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Quiz 3 Today HW Set 4 will be posted What we are covering today
Quantification in Chromatography Mass Spectrometry
Quantitation in Chromatography
Overview Performance Measures Detector Response Levels of Detection and Quantification Data Smoothing Integration Calibration Methods
Quantitation in Chromatography
Performance Measures
Precision
How reproducible a measurement is
Accuracy
How close measured concentration is to true value
Sensitivity
The ability to measure small concentrations or amounts of analyte
Selectivity
Can be an issue in quantification when overlapping/interfering peaks occur
% Recovery
% of analyte added to sample that is measured in sample
Quantitation in Chromatography
Detector Response
Concentration Type vs. Mass Flow Type
In concentration type, signal depends on analyte in sample cell; so generally flow independent In mass flow type, signal depends on mass transport to detector (e.g. in FID without compounds entering flame, no signal will result) Note: for some mass flow (HPLC-ABDs and HPLC-MS) transport efficiency depends on liquid flow so signal is not directly proportional to flow rate
Concentration Detector Mass Flow Detector
Time
Time
Quantitation in Chromatography
Detector Response
Concentration Type - examples:
PID (GC) UV-Vis (HPLC) Fluorescence (HPLC)
Quantitation in Chromatography
Detector Response
Detector Signal
Depends on concentration of analyte or mass of analyte reaching detector Most (but not all) detectors give linear response over portion of detectable range
Detector Noise
Present in all detectors High and low frequency types
Ability to Detect Small Quantities Depends on Signal (Peak Height) to Noise Ratios
Quantitation in Chromatography
Levels of Detection and Quantification
Noise can have high and low frequency parts Ways of defining noise
peak to peak (roughly 5) standard deviation (more accurate way)
Quantitation in Chromatography
Levels of Detection and Quantification
Limit of Detection (LOD):
minimum detectable signal can be defined as S/Npeak-to-peak = 2 to 3.3 minimum detectable concentration = concentration needed to get S/Npeak-to-peak = 2 or S/ = 3.3 Calculate as 2N/m where m = slope in peak height vs. conc. calibration plot Minimum detectable quantity = (minimum detectable conc.)(injection volume) Calculated in similar fashion as LOD Lowest concentration to give an reasonable conc. (e.g. can be auto-integrated using software) Typically 5LOD
Quantitation in Chromatography
Data Smoothing
Data should be digitized with a frequency ~20/peak width High frequency noise (where fnoise >> fsignal) can be removed by filtering
see example below note: overfiltering results in reduction of signal and loss of resolution overfiltering result also can occur if detector response is too slow (or cell volume is too large
response
Quantitation in Chromatography
Integration
Integration of peak should give:
Difficulty comes from determining if a peak is a peak (or just noise), and when to start the peak and end the peak. Can use auto integration or manual integration
peak height peak area peak width (often just peak area/peak height)
we want to pick up this peak but not these noise spikes
Quantitation in Chromatography
Integration
Other issues in integration (besides noise peaks)
start and ends to peaks how to split overlapping peaks
Quantitation in Chromatography
Integration Peak Height vs. Peak Area
Reasons for using peak area
peak area is independent of retention time (assuming linear response), while the peak height will decrease with an increase in retention time peak area is independent of peak width, while the peak height will decrease if the column is overloaded (non-linear response)
Quantitation in Chromatography
LOD/LOQ example Determine the LODs and LOQ for the following example. Determine it for the 4.6 min peak if the concentration is 0.4 ng L-1. Use the 3.3 and 2N LOD defintions.
Quantitation in Chromatography
Calibration Methods
External Standard
most common method Area standards run separately and calibration curve prepared samples run, from peak areas, concentrations are determined best results if unknown concentration comes out in calibration standard range Common for GC with manual injection (imprecisely known sample volume) Useful if slow drift in detector response Standard added to sample; calibration and AX/AS sample determination based on peak area ratio F = constant where A = area and C = conc. (X = analyte, S = internal standard) Concentration
Internal Standard
AX / AS C X / CS
Conc. X (constant conc. S)
Quantitation in Chromatography
Calibration Methods
Standard Addition
Used when sample matrix affects response to analytes Commonly needed for LC-MS with complicated samples Analyte Standard is added to sample (usually Concentration in multiple increments) Needed if slope is affected by matrix Concentration is determined by extrapolation (= |X-intercept|) Used when actual standard is not available Should use structurally similar compounds as standards Will work with some detector types (FID, RI, ABDs)
Area standards in water
Surrogate Standards
Concentration Added
A mX b 0
X b/ m
Quantitation
Useful for determining losses during extractions, derivatization, and with matrix effects
Some Questions/Problems
1. 2. 3. 4. Does increasing the flow rate improve the sensitivity of a method? Does the use of standard addition make more sense when using a selective detector or a universal detector? Is a matrix effect more likely with a simple sample or a complex sample? Why is the internal standard calibration more common when using manual injection than injection with an autosampler?
Quantitation
Some Questions/Problems
5. A scientist is using GC-FID to quantitate hydrocarbons. The FID is expected to generate equal peak areas for equal numbers of carbons (if substances are similar). Determine the concentrations of compounds X and Y based on the calibration standard (1octanol). X = hydroxycyclohexane and Y = hydroxypentane.
Quantitation
cC6-OH 299 ?
cC5-OH 1839 ?
Quantitation
Quantitation
Mass Spectrometery
Overview Applications of Mass Spectrometry Mass Spectrometer Components GC-MS LC-MS Other Applications
Chromatographic Detectors
generally most desired type since this allows resolution of overlapping peaks
Mass Spectrometery
Applications
Purposes of Mass Spectrometry
Quantitative Analysis (essentially used as any other chromatographic detector)
Advantages:
selective detector (only compounds giving same ion fragments will overlap) overlapping peaks with same ion fragment can be resolved (through deconvolution methods) semi-universal detector (almost all gases and many solutes in liquid will ionize) very good sensitivity
Disadvantages
cost requires standards for quantification
Mass Spectrometery
Applications
Purposes of Mass Spectrometry - continued
Qualitative Analysis/Confirmation of Identity
With ionization method giving fragmentation, few compounds will produce the same fragmentation pattern Even for ionization methods that dont cause fragmentation, the parent ion mass to charge data gives information about the compound identity. Some degree of elemental determination can be made based on isotopic abundances (e.g. determination of # of Cl atoms in small molecules). Additional information can be obtained from MS-MS (further fragmentation of ions) and from high resolution mass spectrometry (molecular formula) if those options are available. Mass spectrometry allows analysis of the % of specific isotopes present in compounds (although this is normally done by dedicated instruments) An example of this use is in drug testing to determine if testosterone is naturally produced or synthetic
Isotopic Analysis
Mass Spectrometery
Instrumentation
Main Components:
Ion source (more details on subsequent slides) Analyzer (more details on subsequent slides) Detector: most common is electron multiplier
Detection Process: Ion strikes anode Electrons are ejected Ejected electrons hit dynodes causing a cascade of electron releases Current of electrons hitting cathode is measured
Anode
Dynodes
M+
Cathode
e- e
Mass Spectrometery
Instrumentation
Ion Sources
For Gases
Electron Impact (EI):
gas stream
+ M e- e
-
electrons from heated element strike molecules M + e- => M+* + 2e M+ is the parent ion Because M+* often has excess energy, it can fragment further, usually producing a smaller ion and a radical Fragmentation occurs at bonds, but electronegative elements tend to keep electrons
CH3-Br+*
CH3+ + Br
CH3 + Br+ Main fragment Minor or unobserved fragment
Mass Spectrometery
Instrumentation Ion Sources
For Gases
Chemical Ionization (CI):
Can produce positive or negative ions First, a reagent gas reacts with a corona discharge to produce a reagent ion: CH4 => => CH5+ (more likely CH4H+) Then the reagent ion transfers its charge to a molecule: M + CH5+ => MH+ (one of largest peak has mass to charge ratio of MW + 1) Less fragmentation occurs, so more useful for identifying the parent ion