Forensics and the Environment (KHP) Flashcards
What is important for method validation?
- precision, accuracy
- linearity, working range
- specificity/selectivity
- detection limit and quantification limit
- robustness/ruggedness/repeatability + reproducibility
Define qualitative analysis.
- analyte identification on the basis of chemical or physical properties
Describe quantitative analysis.
Determining:
- exact concentration
- if the concentration is above a threshold/limit
- essential to understand how reliable measurements are
- be able to understand and work within limitations of analysis
Describe accuracy and precision.
- Precision = difference between measured values and true value (bias), replication
- Accuracy = certified reference materials (CRMs) or recovery experiments, define the true value
Absolute error = true (accepted) value - measured value
Describe quantification.
Measure response generated by target analyte of unknown concentration e.g. peak areas.
Relate measurement to response produced by standard(s) of known concentration (calibration):
- external standard (target compound)
- internal standard (non-naturally occurring compound)
- method of standard additions (matrix effects)
Describe peak separation and resolution in quantification.
Chromatographic data comprises series of peaks:
- need to relate peak ‘size’ to the amount of analyte
- can only distinguish peaks precisely if they are separated and resolved
Separation - measure of distance between peak centres
Resolution - quantitative description of how well the two components appear as separate peaks
Describe sampling rate of data.
Sampling rate (digitisation) of data is a function of peak width:
- needs to be high enough to caharacterise peaks
- digitisation rate important - double the digitisation rate means samples detected quicker so better resolution
- not just chemistry - also ability of detector to sample enough
Describe external calibration in quantification.
A series of standards are analysed.
- injection volumes etc. must be controlled
- area/unit amount of analyte is calculated via interpolation on a graph
Advantage:
- can use the target compound as a calibrant
Disadvantage:
- differences in conditions of analysis accentuate erros
Describe the type of internal standards that can be used.
- similar to compounds of interest - behave similarly in separation/detector
- do not occur naturally in the sample
- do no co-elute with any other compound
- are stable
Describe internal calibration in quantification.
Include reference material in test solution.
- no need for accurate injection volume - useful for GC+CE
- use response factors to relate amounts of target analyte and internal standard (establish prior to analyte)
Advantage:
- no need to account for losses
- accounts for matrix effects
Disadvantage:
- need to determine response factors
Describe working range and linearity.
- to define this, you need to analyse ≥ 5 standard samples and need to analyse then ≥ 3 times
- R > 0.999 for correct method validation
Describe selectivity and specificity.
- Specificity: the ability of a method to only measure what it is intended to measure
- single component analysis
- Selectivity: the extent to which other substances interfere with the determination of a substance
- multi component analysis
- Rs ≥ 1.5 to ensure no interference
Describe signal-to-noise.
Affects our ability to make a measurement.
- greater influence when signal is small
- detection limits are dependent on both signal intensity and the noise
- noise: determined from datapoints from a selected time range, various methods
Describe detection limit (LD).
- the lowest analyte concentration likely to be reliably distinguished from the blank and at which detection is feasible
- measuring replicates, usually n = 10, of a zero calibrator or blank sample
LoD = S/N > 3
- assume that if analyte is present, it will produce a signal greater than the analytical noise in absence of analyte
Describe the advantages and disadvantages of detection limit.
Advantage:
- simple and quick method
Disadvantage:
- no objective evidence to prove that a low concentration of analye will produce a signal distinguishable from blank
Describe limit of absence.
Measuring replicates, usually n = 20, of a zero calibrator or blank sample.
LoA = meanblank + 1.645 σLoA
Then, LoD = measure dilute solutions of analyte in replicate (n = 20).
LoD = LoA + 1.645 σLoD
Describe quantification limit (LQ).
The lowest concentration at which the analyte can not only be reliably detected, but where some predefined goals for bias and imprecision are met.
LoQ = S/N > 10
LoQ ≥ LoD
How do you assess ruggedness/robustness?
- determine the effect of varying conditions of method by small amounts (e.g. 10%)
- precision/accuracy should be insensitive to minor changes
How do you assess repeatabilty (sr)?
- obtain independent results with everything the same within a short period of time
- best achievable internal precision value given
How do you assess reproducibility (sR)?
- obtain results with different lab, operators and equipment
- total error becomes random
Describe contaminant and pollutant.
- contaminant - substance present in the environment above natural background levels
- pollutant - a contaminant that has a harmful effect on the environment
- all pollutants are contaminants but not all contaminants are pollutants
How can we observe if there is evidence of contamination?
- using site history
- characterising natural abundances
- records - documentary, photographic, sedimentary, etc
- both general and specific
How can we observe who/what caused the contamination?
Via isotopes.
- powerful technique for tracing
- isotopic enrichment/depletion during reactions
- can use mass balance to allocate between multiple sources
- analysis by IR-MS
What can isotope analysis be performed on and why is it useful?
- isotope analysis can be performed on bulk and can also be isotope specific (CSIA)
- can provide additional data to enable discrimination between potential contaminant sources, extent of degredation, etc.
How does compound specific isotope analysis work?
- GC-IRMS
- GC separates the individual compounds
- IRMS measures the isotopic abundance of each compound
- useful for differentiating between different oils
What are the key points in method development for quantification?
- decide if pre-treatment is necessary
- choice of separation technique
- choice of detection method
- method of quantification
- normally by peak area
- calibration procedure
- blanks, number of repeats and estimate of precision
