Using the scanning device the thickness of the layer to be removed can be set by simply selecting appropriate values of the pulse-repetition-rate pulse-length product. Although the maximum penetration into the surface being analyzed may be from 1 to 4 pm greater than the mean eroded depth, the volume of material taken from below the eroded layer is relatively small. A layer with a distinct composition 10 pm thick may be analyzed with a sensitivity of ppm atomic without interference from the underlying layer. Errors due to dilution with the underlying layer do not become significant until the surface layer is less than 0.1 pmin thickness. The sensitivity to surface impurities is very high (limit of detection of the order of 10” atoms/cmz) but they can be identified by their rapid decay with successive scans. The technique can be applied to insulators coated with a conducting layer, and thin conducting films on insulating substrates.
While the scanning device was designed for the analysis of semiconductor materials, it is obviously applicable in any analysis where a flat surface of sufficient area is available and to the analysis of solutions. ACKNOWLEDGMENT
The authors thank the members of the Engineering Division of the Mullard Research Laboratories concerned with the manufacture of the scanning device and the staff of the Metallurgy Laboratory for carrying out the low angle sectioning. The assistance of the Measurement Department of Mullard Mitcham in carrying out the Talysurf measurements is also acknowledged. RECEIVED for review August 25, 1969. 29, 1969.
Accepted December
Internal Standardization in Atomic Emission and Absorption Spectrometry Fredric J. Feldman’ Instrumentation Laboratory, Lexington, Mass. Some of the various factors are studied which affect the resulting signal in atomic absorption and ernission-such as changes in fuel and air flow, matrix composition, and aspiration rate-are extremely difficult to control, and will adversely affect the precision. Internal standardization which effectively reduces or eliminates the variations in these factors allows the high precision measurements of which flame spectroscopy is capable to be attained. Some selection rules and experimental studies for determining the efficiency of an internal standard are presented. Limitations in the use of internal standards are discussed. Hybridization techniques-i.e., simultaneous emission and absorption measurements-can be used for greater latitude in the selection of internal standards. Increases of 2- to 3-fold in precision as well as higher accuracy are found utilizing internal standardization.
THEPRECISION of a measurement in flame spectrometry is dependent on a constant atom population and constant temperature in the area of the flame traversed by the light source. Therefore, instrumentation in which open burner systems are used for sampling, relatively small changes in sample aspiration rate, flame configuration, and air-fuel ratio, adversely affect the stability of signal measurements. These factors combine with random variations in incident light intensity and general background noise to reduce the inherent precision of analytical measurements. Greater precision of analysis is attainable with flame methods than is presently being realized. To achieve this potential improvement in analytical precision, it is desirable to analyze the error or noise sources inherent in the atomic absorption and emission techniques with the view of finding methods to compensate and correct for these errors.
Several of these potential error sources have been identified. The flame and nebulizer constitute a noise area (or error source) which is particularly suspect. Variations in viscosity, (such as sample to sample) sample composition, surface tension, nebulizer and/or air pressure will cause variations in the conversion of the sample into the requisite atomic cloud. Changes in the flame geometry and composition will also affect the absorbance of a given atomic cloud. These variations are some of the factors which limit the accuracy and precision of the flame methods. These factors have been recognized in flame emission spectrometry, where the common compensation technique is the use of an internal standard ( I ) . The application of a n internal standard to atomic absorption is so recent (2, 3) that internal standard selection criteria have not yet been completely evaluated. Reasonable criteria for selection of internal standards should include matching of excitation and ionization energies, similar vaporization rate in the flame so optimum height for the light beam is taken into account, and agreement of chemistry with respect to anion effects as well as flame chemistry. Whereas some of these may apply, early experiments failed to substantiate that any general rules can be utilized for internal standard selection. The exception to this is the alkalies and alkaline earths where the above-mentioned criteria hold fairly well. The present paper describes the utilization of internal standardization techniques to compensate for most of the (1) W. Gerlach and E. Schweitzer, “Foundations and Methods of Chemical Analysis by the Emission Spectrum,” Vol. 1, p 2,
Voss, Adam Hilger, London, 1929. (2) L. R. P. Butler and A. Strasheim, Spectrochim. Acta, 7, 1207
(1965). Present address, Beckman Instruments, Clinical Instruments Operations, Fullerton, Calif. 92634
(3) F. J. Feldman, J. A. Blasi, and S. B. Smith, Jr., ANAL.CHEM., 41, 1095 (1969).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 7, JUNE 1970
719
ROTATING MODULATOR
Figure 1. Block diagram of the IL 153, Atomic Absorption Spectrophotometer
being used. The instrument is then set in the internal standard mode and readings are taken directly. Fc 365.9
0 0
nm
I
I
1
I
I
10
20
30
40
50
PPM
Figure 2. Relationship between precision and concentration for iron in lube oil
error sources associated with the flame and nebulization processes. EXPERIMENTAL Reagents. Analytical reagent grade chemicals were used without further purification. Distilled deionized water was used throughout. Apparatus. All atomic absorption measurements were made with an Instrumentation Laboratory atomic absorption spectrometer (Model 153). The instrument was operated in either the internal standard (see below) or single channel mode. Operation of this instrument has been previously described (3). Figure 1 illustrates the optical layout of the instrument used for this evaluation. Two hollow cathode devices are excited by square wave modulation at two separate frequencies. The light from the hollow cathode devices are combined, passed through and around the flame, and then detected at two photo detectors. Thus, two separate spectrometer systems are incorporated with the flame as a common reference point. A change in the flame or nebulizer may be monitored at each channel. The intensity signals from the two detectors are demodulated at the correct frequency for each lamp by synchronous demodulation with a “lock-in” type amplifier. The signals are corrected in a pair of amplifiers so that the response is linear in concentration for the two analyzed elements. The concentration signals are then ratioed to yield the internal standard compensated analysis. Procedure. A known amount of a n internal standard element is added to all solutions including the blank. The internal standard channel is then adjusted by means of the scale and calibration controls to give a pre-set absorbance. This is to make the numbers of the digital readout the same when either single channel or internal standardization is 720
ANALYTICAL CHEMISTRY, VOL. 42, NO. 7, JUNE 1970
RESULTS AND DISCUSSION The application of a n internal standard to atomic absorption spectrometry implies the requirement of being able to measure two independent elements simultaneously in the flame. This requirement dictates certain instrumental features. The major feature is that the light from the two channels must be exactly superimposed in the flame. When the samples are analyzed, the signal from the internal standard element is ratioed to the signal from the element of interest, Since both elements are being analyzed in the identical portion of the flame, the fluctuations caused by the nebulizer and the flame affect both elements simultaneously. The ratioing thus corrects for that portion of noise due to variations in the flame and nebulizer. There are a t least three possible instrumental sources of error. These are photon or electronic limitations, nebulization variations, and changes in the flame condition or geometry. The first category contains the instrumental sources of noise imposed by photon-limitation or shot noises. This limitation is due to the random (statistical) arrival of photons at the photocathode which induces noise into the analytical system. It is within the state of the art to design the electronics so that it contributes no significant additional noise, The flame and nebulizer variations are stated separately t o emphasize that variations in these parameters manifest themselves in different ways. Nebulization efficiency or the amount of sample actually reaching the flame is affected by variations in sample viscosity, sample surface tension, sample temperature, oxidant and fuel temperature, air or gases dissolved in the sample, minor variations in the air pressure used to aspirate the sample, sample height, and sample chemistry such as protein chain formation affecting flow through the capillary. Each of these parameters affects the rate of delivery of the sample to the flame. The flame may change in geometry, hence the light absorption path will become longer or shorter. The flame may also change in relative fuel or air richness causing a corresponding change in flame temperature as well as atom and electron populations. It is interesting to analyze the precision cs. concentration curve to study the effect of photon limitation. The noise a t the base line or a t high absorbance levels is due primarily to the random arrival of photons a t the photocathode. This base-line noise adds uncertainty to the absorbance due to the analyte and a t low absorbances the base-line noise dominates the total signal and at high absorbances this base-line noise is low relative to the total absorbance. Figure 2 illustrates a precision OS. concentration plot of iron in lubricating oil. As one would predict, from these and other
Table I. Precision and Noise Measurements EXPERIMENTAL DATA cu Ca Base-line stability 0.0003 Absorbance 0.0003 Absorbance Base-line % at 0 . 3 Absorb. 0.1% 0.1% R.S.D.at 0 . 3 Absorbance 0.3% 0.6%
y 0.2 8
w
[r
s:m
ERROR CALCULATIONS R.S.D. cu Ca Noise source 0.1 0.1 a. Electronics 0.27 0.27 6. Nebulizer Nil 0.52 c . Flame d. Total (a2 b2 c2)l/* 0.3 0.6
a
0.1
-
+ +
RELATIVE FUEL -AIR RATIO
considerations, the precision becomes poorer a t lower concentrations. This actual figure closely follows the theoretical curve. The curve increases at low concentrations even though the base-line noise (Le. the photon limitation noise) decreases (due to a higher light flux) since the base-line noise becomes a greater percentage of the total signal. An example can be given to clarify this point. Assume a photon flux of 106. In the case of high absorbance I can be lo5and at a low absorbance 9 x 105. Since precision is the NIS ratio and the noise is a function of I , the following precision can be estimated. High absorbance
s
= 1,
9 N
x
=
-I 105
N
=
106 - 105 =
dy=
4 1 0 5=
-
3 x 102 3 x 102 NIS = ___ 9 x 105 3- .x. 10-4 R.S.D. = 3 x 10-4 x 100 = -0.03%
Low absorbance
s = z o - I = 1069 x 105 = 105 N = d 9 = -103
,-dT=
x
105
R.S.D.
= lov2 X loo= 1%
If the loss of precision in a given analysis is due to low absorbance--i.e., absorbances near the detection limit-the internal standard would not be expected to help. As one goes to higher absorbances, the base-line noise becomes a minor portion of the precision limitation. It is this region where the internal standard helps to the greatest extent. In order to differentiate nebulizer from flame originated noise, it is necessary to compare elements with divergent flame sensitivities, such as copper and calcium. Flame sensitivity is used to connote the dependence of an element to the flame condition--i.e., if the absorbance of an element changes markedly when the Aame condition changes from lean to rich, the element is flame sensitive. Both of these elements have approximately the same absorptivity, but vary in the precision of analysis. It is inferred that the difference in precision is due to the difference in flame sensitivity. Calcium ( 4 ) is known to be very sensitive to flame variations in the acetyleneair flame, while copper is quite insensitive to these variations in this temperature range (4). (4) G . N. Bowers and F. J. Feldman, 20th National Meeting, Amer. Assoc. Clin. Chemists, Washington, D.C., Aug. 1968, unpublished
data.
Figure 3. Relationship between absorbance and fuel-air ratios
Table I compares some analytical observations on calcium and copper and includes error calculations. The difference between base-line contribution and the observed R.S.D. in the case of copper is attributed to variations in the nebulization rate. Calcium, which is also subject to the variations in nebulization rate is also sensitive to variations in flame temperature-Le., fuel-oxidant ratio changes. The noise sources are assumed to add as the root-mean square values (Table I). Using copper data, the contribution due to nebul'ization-sample flow variations is estimated at 0.27%. This value is calculated by using the observed R.S.D. and assuming a 0.1 % R.S.D. for the electronics and no flame contribution. The variations in the R.S.D. for calcium due to flame temperature may be calculated by assuming a nebulizer contribution of 0.27 % R.S.D. as for copper since the nebulization contribution should be the same for both elements. The calculation indicates that an additional 0.52 R.S.D. is due to flame temperature variations. An internal standard element for copper analysis should be insensitive to flame temperature variations and would be expected to correct for nebulization rate changes and flame geometry (Le., path length) changes. In a similar manner calcium analysis benefits from the selection of an internal standard which is flame sensitive as strontium (4). Such an internal standard would be expected to correct for flame temperature fluctuations as well as the aforementioned sources of variation. Therefore, the internal standard for a particular element must have essentially the same relative properties with respect to the matrix and flame. These can be determined experimentally by running absorbance measurements on the elements as a function of the flame composition, etc. The elements with curves that have similar slopes that can be essentially superimposed can be used as internal standards. The selection criteria discussed in the introduction apply only to the alkalies and the alkaline earth. For example, Figure 3 illustrates the aforementioned measurements for calcium, strontium, and copper. Therefore, it would be expected that strontium would be a good internal standard for calcium and a poor one for copper. The effect of copper and strontium as internal standards for calcium is illustrated in Figure 4. The drastic decrease in the absorbance ANALYTICAL CHEMISTRY, VOL. 42, NO. 7, JUNE 1970
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I
Table 11. Internal Standardization Using Hybrid Techniques Mode of oDerationaElements Flame condition NE E/A E/E Cd/Mn Oxidizing N.C. N.C. 71 N.C. 89 Reducing N.C. 100 N.C. 98 Oxidizing Ca/Sr N.C. 47 14 25 100 46 57 Reducing 22 Oxidizing 89 N.C. 46 Sr/Ca Reducing 46 31 41 97 Oxidizing 32 98 37 32 Al/Cr Reducing N.C. 69 20 45 36 N.C. 91 Oxidizing 72 Co/Ni N.C. 19 71 Reducing 58 a Correction for a 15 change in signal in a nitrous oxide-
0 w
0
z
3 -20 -
8
4 -40
ae
-
-60 -
0
RICH
LEAN
AS
-
ASr
Figure 4. Relationship between change in absorbance and fuel-air ratios
acetylene flame, e.g. AS Where AS = change in signal without internal standard AS1 = change in signal with internal standard ~ 1 values 1 in percent, A = absorption,E = emission, N.C. correction.
of calcium when the flame was changed from a rich to a lean condition in the absence of a n internal standard was corrected to a great extent when strontium was used as the internal standard. The expected result of essentially no correction occurred when copper was used as the internal standard element for copper. Although flame conditions d o not normally change to the degree shown in this figure, observable changes do occur in the flame conditions. It is the effect of these changes that is compensated for by the internal standard technique. A series of calcium determinations in serum utilizing a strontium internal standard gave a precision of 0.21 % R.S.D. A direct measurement under the same conditions yielded a R.S.D. of 0.48%. The above mentioned results are from a n air-acetylene flame and for absorption measurements. Similar corrections occur in nitrous oxide-acetylene flames as well as in emission. The selection of internal standards for nitrous oxide-acetylene flames can also be done in a similar manner. The effect of internal standards using different modes of operation in a nitrous oxide-acetylene flame is illustrated in Figure 5. In all of the experiments, a 15 percent change in signal was caused by adjusting the fuel-air ratio. The elements were measured by emission and absorption utilizing emission and absorption internal standards for comparison. The optimum correction would then be a 0 % change in signal. A series of these experiments are summarized in Table 11. The construction of similar curves for other elements can be used to determine the optimum internal standards and the
type of measurement, such as emission or absorption, t o use in each case. Thus by utilizing simultaneous emission and absorption measurements, determining elements that track each other can be done with more versatility. In many cases, as in those above, hybridization of the two techniques is necessary for maximum precision. It must be emphasized that in selecting a n internal standard the major criterion to be satisfied is the relative flame sensitivity which should include all parameters affecting the element's absorbance in the flame. For most practical purposes, any element will correct for nebulization and flow changes. It should also be considered that the use of an internal standard call actually cause the precision to be poorer. If the internal standard element were t o be affected to a greater extent by the sample matrix than the analyte, this would cause poorer precision than a direct measurement as would the use of a n internal standard hollow cathode lamp with a larger noise, drift, or a small output leading to a larger electronic noise. An examination of internal standardization on various nebulization factors was investigated. Figure 6 illustrates a simultaneous recording of the effect of internal standardization on the change in aspiration rate. In this case, manganese is used as the internal standard for copper. The copper curve is obtained by slowly adjusting the aspiration rate to a maximum setting. This is accomplished by turning in the differential screw on the nebulizer. The step-wise like curve re-
1
2
3
RELATIVE FUEL- AIR RATIO
Figure 5. Relationship between change in signal and flame type for cadmium-manganese ratio in emission and absorption
P
.i 'T d Qtidizinq
722
ANALYTICAL CHEMISTRY, VOL. 42, NO.
=
7, JUNE 1970
I
Stoich FLAME TYPE
. -. L-
n
Reducing
N~
co/ sr
I
t
/
2
Ullrosonic Nebulization
- 60%
power
COPPER
P Sr
0
Stoichiometric flome
- Air -C2'H2
I min
TIME
-
(4sec. time constant)
Figure 7. Simultaneous recording of calcium-strontium ratio utilizing ultrasonic nebulization ASPIRATION RATE (ml/min)
Both curves give rise to the same absorbance, Sr curve shifted downward for easier comparison
Figure 6. Relationship between absorbance and aspiration rate for copper COPPER / MANGANESE
sulted each time the nebulizer is turned further in, and as would be expected, the absorbance of copper increases and finally goes off the chart. The copper us. manganese curve shows very little change, demonstrating the correction of the internal standard. The curve is expanded and, under normal operating conditions, practically no change would be observed. A simultaneous recording of absorbance curves comparing internal standardization to a direct measurement when using ultrasonic nebulization is illustrated in Figure 7. The power setting used represents an unstable nebulizing condition. Referring to the strontium curve which is recorded on the same scale as calcium, it is evident that the absorbance is changing drastically, giving rise to an extremely noisy signal. This is evident in the visual observation of the flame where it seems to pulse in an alternate red and blue color. When strontium is used as the internal standard, a relatively smooth curve is obtained indicating that although calcium is also shifting like this, the ratio stabilized the signal. An example of some competing mechanisms is illustrated in Figure 8. In this experiment a copper solution was made increasingly more viscous by the addition of glycerin. However, at low glycerin levels, rather than decreasing the signal by a viscosity effect the converse is observed and the signal increases. This is presumably due to the well documented organic solvent effect (5). When more glycerin is added to the solution, this effect is counter balanced and a decrease in the total absorbance is finally noted. In the top series of curves using manganese as the internal standard for the copper, the measurements are all essentially the same magnitude except in the case of 12 glycerin. This anomaly probably occurs because of a greater organic solvent effect on the copper than on the manganese. It is well known that temperature affects the mass rate of
flow and therefore will affect the absorbance (6). In a set of experiments in our laboratory in which iron was measured to determine total hemoglobin in blood, it was noticed that different values were being obtained for the standard on different days. This was finally traced to the fact that the standards were kept in a refrigerator and were taken out for measurement and kept at room temperature for different lengths of time. Therefore, the standard and the sample were a t different temperatures. It was thought that an internal standard should easily correct for this effect. Manganese was added to both standards and samples, and the ratio of iron to manganese was taken rather than direct iron measurement.
( 5 ) F. J. Feldman, R. E. Bosshart, and G. D. Christian, ANAL. CHEM., 39, 1175 (1967).
(6) F. J. (1968).
0.4
w O
f
8 a
C
Cu I ppm Mn 10ppm
0.4
C
1
I
I
0
12
I
I
20 27 % GLYCERINE
I
33
Figure 8. Effect of glycerin on absorbance of copper and copper-manganese ratio
Feldman and G. D. Christian, Carl. Spectrosc., 13, 139
ANALYTICAL CHEMISTRY, VOL. 42, NO. 7, J U N E 1970
723
Figure 9. Effect of temperature on absorbance of iron in dilute whole blood
0.70
-
0.65
-
DIRECT
e* 0.60a
H m
-
* 0.55 -
12
- -
INTERNAL STANDARD
-
1
0
BLOOD Fe Mn
I
I
14
16
a 5 Ng/ml KX)pg/ml
I
I
I
I
I
18
20
22
24
26
TEMP 'C
Table 111. Elements Found Useful as Internal Standards Metal determined Internal standard Ca Sr AI Cr Fe Au/Mn Mn Cd cu Cd, Zn, Mn Cd Mn Zn Mn, Cd Pb Zn V, Cr Si V Cr Ni Cd Cr Mn Cd Mi3 co Cd Na Li K Li Au Mn Mo Sn a E = Emission, A = Absorption.
This gave rise to results that were more precise and more accurate. The results of the experiments are shown in Figure 9. Note the large change in absorbance (0.1) of the iron a t 14" compared t o 25 "C. In some recent work by Feldman and Christian (8),it was noted that just a 5' change in temperature can cause a n error in the analysis of 10 percent. It would appear that internal standardization could correct for these types of errors. Other advantages are also obtained with internal standardization. If the internal standard is added a t the initial weighing of the sample, the internal standard will also correct for sample dilution errors since the internal standard element and the sample will both be subject t o the same dilution errors. An investigation of several different analyses was made in a n attempt to demonstrate the advantage of internal standardiza-
724
ANALYTICAL CHEMISTRY, VOL. 42, NO. 7, JUNE 1970
Table IV. Precision of Atomic Absorption Analyses Utilizing Internal Standards % R.S.D.4 Metal Internal determined Matrix Direct standard Ni Stainless 0.51 0.18 Steel Mn 0.83 Aluminum 0.58 alloy Fe Aluminum 1.55 0.71 alloy Aluminum 0.43 0.77 Mg alloy Cement Ca 0.37 0.79 Fe Whole 0.49 0.21 blood AI Cryolite 1.35 0.61 Gold alloy 0.85 Au 0.22 a The statistics shown here are based on a series of 10 sequential analyses. The instrument was used in a normal laboratory mode with periodic confirmation of base-line calibration. The samples were prepared according to a normal chemical dissolution method as dictated by the matrices.
tion. Table I11 enumerates the elements that were found useful as the internal standard for the particular metal determination. Table IV summarizes some of the improved precision data recorded using internal standards. It is the experience of our laboratory that internal standards usually improve the precision of analysis approximately twofold. In some cases as much as a four- t o fivefold improvement has been noted. Further work is under way at the present time to elucidate more of the selection criteria for internal standards t o apply both to atomic emission and absorption spectrometry.
RECEIVED for review February 7, 1969. Resubmitted February 4,1970. Accepted April 6,1970.
