1
Project SESKUPIT
Masaryk University, Faculty of Science
Manual for laboratory courses:
Methods of sample decomposition: cryogenic grinding
microwave decomposition
Solution analysis: ICP OES and ICP MS
Laser Ablation based methods: LA-ICP-MS
Laser-Induced Breakdown Spectroscopy LIBS
K. Novotný, A. Hrdlička, V. Dillingerová, M. Tvrdoňová, E. Pospíšilová
2017
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Methods of sample decomposition: cryogenic
grinding microwave decomposition
Solution analysis: ICP OES and ICP MS
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Determination of copper in a blue print by ICP-OES
Nowadays, analytical chemistry encounters the necessity of quantitative determination of elements
in samples of all kind of states. Liquid and gas samples can be easily carried into flame or analytical
plasma such as widely used inductively coupled plasma (ICP). If there is no possibility, or we just
don’t want to bring samples directly into excitation source by spark or laser ablation, it is necessary
to transform solid and suspense samples into liquid solution. One of the common ways is to dissolve
the sample in acid solution or in a mixture of acids with addition of other reagents. However, the
correct procedure depends on the type of sample. Visible dissolution of sample does not necessarily
mean that the sample was quantitatively dissolved or that there wasn’t loss of one of the sample’s
component. Efficiency and the speed of the decomposition can be increased for instance by
increasing temperature using a heating source or even better by microwave decomposition system.
Furthermore, it’s much easier to dissolve powder sample with small particles, than monoliths such as
stones or metals. For this purpose, there is more than a one method; most common are blending or
grinding. Grinding and homogenisation of sample are often necessary steps in sample preparation of
tablets before X-ray analysis or laser ablation with ICP.
This task concerns the use of modern methods of a microwave decomposition and a cryogenic
grinding for a solid sample preparation and analysis of this sample using ICP. A paper with a blue
colour ink from basic PC printer is a good sample to demonstrate microwave decomposition as well
as cryogenic grinding for copper determination in this ink.
Cryogenic grinding for sample preparation
Cryogenic grinding is based on cooling samples to
cryogenic temperatures using liquid nitrogen, then
pulverizes them by magnetically shuttling a steel
impactor back and forth against two stationary
end-plugs in a closed vial. This method makes it
possible to grind even materials sensitive to heat or
materials that are impossible to grind at normal
temperature such as thermoplastics, hairs, textile
or organic tissues. Cryogenic grinding is mostly
suitable for materials that are soft and flexible at
room temperature. Extremely low temperatures
during the grinding suppress recrystallization and
leads to finer grain structures.
Major advantages of cryogenic grinding are higher
yield of fine particles in target range and uniform
particle size distribution.
Fig. 1 Cryogenic mill 6775 Freezer/Mill
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Procedure:
1.) Sample preparation
Blue square is printed on paper by an ink printer. Area of the printed
colour is 10x10 cm. This sample is then cut into small squares
ca. 1x1 cm and put into grinding vial (Fig. 2) with a steel impactor.
Along with the sample it is necessary to prepare a blank sample; in
this case it is pure unprinted paper. Given the fact, that grinding takes
about 25 minutes, it is best to prepare blank sample during the
sample grinding.
2.) Grinding
Vial with the sample is placed into the mill in the vial slot. It is essential
to check the amount of liquid nitrogen in the container (the level of
nitrogen cannot drop more than 2 cm below the top edge of the
container). Afterwards, the container is closed and secured with rubber
clip. Programme for paper grinding is started from the screen.
Parameters for paper grinding are shown in table 1.
3.) Sample removal
When the programme finishes, the freezer mill is opened and the vial is
removed using magnetic pen. Be careful when handling the vial, its
metal parts are frozen to cryogenic temperature. Then the vial is
opened using opener (Fig. 3) by removing the plug. Grinded sample is
then spilled on filter
paper and placed into
exicator.
4.) Microwave decomposition
Microwave decomposition is commonly used for
sample dissolution prior to elemental analysis
using methods ICP-MS, ICP-OES or AAS.
Advantages of microwave decomposition are
shortening of the time of the analysis, reducing of
the amount of acid needed, reducing of the risk of
contamination or decomposition of samples, that
are difficult to decompose. Microwave
decomposition can be either in open system (at
atmospheric pressure) or closed system. In open
systems, the boiling point of the acid establishes
parameter value
Precooling 15 min
Grinding 1,5 min
Cooling 2 min
Nr. of cycles 3
rate 12 cps
Tab. 1 Grinding parameters
Fig. 2 Grinding vials
Pic. 3 Grinding vial opener
Fig. 4 Microwave system ETHOS ONE
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the maximum decomposition temperature, whereas in closed systems is maximum limited to 250°C.
Usually, different mineral acids are used for sample decomposition (e.g. HCl, HNO3, HF, H2SO4…) or
hydrogen peroxide. Vials for decomposition are made of inert material (such as teflon) which are
transparent for microwave radiation. Heating by microwave radiation depends on type of the
sample, therefore it is necessary to monitor temperature of samples. Decomposition will be carried
out in microwave decomposition system ETHOS ONE (Fig. 4).
Principles of working with microwave decomposition system
 Vials must be clean and dry, therefore it is necessary to use gloves when operating.
 Vials and their caps are numbered, it is necessary to keep sets together.
 Output from microwave system leads to hood, which must be always on.
 Vial number 1, reference vial with thermal sensor, must always be at position 1. In this vial,
temperature is measured, therefore it is crucial to use it for a sample, not a blank.
Reagents
6ml HNO3 (65%), 2ml H2O2 (30%)
Programme
Step Time Energy T1 T2
1 00:15:00 200 ramp time
2
3
00:15:00
00:10:00
200
-
decomposition
cooling
Tab. 2 Parameters for temperature programme
Procedure:
1.) Sample preparation
0,1 g of sample is put into decomposition vial
and using acid. (Fig. 5). Analogously, blank
sample is put into second vial. Both vials are
put into safety cover and then the whole
segment is closed (Fig. 6.). Using torque
wrench the pressure gauge is tightened until
it clicks. Now the segments are ready to be
put into microwave system. It is necessary to
put there at least 3 vials, so the system is
balanced. Therefore, we use same amount of
acids in the 3rd
vial. All 3 segments are evenly
embedded into microwave system.
Fig. 6 Segment for decomposition of one
sample
Fig. 5 Vial for
microwave
decomposition
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Choosing a position on Samples touch
screen (Fig. 7) will rotate the disk so that the
chosen position is in the front. Reference
vial is placed in the position 1 as the last
one. After placing all segments except the
reference one, an upper disc is placed on
top of them as shown on Fig. 8. Then the
reference segment is placed and the
thermal sensor is connected via cable.
Lastly, the upper disc is fitted onto all
segments so that all segments are fixed.
2.) Programme settings
It is important to check all boxes on the
home screen exactly as is shown on Fig. 9.
Be sure to check the TWIST button, without
it the cable of thermal sensor would be torn
up. It is possible to check the correct
rotation of the rotor using button with a fan.
(Fig. 10).
Fig. 7 Sample screen
Fig. 8 Segments with upper disc
Output of thermal sensor
Upper disc
Segment with sample
Bottom disc with
positions
Fig. 9 Settings home screen
Fig. 10 Rotation control button
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Setting of the thermal programme is done
in the Method tab. Table is filled with
parameters for microwave decomposition
programme. Step 1 is the time of the
gradient increase of the temperature until
the required temperature is reached. Step
2 is the time of stable temperature. The
graph above the table shows temperature
profile of the programme. Using save
button is the method saved. If the method
was already created, it is possible to load it
from saved files using load button.
To launch the programme the Start button
(in the Run tab) is pushed. The progress of
the decomposition is shown on the Run
screen. (pic. 12). The door is automatically
locked at 80 °C. after the decomposition it is
necessary to wait until the temperature of
the samples drops to about 50 °C. After that
it is possible to remove them from
microwave and open them using torque
wrench. Samples are then quantitatively
transferred into 25 ml volumetric flasks.
Inductively coupled plasma optical emission spectrometry (ICP-OES)
ICP-OES is an analytical method based on the determination of the intensity of emitted light from
excited atoms and ions. Sample is introduced into the ICP as liquid form using nebuliser. Inside of
ICP, aerosol is evaporated and dried. The plasma excites atoms, which leads to photon emission and
ionization. Deexcitation causes radiation at the characteristic wavelengths of the elements involved,
and its intensity is measured with detector. Emission spectra of atoms an ions offers qualitative
information (wavelength) as well as quantitative information (intensity).
 Atomisation and excitation source
The excitation source must desolvate, atomize, and excite the analyte atoms. As atomisation
and excitation source in ICP-OES is used electrical discharge, that creates inductively coupled
plasma. An inductively coupled plasma can be generated by directing the energy of a radio
frequency generator into a suitable gas, usually ICP argon. This plasma has high electron
density and temperature (10000K) and its energy is used in the excitation-emission of the
atoms from the sample.
Fig. 11 Method tab
Fig. 12 Run tab
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 Optics of spectral instrument
Electromagnetic radiation is emitted during deexcitation of particles inside of the plasma.
This radiation consists of analytically important radiation of atoms and ions of the sample
and radiation of non-dissociated molecules (OH, CO, N2…), which does not carry analytical
information and therefore creates noise. For analytical purposes, it is necessary to split this
polychromatic radiation to individual components (emission lines), which are characteristic
for elements. For this purpose, a dispersive element is used, such as prisms, gratings or
filters. This component is one of the most important part of an ICP optical spectrometer,
since it is their quality that has the major impact on resolution. Prisms and filters don’t
achieve resolution needed for ICP-OES and therefore only gratings are used. Based on the
type of grating, ICP-OES spectrometers can be divided into 3 groups:
 spectrometer with planar diffraction grating Czerny-Turner
 spectrometer with concave diffraction grating Paschen-Runge
 spectrometer with echelle grating
Deretmination of Cu in the sample will
be carried out using ICP spectrometer
iCAP 6500 Duo (Thermo Scientific). ICP
spectrometer contains echelle
monochromator with grating with 52,9
grooves per mm. This system offers
simultaneous measuring of spectrum in
the range 166 –847 nm.
Procedure:
1.) Sample and calibration standards preparation
Calibration standards with concentration of Cu2+
1 mg/l, 0,5 mg/l a 0,1 mg/l a 0 mg/l is
prepared from calibration standard Astasol (cCu = 1 g/l). It is important to add the same
amount of acid to each calibration solution as was added to sample. Samples don’t need to
be adjusted anymore.
Fig. 14 ICP Spectrometer ICAP 6000 series
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2.) Method settings
Spectrometer is controlled using
programme iTEVA (Pic. 15). At the bottom
bar choose lock icon, which opens
„Interlocks“ window, where actual state
of spectrometer is shown (communication
with PC, exhaust system,…) including
actual temperature of detector (Fig. 16).
If any of Interlocks is red and the
temperature of the detector is
-45°C, it is possible to
start plasma.
Icon of the burner will open window with
the table „Plasma status“ (Fig. 17), where
the button Plasma On is chosen. On the
home screen of iTEVA choose the tab
Analyst. New method is created as follows:
Method => New
Fig. 15 Home screen of iTEVA
Fig. 16 Instrument status
Fig. 17 Plasma status
Fig. 18 Periodic table for the choice of elements
Fig. 12 Selection of Cu lines
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Fig. 20 Method settings tabs
Window with a periodic table is opened (Fig. 18.). Measured lines of Cu are selected after the
selection of measured element – Cu. (pic. 19.). Although, individual lines are sorted based on
their intensity, it is necessary to control interferences (right part).
From method settings tabs (Fig. 20.)
choose Analysis Preferences (Fig. 21.)
and select axial view, which is often
used for determination of elements
with low content in samples with
simple matrix. Axially viewed plasma
system looks down the central
channel of the plasma and collects all
the analyte emission over the entire
length of the plasma. In radial plasma,
spectrometer views the analyte
emission from the side of the plasma
through the background argon
emission.
In Source Settings (Fig. 22.) set up
following parameters:
Parameter Value
Flush pump rate 75 rpm
Analysis pump rate 25 rpm
Pump stabilisation time 5 s
Nebuliser gas flow 0,65 L/min
Tab. 3 Parameters for measurement
Fig. 21 Analysis preferences
Fig. 22 Source settings
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Pic. 27 Publisher icon
For each standard, set its
concentration in Standards
window (Fig. 23.). If needed,
using Add button it is possible
to add more standards. As
next step save the method
and choose Analyst tab.
3.) Calibration and analysis
Fig. 24 Upper bar with icons
Open the calibration window (Fig. 25.) by pressinng
a button with purple beaker (Fig. 24.) at the upper
bar. Immerse the nebulizer sample tube into
calibration blank and start the measurement by
pressing Run button. Analogously measure the
calibration solutions. After measuring all the
calibration standards finish the measurement by
pressing the button Done. Analysis of sample is
started by pressing the green beaker (Fig. 24.). It is
good to check stability when measuring large
number of samples by measuring one of the
standards at the end of the measurement again.
4.) Data export
Fig. 23 Standards settings
Fig. 25 Calibration window
Pic. 26 Selection of samples
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Measured data are exported using the Publisher icon (pic. 27.), right below the Analyst icon.
New report => Sample report => OK
In Sample query window (pic. 26.) set the date of measurement and select method used. Select
analysed samples from the list for which data are to be exported. Select the sample and press
OK.
Data file is exported as follows:
Export => Excel => Save
Data evaluation
The precision of the result is limited by the random errors. Random errors in experimental
measurements are caused by unknown and unpredictable changes in the experiment. Random errors
often have a Gaussian normal distribution. The mean m of measurements of the same quantity is the
best estimate of that quantity, and the standard deviation s of the measurements shows the
accuracy of the estimate. When number of measurements is small n << 10, instead of standard
deviation is calculated from range R:
𝑠 𝑅 = 𝑘 𝑛 ∙ 𝑅
Where kn is Dean-Dixon coefficient (k3 = 0,5908).
Confidence interval
Confidence intervals consist of a range of values (interval) that act as good estimates of the unknown
value. Dean-Dixon tailored a method of the calculation of confidence interval for small sample size
datasets:
𝐿1,2 = 𝑥̅ ± 𝐾 𝛼 ∙ 𝑅
where Kα is critical value of Lord distribution for confidence level α.
(for α=0,05 k3 = 1,304)
Limit of detection and limit of quantification
These limits are calculated from value 𝑥̅ 𝐵 (mean of 10 measurements of blank) and the standard
deviation 𝜎̅ 𝐵 (also calculated from 10 measurements of blank).
LoD = 𝑥̅ 𝐵 + 3𝜎̅ 𝐵
LoQ = 𝑥̅ 𝐵 + 10𝜎̅ 𝐵
Hypothesis testing
Two-sample test for a difference in mean is used when measuring sample by 2 different methods.
When the number of measurements (nA = nB = n) is most often done by simplified Student t-test. For
small size datasets is commonly used the Lord test:
𝑢 =
|𝑥̅ 𝐴 − 𝑥̅ 𝐵|
𝑅 𝐴 + 𝑅 𝐵
If the value of u is greater than critical value of Lord test at the chosen significance level α, the
difference in means is statistically significant. In that case the results from different measurements
cannot be considered as identical (for α=0,05 uα = 0,636)
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Fig. 1: Schema of ICP-MS. (http://www.chem-agilent.com/contents.php?id=53926)
Determination of copper in a blue print by ICP-MS
Principle of ICP – MS
Liquid sample is transformed to the aerosol by nebuliser and transported to the inductively
coupled of plasma with its temperature between 6 000 and 10 000 K (depending on plasma
region). Here is aerosol dried, evaporated, atomized and ionized. In ICP the ionization occurs
into the first degree only, except alkali metals and rare earth elements. Ions created in the ICP
source enter into mass spectrometer via interface system. The interface consists of sampler
and skimmer cones and there is an effective separation of argon flow from atoms and ions
flow occurred here. First the plasma beam (ions and atoms from the sample and carrier gas)
go through the sampler cone into pre-vacuum space with pressure of 100 Pa, approximately.
The supersonic expansion occurs with subsequent cooling of the plasma beam. Then it enters
into ion optic via skimmer. The ion optic serves to focusing of the beam and removing of
photons and non-charged atoms that would increase the background level of the detector.
Focused ion beam enters into collision-reaction cell filled with He that suppress possible
polyatomic interferences. Then the ions are separated in the quadrupole analyser according to
their mass to charge ratio (m/z) and ions with selected m/z exit from the analyser and hit the
electron multiplier that generates signal (Fig. 1). For more details of ICP see instruction LA-
ICP-MS.
The aim of this practicum is:
a) to quantitative deremine the total content of copper in ink (blue color)
b) to prepare calibration standards for determination of copper
c) to learn how to measure on ICP-MS
d) to calculate the limit of detection (LOD)
e) to compare the LOD with another solution analysis method ICP-OES.
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d)
a) c)b) d)
Fig. 2: Turning on of LA-ICP-MS, a) argon b) cooling, c) helium into collision cell.
a)
c)
d)
b)
Fig. 3: ICP-MS configuration. Fig. 4: Set up of method for ICP-MS.
a)
b)
Fig. 5: Ignite the plasma.
 Turning on the spectrometer:
Turn the argon (Fig. 2a) valves on the wall so that the machines have access to a flow of the
gases. Turn on the cooling system (Fig. 2b). Now open the valve on the helium cylinder (Fig.
2c). Finally, turn on the computer.
First of all, set up measurement of solution analysis → open the program Configuration, →
Sample introduction - Type click Peristaltic Pump (Fig. 3a). Now click Miscellaneous
(Fig. 3b) → SC Cooling (Fig. 3c) → OK (Fig. 3d) → OK. Then open in PC program ICP-MS-
Top.
 Analysis process:
a) Ignite the plasma
.
In order to start ICP move to the ICP-MS-Top
program and click on the plasma on icon (Fig. 5).
Wait for 45 minutes after the plasma is ignited to
start measurements. Use this icon and set up
the program for solution analysis → file → Load
Tuning Parameter File → He_roz.U → OK.
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Fig. 6: Program of peristaltic pump.
Fig. 7: Setting the acquisition method
a)
d)
b)
c)
b) Set up the method of analysis
In folder data and methods create own folder with
analysis date. Next set up the parameters of
method analysis ICP-MS-Top: Click to upper
icon Methods → Edit entire methods (Fig. 4a) →
select Method information a Aquisition → ok →
create a description of the analysis being performed
(copper amount determination in ink) →
Acquisition mode – spectrum (Fig. 4b) → ok →
periodic table (Fig. 7a) select 63
Cu and 66
Zn –
internal standard (Fig. 7b) → integration time
(Fig. 7c) 63
Cu fill 0.1 s → OK → Peristaltic pump → Uptake Speed: 0.50 rps, Uptake
Time: 40 sec, Stabilization Time: 10 sec.
Now click DataAnalysis (Fig. 7d) → Main panel → now is opening the program Online
ICP-MS Data Analysis.
c) ICP-MS Data Analysis
Once the data analysis program opens select the folder icon with a plus mark (Fig. 8a, here will
be saved the results) → click data → name the folder → create (Fig. 8b).
Now go to the DA Method (Fig. 8c) → click edit → Analys. Mode. → select Spectrum (Fig. 8d).
Then click the green arrow icon. (Fig. 8e) → Load List with element of interest → Methods and
select the name of the folder → now you see the elements of interests → click the green arrow
icon again (Fig. 8e)→ set up the units (ppb) and concentration of standards (0, 100, 500, 1000
Fig. 9a) → Return to Batch-at-a-Glance (Fig. 9b) → yes.
c)
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Fig. 9: Setting the calibration parameters.
a)
b)
a)
Fig. 8: Setting the measurement parameters in ICP-MS Data Analysis.
a) c)
d)
e)
b)
c)
a)
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Fig. 10: Start Run.
 Preparation of calibration standards
We will use the methods of calibration curve for quantification of copper in ink (with respect
to Zinc as internal standard). It is necessary to utilize internal standard because of matrix
effect which can lead to signal distortion. It is chosen according these criteria: a) similar atom
mass b) it is not present in the sample c) degree of ionisation in ICP is similar.
Prepare copper calibration standards with concentration 0, 100, 500 a 1000 µg/l in 100 ml
volumetric flask from stock solution with concentration Cu 1 g/l. Next prepare zinc internal
standard with concentration 200 µg/l from stock (1 g/l). Fill the values in the table 1.
Cu
Concentration [µg/l] 0 100 500 1000
Pipetted volume[µl]
Zn (internal standard)
Concentration [µg/l] 200
Pipetted volume [µl]
 Samples
Sample which was prepared in cryogenic mill and after microwave mineralization is
necessary to dilute 10x.
 Start run
Now start run in program ICP-MS-Top → Method → Run →
Browse (find your folder) → OK → name your measurement
(only 7 letters) → in Sample Type click CalStd (Fig. 10) →
Cal Level → 1 (measure the lowest concentration calibration
standard) → Run Method → put the tube in your sample →
OK. Mearsure all calibration standards according this
procedure and increase the Cal Level. Then measure blank
(10 x) and your sample (3x). See instruction ICP-OES for
statistical evaluation.
 Data export
Export the table of your masured data (Fig 11) in program ICP-MS Data Analysis → file →
export → export table → save in your own folder.
Table 1: Calculation of addition to calibration and internal standards.
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 Evaluation:
Calculate the concentration of copper with respect to dilution, blank and internal standard
(zinc) and convert it to the amount of copper (mtot) in [µg] in ink on whole paper surface (100
cm2
) and then to the amount of copper in one shot of laser (mCu) [fg] (see instruction for LA-
ICP-MS).
Correction of copper concentration with respect to internal standard.
𝑐 𝑟
𝐶𝑢
=
𝑐 𝑟
𝑍𝑛
𝑐 𝑛
𝑍𝑛 ∙ 𝑐 𝑛
𝐶𝑢
WHERE:
cr
Cu
real copper concentration Cu
cr
Zn
real zinc concentration – average of measured (Fig. 11).
cn
Cu
concentration of copper which you measured in sample
cn
Zn
concentration of zinc which you measured in sample
Fig. 11: data export.
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Total copper content [µg] in ink on whole paper surface (100 cm2
):
𝑚 𝑡𝑜𝑡 =
𝑐 𝑟
𝐶𝑢
∙ 𝑉
𝑚 𝑀𝑊
∙ 𝑚 𝑐𝑟𝑦𝑜
Where:
mtot total copper content [µg]
V sample volume in volumetric flask
mMW sample weight before microwave decomposition
mcryo sample weight before cryogenic decomposition
Now convert it to amount of copper in one shot of laser mCu [fg] see instruction LA-ICP-MS,
quotation 2 and then to [fg / cm2
]. Compare it with solution analysis by ICP-OES via statistic
evaluation (see. Instruction ICP-OES). LOD convert to the paper surface.
Lab report has to contain:
 Calculation of copper concentration in sample.
 Total content of copper (include the calculations) in ink on whole paper surface in
µg/cm2
.
 Statistical comparison of two solution analysis methods (ICP-MS a ICP-OES) via
LOD.
 Evaluate your results.
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Laser Ablation based methods: LA-ICP-MS
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Imaging of elements in biological tissues by means of
LA-ICP-MS
Imaging of elements in biological tissues is one of the most frequent application laser ablation
technique combined with mass spectrometry of inductively coupled plasma (LA-ICP-MS). In
the last decades, this analytical technique was developed as a powerful elemental bioimaging
method with very good limits of detection (sub µg/g) and high spatial resolution (micrometer
scale). It can open the way to an understanding of the transport of essential or trace elements
such as Cu, Zn or Pt from cytostatic drugs during the treatment period.
The aim of this practicum is:
f) to optimize the lateral distribution (a choice of suitable scan speed)
g) preparation of set of calibration standards based on an agarose gel
h) to image and quantify the distribution of Cu, Zn and Pt in tumour tissue from a mouse
treated by platinum cytostatic drug
i) to calculate the limit of detection (LOD)
j) to compare the LOD with another imaging method – LIBS.
1) Principle
The LA-ICP-MS setup consists of laser ablation system used for creation dry aerosol removed
from the sample and ICP-MS used for the elemental analysis of the ablated material. .
Focusing laser beam interacts with the sample surface, then the ablated material is transported
by a carrier gas (He) as a dry aerosol to the ICP-MS. The plasma is a high energetic source
and the ablated material is atomized, excited and ionized. Resulting ions enter into the mass
spectrometer where are separated according to their mass to charge ratio (m/z) and detected
by the electron multiplier.
Laser ablation
There are several processes when the laser beam interacts with the sample surface. The
sample surface is sharply warmed even in particular depth under the sample. A sudden
increase in pressure cases an explosion and a destruction of the surface layer (Fig. 1a) and
evaporated and atomized material absorbs further laser radiation with subsequent creation of
excited atoms and ions from the sample and surrounding gas which results in a creation of
laser induced plasma and ablated material. The particle size distribution of the ablated
material is dependent on laser ablation parameters such as are wavelength of the laser
radiation, its laser beam fluence and repetition rate. In most cases the particle size distribution
has bimodal distribution (Fig. 1b) when the largest amount of particles is in range of tens nm.
This particles are created at laser ablation process and was not agglomerate into larger
particle. The second maximum of the distribution is about hundreds nm and this particles
22
were created by agglomeration of smaller particles. The large particles are not atomized
completely in comparison with the smaller one and it results in changes of ICP-MS signal.
Fig. 1: a) Laser interaction with sample surface
http://blogs.maryville.edu/aas/files/2013/06/ablation_diagram.jpg
b) Particle size distribution formed by laser ablation
(https://media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fs41598-017-
03275-x/MediaObjects/41598_2017_3275_Fig4_HTML.jpg)
c) Inductively coupled plasma
(https://chem.libretexts.org/@api/deki/files/100468/icp-
use.png?revision=1&size=bestfit&width=309&height=451)
d) Dependence of ionisation degree to ionisation energy
(http://slideplayer.cz/slide/4108925/12/images/9/Ioniza%C4%8Dn%C3%AD+energie+(eV).jpg)
a)
c)
d)
Ionisation energy [eV]
Ionisationdegree[%]
b)
Sample with
carrier gas
23
Inductively coupled plasma
Aerosol of the sample created by laser ablation is transported by carrier gas into the plasma torch,
where (in high frequency magnetic field) argon plasma is created. Argon plasma is formed by plasma
and auxiliary gas (Fig. 2c). Plasma is said to be the fourth state of matter due to its properties, which
are very different from liquid or gas.
Definition:
Plasma is quasineutral gas of charged and uncharged particles, exhibits collection behaviour.
Collective behaviour is a movement of particles depending on local conditions and the state of plasma
in remote area.
Even though plasma can be created from any gas, noble gases are preferred thanks to their simple
spectra, to not creating stable compounds and not dissociating the atoms. They have high of ionisation
energy (Ar – first ionisation energy 15.8 eV) as well which leads to capability of ionisation of each
element except for He, Ne and F (see Fig. 1d). Plasma temperature ranges between 6 000 – 10 000 K,
depending on plasma region. Electron concentration in ICP is in range of 1020
– 1021
m-3
which is
significantly higher amount than in flame (1014
– 1017
m-3
).
Mass spectrometer
Ions created in the ICP source enter into mass spectrometer via interface system. The interface
consists of sampler and skimmer cones and there is an effective separation of argon flow from
atoms and ions flow occurred here. First the plasma beam (ions and atoms from the sample
and carrier gas) go through the sampler cone into pre-vacuum space with pressure of 100 Pa,
approximately. The supersonic expansion occurs with subsequent cooling of the plasma beam.
Then it enters into ion optic via skimmer. The ion optic serves to focusing of the beam and
removing of photons and non-charged atoms that would increase the background level of the
detector. Focused ion beam enters into collision-reaction cell filled with He that suppress
possible polyatomic interferences. Then the ions are separated in the quadrupole analyser
according to their mass to charge ratio (m/z) and ions with selected m/z exit from the analyser
and hit the electron multiplier that generates signal.
24
Fig. 2: Instrumentation of laser ablation with inductively coupled plasma and mass spectrometry
(LA-ICP-MS). (D. Günther, B. Hattendorf, TrAC (2005), 24(3), 255-265).
2) Experimental Procedure:
 Preparation of calibration standards
Prepare 5 calibration agarose gel standards with addition of standard solution according
the table 1.
Agarose gel
Addition of standard a b c d e
Pt [µg ∙ g-1
] 0 1 2 10 20
Cu, Zn [µg ∙ g-1
] 0 10 20 50 100
Prepare the working solution (100 ml) that contains 100 mg ∙ l-1
of Pt, Cu, Zn from a stock solutions 1
with concentration of 1 g ∙ l-1
. Calculated volume [µl] of the addition from working solution 2 add to
0.1 g agarose and add up to 5 ml of MQ water in small beaker. Then put it on the magnetic hotplate
stirrer and boil it weakly under constant stirring until it becomes homogeneous (5 min, approx., see
Tab. 1: addition of standard to agarose gels.
25
Fig. 3). 100 µl of mixture pipette on the small glass slide and let to gel. Then attach the 5 small glasses
to the large glass slide with the double sided adhesive tape.
1
2
3
4
5 6
7
8
9
1 10
2
3
4
5 6
7
8
9
11
Agarose gel
Standard addition a b c d e
Pt [µl] Cu,
Zn [µl] Water
[ml] 5
Fill the calculated amount of working solutions into Tab.2.
 Sample handling
Remove the sample holder from the ablation chamber and put the sample
of the tissue and glass with calibration standard on it (Fig. 4) and attach it
with the double sided adhesive tape to secure against a movement of the
sample in ablation chamber during laser ablation.
Solution 1 Solution 2
Agarose
gel
? µl
10 x diluted
? µl
1 g ∙ l-1
Tab. 2: Calculated standard addition [µl] to agarose gels.
Fig. 3: Preparation of calibration standards.
Fig. 4: Sample handling.
26
a) c) e)b) d)
Fig. 5: Turn on of individual parts of instrument LA-ICP-MS, a) argon, helium, b) cooling, c)
helium to collision-reaction cell, d) and e) laser.
3) LA-ICP-MS Measurement
Turning on the machines:
Turn the argon and helium (Fig. 5a) valves on the wall so that the machines have access to a flow of
the gases. Turn on the cooling system (Fig. 5b). Now open the valve on the helium cylinder (Fig. 5c).
In order to turn on the laser, flip the switch on the back of the machine
(Fig. 5d) and turn the key found under the machine (Fig. 5e). Turn on the flow meter. Finally, turn on
the computer.
Now open the desktop programs New Wave Research – Laser Ablation, ICP-MS-Top, and Smart
Control. On the Smart Control system select file, open, and then find the serial number of the
flowmeter. Now click OK. On this display 100% flow is a flow rate of 2.00 L/min.
The air in the ablation chamber must be flushed after the ablation chamber is opened. In order to do
this, go to the New Wave Research program and switch the system from bypass mode to purge mode
as shown in figure 6.
Fig. 6: Switching the mode from bypass to purge to online.
27
Now move to the Smart Control program and increase the Helium flow to 50% (1.00 L/min). Allow
the ablation chamber to be washed for approximately 1 minute and just before the ablation chamber
moves from the purge mode to the online mode, reduce the He flow to 1%.
ICP-MS-Top
In order to start ICP move to the ICP-MS-Top program and click on the plasma on icon. The plasma
igniting should be visible on the Mass Spectrometer (Fig. 7). Wait for 45 minutes after the plasma is
ignited to start measurements. During the waiting period, it is a good idea to start setting up the
methods.
Fig. 7: Ignited plasma in the Mass Spectrometer.
In the folder data and methods create new folder and name the folder after the analysis date. Now the
ICP-MS-Top measurement conditions must be set.
Methods → Edit entire methods → select Method information a Aquisition → ok → create a
description of the analysis being performed → Time Resolved Analysis → ok → periodic table (select
the elements being analyzed) → the integration time for each element should be as follows: Carbon
0.01s, Silicon 0.1s, Copper 0.1s, Zinc 0.1s, Platinum 0.3s. → Real Time Plot select extract → Time
window → 500 s → OK → Aquisition → 9990 s (max. time analysis) → OK.
ICP-MS Data Analysis
Once the data analysis program opens select the folder icon with a plus mark. Click data. Name the
folder. Click create. Now go to the DA Method tab. → click edit → Analys. Mode. → select
Timechard (Fig. 8). Then click the green arrow icon. → Load List with element of interest →
Methods and select the name of the folder (Fig. 8). Click Batch-at-a-glance on the right → yes.
28
Fig. 8: Editing the data analysis information. Fig. 9: Selecting the correct folder for data analysis.
New Wave Research
Now move to the New Wave Research software. Set the frequency to 10 Hz (fig. 10a), the laser beam
size to 100 µm (fig. 10b), the energy to 96 % (cca 8 J/cm2
) (fig. 10c).
Now it is necessary to prepare an ablation pattern for each sample. Using the microscope find the
outline of the tissue sample and note the leftmost, rightmost, topmost, and bottommost points of the
tissue. Now click tools, select line and create a line on the page (left click, drag, left click, right click).
On the left-hand side of the screen under scan patterns select the line just created and right click (Fig.
11a). Now click on edit endpoints (Fig. 11b). Edit the X coordinates to be the leftmost and rightmost
points. Edit both the Y coordinates to be the topmost point. Click OK. Now right click the line again
and select properties. Change the scan speed to 200 µm/sec
(Fig. 12a), the output energy to 96 % (Fig. 12b), the spot size to 100 µm (Fig. 12c), and then check
Default (Fig. 112d). Click OK (Fig. 12e).
Fig. 10: a) Frequency, b) laser beam size, c) energy percentage
a b c
29
Right click the line again and select Duplicate Scans. Each line should be 0.11 mm apart, so based on
the length of the tissue determine the number of copies necessary to create the grid (Fig. 13). Enter this
number and click OK. Make sure that the grid encompasses the entire tissue area.
One line for each calibration standard must also be created. Each line should be at least 2 mm in width
of the calibration gel.
Now go back to ICP-MS-Top software and select Method and then Run. Choose where the results
should be saved. Click OK. Name the measurement (only use 7 letters). Click Run Method. The ICPMS-Top
software will start collecting data and should show data looking similar to figure 14.
In the New Wave Research program click Run Experiment and select Enable Laser During Scans.
Change the laser warm up time to 5 seconds. Then click Run.
a
b
a
b
c
d e
Fig. 13: Adjusting the copy pattern.
Fig. 11: a) Selecting the line, b) edit
size, endpoints
Fig. 12: a) scan speed, b) output energy, c) spot d)
select default, e) OK
30
Once the laser has gone through the entire ablation pattern click Stop Run in the ICP-MS Top
program.
Importing the Results:
Using the ICP-MS Data Analysis program and select File → import samples, select the folder made
earlier, and click OK. On the chart below right click and select Tabulate Chart and then select csv
Data (Fig. 15).
Fig. 14: ICP-MS-Top software after starting to run the method.
Fig. 15: Converting the data into csv file.
.
31
The data collected should look similar to the data in figure 16.
Turning off the device:
In the ICP-MS-Top program click the plasma off icon at the top left portion of the screen. Now close
all programs and shut down the computer, the cooler, and the laser. The gases must also be switched
off.
Creating the maps:
Start by opening the Laser Ablation Tool program. Click load data from selected file and choose the
csv file. Click next. Now sync one of the elements by setting the start time of the measurement. Next
set the rising and falling steps as well as the period of signal. Continue adjusting these settings until
the intervals on the graph turn gray, as shown in figure 17.
Click next and set the speed of the laser and the distance between the two laser tracks (the distance
should be 110 µm). Then click finish.
To save the collected data click the XLS icon at the top left corner, name the file, and save it.
Now open the file in excel. There should be an excel sheet for each element. Select an element and
highlight all of the data. Then, go to insert and under graphs click on templates. Select the template
with the icon shown in figure 18.
Fig. 16: Data in converted from the ICP-MS data analysis to csv format.
32
Fig. 17: Using the laser ablation tool to sync each element.
Fig. 18: Selecting an appropriate template for the map.
33
Fig. 19: Correction of picture aspect ratio.
Once the graph is created, right click, click move graph → new sheet → and then name the graph.
Next, right click on the edge of the graph and click on format axis. Adjust the minimum and maximum
values to create an appropriate range for the legend. The main unit should be one order lower than the
maximum.
Now, use the cutting tool on the computer to cut out the graph from the excel sheet. Save the image
and then open it with the program XnView (any other enabling basic image processing). It is
necessary to adjust the aspect ratios of image (see excel µm x µm) → picture → size → (Fig. 19) and
save it. Create panoramic picture with scale → panorama → add (Fig. 20a) → select our picture →
add (Fig. 20b) → add scale picture as well → ok (Fig. 20c) → create (Fig. 20d).
34
Fig. 21: Description of scale.
At the end insert the describtion of scale (units, name of sample, etc.) → picture → add text → write a
text (Fig. 21) → ok → use the mouse to select the position of text in picture → save picture.
Fig. 20: Creation of panoramic picture.
b)
c)
a)
d)
35
EVALUATION:
 There are 2 very important parameters which have to be optimize before you start to measure
the real samples. Scan speed [µm ∙ s -1
] influence the a) relative broadening Δwrel (equation 5) end
thereby lateral resolution and b) time of analysis. One of the other important parameter is c)
sensitivity which is expressed by limit of detection (LOD). You will calculate LOD1 of Cu in ink to
compare LIBS and LA-ICP-MS and LOD2,3,4 of tissue sample for each determined element (Cu, Pt,
Zn).
 Amount of copper [fg] in one shot of laser:
It is known the total copper content (mtot) on the paper (Stot - Fig. 23) determined by solution
analysis via ICP-MS. Now convert it to the copper amount in one shot of laser (mCu) according the
equation (1) and (2). Diameter of laser spot is 100 µm.
𝑆 𝑝 = 𝜋𝑟2
= 3,14 ∙ 502
= 7854 𝜇𝑚2
(1)
𝑚 𝐶𝑢 =
𝑆 𝑝 ∙ 𝑚 𝑡𝑜𝑡
𝑆 𝑡𝑜𝑡
(2)
 Limit of detection (ink):
Calculate for each scan speed and compare (fig. 22).
𝐿𝑂𝐷1 =
3 ∙𝑆𝐷∙ 𝑚 𝐶𝑢
𝐼 𝐶𝑢
(3)
Fig. 22: Different scan speed line by line.
Sp
w
80 µm ∙ s-1
100 µm ∙ s-1
150 µm ∙ s-1
200 µm ∙ s-1
300 µm ∙ s-1
400 µm ∙ s-1
500 µm ∙ s-1
1000 µm ∙ s-1
100mm
Stot
100 mm
Fig. 23: Original paper size.
36
WHERE:
SD is standard deviation of background (10 values of background – intensity of copper before the
starting ablation).
ICu is average intensity of Cu (5 values of intensity in ink line during the ablation)
mCu is amount of copper [fg] in one shot of laser (diameter of laser spot 100 µm).
Convert LOD1 to [fg/µm2
].
 Limit of detection in tissue sample:
𝐿𝑂𝐷 𝑍𝑛 =
3 ∙𝑆𝐷
𝐺𝐿 𝑍𝑛
(4)
WHERE:
SD is standard deviation of background (10 values of background – intensity of zinc before the
starting ablation).
GLZn is guideline of calibration curve (the average amount of the line intensities is plotted to y-axis
and concentration of added amount [µg/g] of element of interest to x-axis).
 Relative broadening (ΔWrel):
Lateral resolution of imaging is quantified as relative broadening (Δwrel) of width ink line (w) –
(fig. 22) measure it by microscope in program New Wave Research - Laser Ablation System,
∆𝑊𝑟𝑒𝑙 =
∆𝑤 𝑎𝑝𝑝− 𝑤
𝑤
∙ 100 (5)
where wapp is measured width line and w is real width of the ink line (Fig. 24).
37
Lab report has to contain:
 Pictures of quantitative determination of copper and zinc distribution in tumour tissue.
 Calculation of LOD1 [fg/µm2
], LODZn,Cu, Pt [µg/g] and relative broadening ΔWrel [%]
 According LOD1 and ΔWrel compare two imaging method LA-ICP-MS and LIBS and with
LOD1 compare method of solution analysis (ICP-MS and ICP-OES) as well.
Fig. 24: Record of line scan speed 200 µm/s and the laser spot diameter 100 µm together with
determination of apparent width Wapp which was obtained as the trend intersections of line 1 and 2 and
trend lines 3 and 4, respectively. These intersections indicate the onset and end points of 63
Cu signal
intensity in ink line.
38
Laser-Induced Breakdown Spectroscopy LIBS
39
Determination of Cu in a blue print by LIBS technique
Principle
Laser-Induced Breakdown Spectroscopy (LIBS) is a method of atomic emission
spectroscopy which uses radiation of microplasma created by the interaction of focused laser
beam with the sample surface.
Laser beam reaching the sample surface causes rapid heating of the material and
subsequent ablation. Small amount of the material is being released (in the order of tens to
hundreds of ng) and a radiation of microplasma created is detected and evaluated. After the
end of the pulse microplasma is expanding and continuous bremsstrahlung is emitted. After
a moment in the order of hundreds of ns microplasma starts to cool down rapidly, the
intensity of bremsstrahlung decreases and characteristic emission lines of the atoms and
ions are left in the spectrum. It is possible to determine the elemental composition of the
sample based on matching the lines observed to particular elements (Fig. 1).
Fig. 1 Scheme of the processes in Laser-Induced Breakdown Spectroscopy [1]
The method doesn’t require any sample preparation and it is possible to measure in the air at
the atmospheric pressure. Possibility of remote measurements is among the advantages of
this method, either with so called remote LIBS using an optical fibre or stand-off LIBS with a
telescope. LIBS is microdestructive and, therefore, usable when other methods of sampling
are too destructive.
Thanks to a good spatial resolution depth-resolved analysis (Fig. 2) can be carried out and
maps of elemental distribution (Fig. 3) can be created. During the depth-resolved analysis
the laser pulse is repeatedly directed to one point, one spectrum is acquired for each pulse
and intensity of significant line as a function of number of pulses is observed. Depth profiling
can be used e.g. to distinguish layers of multi-layered surfaces. Elemental distribution
analysis is carried out in a pre-set grid of points in the area of interest. One pulse is directed
to one point of the grid and a spectrum is acquired. Elements present in the area of interest
can be given different colours and illustrative maps of their distribution can be created.
Fig. 2 Example of the depth profile of a three-layered sample Fig. 3 Elemental distribution of Al
40
Instrumentation
Modified ablation system New Wawe UP-266 MACRO (Fig. 4) is equipped with a solid-state
Nd:YAG laser operating at fourth harmonic frequency (~ 266 nm). The sample holder can be
moved in x and y axes using a software, while the sample is recorded with a camera. That
enables scanning of the sample surface. Measurements are usually carried out in the air at
the atmospheric pressure. However, the environment of the sample and the pressure can be
adjusted by using the ablation cell. Emission of microplasma is recorded and transmitted
through an optical fibre to a Czerny-Turner monochromator (Jobin Yvon, TRIAX 320,
Francie) and it is detected by an intensified CCD (iCCD) camera (PI-MAX 3, Princeton
Instruments, USA). The intensity of the signal is then evaluated as a function of wavelength
and can be further elaborated.
Fig. 4 New Wawe UP-266 MACRO
Turning-on:
1) Turn on the power supply of the laser system UP-266 MACRO
2) Turn on the laser UP-266 MACRO with a 0/I switch in the back
3) Turn on the Dell computer (black one)
4) Turn on the MEO laser program
5) Turn on both delay generators by turning the POWER switch to ON
1) 3)
2)
4)
41
6) Turn on the pulse generator and divider, both values are pre-set to 005 (~ 2 Hz)
7) Turn on the source and the control unit of the detector using the 0/I switch in the back of
the source
8) Turn on the HP computer (silver)
9) Turn on Winspec program, choose Restore to last setting.
10) Turn on the ICCD camera using the ON/OFF switch in the back of the detector
5)
6)
7)
42
Analysis procedure
1) Sample preparation
Cut out the printed sample. Adjust the sample to the base using a double-sided adhesive
tape. Place the base onto the sample holder. Adjust the sample using the software so it is in
the middle and parallel to the axes in the software.
Turn on the light above the sample using the Ring mode
Focus the sample surface using the micro-shift screw.
Sample holder control:
2) Experimental set-up
Micro-shift screw
for up and down
movement
Sample holder
with the screws
to set the leaning
angle of the
sample
Revolving screw
for focusing
Arresting screw
- first loosen it,
set the sample,
then tighten it
again. Secure the
sample holder
with your hand
the whole time!
43
Parameters of the spectrometer and the detector are set up first using the HP computer.
Characteristic spectral lines of copper lie at 324.7 nm and 327.4 nm. Set the wavelength in
the Winspec program to 327 nm. Choose Spectrograph → Move…→ Move to: 327 nm.
Grating n. 2 (2400) and a 0,6 slit are already pre-set.
Choose the blue Pulse button and set the Gate Width to 5 µs and the Gate Delay to 0,5 µs.
Press Apply.
Choose Acquisition → Experiment Setup… → tab Main → set Number of Spectra to 1800.
44
… → tab Data File → set the Name: SESKUPIT in
the file C:\Program Files\Princeton Instruments\Winspec\Cviceni SESKUPIT
Go to the Dell computer. Set the crater diameter size to 780 µm in the MEO laser program.
Set the laser energy to 80%.
In the MEO laser program choose File → Open Experiment… → open rastr 30x30
seskupit.LAE
45
Choose Properties and check the parameters setting according to the table
Set the position of the red axes cross according to the picture. Choose Options → Move
Scans to Crosshairs. The sample should be approximately in the middle of the raster.
This value has no impact on the frequency, as
the laser is connected to the pulse divider.
46
47
3) Measurements
In the Winspec program press the green button ACQ (Acquire).
Defocus the sample using the revolving screw
In the MEO laser program choose Run Scans → tick Manually Control Valves and Enable
Laser During Scans
!!!ALWAYS PUT ON PROTECTIVE GLASSES BEFORE TURNING THE LASER ON!!!
Choose Run
Measurement time ~ 25 minutes
Revolving screw
for focusing
48
Fig. 5 Raster used for surface mapping – there are 30 points of ca. 100 µm in diameter in each line, arrows show
the direction of measurement of individual points
49
4) Data export
In the Winspec program choose the Tools tab → Convert To ASCII
Choose Choose Files → Oblast hledání: C:\Program Files\Princeton
Instruments\Winspec\Cviceni SESKUPIT → Choose and open the last file.
Choose Choose Output Directory → C:\Program Files\Princeton
Instruments\Winspec\Cviceni SESKUPIT
50
Choose Convert To ASCII for selected files. Check the creation of the file SESKUPIT.txt in
C:\Program Files\Princeton Instruments\Winspec\Cviceni SESKUPIT
5) Data elaboration
Copy the SESKUPIT.txt file to the desktop. Open the SESKUPIT.xlsx file on the desktop →
choose the sheet Original data → choose the tab Data → in the first section Načíst externí
data choose Z textu → import the SESKUPIT.txt file from the desktop
51
Do not make any changes in Průvodce importem textu (1/3) and (2/3) and choose Další →
in Průvodce importem textu (3/3) choose Dokončit → in the table Importovat data check the
data location and choose OK
The system adds a vacant even-numbered line with no information to the data file. These
even lines need to be removed. Click to A1 box in Original data sheet and press
Ctrl+Shift+S. Data from odd-numbered lines will be copied to 1. smazání řádků.
52
Copy the 324.7 nm column from 1. smazání řádků with the maximum signal intensity to 2.
rozdělení řádků.
Regarding the course of the raster measurements (Fig. 5), it is necessary to transpose 30
values from the column to the line, while it is necessary to both transpose and reverse the
order of the next 30 values (change of the course in the raster in every second line) and
repeat until the end.
Click to A3 box and press Ctrl+Shift+L for transposition of the odd-numbered lines.
Analogously press Ctrl+Shift+P for reversal and transposition of the even-numbered lines.
Final data for elemental map creation will be displayed in Finální tabulka.
53
Mark all values in the table → choose tab Vložení → Grafy → Šablony → choose template
according to the picture
Adjust the vertical axis of the elemental map so the maximum value approximately
corresponds to the maximum intensities.
54
In the MWSnap (camera icon on the toolbar) choose Libovolný úhelník and mark precisely
the area of the map → save the image to the desktop → open the image in the XnView
program
In the XnView program choose the tab Nástroje → choose Panorama…
55
In Panoramatický obrázek choose Přidat… → in Zvolte soubory choose the saved map and
choose Přidat, then choose škála 1 and Přidat → press OK → back in Panoramatický
obrázek add selected files, then click on Vytvořit…
Choose file Obrázek → Přidat text → add legend accorging to the picture → copy the final
map to the lab report
56
EVALUATION:
Visually compare the elemental maps acquired by both techniques (LIBS a LA-ICP-MS) and
describe the differences.
For further comparison of both techniques calculate the detection limit (LOD) and lateral
resolution (according to Δwrel).
Limit of detection calculation:
𝐿𝑂𝐷 =
3 ∙ 𝑆𝐷 ∙ 𝑚 𝐶𝑢
𝐼 𝐶𝑢
Where:
SD standard deviation of the background (measured on the plain paper without print)
ICu average intensity of Cu calculated using at least 5 values in the area of blue print (see
LA-ICP-MS instructions)
mCu weight of Cu in fg on the crater surface of 100 µm in diameter (see Solution analysis
instructions)
For the calculation of copper weight per crater surface go as follows:
The content of Cu (mcelk) in the whole surface (Scelk) of the printed square is related to the
crater surface calculated using the laser beam diameter of 100 µm (crater surface is actually
bigger than the laser beam diameter, we will, however consider it equal to simplify the
calculations).
𝑆 𝑝 = 𝜋𝑟2
= 3,14 ∙ 502
= 7854 𝜇𝑚2
𝑚 𝐶𝑢 =
𝑆 𝑝 ∙ 𝑚 𝑐𝑒𝑙𝑘
𝑆𝑐𝑒𝑙𝑘
Final limit of detection related to the crater surface [fg] will be related to the unit surface
[µm2
].
Relative enlargement of the printed line (ΔWrel):
𝛥𝑤𝑟𝑒𝑙 =
𝛥𝑙 − 𝑙 𝑟
𝑙 𝑟
∙ 100
Where:
Δl apparent width of the printed line from the acquired map [mm]
lr real width of the line (measured in the MEO laser program)
57
Fig. 6 Apparent width estimation
For calculation of the relative enlargement of the printed line see LA-ICP-MS imaging
instructions using the graph (Fig. 6).
Lab report will contain:
 Images of Cu distribution in the letter A acquired by LA-ICP-MS and LIBS
 Calculations of relative enlargement of the printed line and limit of detection
 Comparison of the sensitivity and lateral resolution of both techniques based on these
parameters
 Comparison of solution analysis (ICP-OES a ICP-MS) and imaging methods (LA-ICPMS
a LIBS) based on LOD related to the unit surface
References:
[1] What is LIBS? [online]. [accessed 2016-09-05]. Accessible from:
http://www1.uwindsor.ca/people/rehse/15/what-is-libs
[2] Fyzikální princip LIBS [online]. 2014-11-09 [accessed 2016-10-10]. Accessible from:
http://libs.fme.vutbr.cz/index.php/teorie/fyzikalni-princip-libs-zaklady
0
2000
4000
6000
8000
10000
12000
14000
126.5 127 127.5 128 128.5 129 129.5 130 130.5 131 131.5 132
I[A.U.]
l [mm]
ab
c d
l