Schedule. Draft Section of Lab Report Monday 6pm (Jan 27) Summary of Paper 2 Monday 2pm (Feb 3)

Transcription

1 Schedule Assignment Due Date Draft Section of Lab Report Monday 6pm (Jan 27) Quiz for Lab 2 Peer Review of Draft Complete Lab Report 1 Tuesday 9:30am Wednesday 6pm Friday 6pm Summary of Paper 2 Monday 2pm (Feb 3)

2 Experimental Design and Quality Control in Instrumental Analysis Analytical method validation and performance Reference: Harris, D.C. Quantitative Chemical Analysis (7th ed.), Chapters 1, 3, 4, 5

3 Method suitability What are the chemical/physical properties of the sample? What accuracy is required? How much sample is available? What is the expected range of analyte concentration? Are there matrix interferences? How many samples are to be analyzed?

4 What is a standard method? Thoroughly validated analytical procedure for a specific analyte in a specific sample matrix Validated by multiple laboratories Specified by governmental or professional organizations Examples; NIOSH 3500, formaldehyde by visible spectroscopy EPA 505, Rev 2.1 Organohalide Pesticides and PCBs by microextraction and GC

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6 Errors in quantitative analysis Random errors (increase variability) Arise from the effects of uncontrolled variables Error in reading the scale on an instrument (gauge vs. digital) Random electrical noise Systematic errors (introduce bias) Contamination in lab or during sampling (+) Calibrants incorrectly prepared (+) or (-) Failure to completely extract analyte from sample (-)

7 Accuracy and Precision Accuracy of a measurement: Closeness of an experimental value (x i ), or the mean of a set of measurements, to the true value, µ. Often expressed by the absolute error, E. e.g. a 50 ml burette is accurate to ± 0.1 ml Or percent error, %E = (x i - µ) / µ Precision of a set of measurements: Refers to the agreement among results Usually is expressed by the standard deviation, s.

8 What do accuracy and precision look like? Good accuracy Poor precision Poor accuracy Good precision Good accuracy Good precision

9 Significant figures! The number of significant figures is the minimum number of digits needed to write a given value, without loss of accuracy. Therefore: The choice of the number of figures used to express your results is not arbitrary The accuracy of an assay denotes the appropriate number of significant figures for reporting your results.

10 Accuracy and Significant figures! Example 1 An analytical balance is reported to have an accuracy of ± 1 µg. Which of the following values are correctly reported? 0.75 µg µg 6 µg Example 2 A method for analysis of chloroform by gas chromatography is reported to have an uncertainty of ±10%. How should the following values be correctly reported? ng/ml 82.6 ng/ml 1753 ng/ml

11 Precision and Significant figures! Example 3 Duplicate extracts of acetamiprid from air filters have an average (x )of µg/ml and a standard deviation (s) of µg/ml. How many significant figures should be reported for the extract concentration? Three: 168 ±4 µg/ml Four: ±3.6 µg/ml Six: ±3.621 µg/ml Relative Standard Deviation, %RSD = s / x = 2 %

12 Calibration For most analytical methods, the relationship between analyte concentration and instrument response must be defined, in order for the analyte concentration in unknown samples to be evaluated. This is achieved by measuring instrument response for a series of samples (called standards or calibrants ) of known concentration. A plot of instrument response vs. analyte concentration is a calibration curve

13 Calibration curves Instrument response Instrument response mg injected mg injected Calibration looks great in left panel, r 2 =0.9989, but expansion (right panel) indicates problems with low concentration samples.

14 Finding the best straight line Use least squares regression Fit to the form y = mx + b Calculates uncertainties for slope (m) and intercept (b) Calculates a correlation coefficient (r) A measure of goodness of fit Typically report r 2 Want r 2 to be greater than 0.99

15 Effect of calibration equations on Uncertainty (Percent Error) 60.00% 40.00% 20.00% 0.00% % % % linear, no intercept 1/x2 weighted, w/ intercept % %

16 Linearity and range Linearity The ability of a method to generate results that are directly proportional to analyte concentration Range The interval of analyte levels over which the analytical method has been validated Area Counts FLD excitation 275nm and emission 315 nm y = x x 2000 R 2 = y = x x 1500 R 2 = Area Counts ng Tyr HPA Poly. (Tyr) Poly. (HPA) FLD excitation 275nm and emission 315 nm y = x R 2 = y = x R 2 = ng Tyr HPA Linear (Tyr) Linear (HPA)

17 Confidence intervals The confidence interval (CI) is an expression stating that the true value (µ) is likely to lie within a certain distance of the measured value: µ = ts x ± n Where: s = measured standard deviation n = number of observations t = student s t-test (from tables), typically specified as a 95% CI

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19 Tests of significance: t-test Comparison of the experimental mean with the true value Statistically we are testing the null hypothesis, i.e. that two sets of measurements are NOT different If t exp > t table If t exp < t table significant difference No significant difference

20 Specificity Specificity is a measurement of the degree to which the analyte of interest can be distinguished from other compounds in the sample Analytical methods vary in their specificity. Ionization detectors (relatively non-specific) Chromatography + UV detection (more specific) Chromatography + tandem mass spectrometry (most specific)

21 HPLC analysis of tyrosine metabolites in urine extract

22 Limit of detection/sensitivity The limit of detection (LOD) is a measure of how sensitive the analytical method is. LOD = the smallest quantity of analyte that is significantly different from the blank. A variety of methods exist to measure LOD: LOD = 3 x Signal/Noise LOD = (t x s)/m where t = student s t-statistic m = slope of calibration curve s = standard deviation of n 7 measurements made near the LOD

23 Limit of quantitation The limit of quantitation (LOQ) is the smallest quantity of analyte that can be measured with acceptable accuracy and precision. A variety of definitions exist for LOQ: LOQ = 10 x S/N LOQ = (10 x s)/m LOQ is determined by n 7 replicate analyses of a samples with a concentration near the LOQ LOQ should be reported as a concentration, with the associated precision and accuracy of the measurement

24 How is QA/QC incorporated into Sampling level: chemical analysis? Choose sampling techniques that will meet the needs of your analysis Collect sufficient samples (and replicates) in the field to adequately characterize the environmental contaminant being sampled Include field blanks to quantify the potential for contamination in the field

25 How is QA/QC incorporated into chemical analysis? Analysis level (as a client): Choose a reliable, certified laboratory Ensure the analytical method to be used is suitable Submit blind samples to the lab including blank and positive control samples Ensure adequate documentation of services to be provided Ensure appropriate chain of custody procedures

26 How is QA/QC incorporated into chemical analysis? Analysis level (as a lab or analyst): Ensure the analytical method and sample preparation procedures to be used are suitable. Include assay & instrument blanks and duplicates to evaluate contamination and precision for each component of the analytical procedure Ensure adequate documentation of services to be provided Ensure appropriate chain of custody procedures Participate in accreditation and performance evaluation programs

27 Assessing recovery Oftentimes, we are unable to extract or recover all of the analyte from a given matrix. If this incomplete recovery is not accounted for it would cause a systematic (negative) bias in our results. Recovery can be assessed as follows: Add a known amount of analyte to a clean sample or extract. Percent Recovery = 100 Cspiked sample C C added unspiked sample Use a surrogate compound ( recovery standard ) to assess recovery Determine analyte concentration in a known sample

28 Standard Reference Materials (SRMs) The ultimate known sample SRMs are typically environmental or biological samples of consistent composition in which the analyte concentration has been reliably determined by multiple laboratories using a variety of acceptable analytical methods Examples: Arsenic compounds in human urine Heavy metals in mussel tissue Polyaromatic hydrocarbons in air particulate Analysis of an SRM allows you to test yourself against a defined standard, and hence to prove that your lab is generating reliable data

29 Specific QC samples Calibrants at least 5, spanning the full range of analyte concentrations reported (not including zero) Blanks field blanks, lab blanks, instrument blanks Duplicates instrument duplicates and sample duplicates duplicates for 10-15% of samples analyzed Positive control or spiked matrix samples

30 Laboratory performance validation Certification by professional bodies ISO 9001 American Industrial Hygiene Association Participation in proficiency testing programs Participation in round-robin or intercomparison exercises

31 Advanced Quantification methods: Standard addition In standard addition, known quantities of analyte are added to the unknown. From the increase in signal, we deduce how much analyte was present in the original unknown. Standard addition is particularly appropriate to account for matrix effects, i.e. where the composition of the sample affects the analytical signal

32 Advanced Quantification methods: Standard addition

33 Advanced Quantification methods: Internal Standards An internal standard (ISTD) is a compound that is different from the analyte, but chemically similar to it, that is added to the sample at a known concentration. The concentration of analyte is calculated as a ratio of the signal generated by the ISTD: Conc Analyte = Area Analyte Area Conc ISTD ISTD An ISTD can correct for certain errors, including: Drifting instrumental response Volumetric errors Losses of analyte during sample prep.

34 Sampling Protocol for ENV H 432 Choosing a good sample location What range of concentrations is expected and detected by the field method? What is the detection limit of the instrument in the laboratory? How much sample mass is necessary to collect for detection in laboratory? Collect enough sample for lab analyses Duplicate sampling allows measurement of reproducibility (Precision)

35 Sampling Protocol Choosing a representative sample Is the sampling location homogeneous with respect to the analyte? Analyte concentrations may vary in a water column (lake), soil core, paint layer, particle size fraction Pooling samples from different locations will correct for spatial variability of the analyte concentration Choosing a relevant sample Does the sample matrix capture the route of human exposure to the analyte? Compliance monitoring follows a preset map of sampling sites in order to detect contamination

36 Sampling Protocol Do the collection vessels or filters irreversibly sorb the analytes? Glass containers best for organic compounds Plastic containers for metals (inorganics) How do you monitor contamination during sampling? Field blank detects any possible contamination How should the samples be stored prior to laboratory analysis? Analyte stability can be affected by storage temperature, light, humidity, air

37 Analyte Stability Are there any reactions that need to be halted at the moment of sampling? For analysis of disinfection byproducts in drinking water samples : Quench chlorine to prevent further chlorination Add biocide (NaN 3 or Boric Acid) to prevent biodegradation Will the analytes be more stable (less volatile) at a certain ph? ph buffer (ph 5 for trihalomethanes) Acidify for metals

38 Analyte Recovery (QC) Field and Laboratory Blanks Analyte-free samples used to detect contamination Matrix-Spiked Samples and Blanks A known amount of analytes spiked into samples and blanks in order to measure Percent Recovery* R = 100 (A B) / C A = measured concentration in the fortified sample B = measured concentration in the sample C = fortifying concentration (spiked level) Also known as fortified samples and blanks * EPA version of earlier equation, with better defined variables

39 Analyte Recovery (QC) Quality Control Standards added to sample Surrogate Standard (prior to lab sample prep) Compound with similar properties to analyte, used to monitor percent recovery through sample prep steps Sometimes called the internal standard (definition inconsistent) Internal Standard (prior to instrumental analysis) Compound that is different from the analyte and is added to the final extract (or extraction solvent) to account for variations in instrument response Internal Standard Calibration draws a best-fit line through the response ratio of the analyte to the internal standard (Area Analyte /Area IS ) vs. the analyte concentration (C Analyte )