frame scans. The slight flame background, and tube diode leakage current was automatically removed by accumulating 150 frame scans of distilled, deionized water into memory B. The A minus B mode gave the resultant integrated intensity profiles for potassium, lithium, sodium, and calcium. For this analysis, and all others, the peak height measurements were manually obtained off the OMA console. The cursor spot was moved to peak maximum and the magnitude of that channel read off the light emitting diode display. The background was then averaged for ten neighboring channels and subtracted from the peak magnitude. These background corrected intensities for sodium, potassium, and calcium were divided by the lithium intensity value for each internal standard run. Data obtained for the unratioed runs were simply background corrected intensities. The samples were calibrated by using the curves obtained for the aqueous standards’ concentration vs. intensity or intensity ratio plots. Each sample and standard was run three times. The average of those three is reported as the sample concentration value, and the average standard deviation for that element in all three serum samples is given as the precision measurement. MICRO INJECTION MODE. The instrumental setup for this method is identical to the direct aspiration mode except for the use of a micro sample introduction system (14). This sampling system consists of a 1-1. reservoir containing a 20% (v/v) isopropanol carrier solution placed approximately 20 inches above the table. A sample introduction port was designed to allow the injection of microliter volumes into the carrier solution stream which is fed directly into the burner system.
Table I. Experimental Facilities Burner External optics
Entrance slit Spectrometers
Detectors
Optical multichannel analyzer Readout Flow meters Syringe
ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976
Varian Techtron 5-cm high solids slot burner for nitrous oxideacetylene. Five-cm diameter Supracil lens with 12.5-cm focal length. Lens stopped down to 2.6-cm diameter o n the 0.5-cm spectrometer. Five-cm diameter Supracil lens with 10.0-cm focal length o n the 0.3-m spectrometer, stopped down to 3.0 cm. Jarrell-Ash, Model 12-080, variable 0-2000 y m straight edged slit, used on both spectrometers. Jarrell-Ash, Model 82-000, 0.5-m Ebert Mounting scanning monochromator with 590 grooves/mm grating blazed for 4000 8.Reciprocal linear dispersion 32 &mm in first order. Princeton Applied Research Model 1208, 0.3-m modified Ebert Mount Polychromator using 590 grooves/ mm grating blazed for 3000 8. Reciprocal linear dispersion 5 6 8/ m m in first order. UV sensitive silicon diode vidicon, Model 1205F, SSR Instruments Co. Silicon Intensified Target Vidicon, Model 1205D, SSR Instruments Co. Model 1205A, SSR Instruments Co. Tektronix Oscilloscope, Model 6 0 4 Monitor. Brooks Full-view Rotameters calibrated for nitrous oxide and acetylene, Brooks Instrument Co. Hamilton Co., constant rate microliter syringe Model CR 700-200.
Table 11. Analytical Conditions .Analysis performed Parameters
Flame Fuel, C,H,; pres., 8 psi flow, c m 3 min-’ Oxidant, N,O; pres., 30 psi flow, om3 min-’ Observation height Distance above burner top, m m Spectrometer System Spectral window, n m Slit width, p m Filters Solu te/solvent
Electrolytes via direct aspiration
Electrolytes via injection m o d e
3250 4750
3250 4750
3000 5500
3000 5500
7
7
6
6
0.5 m 807-847 75 I,/CCl, Cu SO ,/H, 0
Detector Readout Vidicon tube type Accumulation cycles Sample dilutions A m t of sample or standard, ml Amt of int std soln, ml Int std elem. final concn, pg ml-’ Ioniz. buf. elem. final concn, p g ml - 1
SIT 150 1.0 2.0 Li; 1000 Cs; 7000
Table 111. Comparison of Analytes and Internal Standards0 El ement
Li
Function
Internal standard Na Analyte K Analyte Ca Analyte Mn Internal standard Mg Analyte a Ref. 17.
0.5 m 807-847 75
1st ionization Av sol potential. concn. Mg/ml cm
Analytical line, n m
Transition levels, cm-’
812.65
14904-27206
43487
1000
819.48 404.41 422.67 279.48
16973-29173 0-24720 0-23652 0-35770
41450 35010 49305 59960
1100 60 33 40
285.21
0-35051
61669
7
-’
A 200-wl volume was utilized as the sample size. At the moment of solution injection the accumulation button for memory A was engaged for 500 frame scans, storing the entire signal pulse in memory A. Storage of 500 scans of the carrier solution background in memory B for subsequent subtraction provided the integrated injection pulse intensities. The raw data were obtained and reduced to sample concentrations in the same manner as described in the direct aspiration case. Determination o i M g i n S e r u m with a M n Internal Standard. For these analyses the 0.3-m polychromator with the UV sensitive
vidicon tube was used. The spectrometer’s 70-nm window was centered at 303 nm, just shy of the 308-nm OH band system. At this position, both the magnesium 285.2-nm line and sodium 330.2, 330.3-nm doublet were monitored. The aqueous standards, containing only magnesium, and the serum samples were diluted with internal standard-buffer solution as described earlier. The calibration curve was obtained by direct aspiration into the flame for 150 accumulation cycles. The intense flame background was removed by the storage of 150 scans of distilled deionized water in memory B. Peak intensities for the magnesium line and unresolved manganese triplet were obtained from the OMA console as previously explained. The standards’ intensity, or ratio measurements, were plotted vs. concentration for the analytical curve. The serum samples required a slightly different spectrum acquisition method because of the presence of a severe spectral interference. To remove the interference caused by the presence of high sodium concentrations in the serum, the technique of spectral stripping ( 1 8 ) was employed. The sample was aspirated for 150 frame scans stored in memory A. The A minus B display mode was selected, and the ultrapure sodium stripping solution was aspirated
I, I cc14 CuSO,/H,O
SIT 150 0.5 1.0 Li; 1000 Cs; 7000
Magne\iuni in serum
0.3 m 268-338 40
Magnesium in Bovine Liver
0.3 m 268-338 50
Ni Cl,/H,O
uv
uv
1.0 2.0 Mn; 40 cs; 1000
Mn; 50 c s ; 2000
150
300
...
into the flame. The concentration of sodium in the stripper was greater than the upper limit of the serum sample concentrations. This was determined by real-time observation of the sodium line for the serum, where the stripper was brought to a concentration which yielded an intensity which was just greater than the serum intensity. The same stripping solution was used for all samples. The spectrum of the stripper was then subtracted from the sample by enabling the “B accumulate” button until the sodium doublet had disappeared from the sample spectrum. At this point, the subtraction of the sodium solution spectra was halted, and a distilled water spectrum was accumulated in B until the preset hold was reached. Triplicate runs for each sample were compared with the previously determined calibration curve. The intensity ratio and intensity values were obtained in the same manner as before. Where spectral stripping was not utilized, the sample data were obtained in the same manner as the standards. Determination of Mg i n Bovine Liver Using a M n Internal S t a n d a r d . The same polychromator system was employed in this analysis, only the radiation from the flame did not pass through any filter. Simple aqueous magnesium calibration standards were used where the internal standard and ionization buffer were added to the solutions as they were prepared. The standards were directly aspirated into the flame, as were the samples. Spectral stripping was not required. The comparison of magnesium intensities facilitated the analysis without the internal standard, and the magnesium-to-manganese background corrected intensity ratio was used for the internal standard analysis. Three runs for each sample were used to determine the average value and relative standard deviation of the values obtained.
RESULTS AND DISCUSSION The Detector System. The application of a vidicon flame spectrometer t o internal standard measurements has previously been mentioned (9, 19). The present study details the advantages a continuous multichannel spatial detector provides for making intensity ratio measurements. The continuous nature of the vidicon tube allows spectral lines to be simultaneously monitored without the multiple slit alignment problem. This feature also provides individual peak maxima and background measurements to be easily obtained. T h e incorporation of internal standard elements into t h e vidicon wavelength window does not require any modification of t h e detection system. Multiple spectrometer systems a n d small direct readers (5, 6) can monitor only a limited number of intensity channels. Utilizing internal standards in multielement analyses would neces-
ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976
321
.-
30r
l3
25H
=
151
\
I
3000
>q 5000
Cesium Concentration, pg
7000
ma-‘
Figure 2. Buffering of potassium in a serum matrix
sarily reduce the number of analytes which can be simultaneously monitored. Because internal standard lines of comparable excitation energy are generally in close wavelength proximity to the respective analyte lines, the vidicon system is limited only by the possibility of spectral overlap. This detector system provides the opportunity for multiple analyte-internal standard methods in flame spectrometry. The nature of the data obtained from a vidicon detector makes it easily amenable to computerization. The OMA console being a hard wired computer system permits the use of signal averaging techniques to improve the precision of measurement, and to allow the removal of practically all types of spectral interferences. A recently completed computer interface provides for a more rapid data acquisition and manipulation system facilitating a more extensive use of the ratio method in flame spectrometric analyses (20). The examples which follow explore the capabilities of the vidicon-OMA system as they apply to the analysis of real samples. The Matrix Problem. The precision and accuracy of a given set of flame spectrometric analyses are often dictated by the composition of the sample matrix. The detailed problems caused by matrix effects are frequently difficult to solve. However, the analyst can usually succeed in attaining quantitative results by employing either of the following compensation techniques. When the signal obtained from the element of interest is sufficiently strong, the sample matrix is simply diluted to the point that the interference becomes negligible. If the analyte signal is relatively weak, the interference effect cannot be diluted out, and thus calibration standards must be modified to adequately duplicate the interference effect. These compensation methods are easily applied to single element determinations. In extending the capability of flame spectrometry to multielement analyses, a difficulty arises when elements of drastically differing signal strengths in the sample are to be measured simultaneously. The intensity of the weak signal would no longer be analytically useful if the matrix is diluted to minimize its effect. Conversely, the matrix duplication method when applied to all of the elements to be determined becomes extremely complex, and time consuming. This type of matrix problem was observed in a recent paper detailing the analysis of sodium, potassium, and calcium in a serum matrix (14). A 200-p1 direct injection of serum into the carrier stream of an ionization buffer solution resulted in a signal enhanced 5-10% over the aqueous standards’ response. T h e interference probably was caused by the proteins affecting solution nebulization and atomization parameters. The actual analysis method incorporat322
ed the use of well characterized reference sera as calibration solutions. This matrix duplication method gave excellent results; however, the lack of availability and expense of the reference sera precludes its routine use. The Internal Standard Method. Selection Criteria. Internal standard measurements with the proper analyte and internal standard pairings are capable of compensating for numerous sample matrix interferences, as well as instrumental variations. The extent of matching elemental parameters determines the eventual success of the technique. The selection rules as outlined by Barnett et al. (7) provide a means for evaluating the utility of an internal standardanalyte pair. This section of the report discussed the application of these criteria to the serum matrix interference problem, and the choice of an internal standard for making ratio measurements. The element chosen to be added to the sample should be “negligibly low” a t its natural occurrence within that matrix. In applying lithium as the internal standard for sodium, potassium, and calcium in the CDC samples, the highest serum lithium concentration was 20 pg ml-I. After dilution, this amount was just a t the detection limit for the lithium line used as the internal standard line. The actual internal standard concentration was maintained a t 100 times the original sample concentration to limit its effect. This high concentration of lithium added to the samples and standards amplified the requirement that the internal standard be added in a high state of purity. The purity must not only be with respect to the analytes monitored by the internal standard, but also additional elements to be analyzed within the same sample preparation. Ionization energies of the analyte and internal standard must be relatively close. The Table of Comparison, Table 111, lists the first ionization potentials of all the elements in this study. Special attention was given to the difference between lithium and potassium. The lithium internal standard could not be expected to compensate for the appreciable ionization of potassium in a nitrous oxide-acetylene flame. The potassium line intensity did not plateau over the entire range of cesium ionization buffer concentrations which the high solids burner allowed. Thus, an alternate method of optimization was employed. The sodium concentration in human serum might vary between 2000 and 5000 +g m1-I. The relative deviation in intensity of a 100 pg ml-l potassium solution containing 2000 pg ml-I from a solution containing the same amount of potassium and 5000 pg ml-I sodium was monitored vs. the concentration of cesium also present in each. The results of this experiment are displayed in Figure 2 where the percent deviation between the two potassium intensity values falls to zero a t 7000 pg ml-’ of cesium. The partial buffering capability of the sodium present aided in reducing the amount of ionization buffer required. In choosing the internal standard line, a critical factor is the matching of excitation energies for the analyte-internal standard pair. The degree to which the excitation energies should match to maintain a constant minimal error in the intensity ratio varies directly with the fluctuation in source temperature, and inversely with the square of the temperature of that source. Thus, when employing “low” temperature flame-type sources, the matching of excitation energies becomes crucial. Two experiments were performed to evaluate the extent of match for separate flame condition changes with the analytes used in this report. Table IV lists the intensity fluctuations observed for a change in acetylene flow of 200 cm3 min-l. The signal variations a t a constant fuel flow were less than 2%. Sodium and lithium are shown to be well matched, but the sensitivities of calcium
ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976
t
Table IV. The Compensation for Flame Conditions Signal m o n i t o r e d
Re1 std dev of signal observed over a 200 cm3 min-’ change in the acetylene flow rate, %
El gj W
IN^
INa/lLi I K
2.6
1.1
Ica/I~i
IKiILi
7.1
6.1
5.6
4.7
0.8
I-
z
Table V. Matrix Compensation by the Internal Standard K -
Na I-
a J W
0O O
2
4
2 6
8
1
Ca
Abs Av Abs Av Abs Av dev, %a dev, %b dev, 7c dev, 76 dev, Yc dev,
Sample introduction
0 4
5%
Direct aspiration with int std 1.0 -1.0 5 . 3 -5.3 4.4 +4.4 w i t h o u t i n t s t d 11.2 -11.2 14.0 14.0 16.2 -16.2 Micro injection With int std 0.8 +0.4 0.9 0.0 0.9 -0.7 7.0 +7.0 4.0 +2.5 7.7 t7.7 Withoutint std a Average of the absolute values of the relative deviations. b Average of the relative deviations.
L0
DISTANCE ABOVE BURNER TOP, rnrn
Figure 3. Magnesium flame profile with and without internal stan-
dardization 0 Magnesium profile. 0 Magnesium-manganese intensity ratio
standards into the flame. A summary of the results are found in the “direct mode” section of Table V. Here the accuracy of the methods with and without internal standardization are Dresented. In all cases. the absolute errors illustrated a bia‘s; without the internal standard, the viscosity of the sample solutions were such that all values were low. When the ratio method was utilized, the sodium results were excellent, but the expected poor potassium and calcium results were obtained. T o overcome these effects, a modified dilution technique was employed. The injection mode of analysis with a 2OO-kl sample volume allowed sufficient sensitivity for the detection of the weaker potassium lines while providing for some additional matrix dilution. Also in Table V is a summary of the results of the injection mode analysis of sodium, potassium, and calcium. The enhancements previously observed (14) are partially reduced due to the 1:2 sample dilution, yet one still finds about a 5% error when the internal standard is not used. Use of the ratio method reduces the injection method error to the point that the average deviations are all less than 1%.Table VI provides a more detailed comparison of the results obtained via the two sample introduction methods utilizing the internal standard. The precision of measurement for these methods also deserves some discussion. A comparison of the injection mode precision with and without the ratio technique shows the
and potassium to fuel flow variations were not compensated by the referenced lithium transition. T h e relative standard deviations are for nine measurements taken over the fuel flow range. A flame profile for magnesium displays the extent of match for the magnesium-manganese pair. The elemental properties found in Table I11 suggest the experimental facts shown in Figure 3. The average of each profile’s data points was normalized to one, and each intensity was then made a fraction of that average. While the unreferenced magnesium signal varied by over 100% in the 9-mm profile span, the ratioed signal’s fluctuation was only 10%. Relative standard deviations of the eight values went from 35.5 to 3.7% when the internal standard was applied. These experiments imply that those matrix interferences which are primarily due to flame condition variations would be effectively compensated in the sodium-lithium and magnesiummanganese pairs. Both the calcium-lithium and potassiumlithium cases should still exhibit some degree of matrix interference, requiring additional compensation methods.
Determination of N a , K , and Ca Using a Li Internal S t a n d a r d . Two methods were utilized in evaluating the effectiveness of the internal standard in the actual analyses of bovine sera electrolytes. T h e initial analysis utilized the direct aspiration of a 1:2 dilution of serum and aqueous
Table VI. Analyses of Na, K, and Ca in Serum Using a Li Internal Standard ~ Specimen No.
_ CDC
_ N a_ Injection
K
Direct
CDC
Injection
174-13 Valuer SDa, 2 8 3 2 - 1 2 2 8 1 4 - 23 2783 i 72 1 0 9 i 2 l l O i 7 pg ml Dev, % 0.6 1.7 ... 0.9 374-13 Value t SD, 3161 i 1 2 3179 i 23 3148 i 72 187 i 2 188 z 7 pg ml-‘ Dev. % ... 0.6 0.4 ... 0.5 574-i3 Value = SD, 3517 I1 2 3564 x 23 3489 i 72 267 i 7 263 i 7 pg ml-’ Dev, 7% ... 1.3 0.8 ... 1.5 a The average standard deviation obtained for all three samples.
Ca Direct
CDC
105r 6
Injection
5 1 . 5 i 1.0
3.7
256
2
4.1
i
1.7
90.8
i
...
1.0
88.6
i
2.4
55.61 5.7 8.0
0.4
172 2 6
8.0
51.7
Direct
1.7
92.8 z 5.7
2.0
6 130.1 i 1.0 130.1 t 1.7 134.3 = 5.7
...
0.0
3.2
ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976
323
'' 1 0.6
'10
08
06
04
02
'70
0
08
06
04
02
0
FRACTION OF I N I T I A L I N J E C T I O N VOLUME
Figure 4. Compensation for microsample volume changes (a)Without the internal standard: (A)potassium, (0) calcium, (0) sodium. ( b )With a lithium internal standard: (A)potassium, (m) calcium, ( 0 )sodium
sodium relative standard deviation improves from 0.9 to 0.7%, calcium goes from 2.6 to 1.9%, but potassium precision degrades slightly from 3.3 for the unratioed to 3.7% for the internal standard injection method. The noise component of the low concentration potassium signal contributes significantly to this observation. When discrete microsampling systems are used for spectrometric analyses, the limit of the method's precision is most often dictated by the reproducibility of the microsampler. The internal standard method is capable of compensating for this difficult problem. Since the signals generated by a microsampling system are time variant, a n integrating spatial detector system i s required. In Figure 4, the vidicon system was used t o obtain the line intensities for sodium, potassium, and calcium as the volume of a serum sample was varied from 200 to 50 pl. The open data points in Figure 4a detail the single injection relative intensities for each element a t normalized injection volumes (200 ~1 = 1.00). When intensity ratios are utilized for the same volume changes, the closed data points in Figure 4 b yield an almost uniform response for sodium and potassium. The marked deviation of the calcium signal with and without the internal standard is attributable to the self-absorption phenomena. As less sample volume is used, a more dilute solution enters the flame with a proportionate decrease in the amount of self-absorption encountered. Thus, the smaller the amount of self-absorption, the larger the internal standard ratio. One of the criteria for selecting spectral lines used in internal standard analyses states that selfabsorption should be avoided. The sodium and potassium results exemplify the method's strength. A decrease in sample volume from 200 to 50 pl results in a signal change of only 8%.
Determination of M g Using a M n Internal S t a n d a r d . The excellent matching characteristics between magnesium and manganese facilitates the analysis of serum and bovine liver samples. The rapid analysis of magnesium in serum can prove to be one of the most important tests in a clinical laboratory. Current methods employ the technique of atomic absorption to good advantage. Atomic absorption adequately resolves the 0.07-nm analyte-interferent line separation. The sensitivity of the technique allows for matrix dilution, and the addition of releasing agents to compensate for matrix effects (21). As one attempts to analyze for magnesium in serum using conventional flame emission spectrometers, two factors immediately inhibit the analysis. T h e detectability of the element in emission does not permit a dilution sufficient to eliminate matrix interferences. Also, a significant 324
ANALYTICAL CHEMISTRY, VOL. 48,
NO. 2,
spectral interference is found with a secondary sodium .line on bench top spectrometer systems. A specially designed high-resolution scanning spectrometer has been used for serum magnesium measurements (ZZ), but the practical use of the emission mode of analysis is extremely difficult with conventional systems. Matrix compensation has been accomplished a t low dilutions with well matched analyte-internal standard pairs. The flame profile experiment displayed the potential which the manganese 279.5-nm line has for compensating flame variations observed with the magnesium 285.2-nm line. In a recent article, we have described a rapid, simple, and routine method for the removal of all types of spectral interferences which may occur in flame spectrometric measurements (18). The direct application of this technique to the spectral interference of sodium in the magnesium analysis was used in conjunction with the internal standard method to obtain excellent results for the serum magnesium determination. The spectral window employed simultaneously viewed the manganese internal standard line, the magnesium analyte line, and the sodium monitor line. A spectrum obtained by aspirating the 1:2 diluted serum solution into the nitrous oxide-acetylene flame for 150 accumulation cycles and displaying memory A is shown in Figure 5a. T h e intensity of the sodium line has been attenuated by the nickel chloride filter, which also changes the OH band system profile. Figure 56 is the result of the spectral stripping analysis run, where a sodium stripping spectrum has been subtracted from the sample spectrum until the monitor line disappears into the background, and a distilled water spectra removed the remaining dark current and flame background. The results for the analyses of magnesium in CDC sera are listed in Table VII. The application of multichannel spectrometric techniques which correct for the two types of interferences is shown as one progresses from using only a n internal standard (IS), to using only spectral stripping (SS),to finally combining both spectral stripping and internal standardization (SSIS). The analysis IS displays the average magnitude of the sodium line overlap interference a t 40%. The values obtained by method SS, direct aspiration with spectral stripping, parallel the viscosity effect data reported in the earlier analysis. The results of the SSIS analysis convey the inherent accuracy and precision attainable with this type of system, with the utilization of these two methods of interference compensation. The flame analysis of solid samples requires dissolution and dilution procedures which are susceptible to manipula-
FEBRUARY 1976
I
A-B
N I
0
E$$
E
??-
N
X
>
k v, z W
I-
z
H W
L
I-
a
1 W
U
I
I
i 3 WAVELENGTH, nm
330
330
3
Figure 5. ( a )Spectrum of serum in memory A. (6) Resultant stripped serum spectrum ~
~~
Table VII. Analyses of Mg in Serum Using a Mn Internal Standard Analysis Specimen No. 114-39
-
Value c SD, pg ml-l Dev, % 514-39
8.15 i 0.60
24.8 i
0.6
8.49 c 0 . 3 0 3.0 24.9
-
0.3 0.4 t
I sc
SSb
-
Value + SDd, pg ml-I Dev, 70
314-39
SSlSa
CDC
*
*
6.15 1.3 -22.9
1 3 . 0 3.4 +48.6
*
22.8 1.3 -8.0
3 4 . 8 f 3.4 +40.3
Value r SD, pg ml-‘ 42.3 i 0.6 4 0 . 9 c 0.3 38.3 I 1 . 3 Dev, % 2.9 -9.5 a Analysis using spectral stripping and internal standardization. b Analysis using only spectral stripping. only internal standardization. d The average of the standard deviations for all three samples.
tive errors. T h e ratio method can substantially reduce these errors, and speed up the repetitive analysis procedure as well. T h e determination of magnesium in NBS Bovine Liver (SRM 1577) was designed to evaluate the dilution error compensation capabilities which the internal standard method offers. A small aliquot of manganese was added along with the dissolution acids when the samples were placed in the Teflon-lined bombs. As ashing was complete a control sample (No. 1) was completely transferred, and diluted to volume for analysis. The other sample (No. 2) was partially transferred to the flask, and then diluted by an unknown amount. Spectral stripping was not required because of the lower sodium concentration in this matrix. T h e analytical results obtained for both samples are shown in Table VIII. Internal standardization is again shown to improve the accuracy and precision for both samples. The 37% manipulation error purposely inflicted upon sample 2 was totally compensated by the internal standard. T h e slightly high result (1.7%) could be due to the slight curvature exhibited by the magnesium calibration line on the 5-cm slot burner. T h e application of the ratio method to the routine flame analysis of metals in a solid or liquid matrix could result in a considerable time savings with no loss in accuracy. T h e samples need only to be carefully weighed and the internal
59.5 ? 3.4 +40.6 c
Analysis using
Table VIII. Analysis of Mg in NBS Bovine Liver Sample No.
NBS “Inforination”
With IS
W i t h o u t IS
605
601
595 1.3
1
Value, pg g-’ RSDa, % Devb, %
1.0 0. I
1.I
2
Value, pg g - l 605 6 15 381 3.3 4.3 RSD, % Dev, % 1.7 37.0 a Relative standard deviation of three measurements. b Relative average deviation of three measurements. standard accurately added to each prior to dissolution. T h e ashed sample can then be transferred and diluted without the tedious washing and careful dilutions required for a non-ratioed sample. A well chosen internal standard will also compensate for matrix interferences still present in the sample measurement step. The only requirement is that the calibration curves be linear for the lines used a t the concentration regions covered.
CONCLUSIONS There are several advantages in applying internal standard methods to simultaneous multielement analyses with ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976
325
(6) J. Pybus, F. J. Feldman, and G. N. Bowers, Jr.. Clin. Cbem., 18, 996 (1970). (7) W. B. Barnett, V. A. Fassel, and R . N. Kniseley, Spectrocbim. Acta. Part 8, 23, 643 (1968). (8) W. B. Barnett, V. A. Fassel, and R. N. Kniseley, Spectrocbim. Acta, Part B, 25, 139 (1970). (9) K. W. Busch, N. G. Howell, and G. H. Morrison, Anal. Chem., 46, 575 (1974). (10) M. J. Milano, H. L. Pardue, T. E. Cook, R. E. Santini, D. W. Margerum, and J. M. T. Raycheba, Anal. Cbem., 46,374 (1974). (11) K. W. Jackson, K. M. Aldous, and D. G. Mitchell, Spectrosc. Lett., 6, 315 (1973). (12) G. Horlick and E. G. Codding, Anal. Cbem., 45, 1490 (1973). (13) D. 0. Knapp, N. Omenetto, L. P. Hart. F. W. Plankey. and J. D. Winefordner, Anal. Cbim. Acta, 89, 455 (1974). (14) K. W. Busch, N. G. Howell, and G. H. Morrison, Anal. Cbem., 46, 1231 (1974). (15) K. W. Busch and G. H. Morrison, Anal. Cbem.. 45, 712A (1973). (16) J. A. Dean and T. C. Rains, "Standard Solutions for Flame Spectrometry", in "Flame Emission and Atomic Absorption Spectrometry", Vol. 2, J. A. Dean and T. C. Rains, Ed.. Marcel Dekker, New York, N.Y., 1971, p 327. (17) W. F. Meggers, "Tables of Spectral-Line Intensities, Part I", National Bureau of Standards Monograph 32, US. Government Printing Office, Washington, D.C.. 1961. (18) K. W. Bush, N. G. Howell, and G. H. Morrison, Anal. Chem., 46, 2074 (1974). (19) T. E. Cook, M. J. Milano, and H. L. Pardue, Clln. Cbem., 20, 1422 (1974). (20) J. D. Ganjei. N. G. Howell, J. R. Roth, and G. H. Morrison, Anal. Chem.,
a vidicon spectrometer system. The experimental results have shown how the ratio technique adequately compensates for matrix effects, thus improving the accuracy and precision of flame measurements. Trace elements can then be determined with minimal sample alteration or matrix duplication.. The system's versatility makes it amenable to a wide range of applications without lengthy instrumental modifications. Incorporating a computer interface with the vidicon system will reduce the additional time required for internal standard calculations and enable rapid experimental data interpretation. While the analyses presented in this report are not intended for adoption as routine clinical procedures, the impressive capabilities of the multichannel vidicon spectrometer in applying the internal standard method merit considerable attention.
ACKNOWLEDGMENT The authors thank J. H. Boutwell and D. D. Bayse of the Center for Disease Control, Atlanta, Ga., for supplying the samples of analyzed bovine serum, and K. W. Busch of Baylor University for his help and advice.
in press.
LITERATURE CITED (1) W. Gerlach. Z. Anorg. Allg. Cbem., 142, 363 (1925). (2) J. W. Berry, D. G. Chappell. and R. 8. Barnes, ind. Eng. Cbem., Anal. Ed., 18, 19 (1946). (3) H. M. Bauserman and R. R. Cerney, Jr., Anal. Cbem., 25, 1821 (1953). (4) R. J. Schlesinger. R. A. Lesonsky, and R. Lottritz. Clin. Chem., 18, 1005 (1972). (5) F. J. Feldman, Anal. Cbem.. 42, 719 (1970).
(21) E. Berman, Appl. Spectrosc., 29, 1 (1975). (22) R. L. Warren, Analyst(London), 90, 549 (1965).
RECEIVEDfor review August 7 , 1975. Accepted November 12, 1975. This work was supported by the National Institutes of Health, Grant No. 5 R01 GM 19905-03.
Characteristic Noise Spectra of Some Common Analytical Spectrometric Sources Yair Talmi," Ronald Crosmun,2 and N. M. Larson Oak Ridge Nafional Laboratory, Oak Ridge, Tenn. 37830
This study deals with the noise characteristics of various analytical spectrometric systems, including primary plasma excitation and flame sources. The noise power spectra of these spectrometric sources have been obtained digitally using a dedicated Fourier processor which calculated the power density functions by direct Fourier analysis. in addition, a quantitative estimate of the magnitude of these noise fluctuations was also obtained. In the Discussion section, an attempt has been made to isolate the few experimental factors that are most dominant in the generation of noise in spectrometry sources and to determine their origin. The analytical implications of the noise phenomena are also dlscussed. In particular, the merits of signal modulation techniques and multiplex transform techniques, e.g., Hadamard transform spectrometry, are reevaluated, based on the experimental noise data obtained in this study.
The original purpose of this study, which deals with the magnitude and characteristics of the noise phenomena inCurrent address, Princeton Applied Research Corp., P.O. Box
2565, Princeton, N.J. 08540.
* Current address, E. I. du Pont, Wilmington, Del. 19898.
326 * ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976
volved in the operation of various spectrometric systems, was to evaluate the applicability of Fourier and Hadamard Transform Spectrometers (HTS), to the uv-visible spectral range. The sensitivity of any spectrometric system is limited by noise originating in either the sampling device, the source, or the detection system. Most spectrometric sources are thermal; Le., they are characterized by a photon flux whose time variability obeys Poisson statistics. Similarly, the output signal of photomultiplier tubes ( P M T ) , universally accepted as uv-visible detectors, also obeys this statistical mode. This behavior of P M T s is a direct result of their quantum efficiency being less than one. Thus, in uv-visible spectrometric studies, in which the P M T is operated in the d c mode (not saturated and not as a photon counter) the signal to noise ratio (S/N) is proportional to the square root of the signal regardless of whether the detector or the spectrometric source is the dominant noise source. Conversely, in infrared (ir) spectrometry, the detector is nearly always the dominant noise source. These detectors operate in an indirect mode; e.g., absorption of heat causes a change in the electrical conductivity of a crystal, a chnage in gas volume, etc. Their noise level is proportional to the square root of the detector area rather than the signal. These de-
