Chem.

230 10/30 Lecture

Announcements
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

flow on flow off

Time

flow off flow on

Time

Quantitation in Chromatography
Detector Response
Concentration Type - examples:
PID (GC) UV-Vis (HPLC) Fluorescence (HPLC)

Mass Flow Type - examples:

FID (GC) NPD (GC)

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)

Signal = peak height

peak to peak noise

high frequency component

low frequency component

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

Limit of Quantification (LOQ):

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

Difficult to remove noise with frequency similar to or lower than peaks

550 500

response

450 400 350 300 15 17 19 time 21 23

Raw Data Filtered Data Excess Filtering

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)

Reasons for using peak height

Integration errors tend to be smaller if samples are close to the detection limits

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

Additional (Recovery Standards + Questions) Recovery Standards

Principle of use is similar to standard addition Standard (same as analyte or related compound) added to sample, then measured (in addition to direct measurement of sample)
%recovered amount recovered100 amount total - amount unknown 100 amount expected amount expected

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.

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Compound Area Conc. (ug mL-1)

1-octanol 3520 10.0

cC6-OH 299 ?

cC5-OH 1839 ?

Some More Questions/Problems

6. A chemist is using HPLC with fluorescence detection. He wants to see if a compound co-eluting with a peak is quenching (decreasing) the fluorescence signal. A set of calibration standards gives a slope of 79 mL g-1 and an intercept of 3. The unknown gives a signal of 193 when diluted 4 mL to 5 mL (using 1 mL of water). When 1.0 mL of a 5.0 g mL-1 standard is added to 4.0 mL of the unknown, it gives a signal of 265. What is the concentration of the unknown compound and is a significant quenching (more than 10% drop in signal) occurring?

Quantitation

Some More Questions/Problems

6.7. A chemist is testing an extraction process for removing DDT from fish fat. 8.0 g of fat is first dissolved in 50 mL of 25% methylene chloride in hexane. The 50 mL is divided into two 25 mL portions, one of which is spiked by adding 2.0 mL of 25.0 ng mL-1 DDT. Each portion is run through a phenyl type SPE cartridge and the trapped DDT is eluted with 5.0 mL 100% methylene chloride. The methylene chloride is evaporated off, and the sample is redissolved in 0.5 mL of hexane and injected onto a GC. The un-spiked sample gives a DDT conc. (in 0.5 mL of hexane) of 63 ng mL-1, while the spiked sample gives a DDT conc. of 148 ng mL-1. What is the % recovery? What was the original conc. of DDT in the fat in ppb?

Quantitation

Mass Spectrometery
Overview Applications of Mass Spectrometry Mass Spectrometer Components GC-MS LC-MS Other Applications

Mass Spectrometery Applications

Direct Analysis of Samples
Most common with liquid or solid samples Reduces sample preparation Main problem: interfering analytes

Off-line Analysis of Samples

Samples can be separated through low or high efficiency separations More laborious

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