Forensics and the Environment (KHP)

Question 1

What is important for method validation?

Answer 1

• precision, accuracy
• linearity, working range
• specificity/selectivity
• detection limit and quantification limit
• robustness/ruggedness/repeatability + reproducibility

Question 2

Define qualitative analysis.

Answer 2

• analyte identification on the basis of chemical or physical properties

Question 3

Describe quantitative analysis.

Answer 3

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

Question 4

Describe accuracy and precision.

Answer 4

• 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

Question 5

Describe quantification.

Answer 5

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)

Question 6

Describe peak separation and resolution in quantification.

Answer 6

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

Question 7

Describe sampling rate of data.

Answer 7

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

Question 8

Describe external calibration in quantification.

Answer 8

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

Question 9

Describe the type of internal standards that can be used.

Answer 9

• 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

Question 10

Describe internal calibration in quantification.

Answer 10

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

Question 11

Describe working range and linearity.

Answer 11

• to define this, you need to analyse ≥ 5 standard samples and need to analyse then ≥ 3 times
• R > 0.999 for correct method validation

Question 12

Describe selectivity and specificity.

Answer 12

• 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
  • R_s ≥ 1.5 to ensure no interference

Question 13

Describe signal-to-noise.

Answer 13

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

Question 14

Describe detection limit (L_D).

Answer 14

• 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

Question 15

Describe the advantages and disadvantages of detection limit.

Answer 15

Advantage:

• simple and quick method

Disadvantage:

• no objective evidence to prove that a low concentration of analye will produce a signal distinguishable from blank

Question 16

Describe limit of absence.

Answer 16

Measuring replicates, usually n = 20, of a zero calibrator or blank sample.

LoA = mean_blank + 1.645 σ_LoA

Then, LoD = measure dilute solutions of analyte in replicate (n = 20).

LoD = LoA + 1.645 σ_LoD

Question 17

Describe quantification limit (L_Q).

Answer 17

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

Question 18

How do you assess ruggedness/robustness?

Answer 18

• determine the effect of varying conditions of method by small amounts (e.g. 10%)
• precision/accuracy should be insensitive to minor changes

Question 19

How do you assess repeatabilty (s_r)?

Answer 19

• obtain independent results with everything the same within a short period of time
• best achievable internal precision value given

Question 20

How do you assess reproducibility (s_R)?

Answer 20

• obtain results with different lab, operators and equipment
• total error becomes random

Question 21

Describe contaminant and pollutant.

Answer 21

• 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

Question 22

How can we observe if there is evidence of contamination?

Answer 22

• using site history
  
  • characterising natural abundances
  • records - documentary, photographic, sedimentary, etc
  • both general and specific

Question 23

How can we observe who/what caused the contamination?

Answer 23

Via isotopes.

• powerful technique for tracing
• isotopic enrichment/depletion during reactions
• can use mass balance to allocate between multiple sources
• analysis by IR-MS

Question 24

What can isotope analysis be performed on and why is it useful?

Answer 24

• 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.

Question 25

How does compound specific isotope analysis work?

Answer 25

• GC-IRMS
  
  • GC separates the individual compounds
  • IRMS measures the isotopic abundance of each compound
• useful for differentiating between different oils

Question 26

What are the key points in method development for quantification?

Answer 26

• 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