Chem. 231 – 3/11 Lecture

1. Chem. 231 – 3/11 Lecture

2. Announcements I Set 1 Lab Reports – due 3/13 Homework Set 3 – due today Quiz 3 (last quiz) – today Set 3 Labs Pass out handout Will Start after Spring Break Remaining Discussion Schedule (tentative) Today (finish practical aspects of GC/HPLC) Mar. 18 (remaining topics and discussion of set 3 labs)

3. Announcements II Remaining Discussion Schedule (cont.) April 1 (review for final exam) April 8 (Final Exam) April 29 (Set 3 Lab Presentations) Today’s Discussion Today’s Lecture Practical Aspects of GC (parts not covered previously) Practical Aspects of HPLC

4. Practical Aspects of GCMore on Matching Sample/Analytes to Equipment Injectors – other than split/splitless gas samples: can do direct injections with split/splitless, but lack sensitivity specialized methods (injection valves, trap systems) water samples: purge and trap most common SPME methods: wide variety of applications with standard GC injector limited to moderately volatile compounds in head space methods or moderate to low volatility in immersion methods

5. Practical Aspects of GCMore on Matching Sample/Analytes to Equipment Columns format: capillary columns generally preferred over packed columns (much higher resolution possible) exceptions: prep separations, specialized stationary phases, thermal conductivity detectors (hydrocarbon free analytes) capillary column dimensions: smaller diameter gives better resolution but has less capacity and is somewhat slower (for a given length) MS requires small to medium diameters, while FID responds to analyte flux (somewhat greater signal for larger diameters) length governed by needed resolution vs. time for analysis thinner films give better resolution and lower capacity thinner films more useful for higher boiling point compounds 2D formats can give benefits of high resolution without the cost of excessive time

6. Practical Aspects of GCMore on Matching Sample/Analytes to Equipment Columns stationary phase: typically want column polarity to match analyte polarity not required, but advantageous to trap good compounds and pass interferences exception can occur if at upper end of column’s temperature limit less volatile compounds require more robust columns (high temperature limit is usually due to column bleed)

7. Practical Aspects of GCMore on Matching Sample/Analytes to Equipment Detectors Universal (FID, TCD, MS): TCD for non-FID detectable gases (e.g. N2O) FID allows surrogate standard calibrations + is very common MS is useful as both universal and selective detector MS selected if qualitative analysis also needed on unknown compounds

8. Practical Aspects of GCMore on Matching Sample/Analytes to Equipment Detectors – cont. Selective: Generally selected for more complex samples (although all analytes must be detectable) where selectivity is needed Most are sensitive (good for trace analysis) ECD – for electronegative elements (halogens plus some oxygen and nitrogen-containing compounds) Element specific detectors (e.g. NPD, FPD) for specific elements (N, P, and S)

9. Practical Aspects of GCQuestions Pyrethroids are natural pesticides (no N or P atoms) with relatively high boiling points. There are about 15 types. If one is interested in measuring pyrethroids in environmental samples (moderate complexity and concentrations are low), what type of column, injection, and detector should be used? A student is using a GC-FID with a 0.25 mm x 30 m DB-5 column and split/splitless injector to identify relatively volatile reaction products (a mixture of esters) in a hexane solvent. With a split ratio of 1:10, the peak shape is not very good leading to peak overlap. Sensitivity is not an issue. How could the separation be improved?

10. Practical Applications of HPLC Column Selection – Stationary Phase Normal Phase vs. Reversed Phase Solute must be soluble in mobile phase (this may rule out the most polar compounds from silica based normal phase and the most non-polar compounds from reversed phase) In complex samples, more retention is desired to remove analytes from a mass of less retained compounds (this makes reversed phase undesirable for highly polar compounds and normal phase undesirable for non-polar compounds) Silica vs. Silica Bonded Phases Silica is undesired because it requires long stabilization times Bonded silica can bleed, particularly outside stable pH range (~2 to 8)

11. Practical Applications of HPLC Column Selection – Stationary Phase – cont. Silica Particles vs. Other Backbones Silica production (e.g. cost of controlled particle sizes) and column performance (efficiency, capacity, pressure range, etc.) is generally superior to other columns Silica often has more limitations in terms of useful conditions (temperature and pH) vs. other packing material Column Selection – dimensions packing material size small for better resolution but also results in greater backpressure small particles often selected for fast separations with short columns must minimize other sources of extra column broadening

12. Practical Applications of HPLC Column Selection – dimensions – cont. Column length longer (e.g. 250 for 5 mm particles) for better resolution but also slower analyses small particles often selected for fast separations with short columns Column diameter prep size (>4.6 mm diameters) conventional size (~4.6 mm diameter) narrow or micro bore (< 4.6 mm diameter) primarily used to increase mass sensitivity (most detectors show increase mass sensitivity but decreased concentration sensitivity at lower flow rates) particularly good for some detectors (MS and ECD) must minimize extra-column broadening

13. Practical Applications of HPLC How to minimize extra column broadening Make sure connections are good Reduce dispersion in injection Minimize connecting tubing lengths and diameters and connections Gradients help with pre-column dispersion Use fast detectors void volume good connection bad connection

14. Practical Applications of HPLC Detector Selection Universal Detectors RI – most universal but least sensitive UV detection – large range of detectable compounds, but can not use with surrogate standards for calibration Aerosol based – good for non-volatile compounds MS – not as universal as with GC due to ionization difficulties Selective Detectors ECD – applicable to a wide range of compounds, very sensitive, but requires high % water in mobile phase Fluorescence – good sensitivity and selectivity, but to a small range of compounds MS – also not as selective as GC

15. Practical Aspects of HPLCQuestions A chemist has been using a 5 mm, 150 x 4.6 mm C18 column to separate and detect 5 compounds in a 15 minute run. He wants to improve the speed and switches to a 3 mm, 100 x 4.6 mm C18 column. If run at the same flow rate and using the same mobile phase composition, should the resolution improve (theoretically)? How much faster will the run be? What could cause an observed decrease in resolution? If two compounds from a sample are found to co-elute with a k value of 5, what can be done to improve selectivity? For example guaiacols and corresponding syringols typically have small separation factors (a values)