Lecture 8: Evaluating Separation Flashcards
Why is an ideal sample prep method important?
To minimise matrix effects, the key to sensitive analysis and quantification
recovery
ratio of measured concentration to the expected concentration of the analyte after extraction and preparation.
matrix effects
influence of the co-extracted components on the ionization and detection of the analyte
what are the different types of quechers workflows?
spike after extraction
spike before extraction
quality control samples
briefly give the SBE quechers workflow
spike with analyte and internal standard in solvent
extraction: add ACN, MgSO4 and buffer salts
dSPE then evaporate to dryness
reconstitute in mobile phase
briefly give the SAE quechers workflow
spike with solvent
extraction: add ACN, MgSO4 and buffer salts
dSPE then evaporate to dryness
reconstitute in mobile phase and anlayte and IS
quechers example workflow for QCs
spike with analyte, IS and solvent
which quechers is ideal for minising matrix effects?
SAE and QC
how are SAE and QC useful for matrix effects (interference)?
o Extract ‘blank (analyte-free)’ matrix and spike with target analyte after extraction, prior to analysis
o Compare with a non-extracted solvent-based quality control of the same spike concentration.
peak area is determined by…
integrating area under curve of chromatographic peak
equation for matrix effects
%ME= peak area SAE/peak area solvent QC. x100
explain the matrix effects % meanings
100% = no matrix interference
> 100 = signal enhancment
<100 = signal suppression
best quechers method for recovery
SBE and SAE
SBE and SAE for recovery
o Spike ‘blank (analyte-free)’ matrix with target analyte and extract)
o Compare with a SAE quality control of the same spike concentration.
o Important to do replicate samples – key to have repeatable recovery before high recovery!
% recovery equation
peak area SBE/peak area SAE. x100
process efficiency
overall efficiency of prep method
how is process efficiency calculated
- Non-extracted solvent-based quality control of the same spike concentration and ‘spike before extraction’ (SBE)
%PE = peak area SBE/peak area solvent QC x100
or MExRE. /100
what are the chromatographic parameters?
partition ration
retention time
resolution
capacity (retention) factor
relative retention/selectivity (separation) factor
column efficiency
partition ratio equation
molar conc of solute in stationary phase/its conc in mobile phase
retention time
rate at which analyte migrates through column
capacity (retention) factor (k’)
rate of solute migration through column
capacity factor equation
retention time of sample component/retention time on un-retained component
how can capacity factor be improved?
using more suitable mobile phase composition or stationary phase
relative retention/selectivity factor
ration affinity of each component for the stationary phase
column efficiency
the number of theoretical plates or the number of suitable retentive sites for a component to reside in a column.
efficiency is related to peak broadening and time spent on column; also expressed as column height equivalent to a theoretical plate (HETP):
