Analytica Chimica Acta, 123 (1981) 57-63 o Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

SIMULTANEOUS MULTI-ELEMENT ANALYSIS BY INDUCTIVELY- COUPLED PLASMA EMISSION SPECTROMETRY UTILIZING MICRO-SAMPLING TECHNIQUES WITH INTERNAL STANDARD

HIROSHI UCHIDAa, YUKIHIRO NOJIRI, HIROKI HARAGUCHI* and KEIICHIRO FUWA

Department of Chemistry, Faculty of Science, University of Tokyo, Bunkyo-ku. Tokyo I13 (Japan)

(Received 29th April 1980)

SUTvlMARY

A micro-sampling technique, which requires sample volumes of less than 100 ~1, is used for multi-element analysis by i.c.p. emission spectrometry. One drop of sample solution in a teflon cup is nebulized through a capillary tube. Internal standardization with yttrium improves the precision of measurement_ The method is applied to the analysis of serum and whoie blood samples, after dilution or digestion.

In recent years, there has been great demand for the analysis of samples of limited volumes or weights, especially in the geochemical, biological and clinical fields. Microlitre samples have been used in flame atomic absorption spectrometry for the determination of trace elements [l-5] _ However, only one element can be determined at a time, and simultaneous multi-element analysis would be preferable, in order to save time and sample consumption. For this purpose, inductively coupled plasma (i.c.p.) emission spectrometry is the most suitable state-of-art technique for liquid samples.

Greenfield and Smith [6] first suggested a micro-sampling technique for the multielement trace analysis of oil and blood, using a ~-MHZ i.c.p. as excitation source [6] _ A micro-pipet, which contained 25 ~1 of sample, was connected to a capillary for nebulization into a heated chamber, where the aerosol was evaporated, and then introduced into the plasma. Trace elements in 25 ~1 of serum and whole blood have also been determined by use of a single-channel i.c.p. spectrometer in conjunction with a device composed of a micro-pipet and pinch clamp 173. Injection of small volumes with a peri- staltic pump for the sequential determination of trace element by i.c.p. emission spectrometry has been recommended in order to reduce physical interference from high viscosity of solutions [ 81. A tantalum filament vaporization system has also been examined for determinations at the ng ml-’ level 191.

aOn leave from Industrial Research Institute of Kanagawa Prefecture, Isogo-ku, Yokohama 235, Japan_

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The present paper deals with a simpler introduction system for the simul- taneous multi-element analysis of ~1 samples by i.c.p. emission spectrometry. No special modifications were made to the instrument. Yttrium was used as internal standard in order to compensate for the variation in the amount of sample introduced into the plasma. The method proposed has been applied successfully to the simultaneous multi-element determination of major and minor elements in 50-~1 aliquots of serum and whole blood.

EXPERIMENTAL

Instrwnenta tion and operating parameters The instrumental components and operating parameters for the measure-

ment are summarized in Table 1. The solution was fed to the nebulizer through a teflon capillary tube (30 cm long, 0.45 mm i-d.). The uptake rate of water through the tube was 0.96 ml min-’ at a carrier gas flow rate of 0.66 1 mix’. Tne r-f. power for inorganic acid solutions was usually 1.4 kW while 1.8 kW was used for the analysis of diluted samples of serum and whole blood in order to minimize the interference of concomitant organic compounds [ lOj _ The analytical lines conventionally employed [ 111 were used (see Tables 2 and 3), and the straight lines joining the points for the blank and a high standard were used as calibration graphs.

Aspiration procedure A drop of sample solution was transferred with a micro-pipet into a

sampling cup. The cup was one of a series of holes (10 mm diam., 6 mm deep), made by drilling the plane surface of a teflon rod [ 5,121. The capillary tube was removed from the blank solution, placed quickly on the drop in the

TABLE 1

Instrumentation and operating parameters

1.c.p. spectrometer Jarrell-Ash, Plasma Atom Corp., Model 96-984 SP Output power 1.4 kW and 1.8 kW Coolant gas Argon, 18 I min-’ Plasma gas Argon, 1.0 1 min-’ Carrier gas Argon, 0.66 1 min-’ Nebulizer Cross-flow type Viewing Point 15.0 mm (1.4 kW) and 18.0 mm (1.8 kW) above work coil ?olycliiomator Paschen-Runge type (75cm focal length)

Grating 2400 grooves mm” Linear dispersion 0.54 nm mm-’ (1st order, at 270 nm) Entrance slit width 25 flrn Exit slit width 100 pm (Na and K) and 50 pm (others)

Monochromator Ebert type (50 cm focal length) Integration time 20s Data acquisition system Digital Equipment Company, Model PDP8 (8K memory)

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TABLE 2

Dependence of precision on the sampIing volume with and -.+&out internal standard- ization=

Sampling Internal volume (~1) standard

R.s.d. (%)b

Al Fe Na Ti V Zn

5 No Yes

10 No Yes

20 No Yes

50 No Yes

100 No Yes

Continuous No YeS

22 16 11

7.7 12

6.1 4.2 2.2 1.8 1.0

8.1 11 8.2 7.7 9.0 2.1 14 0.4 1.3 2.8 6.7 30 6.3 6.7 5.5 1.9 27 0.4 1.2 1.7 5.9 19 3.7 4.4 4.5 3.7 20 0.6 1.9 2.4 4.0 12 3.2 3.3 3.5 0.8 10 0.2 0.4 1.1 1.2 5.2 1.2 1.3 1.3 0.5 4.8 0.1 0.3 0.6 1.2 1.6 0.5 0.4 1.2 0.9 1.3 0.2 0.2 0.9

aThe concentration of all elements were 10 Pg g-‘. The lines used were 308.2 nm (Al), 259.9 nm (Fe), 589.0 nm (Na), 334.9 nm (Ti), 292.4 nm (V), and 213.9 nm (Zn). bn = 8.

cup, and, whenever the sample had been aspirated, was immediately returned to the blank solution. Transfer of the capillary between the blank and sample solutions must be very quick, because the blank solution must be continu- ously nebulized between nebulization of the sample solutions to prevent blockage of the nebulizer. When the sample was aspirated into an empty capillary tube, it became scattered in the tube and the nebulization flow ceased.

Chemicals and standard solutions Commercially available standard solutions (1000 fig ml-‘) for atomic

absorption spectrometry (Wako Pure Chemical Co.) were used for the stock solutions of aluminum, calcium, copper, iron, lead, potassium silicon, sodium, titanium, vanadium and zinc. A yttrium stock solution (1000 ccg g-‘) was prepared by dissolving the oxide (Wako Pure Chemical Co., 99%) in nitric acid. Potassium dihydrogenphosphate was dissolved in water for the stock solution of phosphorus. Working standard solutions were prepared by appropriate dilution of these stock solutions. All dilutions and sample preparations were done gravimetrically on a top-pan balance, as this is more accurate than volumetric procedures .when small samples are involved.

Application to serum and whole blood Serum and whole blood were obtained from the hospital in the University

of Tokyo. The whole blood sample contained heparin as an anticoagulant. Commercially available control serum (Wako Pure Chemical Co.), prepared

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TABLE 3

Analytical results (pg g-r) for serum and whole blood

Sample Element Wavelength

(nm)

Sample treatment

Dilutiona Digestionb Digestionb DigestionC

50-111 sample Continuous

Serum

Control sernmd

Whole blood

Na K

Ca Fe cu

Mg Zn P Na K Ca Fe cu

Mg Zn P

Na K Ca Fe cu

Mg Zn P

589.0

766.5 317.9

259.9 324.8 279.1 213.9 214.9

3250 3170

166 162 86.2 85.3

1.47 1.67 1.01 1.07

16.2 15.6 0.64 0.63

104 106 3240 3190

165 167 78.4 77.6

1.38 1.63 1.14 1.18

18.5 18.3 0.84 0.86

109 109

1810 1840

1900 1850 54.2 56.6

567 569 - -

31.2 30.9 6.67 7.02

375 380

3120 3080

167 165 83.4 84.8

1.33 1.30 1.14 1.01

16.6 16.7 0.67 0.77

105 103 3220 3220

161 165 80.0 73.7

1.33 1.30 1.02 1.10

17.4 17.0 0.86 0.86

111 107

1770 1780

1870 1830 52.8 53.5

551 562 0.25 0.28

30.3 31.7 ‘7.11 6.93 -

358 360

alO-fold dilution. hl g digested in the teflon bomb and diluted to 10 g. ‘10 g digested by conventional method. dNa = 3149, K = 160.3, Ca = 96.24, Fe = 0.93, Cu = 1.28 pgg-‘-

by lyophilizing serum to a powder, was dissolved in water, and also analysed. Two methods for sample treatment were investigated for nebulization of 50+1 samples. In one, serum and whole blood were diluted 10 times with water. In the other, 1 g of sample was digested in a teflon-lined bomb 1133, with 3 ml of concentrated nitric acid and 0.5 ml of concentrated perchloric acid at i20°C for 4 h (serum) or 8 h (whole blood). The contents were transferred to a glass bottle, and diluted with water to 10 g. Yttrium was added as internal standard at a concentration of 10 pg g-’ in both cases. For continuous nebulization, 10 g of the blood sample was digested conven- tionally with nitric and sulfuric acids, and diluted to 20 g with distilled water. These sample treatments were carried out at the same time to avoid sample heterogeneity.

Two high standards for calibration were prepared. The first contained

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10 ccg Al g-‘, 10 pg Ba g’, 10 pg Ca g-l, 10 pg Cu g-l, 50 pg Fe g-l, 100 pg K g-‘, 10 erg Mg g-l, 300 pg Na g- I, 10 fig Zn g-l and 10 pg Y g-’ (Yttrium nitrate in water was used for the dilution of sera and whole blood to prevent their coagulation)_ The second standard contained 100 pg P g-‘, 10 clg Pb g-l and 10 gg Si g-l. Water was used as the blank solution.

RESULTS AND DISCUSSION

Figure 1 shows the yttrium (10 ccg g-‘) i.c.p. signals obtained for various sample volumes, using the monochromator (Table 1) attached to the spectro- meter. The peak height for a 100~~1 aliquot is as high as that for continuous nebulization. The relative standard deviation of the peak height recording for 50-~1 aliquots of the yttrium solution was 5.3% (n = 8).

For simultaneous multi-element analysis with the polychromator, the emission intensities were integrated for 20 s, and the analytical data were output after data processing. Table 2 shows the repeatability of the simul- taneous measurement of 10 pg g-’ of aluminum, iron, sodium, titanium, vanadium and zinc, when various sampling volumes were aspirated. Precision became worse as the sample volume decreased. Relative standard deviations of the emission intensities for atomic lines were generally larger than those for ionic lines, especially for the alkali metals.

In ternal standardization Generally, the observed emission intensity includes the net emission

intensity of the element and the plasma background (or blank) intensity; it is also influenced by viscosity and surface tension effects. This is usually corrected by internal standardization. ln the present work, variations in the amount of sample introduced into the plasma, caused by errors in pipetting and by changes in the nebulization rate, significantly influence the precision. These variations can be seen in Table 2.

For internal standardization, a 10 ggg-’ solution of yttrium, whose emission intensity was observed at 371.0 nm, was chosen because of the high sensi-

tivity and stability of the emission signal. In the data treatment for internal

--Y-Y -

5P’ lOpI zvpi 5op/ loopI cmtimals

Fig. 1. 1.c.p. emission signals of yttrium (10 rg g’). The arrows indicate the start of aspiration.

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standardization, the net intensity should be obtained to correct for the matrix effect and pipetting errors, because the background intensities for some elements are not negligible_ For example, the ratio of the net emission Men- sity to the background intensity at 371.0 nm was 5.85 for a 5Oq.d aliquot of 10 pg g-* yttrium solution. The data processing facilities of the i.c_p. spectro- meter used [ll] allow the background intensity at any wavelength, obtained for the blank solution, to be subtracted from the observed emission intensity. However, the corrected output is not provided as emission intensity but as the concentration of the element. Therefore, the background-corrected concentration for each element was divided by that for the 10 pg Y g-’ internal standard.

The precisions obtained by internal standardization are also summarized in Table 2. With the exception of sodium, the relative standard deviations are much decreased when the internal standard is used, and is less than 2% for titanium and vanadium for as little as a ~-PI aliquot.

A problem with the use of yttrium as interna.l standard is spectral inter- ference of the yttrium line at 317.942 nm with the calcium line at 317.933 nm. In the present system, the intensity of 10 pg Y g-’ at the calcium wavelength (317.933 nm) corresponded to that from 0.6 pg Ca g-‘, and this value was subtracted from the measured values for calcium. However, at the concentrations of calcium determined here, the spectral interference of yttrium was insignificant

Application to blood samples Analytical resuks for a serum, control serum and a whole blood sample

are summarized in Table 3. Internal standardization was not used in the con- tinuous nebulization experiments. The results obtained by the dilution, teflon bomb digestion and conventional wet digestion methods are consis- tent. The relative standard deviations calculated from the data for six 50-,ul samples were 2-5s for dilution and teflon bomb treatments. The results obtained for the control serum agree well with reported values, except for calcium, for which low values were found. However, in this laboratory, 74 and 80 pg ml-’ were obtained for the same sample by atomic absorption (422.7 nm) and single-channel i.c.p. emission (393.4 nm) spectrometry, respectively.

The recovery and precision for the determination of yttrium added as internal standard to diluted whole blood are summarized in Table 4. In continuous nebulization, both the recovery and precision become worse as the dilution is decreased, possibly because of variations in nebulization efficiency with the less diluted viscous blood. The nebulizer sometimes blocked and the background emission intensity increased for these samples.

Good recoveries and precision were obtained with’ pulse nebulization of 50-,ul samples. The aspiration time was only 3 s; for the rest of the time, distilled water was aspirated, cleaning the nebulizer and apparently reducing variations of nebulization efficiency. The increase in the background emission

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TABLE 4

Dependence of recovery and precision on the dilution factors in the determination of yttrium (10 rg g-‘) added to whole blood

Dilution factor

5

10 20 50 100

50 ~1 nebuiization

Recovery R.s.d.a

(B) (S)

93.0 6.0

96.7 3.9 101.2 3.2

98.9 2.9 106.6 3.1

Continuous nebulization

Recovery R.s.daa (%) (S)

- -

41.8 30 58.2 20 75.8 13 83.6 1.8

an = 8.

intensity was also negligible, even when the less diluted whole blood was used. The recovery and precision for whole blood with 5fold dilution are acceptable (Table 4). Aluminum, silica, lead, copper and barium were deter- mined in 5fold diluted whole blood, with yttrium as internal standard, and the results were in reasonable agreement with those obtained at greater dilutions.

The method developed is simple and has the advantage of allowing use of a commercially available instrument without modification, provided that internal standardization is used. There is no significant difference in accuracy and precision between nebulization of small volumes and con- tinuous nebulization, and the micro-sampling technique reduces errors from variable nebulization efficiency of viscous samples.

The authors thank Prof. C. Iida for providing the teflon bomb, and the Industrial Research Institute of Kanagawa Prefecture for giving study leave to H.U. This research was supported by Grants-in-Aid for Environmental Science from the Ministry of Education, Science and Culture_

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