Analysis of Blood Serum on the

Liberty Series II ICP OES with the

Axially-Viewed Plasma

Application Note
Inductively Coupled Plasma-Optical Emission Spectrometers

Author Introduction
Andrew Ryan The role of major, minor and trace levels of elements in human health has been an
important area of scientific research. In particular, research on the value of trace
elements to the diagnosis, treatment and prevention of diseases has been vast.

The advent of atomic absorption (AA) techniques and the development of the
graphite tube atomizer (GTA) has provided the means for accurate determination of
all levels of many elements in human body fluids. An advantage of the graphite fur-
nace is the small sample consumption in the determination of trace levels.
Disadvantages of flame AA are that releasing agents or modifiers are necessary and
careful control of the flame stoichiometry is important to overcome chemical inter-
ferences [1]. While the atomic absorption technique offers adequate performance,
in most cases it is a single element technique and is therefore slow.

The inductively coupled plasma mass spectrometer (ICP-MS) offers rapid, highly
sensitive, multi-element determinations. The high sensitivity of ICP-MS means that
samples can be diluted to give a reasonable working volume. Dilution is also
required for ICP-MS because of limitations imposed by the sample matrix. Typically
with ICP-MS, an upper total dissolved solids (TDS) limit of 0.2% in the solution
should not be exceeded to ensure continuous operation for an extended period [2].
At TDS levels in excess of this limit, unacceptable levels of signal instability are
commonly experienced.

Inductively coupled plasma optical emission spectrometry (ICP-OES) also offers

rapid, multi-element determinations. The sensitivity of ICP-OES is lower than that of
either ICP-MS or AA-GTA, but ICP-OES can handle higher levels of TDS than ICP-MS
and is much faster than AA-GTA. Since ICP-OES is able to analyze samples with
higher TDS, more concentrated solutions can be prepared allowing trace elements
to be measured. A disadvantage of ICP-OES for the determination of trace elements
is that sample volumes will often be small and sample consumption for ICP-OES is
typically about 1–2 mL/min. The use of a microconcentric Experimental
nebulizer (MCN) is a convenient way to reduce the sample
consumption. Such nebulizers are obtainable from several Instrumental
suppliers. For example, Glass Expansion Pty Ltd supply
An Agilent Liberty Series II ICP-OES with the axially-viewed
MCNs with free aspiration uptake rates ranging from 100 to
plasma was used for the analysis.
800 µL/min. A Glass Expansion MCN with a free aspiration
uptake rate of 400 µL/min was used in this work. The Liberty Series II ICP features a 40 MHz free running RF
generator, a 0.75 m Czerny-Turner monochromator with a 1800
This report describes the analysis of blood serum using stan-
grooves/mm holographic grating used in up to four orders.
dard quantitative calibration with aqueous standards.
The resolution of the optical system ranges from 0.018 nm in
Viscosity effects of the blood serum solutions were corrected
the 1st order to 0.006 nm in the 4th order.
using scandium (361.384 nm—ionic line) as an internal stan-
dard. Major, minor and trace elements were determined in a The instrument was controlled with a Digital Equipment
single analysis. Corporation (DEC) Venturis computer with an Intel Pentium
processor and Agilent Plasma 96 software running under
A major element in blood serum is sodium, which is an easily
Microsoft Windows 95 operating system.
ionized element (EIE) and has been reported to cause ioniza-
tion interference when present in reasonably high levels. The instrument operating conditions are listed in Table 1.
Ionization interference tends to cause a reduction in signal
intensity with increasing concentration of EIE and the effect Table 1. Instrument Operating Conditions
is prominent at interferent concentrations at or above
100 mg/L. The atomic lines of Na, K and to a lesser extent Ca Power 1.0 kW
(422.673 nm) exhibit signal enhancement with increasing con- Plasma gas flow 15.0 L/min
centrations of EIE. The effect can be easily minimized or elimi- Auxiliary gas flow 1.5 L/min
nated on a radially-viewed ICP-OES by adjusting the viewing Spray chamber type Glass cyclonic
height. For the more sensitive axially-viewed ICP-OES, many Torch Standard axial torch with 2.3 mm id injector
reports of interferences due to EIE have been described [3–5].
Nebulizer High flow microconcentric nebulizer (Glass
Reducing the nebulizer pressure and increasing the RF power Expansion Pty Ltd ), free aspiration uptake rate
has been reported to reduce ionization interference on the 400 µL/min
axially-viewed ICP-OES [3]. Scandium as an internal standard Nebulizer pressure 300 kPa
has also been found to compensate for part of the signal Pump tube Inlet - PVC, orange-green, 0.38 mm id
depression [4,5]. Generally, when analyzing samples that con- Cs solution inlet, orange-blue, 0.25 mm id
tain high levels of EIE, it is recommended that all standards Outlet - PVC, black-black, 0.76 mm id
have similar levels of EIE added (matrix matching). Pump speed 15 rpm
Sample uptake rate 160 µL/min
An alternative is to saturate the plasma with a high concen-
tration of another EIE such as cesium. Therefore, the effect of Integration time 3 seconds for Ca, Cu, Fe, K, Mg, Na, P, S and Zn
5 seconds for Al and Mn
adding cesium as an ionization buffer to the standards and
No. of replicates 3
samples was also investigated.
Sample delay time 20 seconds
Cesium was chosen as an ionization buffer as it has a low Stabilization time 15 seconds
energy of ionization, is not very sensitive by ICP-OES and, Fast pump On
therefore, spectral interference is generally not a problem.
Upward curvature limit 125%
Cesium chloride is available in a very pure form and does not
Background correction Polynomial plotted background for Ca, Cu, Fe, K,
build up in the torch injector tube as readily as other alkali
Mg, Na, P, S and Zn
salts.
Offpeak background correction for Al and Mn
The accuracy and validity of the method was assessed by the 0.015 nm left of peak
use of Nycomed Pharma “Seronorm Trace Elements Serum 0.015 nm right of peak
batch no. 311089”. PMT voltage 650 V

2
For the determination of sulfur, an Auxiliary Gas Module-2 taken when removing the stopper to avoid loss of dried mater-
(AGM-2) is required. The AGM-2 provides a nitrogen purge for ial. The vial was closed and allowed to stand for 30 minutes.
the monochromator to extend the working wavelength range The contents were completely dissolved by swirling gently.
from 189 nm down to 175 nm. The default grating order was Shaking of the vial will result in the formation of foam. Long
used for all lines with the exception of the Al 396.152 nm line term contact between the liquid and the rubber stopper
where the order was changed from 1st to 2nd order because should be avoided, particularly for the determination of zinc or
of the presence of spectral interference from the blood serum aluminium, to prevent contamination from the rubber stopper.
matrix.
Three solutions with dilution factors of 5, 20 and 100 were
Standard Preparation prepared in 1% HNO3 and 0.01% Triton X100.
Aqueous standards were prepared from Custom-Grade Multi- Scandium was added to each solution as an internal standard
element Solution Var Cal 2 (Inorganic Ventures, Inc.) and from with a final concentration of 0.5 mg/L.
1,000 mg/L and 10,000 mg/L single element standards
(Spectrosol, BDH Chemicals). The standards were made up in For the study of the effect on the addition of cesium as an
18 MΩ Milli-Q water with 1% v/v high purity HNO3 ionization buffer, 2% w/v Cs as CsCl was added online to all
(Mallinckrodt, AR SELECT PLUS) and 0.01% v/v Triton X100 solutions by pumping the solution into a “T” piece just before
prepared from a 1% w/v Triton X100 solution. Scandium was the nebulizer. The optional three channel pump was utilized
added to each solution as an internal standard with a final with one channel used to introduce the cesium solution. It is
concentration of 0.5 mg/L. possible to add the internal standard to the cesium solution
instead of each individual solution.
The following calibration standards were prepared.
Table 2. Calibration Standards Results and Discussion
Standard No. Concentration (mg/L)
Wavelength Selection
Standard 1 20 µg/L Al, Cu, Fe, Mn and Zn
1.3046 mg/L P Wavelength selection was based on the sensitivity of the line
6.6752 mg/L S and the concentration of elements in each of the solutions.
Standard 2 100 µg/L Al, Cu, Fe, Mn and Zn For most lines, spectral interference did not appear to be a
6.5228 mg/L P major problem.
33.376 mg/L S
Standard 3 0.4 mg/L Ca and K Ionic and atomic lines were selected for Ca and Mg so the
0.1 mg/L Mg effect of ionization interference on the two emission line
10 mg/L Na types could be observed. Any variation in the results would
Standard 4 2 mg/L Ca and K also show the presence of spectral interference.
0.5 mg/L Mg
50 mg/L Na The K 766.490 nm line is known to be subject to spectral
Standard 5 10 mg/L Ca and K interference from Mg, so the K 769.896 nm line was also
2.5 mg/L Mg selected. The concentration of K in this sample was approxi-
250 mg/L Na mately 8.5 times that of Mg, and consequently the Mg spec-
(for Na 330.237 nm line only)
tral interference on the K 766.490 nm line was expected to be
Rinse and calibration blank solutions were prepared from negligible. This expectation was confirmed when similar
18 MΩ Milli-Q water with 1% HNO3 and 0.01% Triton X100 results were found for K at both lines.

Sample Preparation For Cu, the 324.754 nm or 327.396 nm emission lines are gen-
Solutions were prepared from Seronorm Trace Elements erally used, although the 327.396 nm line is preferred. For
Serum, batch no. 311089. both Cu lines, a small OH emission line from the aqueous
matrix is observed and is more prominent with the axially-
The serum was reconstituted by removing the screw cap and viewed plasma than with the radially-viewed plasma. The OH
carefully lifting the rubber stopper—without removing it com- emission line is not resolved from the 324.754 nm line, which
pletely. Air was allowed to enter the vial through the grooves is used in the 2nd order (default or recommended setting),
on the lower part of the stopper. The stopper was removed whereas the OH emission line is almost completely resolved
and 3.00 mL of 18 MΩ Milli-Q water was added. Care must be from the 327.396 nm line, which is used in the 1st order.

3
Figures 1 and 2 show the wavelength scans for the Cu Blood Serum Analysis
327.396 nm and 324.754 nm emission lines in blood serum
The analysis consisted of a single Seronorm Trace Elements
diluted by a factor of 20.
Serum, batch no. 311089, that was diluted 100-fold for the
determination of Ca, Mg, Na and K, 20-fold for the determina-
tion of Ca, Mg, Na, K, Cu, Fe, P, S and Zn and 5-fold for the
determination of Al and Mn. The elements Ca, Mg Na and K
were determined in the 100 and 20-fold dilution blood serum
solutions because any variation in the result would indicate
the presence of ionization interference.

The analysis was repeated with the addition of Cs as an ion-

ization buffer. The Cs was added by pumping CsCl solution
(2% w/v Cs) into a “T” piece just before the nebulizer.
Figure 1. Wavelength scan for Cu 327.396 nm in the 1st order.
Blood serum contains high levels of sodium and the potential
for ionization interference is high. Matrix matching, such as
having equal amounts of Na in all solutions, would mean that
any signal enhancement or suppression because of ionization
interference would be the same for all solutions. To measure
major, minor and trace levels of elements in blood would then
require multiple analyses because blood serum solutions of
various dilution factors would be necessary with matrix
matched standards to be prepared for each. The aim of this
work was to show that the effect of ionization interference
could be overcome, and therefore allow all levels of elements
to be determined in a single analysis.

Figures 3–10 represent the calibration graphs for the standard

Figure 2. Wavelength scan for Cu 324.754 nm in the 2nd order. solutions displayed in Table 2, with and without the addition
of cesium. Standards 3, 4 and 5 contained varying levels of Ca,
The peak to the left of Cu (327.396 nm) is Sc and causes no K and Na. Sodium was present in concentrations high enough
problems as it is completely resolved. The tail of the OH peak for ionization interference to have considerable influence on
does contribute slightly to the signal of the Cu (327.396 nm) the signals of the other elements in the standard solutions.
but this has been successfully corrected using polynomial
plotted background correction. Figures 3– 6 show the effect of the varying levels of ionization
interference, because of the varying EIE concentration
There is also the option of using the 327.396 nm line in the between solutions, on the atomic lines of K (766.490 nm and
2nd order where the resolution will be improved by a factor 769.896 nm), Na (589.592 nm and 330.237 nm) and Ca
of 2. The sensitivity of the line is only reduced by approxi- (422.673 nm) as signal enhancement has produced upward
mately 30% when using it in second order and the peaks are curvature of the calibration. The addition of Cs nullified the
completely resolved. effect of the varying levels of ionization interference, produc-
ing a more linear calibration. Adding Cs instead of matrix
The Cu 327.396 nm line in the 1st order was selected for the
matching allows all elements to be determined in a single
analysis.
analysis because sample solutions with varying dilution fac-
tors, and varying concentrations of EIE, can be analyzed.

An ionic line for Ca (317.933 nm) was also used and upward
curvature of the calibration was not found. The calibration for
Mg (285.213 nm) atomic line exhibited little, if any, upward
curvature as did the remaining atomic and ionic analyte lines.
This is consistent with other reports [4,5] that the atomic
lines of group I and to a lesser extent, group II elements,

4
exhibit signal enhancement with increasing levels of EIE. The
atomic lines of other elements and all ionic lines tend to
exhibit signal suppression by EIE but the effect is not as
severe.

In the Plasma 96 software, the maximum % error of the slope

of the calibration, which is set in the calibration page of the
method editor, only sets the limit of downward curvature for
each specific element. The maximum % error of upward cur-
vature is set from the switches registry
(\\Varian\ICPAES\Run\Switches.reg) and is applied to all
elements. The upward curvature limit was set to 125% and
the calibration failed if the slope at the top of the curve was
more than 125% of the slope at the bottom of the curve.

Without the addition of cesium, the upward curvature for the

atomic lines of Na, K and Ca exceeded the limits and the cali- Figure 4. Calibration graph for Na 589.592 nm (atomic) with and without
the addition of cesium.
bration failed for these lines.

With the addition of cesium, the effect of ionization interference

was reduced and all elements calibrated successfully.

Even though the calibrations for the atomic lines of Ca, K and
Na failed when cesium was not added, the maximum upward
curvature limit was increased post-run so that the calibra-
tions would pass. By so doing, a comparison of the results
with and without the addition of cesium could be made.

Note that Figures 3–10 show the effect of Cs on the linearity of

the calibration and not the effect on the intensity of the analyte
peak.

Figure 5. Calibration graph for K 766.490 nm (atomic) with and without the
addition of cesium.

Figure 3. Calibration graph for Na 330.237 nm (atomic) with and without

the addition of cesium.

Figure 6. Calibration graph for Ca 422.673 nm (atomic) with and without the
addition of cesium.

5
Figure 7. Calibration graph for Ca 317.933 nm (ionic) with and without the Figure 9. Calibration graph for Mg 285.213 nm (atomic) with and without
addition of cesium. the addition of cesium.

Figure 8. Calibration graph for Mg 279.553 nm (ionic) with and without the Figure 10. Calibration graph for Cu 327.396 nm (atomic) with and without the
addition of cesium. addition of cesium.

The mean results of the triplicate analyses for the determina-

tion of elements in blood serum with and without the addition
of 2% w/v Cs are listed in Table 3.

6
Table 3. Results of the Blood Serum Analysis With and Without the Addition of Cesium
Wavelength Blood serum dilution Measured value without Cs Measured value with Cs Certified value
Element (nm) factor (mg/L) (mg/L) (mg/L)
Ca 317.933 100 94.9 ± 2.0 95.1 ± 0.8 94
Ca 317.933 20 96.4 ± 2.0 95.4 ± 1.7 94
Ca 422.673 100 94.1 ± 0.9 93.9 ± 0.7 94
Ca 422.673 20 103.2 ± 0.6 95.7 ± 1.5 94
Mg 279.553 100 19.0 ± 0.1 20.0 ± 0.2 20
Mg 279.553 20 19.3 ± 0.3 20.0 ± 0.1 20
Mg 285.213 100 19.3 ± 0.3 19.7 ± 0.2 20
Mg 285.213 20 19.9 ± 0.3 19.6 ± 0.1 20
Na 330.237 100 3101 ± 7 3151 ± 45 3080
Na 330.237 20 3314 ± 60 3307 ± 6 3080
Na 589.592 100 3305 ± 49 3166 ± 40 3080
Cu 327.396 20 1.19 ± 0.01 1.24 ± 0.01 1.27
Fe 259.940 20 1.19 ± 0.02 1.28 ± 0.04 1.3
K 766.940 100 151.2 ± 3.0 163.8 ± 2.6 168
K 766.940 20 162.8 ± 0.02 167.0 ± 0.8 168
K 769.896 100 154.5 ± 4.0 168.6 ± 3.1 168
K 769.896 20 164.3 ± 3.4 170.7 ± 0.7 168
P 213.618 20 75.5 ± 0.8 75.5 ± 1.1 –
S 180.731 20 1077 ± 6 1112 ± 17 –
Zn 213.856 20 1.51 ± 0.03 1.58 ± 0.03 1.50
Al 396.152 5 0.090 ± 0.005 0.088 ± 0.012 0.093
Mn 257.610 5 0.0073 ± 0.0003 0.0077 ± 0.0004 0.0073

The effect of ionization interference, particularly on the EIE In comparison, the effect of ionization interference on the Mg
such as K, Na and Ca, is clearly visible from the results dis- (279.553 nm) ionic line and Mg (285.213 nm) atomic line was
played in Table 3. Without the addition of Cs, variations in the small, but still present. Similar results were obtained for both
measured results were found for the atomic lines of Ca the ionic and atomic lines of Mg at the different dilution fac-
(317.933 nm), Na (330.237 nm) and K (766.940 nm and tors, although slight enhancement of the Mg (285.213 nm)
769.896 nm). The results for the 100-fold dilution blood serum line in the more concentrated solution was observed. The
solution were lower than those measured in the 20-fold dilu- addition of cesium appeared to improve accuracy of the result
tion blood serum solution because of the higher level of EIE in for the Mg (279.553 nm) ionic line, suggesting a small amount
the latter, particularly Na. of signal suppression due to ionization interference.

The effect of ionization interference on different line types Some signal depression was also observed for Cu and Fe,
was observed for the Ca atomic line (422.673 nm) and Ca although it was not severe. With the addition of cesium, the
ionic line (317.933 nm). From Figures 6 and 7, it can be seen determined concentrations were closer to the certified values.
that ionization interference had considerable effect on the
calibration of the Ca (422.673 nm) line while the Ca (317.933 With the addition of cesium, Na still appears to be affected by
nm) line remained unaffected. This is reflected in the results ionization interference when the levels of EIE are high. This is
of Table 3 with varying results found for Ca (422.673 nm) and observed for the Na (330.237 nm) as a higher result was
similar results found for Ca (317.933 nm) at the different dilu- found for the more concentrated blood serum solution. It
tion factors, without the addition of Cs. When Cs was added, would therefore be recommended that blood serum be diluted
the measured values were similar for both Ca lines and both by a least a factor of 100 when repeating the analysis to
diluted solutions. determine Na.

7
The determination of sodium was repeated with a 1000-fold
dilution of the blood serum. Standards containing 1 and 5
mg/L Na only were prepared and the Na 589.592 nm line was
used. No internal standard or ionization buffer was added.
Triplicate analyses were done and the average result was
3181 mg/L in the original sample. The analysis was repeated
using a standard high flow concentric nebulizer and the aver-
age result was 3170 mg/L. Both these results are similar to
that obtained for Na (330.237 nm and 589.592 nm) in the 100-
fold dilution blood serum solution with cesium being added.

The measured concentrations of Zn and the trace elements

Mn and Al without the addition of cesium were similar to the
certified values. With the addition of cesium, a slight increase
in the measured concentrations of Zn and Mn were found.
Although the results for Zn and Mn were slightly higher, they Figure 11. Signal stability over one hour for a 20-fold dilution blood serum
still compare well with the certified values. solution.

No certified value was available for P and S in the blood

serum batch that was used for the analysis. The measured Summary
results for P and S were however, very close to the certified
The concentrations of major, minor and trace levels of ele-
values of another Seronorm Trace Elements Serum batch. The
ments in blood serum were determined in a single analysis on
certified concentrations of the other elements for both serum
the Liberty Series II with the axially-viewed plasma.
batches varied only slightly and, therefore, the same could be
assumed for P and S. These two elements did not appear to Aqueous calibration solutions were used and the scandium
be affected by the presence of EIE as similar results were internal standard successfully corrected for the varying vis-
found with and without the addition of Cs. cosity of the sample. Scandium also exhibits signal suppres-
sion because of ionization interference and therefore compen-
Long Term Stability sates for part of the signal suppression of the other elements.

The addition of cesium as an ionization buffer considerably

No extensive evaluation of the long term stability was done
reduced the effect of ionization interference and the need for
with the microconcentric nebulizer. It appeared to operate
dilution, allowing both major, minor and trace constituents to
well with no blockage being evident. Blood serum solutions
be measured in a single analysis.
diluted by a factor of 5 were aspirated continuously for peri-
ods of more than 30 minutes with no blockage being With the addition of cesium, all measured values were in very
observed. Blood serum diluted 2-fold appeared to cause no good agreement with the certified values for the Seronorm
problems. Trace Elements Serum sample, confirming the accuracy of the
method.
A single long term stability run was done by continuously
aspirating a 20-fold dilution blood serum solution and measur- The microconcentric nebulizer performed very well with no
ing the signal for a number of elements at intervals. The blockage ever occurring during the analysis of the blood serum.
reproducibility of the measurements for Ca, Cu, Fe, Mg, Na, S Sensitivity of the microconcentric nebulizer with an uptake rate
and Zn over one hour ranged between 0.6 and 1.0 %RSD. of 160 µL/min was estimated as approximately half that of the
standard high flow concentric nebulizer operating at an uptake
The replicate precision using a 3 second integration time and
rate of 1.5 mL/min. The sensitivity could have been improved
measuring 3 replicates ranged between 0.1 and 1.7 %RSD for
by increasing the uptake rate but 160 µL/min appeared to be a
all elements.
good comprise between sufficient sensitivity, particularly for Al,
and low sample consumption.

8
References
1. M. J. Sommer, M. G. Rutman, E. Wask-Rotter,
H. Wagoner, E. T . Fritsche, “Determination of calcium in
serum samples by AAS using a fuel lean flame”, Varian
Australia Pty. Ltd., Mulgrave, Victoria 3170, Australia,
Varian AA At Work No 117, March 1995.
2. G. Hams, S. E. Anderson, “Rapid and simple determina-
tion of trace elements in clinical samples by ICP-MS. Part
1: Whole blood: As, Cd, Mn, Pb and Se”, Varian Australia
Pty. Ltd., Mulgrave, Victoria 3170, Australia, Varian ICP-
MS At Work No 15, May 1997.
3. C. Dubuisson, E. Poussel, J-M. Mermet, “Comparison of
axially- and radially-viewed inductively coupled plasma
atomic emission spectrometry in terms of signal-to-back-
ground ratio and matrix effects”, Journal of Analytical
Atomic Spectrometry, 1997, 12, 281-286.
4. I. B. Brenner, A. Zander, M. Cole, A. Wiseman,
“Comparison of axially- and radially-viewed ICPs for
multi-element analysis—Effect of sodium and calcium”,
Journal of Analytical Atomic Spectrometry, 1997, 12 897-
906.
5. A. Ryan, “Direct analysis of milk powder on the Liberty
Series II ICP-AES with the axially-viewed plasma”, Varian
Australia Pty. Ltd., Mulgrave, Victoria 3170, Australia,
Varian ICP-ES At Work No 21, August 1997.

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November 1, 2010
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