Chem. 230 – 10/30 Lecture

1. Chem. 230 – 10/30 Lecture

2. Announcements • Quiz 3 Today • HW Set 4 will be posted • What we are covering today • Quantification in Chromatography • Mass Spectrometry

3. Quantitation in ChromatographyOverview • Performance Measures • Detector Response • Levels of Detection and Quantification • Data Smoothing • Integration • Calibration Methods

4. 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

5. 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 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 Mass Flow Detector Concentration Detector flow on flow off flow off flow on Time Time

6. Quantitation in ChromatographyDetector Response • Concentration Type - examples: • PID (GC) • UV-Vis (HPLC) • Fluorescence (HPLC) • Mass Flow Type - examples: • FID (GC) • NPD (GC)

7. Quantitation in ChromatographyDetector 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

8. Quantitation in ChromatographyLevels of Detection and Quantification • Noise can have high and low frequency parts • Ways of defining noise • peak to peak (roughly 5σ) • standard deviation (more accurate way) • Signal = peak height high frequency component peak to peak noise low frequency component

9. Quantitation in ChromatographyLevels of Detection and Quantification • Limit of Detection (LOD): • minimum detectable signal can be defined as S/Npeak-to-peak = 2 to 3.3σ • minimum detectable concentration = concentration needed to get S/Npeak-to-peak = 2 or S/σ = 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 5∙LOD

10. 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

11. Quantitation in ChromatographyIntegration • Integration of peak should give: • peak height • peak area • peak width (often just peak area/peak height) • 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” but not these noise spikes we want to pick up this peak

12. Quantitation in ChromatographyIntegration • Other issues in integration (besides noise peaks) • start and ends to peaks • how to split overlapping peaks

13. Quantitation in ChromatographyIntegration • 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

14. Quantitation in ChromatographyLOD/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 μL-1. Use the 3.3 and 2N LOD defintions.

15. 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 area ratio • F = constant where A = area and C = conc. (X = analyte, S = internal standard) Area Concentration AX/AS Conc. X (constant conc. S)

16. 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) standards in water Area Analyte Concentration Concentration Added

17. 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 direct measurement of sample) Useful for determining losses during extractions, derivatization, and with matrix effects

18. QuantitationSome Questions/Problems 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?

19. QuantitationSome 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 (1-octanol). X = hydroxycyclohexane and Y = hydroxypentane.

20. QuantitationSome 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 μg-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 μg 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?

21. QuantitationSome 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?

22. Mass SpectrometeryOverview Applications of Mass Spectrometry Mass Spectrometer Components GC-MS LC-MS Other Applications

23. 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

24. Mass SpectrometeryApplications 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

25. Mass SpectrometeryApplications 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 don’t 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. Isotopic Analysis 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

26. Mass SpectrometeryInstrumentation 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 Cathode M+ e- e- I

27. Mass SpectrometeryInstrumentation 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 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 + gas stream M e- e- CH3-Br+* CH3+ + Br∙ CH3∙+ Br+ Main fragment Minor or unobserved fragment

28. Mass SpectrometeryInstrumentation 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 CH4∙H+) 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