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Chem. 230 10/30 Lecture

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Announcements

Quiz 3 Today

HW Set 4 will be posted

What we are covering today Quantification in Chromatography

Mass Spectrometry

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Quantitation in ChromatographyOverview

Performance Measures

Detector Response

Levels of Detection and Quantification Data Smoothing

Integration

Calibration Methods

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Quantitation in ChromatographyPerformance 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

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Quantitation in ChromatographyDetector 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 willresult)

Note: for some mass flow (HPLC-ABDs and HPLC-MS) transportefficiency depends on liquid flow so signal is not directlyproportional to flow rate

Time

Concentration Detector

flow off

flow on

Time

Mass Flow Detector

flow off flow on

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Concentration Type - examples:

PID (GC)

UV-Vis (HPLC)

Fluorescence (HPLC) Mass Flow Type - examples:

FID (GC)

NPD (GC)

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Detector Signal

Depends on concentration of analyte or mass ofanalyte reaching detector

Most (but not all) detectors give linear response overportion of detectable range

Detector Noise

Present in all detectors

High and low frequency types

Ability to Detect Small Quantities Depends onSignal (Peak Height) to Noise Ratios

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Quantitation in ChromatographyLevels 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

high frequency

component

low frequency component

peak

to

peak

nois

e

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Quantitation in ChromatographyLevels 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)

Limit of Quantification (LOQ): Calculated in similar fashion as LOD Lowest concentration to give an reasonable conc. (e.g. can be

auto-integrated using software) Typically 5LOD

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Quantitation in ChromatographyData 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

300

350

400

450

500

550

15 17 19 21 23

time

response

Raw DataFiltered Data

Excess Filtering

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Quantitation in ChromatographyIntegration

Integration of peakshould give: peak height peak area peak width (often just peak

area/peak height) Difficulty comes from

determining if a peak is apeak (or just noise), andwhen to start the peak

and end the peak. Can use auto

integration or manualintegration

we want to pick

up this peak

but not these

noise spikes

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Other issues inintegration (besidesnoise peaks)

start and ends topeaks

how to splitoverlapping peaks

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Peak Height vs. Peak Area Reasons for using peak area

peak area is independent of retention time

(assuming linear response), while the peak heightwill decrease with an increase in retention time

peak area is independent of peak width, while thepeak height will decrease if the column isoverloaded (non-linear response)

Reasons for using peak height Integration errors tend to be smaller if samples are

close to the detection limits

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Quantitation in ChromatographyLOD/LOQ example

Determine the LODs and LOQ for thefollowing example. Determine it for the4.6 min peak if the concentration is 0.4 ngL-1. Use the 3.3 and 2N LOD defintions.

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Quantitation in ChromatographyCalibration Methods

External Standard most common method 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

Internal Standard Common for GC with manual injection

(imprecisely known sample volume) Useful if slow drift in detector response Standard added to sample; calibration and

sample determination based on peak arearatio

F = constant where A = area and C = conc.(X = analyte, S = internal standard)

SX

SX

CC

AAF

/

/

Area

Concentration

AX/AS

Conc. X (constant conc. S)

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Quantitation in ChromatographyCalibration Methods

Standard Addition Used when sample matrix affects

response to analytes Commonly needed for LC-MS with

complicated samples Standard is added to sample (usually

in multiple increments) Needed if slope is affected by matrix Concentration is determined by

extrapolation (= |X-intercept|)

Surrogate Standards Used when actual standard is not

available Should use structurally similar

compounds as standards Will work with some detector types

(FID, RI, ABDs)

Area

Concentration

Added

Analyte

Concentration

0 bmXA

mbX /

standards in water

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QuantitationAdditional (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 directmeasurement of sample)

Useful for determining losses during extractions,derivatization, and with matrix effects

expected

unknowntotal

expected

recovered

amount

100amount-amount

amount

100amountrecovered%

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QuantitationSome Questions/Problems

1. Does increasing the flow rate improve the sensitivity ofa method?

2. Does the use of standard addition make more sensewhen using a selective detector or a universaldetector?

3. Is a matrix effect more likely with a simple sample or acomplex sample?

4. Why is the internal standard calibration more commonwhen using manual injection than injection with anautosampler?

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QuantitationSome Questions/Problems

5. A scientist is using GC-FID to quantitate hydrocarbons. The FID isexpected to generate equal peak areas for equal numbers ofcarbons (if substances are similar). Determine the concentrationsof compounds X and Y based on the calibration standard (1-octanol). X = hydroxycyclohexane and Y = hydroxypentane.

Compound 1-octanol cC6-OH cC5-OH

Area 3520 299 1839

Conc. (ugmL-1)

10.0 ? ?

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QuantitationSome More Questions/Problems

6. A chemist is using HPLC with fluorescence detection. He wants tosee if a compound co-eluting with a peak is quenching(decreasing) the fluorescence signal. A set of calibrationstandards gives a slope of 79 mL g-1and an intercept of 3. Theunknown 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-1standard is addedto 4.0 mL of the unknown, it gives a signal of 265. What is theconcentration of the unknown compound and is a significantquenching (more than 10% drop in signal) occurring?

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QuantitationSome More Questions/Problems

6.7. A chemist is testing an extraction process for removing DDT fromfish fat. 8.0 g of fat is first dissolved in 50 mL of 25% methylenechloride in hexane. The 50 mL is divided into two 25 mLportions, one of which is spiked by adding 2.0 mL of 25.0 ng mL-1DDT. Each portion is run through a phenyl type SPE cartridge

and the trapped DDT is eluted with 5.0 mL 100% methylenechloride. The methylene chloride is evaporated off, and thesample 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) of63 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. ofDDT in the fat in ppb?

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Mass SpectrometeryOverview

Applications of Mass Spectrometry

Mass Spectrometer Components

GC-MS LC-MS

Other Applications

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Mass SpectrometeryApplications

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

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Purposes of Mass Spectrometry Quantitative Analysis (essentially used as any other

chromatographic detector) Advantages:

selective detector (only compounds giving same ion fragmentswill overlap)

overlapping peaks with same ion fragment can be resolved(through deconvolution methods)

semi-universal detector (almost all gases and many solutes inliquid will ionize)

very good sensitivity

Disadvantages cost

requires standards for quantification

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Purposes of Mass Spectrometry - continued Qualitative Analysis/Confirmation of Identity

With ionization method giving fragmentation, few compounds willproduce the same fragmentation pattern

Even for ionization methods that dont cause fragmentation, theparent ion mass to charge data gives information about thecompound identity.

Some degree of elemental determination can be made based onisotopic abundances (e.g. determination of # of Cl atoms in smallmolecules).

Additional information can be obtained from MS-MS (furtherfragmentation of ions) and from high resolution mass spectrometry

(molecular formula) if those options are available. Isotopic Analysis Mass spectrometry allows analysis of the % of specific isotopes

present in compounds (although this is normally done by dedicatedinstruments)

An example of this use is in drug testing to determine iftestosterone is naturally produced or synthetic

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Mass SpectrometeryInstrumentation

Main Components:

Ion source (more details on subsequent slides)

Analyzer (more details on subsequent slides)

Detector: most common is electron multiplier

Anode

Cathode

Dynodes

M+e-e-

I

Detection Process:

Ion strikes anode

Electrons are ejected

Ejected electrons hitdynodes causing acascade of electronreleases

Current of electronshitting cathode is

measured

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Ion Sources For Gases

Electron Impact (EI): electrons from heated element

strike molecules M + e-=> M+*+ 2e-

M+is the parent ion

Because M+*often has excessenergy, it can fragment further,usually producing a smaller ion

and a radical Fragmentation occurs at bonds,

but electronegative elementstend to keep electrons

e-

+

e-

gas stream M

CH3-Br+*

CH3+ + Br

Main

fragment

CH3+ Br+

Minor or unobserved

fragment

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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 tocharge ratio of MW + 1)

Less fragmentation occurs, so more useful for identifyingthe parent ion

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