Sample Analysis Design Isotope Dilution
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1 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
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4 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
5 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
6 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
7 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!
8 Advantages: Sample Analysis Design Isotope Dilution 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
9 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
10 Sample Analysis Design STEP 3 INTERNAL STANDARDIZATION & INSTRUMENT DRIFT CORRECTION
11 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
12 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
13 Example: Sample Analysis Design Internal Standard attempting to quantify U - use Th attempting to quantify most transition metals - use As attempting to quantify REEs - use Re 115 In and 103 Rh are common IS for general use alternatively, you can add several IS to each sample
14 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
15 Procedure for IS use: Sample Analysis Design Internal Standard 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
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18 Advantages: Sample Analysis Design Internal Standard Fluctuations are monitored in each sample/ calibration / blank Disadvantages: Assume that behavior of IS is the same as the analyte
19 Instrumental Drift Correct for instrument drift with: Internal standardization is a common procedure Use of drift corrector solutions (DCS)
20 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
21 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
22 Instrumental Drift Apply a linear correction to samples between DCS solutions DCS 1 + ((DCS 2 - DCS 1 )*F) F = position dependent fraction
23 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
24 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
25 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
26 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
27 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
28 Outlier tests: Sample Analysis Design Background & blanks 1. I know the truth 2. Looks different 3. Statistical proof
29 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
30 Background & blanks Huber Outlier Test take median of all values calculate absolute deviation x i - x m take mean of absolute deviations (MAD) multiply MAD by coefficient (k = 3-5) anything higher than k*mad is rejected as outlier
31 Background & blanks Calculation of Limit of Detection (DL) and Limit of Quantification (QL) Easy way: LOD = 3*STDEV blank ; LOQ = 10*STDEV blank
32 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
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36 Sample Analysis Design PART II
37 Sample Analysis Design Generating high quality, validated results is the primary goal of elemental abundance determinations It is absolutely critical to plan an ICP-MS analysis carefully from sample gathering to final analysis on the ICPMS Always create and follow an analysis design that shall permit you to follow the procedure for future samples, AND know what you did when looking at old data
38 Sample Analysis Design Goal: high quality, validated, quantitative determination of elemental concentrations/isotope ratios This goal can be achieved by: Specificity - only detecting isotope(s) of interest, not interferences, OR making sure that matrix effects do not play a role Sensitivity - can we differentiate the isotope of interest from the background signal? Accuracy - does the analysis represent the real value? Precision - how repeatable is the value?
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40 Sample Analysis Design Sensitivity and Precision: generally defined by how well the instrument is tuned (assuming dilution factor is appropriate) Specificity and Accuracy: generally governed by how well you prepare your sample and set up your analysis method/ parameters
41 Sample Analysis Design Spectroscopic interferences Matrix effects
42 Sample Analysis Design Interference analytical artifact which causes the ion signal in a sample to vary from an idealized (true) signal Interferences cause inaccurate analyses Two main types: 1. Spectroscopic 2. Nonspectroscopic
43 Sample Analysis Design Spectroscopic Interferences in ICP-MS 1. Isobaric overlap 2. Polyatomic ions 3. Refractory oxides 4. Doubly charged ions
44 Sample Analysis Design Isobaric Overlap Two different elements having the same nominal mass E.g., 40 Ca (96.9%) and 40 Ar (99.6%) Monoisotopic elements: B, Na, Al, Sc, Mn, As, Nb, Y, Rh, I, Cs, Pr, Tb, Ho, Tm, Au, Th None have isobaric interferences
45 Isobaric Overlap A few multi-isotope elements have no interference free isotopes: E.g., In In 113 Cd, and 115 In 115 Sn Usually possible to choose an interference free isotope or one that is unlikely to be interfered upon strongly (either due to very low natural abundance of interfering element (isotope) or the interfering element (isotope) is not present in sample)
46 Isobaric Overlap IF an isobaric interference is unavoidable, you can correct for it by measuring the counts on a non-interfering isotope of the interfering element CPS analyte = CPS total -CPS interferent *(X a /X b ) X a = abundance of interfering isotope X b = abundance of non-interfering isotope
47 Polyatomic Interferences More serious than isobaric interferences Result from possible, short-lived combination of atomic species in the plasma or during ion transfer Common recombinants are Ar, H, and O Dominant elements in reagents also form polyatomic interferences - N, S, and Cl
48 Polyatomic Interferences Analyzing deionized water with ICP-MS instrument we have solely H and O present (within the matrix ) Thus, peaks would be visible at masses: 41 ( 40 Ar + 1 H) 41 K 56 ( 40 Ar + 16 O) 56 Fe 80 ( 40 Ar + 40 Ar) 80 Se As well as other minor peaks from the minor isotopes of Ar and O
49 Polyatomic Interferences If you acidify the deionized water with HNO 3 or H 2 O 2, these behave in the same manner as deionized water these acids are considered ideal matrices as they don t add unnecessary polyatomic interferences Acidification with HCl or H 2 SO 4 changes the situation since Cl and S can be used to form polyatomic ions
50 HCl matrix: Sample Analysis Design Polyatomic Interferences 35 Cl + 16 O = 51 V 37 Cl + 16 O = 53 Cr 40 Ar + 35 Cl = 75 As 40 Ar + 37 Cl = 77 Se
51 Polyatomic Interferences H 2 SO 4 matrix: 32 S + 16 O = 48 Ti 32 S + 16 O + 16 O, or 32 S + 32 S = 64 Zn, 64 Ni 34 S + 16 O = 50 Ti, 50 V, 50 Cr 33 S + 16 O + 16 O = 65 Cu
52 Sample Analysis Design These interferences will be present just from the gas and solvent Therefore, if possible, solutions should be prepared with a weak (1-5% v/v) HNO 3 acid matrix Why not deionized water? acid helps keep elements from sticking to sides of test tubes and tubing during transport ionization efficiency is significantly lower relative to HNO 3
53 Polyatomic Interferences The most serious polyatomic interferences are formed from the most abundant isotopes of H, C, N, O, Cl, and Ar So, if polyatomic interferences are present in plain deionized water with no other ions present, what happens in natural (complex) samples/matrices?
54 Polyatomic Interferences Need to know the most abundant elements present in sample Is (are) there any one (or more) elements present that are in very high abundances? If so - then it s likely interferences could be formed from such abundant element(s) E.g., rock samples - high Si
55 Polyatomic Interferences Typically, most polyatomic interferences are only present below mass 82 - since Ar, H, and O are by far the most abundant isotopes in the plasma, they form most interferences Counteractive measures: Proper machine settings (especially nebulizer flow rate and RF power) can minimize formation
56 Polyatomic Interferences If a polyatomic interference is unavoidable, then a correction equation like the one for isobaric interferences can be used (assuming you are certain of the species of the interference)
57 Refractory Oxides Refractory Oxides Occur because of incomplete sample dissociation or from recombination Always occur as an interference as an integral value of 16 mass units above the interfering element MO, MO 2, or MO 3 where M is the interfering element
58 Refractory Oxides Elements with high oxide bond strength are most likely to form refractory oxide interferences Severity is expressed as MO/M as a percentage MO/M should be as low as possible, typically <5% MO/M minimized by adjusting nebulizer flow rate, z-axis position, and RF power
59 Refractory Oxides Si, Ce, Zr, Ti, Sm, Mo, and P all form strong oxide bonds and may have severe oxide interferences Usually monitor the CeO/Ce ratio to make sure it s below 5%
60 Examples: Sample Analysis Design Refractory Oxides LREE on the HREE BaO on Eu MoO on Cd
61 Refractory Oxides Again, correction equations can be applied but these lead to increased error, especially if the interference is large Oxide formation tends to not be stable and the corrections accumulate error quickly If there is a severe oxide interference, it s usually better to try to separate the interference from the analyte
62 Typically, Sample Analysis Design Refractory Oxides MO > MO 2 > MO 3 > MO 4
63 Doubly Charged Ions Form when the 2nd ionization potential is less than the first ionization potential of Ar Typical of alkaline earths, a few transition metals, and some REE Low nebulizer flow rates increase doubly charged ion formation Interference signal is always M/2 where M is the element becoming doubly charged
64 How to avoid spectral MS interferences? Attempt to dry solutions prior to introduction to plasma - remove O and H gets rid of many polyatomic species (i.e. use a desolvating introduction system e.g. DSN 100) Optimize instruments settings so that formation is minimized and corrections are not severe Use simple matrices whenever possible Process samples prior to analysis to isolate elements of interest or remove potential interfering elements; e.g. ion exchange chromatography
65 How to avoid spectral MS interferences? Alternatively - use higher mass resolutions, MR medium resolution or HR high resolution Most overlaps are not exact using medium and high resolution modes Can completely separate the isotope of interest from interfering species However, consequence is loss of sensitivity
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