Calibration Considerations

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					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
            • 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


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       (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)

Page 8
         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

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

              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

           Element   Conc. ppb   Detection   Element   Conc. PPB   Detection
                                   limit                             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

Page 12
          Impurities in Container Materials
                Every Standard needs a Container, but Be Careful

           Material                            Total No. of   Total   Major
                                               Elements        PPM    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


Page 13
   Plastic Packaging Container Purity

                PFA (PERFLUOROALKOXY)
                PP (POLYPROPYLENE)

Page 14
          Laboratory Pure Water

               ASTM Type                        I      II      III      IV

                     Total matter             <0.1    0.1      1         2
                     (mg/L max.)

                   Specific Resist.           15-18    1      >1.0      0.2

                           pH                  NA     NA     6.2-7.5    5-8

               Min. color retention time of    60     60       10       10
                      KMnO4 mins

                    Soluble Silica             ND     ND     10ug/l     high

                   Bacteria Count             0/ml    0/ml   10/ml     100/ml

Page 15
          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.


Page 16
          Contaminates in Nitric Acid from Major Suppliers (ppb)

            Supplier           Metals    Chlorides     Price


            ACS Reagent          2100         100      $56.90/2.5L

            ACS NF               1500         100      $57.40/2.5L

            Trace Metal          1000          80      $66.4/500ml

            Ultrex                 3          100      $213/500ml


            ACS Reagent          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

Page 17
          Clean Laboratory

     •      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

Page 18
   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!


Page 20
              Clean Instruments

             Check parts of the instrument that contact the sample.

          AA Instruments
           Graphite Components
             • Modified Contact Cylinders: Exhibit less carry-
               over 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

      Glass Spray Chambers
      Quartz Nebulizers
      Ryton Spray Chambers
      Teflon(s)
      Polyethylene Sample Tubes
      PEEK
      Alumina Injectors

      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


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 >

     3. Using standard concentrations
        which are way too high, way
        beyond your expected sample
        range, just to get better c.c.

     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

                                                                   10 g/g           0.1 g/g Post-
          Sample ID    Measured Conc            NIST Certified     pre-dissolution   dissolution spike
                       (g/g)                   Value (g/g)       spike recovery    recovery
          SRM 1400     9.10  0.11              9.07  0.12        106%              109%
          SRM 1486     1.207  0.008            1.335  0.014      101%              99%
                        5% relative Error from Certified Value

             Detection Limits for Pb in Calcium Matrix
            Matrix         Nitric Acid                  Calcium Phosphate
            Type              IDL =                      MDL =(3)(matrix)
                         (3)(1% nitric acid)
           Solution        0.001 g/L                       0.005 g/L
            Solid           0.001 g/g                      0.005 g/g

Page 40
   Results for Calcium Tablets

          Sample ID      Mean (ug/g)   %RSD (n=3)     0.05ug/g
                                                    %Spike Recov
          Tricalcium        0.105         0.88           99
          Phosphate -A
          Tricalcium        0.108         0.60           92
          Phosphate -B
          Calcium           0.315         1.03           90
          Antacid -A        0.114         2.84           93

          Antacid -B        0.259         1.28          106

Page 41
   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.
               •   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

Page 42
   More Data Reporting

           Standard Deviation and % Relative Standard Deviation can indicate the
            reliability of the method of measurement. Example:

             MEAN (n=3) SD                %RSD
             27.6 ug/L  0.35ug/L          1.27%

           QC or SRM Measurement Accuracy is commonly expressed as Percent
            Recovery rather than Percent Relative Error. Example:

             MEAN (SD)             Known QC        % Recovery
             19.3 (0.22) ug/L      20.0 ug/L       96.5%

             %Recovery = 100 – (Known-Measured)/Known *100

Page 43
   Useful Sources and Links

         Nation Research Council Canada
         BRAMMER

         American Water Works Asso.



Page 44
   Thanks for Your Time !

Page 45

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