Sample Analysis Design

Solution Mode
 Sample Analysis Design –
Step I – Sample preparation
•  The quality of your data will only be as good as the
‘quality’ of your sample –

–  i.e. did you adequately prepare your sample in the clean lab?

–  With respect to the destruction of matrices for samples requiring

digestion

–  Did you adequately spike samples with the correct internal

standard?

–  Sample handling protocol is extremely important, e.g. weighing

 Sample Analysis Design –
Solid Samples
•  Analyze in solid state via LA-ICP-MS?

•  Analyze in solid state via SIMS – secondary ion

mass spectrometry?

•  Convert into a glass bead and analyze via XRF

– x-ray fluoresence?

•  Take powder and digest into solution with acids?

 Sample Analysis Design-
Liquid (aqueous) samples

–  run as-is?

–  filter then run?

–  dilute then run?

–  acidify, dilute, then run?

 Sample Analysis Design
•  Method of sample preparation also depends upon the
elements of interest

•  e.g. don’t analyze your samples in a hydrofluoric acid

medium if you wish to measure Si abundances – why?

•  Elemental concentration determinations at ultra-trace

level (ppb, ppt) are very susceptible to contamination
during sample preparation and therefore should be
conducted in clean laboratory environments
 Sample Analysis Design –
Clean room environment
•  Laboratory ‘clean’ room is a facility in which the
concentration of airborne particles is controlled
to specified limits.

•  Eliminating sub-micron airborne contamination is

a control-driven process since contaminants are
generated by people, process, facilities and
equipment. Hence, sub-micron particles must be
continually removed from the air.
 Sample Analysis Design –
Clean room environment
•  Typical office building air contains from 500,000
to 1,000,000 particles (0.5 microns or larger)
per cubic foot of air.

•  A Class 100 cleanroom is designed to never

allow more than 100 particles (0.5 microns or
larger) per cubic foot of air. Class 1000 and
Class 10,000 cleanrooms are designed to limit
particles to 1000 and 10,000, respectively
 Sample Analysis Design –
Clean room environment
•  A human hair is about 75-100 microns in
diameter. A particle 200 times smaller (0.5
micron) than the human hair can cause
major disaster in a clean room.

•  Human hair typically concentrates

elements/contaminants such as Pb!
 Sample Analysis Design -
Sources of contamination
•  Facilities:
–  Walls, floors and ceilings
–  Paint and coatings
–  Construction material (sheet rock, saw dust
etc.)
–  Air conditioning debris
–  Room air and vapors
–  Spills and leaks
 Sample Analysis Design -
Sources of contamination
•  People:

–  Skin flakes and oil

–  Cosmetics and perfume
–  Spittle
–  Clothing debris (lint, fibers etc.)
–  Hair
 Sample Analysis Design -
Contamination Control
•  HEPA (High Efficiency Particulate Air Filter) -
Extremely important for reducing contamination -
filter particles as small as 0.3 microns with a
99.97% minimum particle-collective efficiency.

•  Clean room design – requires air flow dynamics

to be the least disruptive as possible – laminar
flow

•  Cleaning!
 Sample Analysis Design -
Contamination Control
•  Purity of reagents, acids, cleanliness of digestion
vessels, sample bottles, etc can dramatically effect
background levels and data quality

•  If possible, use highest purity commercial acids

•  At the minimum - sometimes need to further process

reagents – e.g. acid distillation in our laboratory

•  Digestion vessels made of disposable fluorinated

polymers (teflon, PFTE, PFA, etc)

•  Solutions stored in polypropylene or equivalent

 Sample Analysis Design –
Concentration terminology
•  Concentrations are typically expressed as either
µg/g, or µg/ml (1 µg - microgram = 1 x 10-6
grams), or ppm, ppb, ppt

–  ppm = parts per million = 1 x 10-6 g/g = 1 µg/g

–  (µg = microgram)

–  ppb = parts per billion = 1 x 10-9 g/g = 1 ng/g

–  (ng = nanogram)

–  ppt = parts per trillion = 1 x 10-12 g/g = 1 pg/g

–  (pg = picogram)
 Sample Analysis Design –
Concentration terminology
•  E.g. Zircon – ZrSiO4

–  ZrO2 = 67.2 wt%

–  SiO2 = 32.8 wt%

–  Atomic mass of Zr= 91.224

–  Atomic mass of Si= 28.0855
–  Atomic mass of O= 15.9994

–  % Zr in ZrO2 = 91.224/(91.224 + (15.9994*2)) = 74

–  % Si in SiO2 = 28.0855/(28.0855 + (15.9994*2)) = 46.7
 Sample Analysis Design –
Concentration terminology
•  If 100% = 1,000,000 ppm, then

•  74% Zr (out of 67.2 wt%) = 49.73% or 497,300 ppm (or

µg/g)
•  46.7% Si (out of 32.8 wt%) = 15.32% or 153,200 ppm (or
µg/g)

•  If you are asked to weigh out 0.00001 g of zircon, then

the amounts of total Zr and Si you would have are:

–  Zr = 497,300 µg/g x 0.00001 g = 4.97 µg

–  Si = 153,200 µg/g x 0.00001 g = 1.53 µg
 Sample Analysis Design –
Concentration terminology
•  However, you are asked to prepare a solution of
this zircon sample for ICP-MS analysis in
solution mode;

•  then what would be the minimum dilution

factor required given that the maximal amount
of ion signal intensity allowed is 50 x 106 cps and
the yield for both elements in medium resolution
mode is ~60,000 cps/ppb?
 Sample Analysis Design –
Concentration terminology
•  Maximum allowable concentration is –

•  = Max. count rate/ yield = 50 x 106 cps/ 60,000

cps/ppb
•  = 833 ppb (ng/g)

•  4.97 µg of Zr needs to be diluted into ? ml of 5%

HNO3.

–  4.97 µg = 4970 ng/833 ng/g = ~5.97 g (ml) of 5%

HNO3
 Sample Analysis Design –
Matrix
•  Requires a medium (i.e. acid) that

–  Provides efficient ion transmission (HNO3 > HCl > H2O)

–  Won’t dissolve the glassware of your introduction system – for

example, hydrofluoric acid (HF) would!

–  Is relatively cheap and not deemed extremely hazardous to use

–  Thus, dilute (1 to 5% volumetric) nitric acid (HNO3) is the acid

medium of choice for most ICP-MS analyses
 Sample Analysis Design –
Step 2 – Calibration/Standard Preparation
•  Choice of calibration method dependent
upon several factors:

•  1. potential matrix effects

•  2. number of samples

•  3. consistency of matrix across samples

 Sample Analysis Design –
Step 2 – Calibration/Standard Preparation
•  EXTERNAL CALIBRATION:
•  Prepare a set of standard solutions to cover the
expected range of analyte concentrations

•  Fit a least squares regression line

•  y = mx + b

and calculate analyte concentration in unknowns

 Sample Analysis Design –
Step 2 – Calibration/Standard Preparation
23
Na calib curve (Medium resolution)

1000000

900000

800000 y = 17557x
700000 R2 = 0.9992
600000
cps

500000

400000

300000

200000

100000

0
0.00 10.00 20.00 30.00 40.00 50.00 60.00
conc ppb
 Sample Analysis Design –
Step 2 – Calibration/Standard Preparation
44
Ca calib curve (Medium resolution)

40000

35000 y = 676.92x
30000
R2 = 0.9961

25000
cps

20000

15000

10000

5000

0
0.00 10.00 20.00 30.00 40.00 50.00 60.00
conc ppb
 Sample Analysis Design –
Step 2 – Calibration/Standard Preparation
•  Advantages of External Calibration

–  Easy to prepare

–  Quick

–  Widely used technique

 Sample Analysis Design –
Step 2 – Calibration/Standard Preparation
•  Disadvantages of External Calibration:

•  Need to matrix match calibration solutions and samples

•  If standards containing <2000 ug/ml (ppm) are being

used, then preparing the standards as simple aqueous
solutions using the acid matrix (5% HNO3) employed for
the samples is sufficient

•  HOWEVER, if the samples contain a very high

concentration of one (or more) elements, then this may
not be adequate
 Sample Analysis Design –
Step 2 – Calibration/Standard Preparation
•  Preparation of External Calibration Solutions:

•  Need to evenly space calibration concentrations

•  If the highest concentration is much higher than the rest,

linear regression introduces bias favoring the high point

•  X = independent variable = concentration

•  Y = dependent variable = counts/second

 Sample Analysis Design –
Standard Addition Method
•  Aliquots of spike are added to unknown samples
to increase the ion signal intensities for elements
of interest

•  Typically use at least three aliquots of sample

spiked with evenly spaced amounts of analyte

•  These spiked aliquots of sample are used to

generate a calibration line and calculate the
concentration in the sample
 Sample Analysis Design –
Standard Addition Method
•  S0 = unspiked sample

•  S1 = sample spiked with analyte at concentration x

•  S2 = sample spiked with analyte at concentration 2x

•  S3 = sample spiked with analyte at concentration 3x

•  S4 = so on and so on
 Sample Analysis Design –
Standard Addition Method
AMT

500000
450000
400000
y = 29387x + 279235
350000
R2 = 0.9992
300000
Cps

250000
200000

150000

100000
50000

0
0 1 2 3 4 5 6 7
Concentration (ppb)
 Sample Analysis Design –
Standard Addition Method
•  The concentration of the unknown solution is
then determined by dividing the y-intercept value
by the slope of the sample-spike mixing line.

–  From example on previous slide,

–  Conc. sol’n = 279235 / 29387 = 9.5 ppb

–  If the original sample was a solution, then this is the

concentration of the analyte in question in the solution
 Sample Analysis Design –
Standard Addition Method
•  If the original sample was in solid form that
you digested and subsequently converted
into a solution;

•  then in order to determine the

concentration of the analyte in question,
you must factor in the amount of total
analyte in the solution and the dry weight
of the sample powder
 Sample Analysis Design –
Standard Addition Method
•  If we continue with the same example, the solution has a
concentration of 9.5 ppb, and the original volume of the unknown
solution was 10 ml (g) prior to aspirating some of it into the plasma
for analysis, then the total amount analyte in the solution is:

•  = 10 g x 9.5 ng/g (ppb)

•  = 95 ng, or
•  = 0.095 µg

•  If the amount of powder weighed out was 0.1 g, then the

concentration of the element in question is:

•  Conc. = 0.095 µg/0.1 g

–  = 0.95 µg/g or ppm
 Sample Analysis Design –
Standard Addition Method
•  This method works best if the slope of the
calibration line is not too shallow

–  This will create more uncertainty in the

location of the intersection between the cps of
your unknown and the calibration line
 Sample Analysis Design –
Standard Addition Method
•  For maximum precision it’s necessary that the
amount of sample be the same in each aliquot

•  Also want the amount of spike added to be the

same for each aliquot

•  Amount of spike added should be as small as

possible (usually 0.1 ml to 10 ml total volume)
 Sample Analysis Design –
Standard Addition Method
•  Ideally, the highest spike concentration
should be approximately equal to the
concentration of analyte in the unknown

•  Need to have some idea of the

concentration in the sample prior to
analysis
 Sample Analysis Design –
Standard Addition Method
•  Advantages:

–  Overcomes matrix differences

–  More precise and accurate than external calibration

•  Disadvantages:

–  Requires at least three aliquots for each sample

–  Run lengths become much longer and more
preparation time is required
 Sample Analysis Design –
Isotope Dilution
•  Most accurate and precise calibration method
available

•  Requires analyte with two stable isotopes

•  Monoisotopic elements cannot be determined

via isotope dilution

•  Spike natural sample with enriched isotope spike

of analyte
 Sample Analysis Design –
Isotope Dilution
•  The amount of spike is selected so that
the resulting ratio between spiked isotope
and unspiked isotope is near unity –
maximizes precision

•  Typically use the most abundant isotope

as the reference -- maximizes sensitivity
 Sample Analysis Design –
Isotope Dilution
•  Check isotope ratio in unspiked sample to
determine if the “natural ratio” in the
sample matches with the predicted ratio

•  If not -- interference in acting on one or

both of the isotopes

•  Always attempt to use interference free

isotopes
 Sample Analysis Design –
Isotope Dilution
•  Prepare the spike to desired concentration

•  Add spike as early as possible – after

equilibration of spike and sample you
don’t have to have complete sample
recovery

•  During any stage of the process complete

equilibration is absolutely necessary
 Sample Analysis Design –
Isotope Dilution
•  Analyze the solution on the ICP using many
repetitive scans (to maximize precision)

•  Need to measure isotopic ratios on standards of

a known ratio in order to correct for machine
mass discrimination

•  Use previous equation to calculate

concentrations!
 Sample Analysis Design –
Isotope Dilution
•  Advantages:

–  Most accurate and precise method for quantitative

elemental concentrations
–  Partial loss of analyte during preparation is
compensated for since physical and chemical
interferences are not an issue -- will cancel out as
they will affect each isotope identically
–  Ideal form of internal standardization since another
isotope of the same element is used in this capacity
 Sample Analysis Design –
Isotope Dilution
•  Disadvantages:

–  Generally only applicable to multiple-isotopic

elements
–  Need an enriched isotope spike for the
analyte of interest - not always available or
sometimes at very high cost
–  Need two interference free isotopes
–  VERY time consuming
Sample Analysis Design

STEP 3 – INTERNAL
STANDARDIZATION & INSTRUMENT
DRIFT CORRECTION
 Sample Analysis Design –
Internal Standard
•  Every sample should be analyzed with an
internal standard (IS)

•  What is an internal standard (IS)?

–  element that is added to EVERY sample/ blank/

calibration standard/QA sample/etc., that is not
expected to be in the sample in appreciable quantities
and is not an element of interest

–  use IS to monitor machine drift (both short and long

term) and matrix effects
 Sample Analysis Design –
Internal Standard
•  Choice of IS depends upon which
elements you are quantifying

•  The IS should have similar properties in

the plasma as element(s) of interest

•  ICP-MS: similar in mass/ionization

potential
 Sample Analysis Design –
Internal Standard
•  Example:

–  attempting to quantify U - use Th

–  attempting to quantify most transition metals - use As

–  attempting to quantify REEs - use Re

–  115In and 103Rh are common IS for general use

–  alternatively, you can add several IS to each sample

 Sample Analysis Design –
Internal Standard
•  From previous slide, we assume that
samples have little or no Th, As, or Re

•  It’s important to have an idea of what’s in

your sample prior to quantitative analysis

•  Solid samples can use a naturally

occurring element as IS, provided that you
know the concentration in each sample
 Sample Analysis Design –
Internal Standard
•  Procedure for IS use:

•  Calculate the concentration of the IS in each centrifuge tube – the latter will
contain an aliquot of your sample and an aliquot of the IS

•  Divide the measured ion signal (CPS) by the concentration of your IS to

derive the factor = CPS/ppb

•  Divide CPS/ppb of each tube by the CPS/ppb for those measured for the
blanks since these are not influenced by possible effects due to sample
matrices

•  The latter yields a dimensionless correction factor (I refer to it as a

normalization factor)

•  Use correction factor to adjust analyte counts for drift or matrix effects
 Sample Analysis Design –
Internal Standard

•  Advantages:

•  Fluctuations are monitored in each sample/

calibration / blank

•  Disadvantages:

•  Assume that behavior of IS is the same as the

analyte
 Sample Analysis Design –
Instrumental Drift
•  Correct for instrument drift with:

•  Internal standardization is a common

procedure

•  Use of drift corrector solutions (DCS)

 Sample Analysis Design –
Instrumental Drift
•  Drift Corrector Solutions (DCS):

•  Measure the same solution intermittently

throughout the course of the analytical
session

•  Change in ion signal is assumed to be

linear between each DCS measurement
 Sample Analysis Design –
Instrumental Drift
•  The DCS should contain all elements of
interest and can be matrix matched to
samples

–  Example: use standard reference materials

(SRMs) for DCS
 Sample Analysis Design –
Instrumental Drift
•  Apply a linear correction to samples
between DCS solutions

•  DCS1 + ((DCS2 - DCS1)*F)

•  F = position dependent fraction

 Sample Analysis Design –
Instrumental Drift
•  Advantages of DCS correction:

–  all analytes are monitored for drift

–  nothing added to sample solutions

•  Disadvantages of DCS correction:

–  assume change is linear

–  cannot easily monitor matrix effects

 Sample Analysis Design –
Background & blanks
•  Standard blank - blank used to monitor
polyatomic ion interferences, gas peaks,
and contamination from reagents; used for
background subtraction

•  Procedural blank - blank used to monitor

contamination acquired during all stages
of sample preparation; grinding, digestion,
acidification, powdering, etc
 Sample Analysis Design –
Background & blanks
•  Use of blanks during an analytical session:

•  ALWAYS begin an analytical session with at least one

standard blank

•  Analyze standard blanks periodically throughout the

course of the session in particular to monitor memory
effects

•  Process and analyze at least one procedural blank at

some point during your research study; for its analysis,
it’s preferable to measure it early in order to avoid any
potential memory effects
 Sample Analysis Design –
Background & blanks
•  The more standard blanks that are run
during an analytical session, the more
information you will have with regards to
monitoring change(s) in background levels
throughout the entire session
 Sample Analysis Design –
Background & blanks
•  How to determine “the background”:

•  1. just use the first standard blank

•  2. average all standard blanks

•  3. take median of all standard blanks

•  4. apply statistical analysis to standard blanks

and select some of them
 Sample Analysis Design –
Background & blanks
•  Outlier tests:

•  1. I know the truth

•  2. Looks different

•  3. Statistical “proof”
 Sample Analysis Design –
Background & blanks
•  Option 1 should be avoided - unscientific
and invalid

•  Option 2 is better but only if the

measurement is repeated

•  Option 3 is the best approach, but needs

to be carried out carefully in order to avoid
false negatives and positives
 Sample Analysis Design –
Background & blanks
•  Huber Outlier Test

•  take median of all values

•  calculate absolute deviation |xi - xm|

•  take mean of absolute deviations (MAD)

•  multiply MAD by coefficient (k = 3-5)

•  anything higher than k*MAD is rejected as outlier

 Sample Analysis Design –
Background & blanks
•  Calculation of Limit of Detection (DL) and
Limit of Quantification (QL)

•  Easy way:
•  LOD = 3*STDEVblank;
•  LOQ = 10*STDEVblank
 Sample Analysis Design –
SUMMARY
•  A ‘good’ analytical method will:

•  1. provide the means to calculate an accurate

background level

•  2. allow for correction of instrument drift

•  3. use Internal standardization to monitor matrix effects

•  4. provide some method for monitoring/ correcting

interferences

•  5. Use a proper calibration strategy