Calibration Considerations Using
Atomic Spectroscopy
“We’re not exactly rocket scientists”
And, luckily we don’t have to be.
Page 2
Why do we Calibrate? Direct vs. Indirect Methods
 Direct Measurement Method:
• The Measurement of a Physical Property.
• Instruments rarely need to be calibrated.
• Examples: Weight and Volume Measurements.
 Relative Measurement Method:
• Using an instrument which requires calibration prior
to the measurement.
• Many Dynamic System Variables
• Examples: AAS, Quantitative ICP-OES, ICP-MS.
Page 3
Accuracy and Precision
 Accuracy: Usually expressed as error. The difference
between a measurement and the True Value is its absolute
error (mg/L).
• Accuracy can also be expressed as Percent Relative Error.
• How much error is in a typical ICP determination?
 Precision: Simply the degree of reproducibility of a set of
replicate measurements.
• Precision can expressed by Standard Deviation (SD) or
Percent Relative Standard Deviation (%RSD).
• What is typical precision for a set of GFAAS replicates?
Page 4
Types of Errors
 Determinate Errors:
• Have specific, identifiable, and correctable causes.
• Examples: Contaminated Method Blank, Incorrect Standard
Concentration.
• Usually main source(s) of most error, can be large.
 Indeterminate Errors:
• Random
• Frequently from Multiple Sources
• Examples: Flicker (Nebulizer) Noise in an ICP, Mechanical Vibrations,
Electronic Noise.
• Hopefully small in magnitude
• Usually determines detection limits
Page 5
Sources of Calibration Error
1. Improper Blanks
2. Improperly Prepared Calibration Standards
3. Calibration Curve Algorithm Type
27
Page 6
Blank Control: Do you have a Contamination Problem?
If you are reporting negative answers,
you could have a contamination problem!
 Run lots of different blanks and compare results
• Different sources of water
• Different sources of acids
• Different Flasks
• Different Analysts
 Run blanks overnight and check stability
• Checks cleanliness of the instrument
Page 7
Sources of Contamination
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Page 8
(Post-sampling)
Analytical Containers (Volumetric flasks, pipettes,..)
Storage Containers (bottles)
Lab Reagents (including lab pure water)
Lab Environment (dust)
Analyst (yes, you!)
Instrumentation (carry-over)
Volumetric Flasks … What are they good for?
 Very Accurate, but do you really need them?
 They are NOT for storage!
 They are NOT for digestions!




Clean with 10% HNO3 4 hours
Rinse with lots of lab pure water
Store filled with lab water
Rinse out prior to use

Do you use graduated Cylinders? Why?
 You can use 50mL Autosampler Vials for
accurate volumetric measurements.
Advantage is less potential sample
contamination.
Page 9
You can do Volumetric measurements with a Balance
 1 mL of distilled water weighs
1 gram
 Minimize the number of
container surfaces the
sample touches.
Page 10
Pipettes
Glass Pipettes:
 Very Accurate, but do you really need them?
 Use only larger volumes (>10mL).
 Clean, check delivery. Re-clean
 Store dry, away from dust
 Use Pipettors with disposable tips whenever possible
 Don’t contaminate the tips!
Page 11
How clean are your pipettes?
2% Nitric acid run through 5mL pipets and scanned on ICPMS
Page 12
Element
Conc. ppb
Detection
limit
Element
Conc. PPB
Detection
limit
Ag
2.33
0.0088
Mn
1.72
0.012
Al
6.43
0.13
Na
19.1
0.6
Be
2.62
0.007
Ni
0.96
0.18
Bi
1.07
0.0006
Pb
5.4
0.13
Ca
18.8
2.9
Sn
0.55
0.0033
Co
2.02
0.004
Th
0.24
0.0003
Cr
0.91
0.28
Ti
0.56
0.003
Fe
1.62
0.75
Tl
1.53
0.0075
Mg
2.56
0.016
Zn
9
0.4
Impurities in Container Materials
Every Standard needs a Container, but Be Careful
Material
Total
PPM
Major
Impurities
Polystyrene-PS
8
4
Na,Ti, Al
Teflon-TFE*
24
19
Ca,Pb,Fe,Cu
Teflon-FEP*
25
241
K,Ca,Mg
Polycarbonate-PC
10
85
Cl,Br,Al
Low Density PE-LDPE
18
23
Ca,Cl,K
Polypropylene-PP
21
519
Cl,Mg,Ca
Polymethyl Pentene-PMP
14
178
Ca,Mg,Zn
High Density PE-HDPE
22
654
Ca,Zn,Si
Borosilicate Glass
14
497
Si,B,Na
*TFE-Tetrafluoroethylene
*FEP=FluorinatedEthylenePropylene
Page 13
Total No. of
Elements
Plastic Packaging Container Purity
FEP (FLUORINATEDETHYLENEPROPYLENE)
PFA (PERFLUOROALKOXY)
FLEP (FLUORINATED HIGH-DENSITY POLYETHLYENE)
PMP (POLYMETHYLPENTENE)
PP (POLYPROPYLENE)
HDPE (HIGH-DENSITY POLYETHYLENE)
LDPE (LOW-DENSITY POLYETHYLENE)
Page 14
Laboratory Pure Water
ASTM Type
Page 15
I
II
III
IV
Total matter
(mg/L max.)
<0.1
0.1
1
2
Specific Resist.
(megohm-cm)
15-18
1
>1.0
0.2
pH
NA
NA
6.2-7.5
5-8
Min. color retention time of
KMnO4 mins
60
60
10
10
Soluble Silica
ND
ND
10ug/l
high
Bacteria Count
0/ml
0/ml
10/ml
100/ml
Laboratory Pure Water
The Direct-Q ultrapure water system
produces 18.2 Megohm-cm reagent
water containing less than 30 ppb Total
Organic Carbon directly from potable
tap water. The system is ideal for
scientists needing 5 to 15 L/day of
ultrapure water for the preparation of
culture media, buffers, blanks and
standard solutions.
www.elgalabwater.com/
Page 16
Contaminates in Nitric Acid from Major Suppliers (ppb)
Supplier
Metals
Chlorides
Price
Baker
ACS Reagent
2100
100
$56.90/2.5L
ACS NF
1500
100
$57.40/2.5L
Trace Metal
1000
80
$66.4/500ml
3
100
$213/500ml
4000
80
$51.14/2.5L
ACS NF
NA
500
$55.18/2.5L
Trace Metal
32
NA
$51.4/500ml
Optima
3
NA
$203/500ml
Ultrex
Fisher
ACS Reagent
Page 17
Clean Laboratory
•
•
•
•
•
Page 18
Environment of class 100 (less than 100 particles of 0.3microns per m3)
Walls, ceilings and floors sealed and dust free
HEPA filters mounted in the ceiling
No fuming Acids
All work performed under clean hood
Airborne Contaminants (ug/g)
Page 19
Clean Techniques
•
•
•
•
No jewelry, cosmetics or lotions
Wear gloves, Powder-Free
Cover hair and mouth
Beware of dust, airborne fallout, cover samples
How do you determine if you have a clean lab?
By running blanks!
http://terrauniversal.com/
http://www.aircleansystems.com/
Page 20
Clean Instruments
Check parts of the instrument that contact the sample.
AA Instruments
 Graphite Components
• Modified Contact Cylinders: Exhibit less carryover and cross contamination for samples with
high dissolved solids content.
• UltraClean Graphite Tubes: Deliver
exceptionally low levels of residual
contamination due to extra high-temperature
gas-phase cleaning procedure. Extremely low
traces of Na, Ca, Fe, Al, Si, Ti, Cr, Ni.
Page 21
Clean Instruments
ICP-OES
 Glass Spray Chambers
 Quartz Nebulizers
 Ryton Spray Chambers
 Teflon(s)
 Polyethylene Sample Tubes
 PEEK
 Alumina Injectors
ICP-MS
 Platinum Cones, Injectors
 Quartz Spray Chambers
 Sapphire Injectors
Page 22
Primary Calibration Standards – Match to your Task
 AA Grade
• Single Element Accuracy
• Stability
• Traceability
 ICP Grade
•
•
•
•
•
•
Accuracy
Purity
Stability
Chemical Compatibility
Traceability
Often You can Choose Acid Matrix
 Multi Element
• Reliable if you need lots of elements
• More Expensive
Page 23
Page 24
ppb Standard Stability Study
http://www.ivstandards.com/tech/reliability/part07.asp
A blend of 65 elements from Inorganic Ventures / IV Labs' CMS-SET was prepared at the 0, 2, 10,
and 100 ppb concentration level in 1 % (v/v) HNO3 at the start of the study.
The set consists of the following;
CMS-1 - 10 µg/mL Ce, Dy, Er, Eu, Gd, Ho, La, Lu, Nd, Pr, Sm, Sc, Tb, Th, Tm, U, Yb, Y in 3.5
% HNO3
CMS-2 - 10 µg/mL Au, Ir, Pd, Pt, Re, Rh, Ru, and Te in 3.5 % HCl
CMS-3 - 10 µg/mL Ge, Hf, Mo, Nb, Ta, Sn, Ti, W, and Zr in 3.5 % HNO3 tr. HF
CMS-4 - 10 µg/mL Sb, As, Ba, Be, Bi, B, Cd, Ga, In, Pb, Se, Tl, and V in 3.5 % HNO3
CMS-5 - 10 µg/mL Ag, Al, Ca, Cs, Cr+3, Co, Cu, Fe, Li, Mg, Mn, Ni, K, Rb, Na, Sr, and Zn in
3.5 % HNO3
The LDPE bottles were acid leached with 1% nitric acid for 59 hours at 60 °C. New blends
prepared in the same way were compared to the original preparation at 1, 3, 25, 75, 137, 300, and
375 days.
Page 25
Experiment Results Hg was not stable long enough to measure (minutes).
Au was the next most unstable element, showing instability at the 2, 20, and 100 ppb levels
at 3 days.
Pd showed instability only at the 2 and 10 ppb levels at 3 days.
Pt and Ta showed instability only at the 2 and 10 ppb levels at 137 days.
Ag showed instability only at the 10 and 100 ppb levels at 137 days.
Mo, Sn, and Hf showed instability only at the 2 ppb level at 375 days.
Ir showed instability only at the 2 ppb level at 300 days.
All other elements showed no instability at 2-100 ppb for 375 days, including:
Ce, Dy, Er, Eu, Gd, Ho, La, Lu, Nd, Pr, Sm, Sc, Tb, Th, Tm, U, Yb, Y, Re, Rh, Ru, Te, Ge,
Nb, Ti, W, Zr, Sb, As, Ba, Be, Bi, B, Cd, Ga, In, Pb, Se, Tl, V, Al, Ca, Cs, Cr+3, Co, Cu,
Fe, Li, Mg, Mn, Ni, K, Rb, Na, Sr, and Zn.
Paul Gaines, Ph.D.
Author of Reliable Measurements and other guides
Page 26
How do I know if my Primary Standard is Good?
 Check against a Second Source or SRM
• NIST, NRC Canada, Brammer…
 Check Characteristic Concentration Flame AAS.
• Also can use Sensitivity Check
 Check Characteristic Mass Graphite Furnace AAS (M0).
 For ICP and ICP-MS, you can check (count/sec) Intensity History.
Page 27
Example Proper Calibration Scheme
 Find Linear working Range.
 Find the range of your samples.
Page 28
Flame Cu Calibration for Samples in the 25-50 ppb range
 Calibration not quite good enough. Let’s try something… anything.
 Must meet 0.995 Law
Page 29
Flame Cu Calibration for Samples in the 25-50 ppb range
 Much better, don’t you think?
 0.995 condition satisfied
 What is a little curve fitting among friends?
Page 30
Flame Cu Calibration for Samples in the 25-50 ppb range
 Oh yes, this is the answer: Linear Fit with much higher standards
 0.995 Law more than satisfied, cc=0.997
 Problem solved! Or is it?
Page 31
Flame Cu Calibration for Samples in the 25-50 ppb range
 What is wrong with this picture?
Page 32
Flame Cu Calibration for Samples in the 25-50 ppb range
 What is wrong with this picture?
Page 33
Flame Cu Calibration for Samples in the 25-50 ppb range
 Look at the change one remade standard can make
Page 34
Calibration Mental Mistakes - Review
1. Choosing a curve algorithm to
fit data which you know should
be linear.
2. Being a “slave” to arbitrary
rules like “c.c. must be >
0.995”.
3. Using standard concentrations
which are way too high, way
beyond your expected sample
range, just to get better c.c.
statistics.
4. Being lazy, re- make the
standards and /or run a
second source standard.
Page 35
Example: Lead in Calcium Nutritional Supplements
 Abstract: ICP-MS
Intercalibrated measurements of lead in calcium supplements indicate the
importance of rigorous analytical techniques to accurately quantify
contaminant exposures in complex matrices. Without such techniques,
measurements of lead concentrations in calcium supplements may be either
erroneously low, by as much as 50%, or below the detection limit needed
for new public health criteria. In this study, we determined the lead content
of 136 brands of supplements that were purchased in 1996. The calcium in
the products was derived from natural sources (bonemeal, dolomite, or
oyster shell) or was synthesized and/or refined (chelated and nonchelated
calcium) . The dried products were acid digested and analyzed for lead by
high resolution-inductively coupled plasma-mass spectrometry. The
method's limit of quantitation averaged 0.06 µg/g, with a coefficient of
variation of 1.7% and a 90-100% lead recovery of a bonemeal standard
reference material. Two-thirds of those calcium supplements failed to
meet the 1999 California criteria for acceptable lead levels (1.5 µg/daily
dose of calcium) in consumer products.
 Environ Health Perspect 108:309-313 (2000) .
Page 36
Example: Lead in Calcium Supplements (<1 ug/g Pb)
 Sample Prep; 0.5g sample to 500 mL with acid dissolution.
•
•
•
•
Sample prep may contaminate samples low level Pb
We will need to accurately measure below 1 ug/L for Pb
Check Acid Reagent Blanks
Check Method Blanks – acids plus containers
 Is my instrument clean enough for sub ppb work?
• Replace or clean any contaminated parts, like cones, injector, …
• Check blanks
 What is the best primary standard to use?
 What is Best Calibration Range and Curve Type to use?
 Is a similar matrix SRM available?
Page 37
ICP-MS Calibration for Pb
•Simple Linear Calibration up to 1.25 ug/L Pb
•Second Source QC at 1ug/L; +/- 10%
Page 38
NIST 1486 Bone Meal SRM = 1.335 +/- 0.014 ugPb/g
Page 39
Results for Reference Materials
NIST 1400 - Bone Ash
NIST 1486 - Bone Meal
Sample ID
SRM 1400
SRM 1486
Measured Conc
(g/g)
9.10  0.11
1.207  0.008
NIST Certified
Value (g/g)
9.07  0.12
1.335  0.014
10 g/g
pre-dissolution
spike recovery
106%
101%
5% relative Error from Certified Value
Detection Limits for Pb in Calcium Matrix
Matrix
Type
Page 40
Calcium Phosphate
MDL =(3)(matrix)
Solution
Nitric Acid
IDL =
(3)(1% nitric acid)
0.001 g/L
Solid
0.001 g/g
0.005 g/g
0.005 g/L
0.1 g/g Postdissolution spike
recovery
109%
99%
Results for Calcium Tablets
Sample ID
Page 41
Mean (ug/g)
%RSD (n=3)
0.05ug/g
%Spike Recov
Tricalcium
Phosphate -A
0.105
0.88
99
Tricalcium
Phosphate -B
0.108
0.60
92
Calcium
Carbonate
0.315
1.03
90
Antacid -A
0.114
2.84
93
Antacid -B
0.259
1.28
106
Data Reporting
No analysis is complete until the final results have been correctly calculated
and properly reported. The report should give the best values obtained
and also indicate the probable accuracy or reliability of the results.
A single result can express the degree of uncertainty by the number of
Significant Figures.
•
•
Page 42
For Example; A weight given as 0.5 g implies that a rough type of balance was
used and that the actual weight is between 0.45 and 0.55 g.
Furthermore, any subsequent computation using the 0.5 g weight in the
calculation of a final value cannot contain any more than 1 significant figure.
Obviously, a calculator or computer cannot improve the precision of the
original data!
Expression
# of Sig. Figs.
5.063
4
3600
2
3.600x103
4
0.00123
3
More Data Reporting
 Standard Deviation and % Relative Standard Deviation can indicate the
reliability of the method of measurement. Example:
MEAN (n=3) SD
27.6 ug/L
0.35ug/L
%RSD
1.27%
 QC or SRM Measurement Accuracy is commonly expressed as Percent
Recovery rather than Percent Relative Error. Example:
MEAN (SD)
19.3 (0.22) ug/L
Known QC
20.0 ug/L
% Recovery
96.5%
%Recovery = 100 – (Known-Measured)/Known *100
Page 43
Useful Sources and Links



http://ts.nist.gov/measurementservices/referencematerials/index.cfm
Nation Research Council Canada http://www.nrc-cnrc.gc.ca/
BRAMMER http://www.brammerstandard.com/


http://www.standardmethods.org/
American Water Works Asso. http://www.awwa.org/


http://www.astm.org/
http://www.astm.org/cgibin/SoftCart.exe/SNEWS/MA_2008/index.html?L+mystore+eswo6699



http://www.inorganicventures.com/tech/reliability/
http://www.spexcsp.com/
http://www.highpuritystandards.com/
Page 44
Thanks for Your Time !
Page 45