Introduction to Chromatography: Techniques, Applications, and Grading

1. Chem. 230 – 9/2 Lecture

2. Introduction- Instructor: Roy Dixon • Educational Background in Environmental Analytical Chemistry • Most of my research currently has been in HPLC technology/methodology development and applications • Currently, I’m working on GC analysis of alternative fuels • I expect to improve my knowledge of several parts of chromatography through teaching this class

3. Introduction- Students • Introduce yourself (name/degree plan) • Is there anything specific you expect to get out of the class?

4. Syllabus – Top Part • I would like to have a break mid-way through class (10 or 15 minutes) • Best way for contact is by email • Can arrange other times to meet with students • Course information will be posted on website

5. Syllabus – Text + Other Reading • Text covers instrumental chromatography fairly well (particularly good with HPLC separations) • Many of the pages assigned will only need to be skimmed over (e.g. p. 1-30) • In other areas, additional readings will be assigned (either in folder or posted on website)

6. Syllabus- Course Topics • “Core” Topics • Simple Extractions • Chromatography (emphasizing instrument based methods) • Mass Spectrometry (focus on use as chromatographic detector) • Electrophoresis (emphasizing capillary electrophoresis and related topics) • Selected “Specialized” Topics • Student Presentations and Discussions • I will give some “example” special topics: • GC x GC • Aerosol-Based Detectors for HPLC

7. Syllabus- Notes on Grading • 40 Minute Exams • Previously had 5 quizzes, but switching this semester due to only 14 meetings • Used to fit with breakdown of topics and because of 1 long lecture • Final Exam • Fairly standard exam • About 35% will come from student presentations

8. Syllabus- Notes on Grading (2) • Specialized Topic Presentation - Will go over handout later - Grading based on materials and presentation • Application Paper • Will research an improved method for a specific application (one to several related papers on a topic) • Homework • No text problems; so will give “practice” and “assignment” problems

9. Typical Lecture Style • Announcements given in first few minutes • Use board or document camera for working out example problems (a significant part to the course) • Powerpoint and Handouts will be more common on topics not covered in detail in text • Powerpoint slides will be made available on website

10. Specialized Topic Presentations I • Goal is for students to learn about new or emerging separation technology • Students will be responsible for: • researching topics • making reading material available (little or no photocopying,hopefully) • understanding concepts • preparing homework problems or questions • preparing presentations

11. Specialized Topic Presentations II • Teams of Two • I will give several “example” presentation (first by guest lecturer) • See list of topics on handout, but you may want to modify topic or select your own topic • If changing topic, be sure to clear it with me • Most of the points will be for the presentation (grading key used previously is provided on last page), but there will also be points for preparation of reading materials and homework questions • Section V show the main points needed for the presentation

12. Applications Paper I Goal is for students to research an application of a separation method for a specific application The paper should focus on new developments to improve the separation (e.g. for either better isolation or for better analysis) Students can select application area Some example applications: analysis of chiral compounds from a specific reaction or class of reactions to determine analysis of smoke tracers in atmospheric aerosols analysis of domoic acid in marine mammals

13. Applications Paper II Improvements can be from one paper (should be a significant improvement) or a set of related papers You will also need to research past separation/alternative analysis methods used for the problem Details on the report are given in the handout Some specific requirements will be asked of you (e.g. estimate the cost of the equipment to perform the method).

14. Homework Set 1 • To do before 1st Quiz. • Longer problems are to be turned in Sept. 16. • May add more problems to do for your benefit. • Problems to be turned in should be worked on independently.

15. Separation Purposes • Isolation/Purification/Removal of Compound(s) • Qualitative Analysis • Quantitative Analysis

16. Separations Diagram All Separations Simple separations Non-Partitioning Based on Partitioning Liquid-liquid extraction Filtering Higher Resolution/Instrument Based Capillary Zone Electrophoresis Gas Chromatography

17. Simple Separations/Extractions- Introduction • Assigned text reading: • p. 1-26: light, background reading • p. 27-30: covered in more detail later • Chapter 14 covers extraction + other “simple” separation methods (will cover text in more detail + will add to this in lecture)

18. Simple Separations - Purposes • Main purpose is to remove analyte(s) from interferants • A common purpose is to concentrate analyte(s) • For complicated samples with numerous analytes, simple separation can be used as coarse separation step • Often integrated with sample collection (e.g. filtration of air to collect aerosol particles) or sample modification (derivatization) • Typically insufficient for isolating analytes but needed for reduction of interferants • common main strategies: • “isolation/trapping” of analytes (usually 2 step) • “removal” of contaminants (often single step) • Primary and secondary separations

19. Simple Separations- Examples of Strategies • You want to measure combustion exhaust gases by GC. There are around 15 gases of interest that are present at moderately high concentrations. Water also is present at high concentration and interferes. • You are interested in measuring phenols present in sea-water at very low concentrations by HPLC. Interference by other organics is not a major issue. • You are interested in analyzing oligosaccharides present in glycoproteins by HPLC. There are close to 100 compounds of interest present in the sample.

20. Simple Separations • Almost all separations require more than one discrete phase • The most common separations involve partitioning between two phases X(phase 1) ↔ X(phase 2) • Some types of non-partitioning separations (mostly involving physical separations): • Filtration (removal of solids from gases or liquids) • Centrifugration (removal of solids from liquids) • Membrane based separations (separation based on molecule size or charge)

21. Simple Separations • An effective simple separation requires effective phase transfer plus significant differences in process between analyte and contaminants (good selectivity) • Sample preparation steps often require as much or more analyst time as instrument based analysis (i.e. are labor-intensive) • Preferred processes are: simpler, require less equipment, faster, effective with volumes desired, can be automated • For concentrating samples, it is important that the method can handle large sample volumes, but result in small “processed” volumes

22. Types of Simple Separations

23. Gas – Liquid Separations Sample Air • Gas Sampling • Bubblers • Mist Chambers (show) • Denuders (above for water soluble gases) • Cold Traps Gases trapped on wall coating Aerosols pass to filter Bubbler To filter, pump Denuder

24. Gas – Liquid Separations • Basis for Partitioning • Into water: Henry’s Law KH = [X]/PX where KH is the Henry’s Law Constant KH = f(T), PX = partial pressure of X, [X] = Molar conc. of X • Cold Trap: boiling point temperature

25. Gas – Liquid Separations • Headspace Analysis (GC method) • Sample in vial with liquid and gas phases • Headspace gas withdrawn with syringe for injection into GC (or other device) septum Headspace liquid

26. Gas – Liquid Separations He to waste He in He in • Purge and Trap (GC method) • Aqueous sample or gas sampled trapped in water • Steps: 1) gas purge of water to trap, 2) heating of trap to GC (or other device) Heat applied Trapped analyte To GC

27. Gas – Liquid Separations • Distillation/Evaporation • Evaporation used for low volatility liquid (or solid) • Distillation for collection of volatile analyte in liquid • Partitioning to gas phase based on Raoult’s Law (although non-ideality often occurs) PA = XAPA• where PA = partial pressure of gas A XA = mole fraction of A in liquid PA• = partial pressure of gas A above a pure A liquid • Complete separation of two volatile components is often difficult

28. Simple Separations – Gas/Liquid A is more volatile (lower Tb) • Distillations • Behavior given in T vs X plots • 80% B example • Vapor (condensate) is 30% B • Multistep/stage distillation results in separation • With azeotropes (non-ideal), complete separation is impossible (no less than 15% B in vapor) T Vapor V + L Liquid 100% A Xi 100% B Azeotropic mixture P 100% A Xi 100% B

29. Simple Separations – Gas/Liquid • Many other separations possible • Unusual Separation: Analysis of dissolved SO2 in cloud water (my dissertation project) Counter-flow Virtual Impactor SO2 Probe attached to airplane flying to left Scrubbed air goes to tip and splits into 2 flows SO2 Counter flow out of probe tip keeps gaseous SO2 out Cloud droplets evaporate releasing SO2 which flows to detector Cloud droplets have inertial and make it into probe

30. Some Questions • If it is desired to trap a gas phase analyte in water, what type of values of Henry’s law constants are desired? • For desorption of gases from liquids, what type of values of Henry’s law constants are desired? • How can temperature adjustments be made to improve Henry’s law constants for trapping or desorbing gases? • How can trapping of acetic acid in aqueous solution be improved? What about desorption? • How can trapping of ammonia in aqueous solution be improved?

31. Some More Questions • If produced correctly, biodiesel is made up of fatty acid methyl esters (for fatty acids between 12 and 20 carbons) of low/moderate volatility. Methanol can be a contaminant from its production and is much more volatile. What separation step could be used to separate methanol from more volatile constituents? Would that also work well if analyzing B20 (20% biodiesel/80% petroleum diesel? • Which component will be enriched in the original solution after distillation (based on the phase diagram below)? 100% A X 100% B

32. Still Mome Questions 3. Based on the phase diagram to the right, is it possible to isolate pure A through multi-step/stage distillation starting from 65% B? 4. Is it possible to isolate pure B by removing A through multi-step/stage distillation (starting at same point)? T 100% A X 100% B

33. Extractions – Solid to Liquid • Dissolution • Dissolution Aides • Ultrasound bath • Other mechanical shaking • Soxhlet Extractions (show device) • Extraction speed is often limited by physical process • Extraction from fine grain particles is easier than large solids

34. Extractions – Liquid to Solid • Trapping applications (such as solid phase extraction – discussed later) • Precipitation/Filtration (or centrifugation) • A way to separate components based on one component having lower solubility in a particular solvent or with particular counter ions • Precipitation of ions • Use of polar/non-polar liquids for compounds of variable polarity • Phase separation by filtration or centrifugation most common

35. Extractions – Liquid to Solid • Precipitations • Removal of ionic compounds/highly polar compounds. Through addition of less polar organic solvent (e.g. ethanol) to water • Removal of less polar compounds from organic solvent by adding more polar solvent (e.g. addition of ethanol to CH2Cl2 or water to ethanol)

36. Extractions – Liquid to Solid • Precipitations • Of Ions • Can select counter ion that will selectively precipitate one ion but not the other ion (ion to be precipitated should have lower Ksp, although will also depend on stoichiometry) • Can use Ksp values to calculate how successful the separation will be

37. Extractions – Liquid to Solid • Precipitation Example: Separation of Sr2+ from Ca2+: An examination of Ksp shows smaller Ksp for SrSO4 vs. CaSO4 (3.2 x 10-7 vs 2.4 x 10-5) If a mixture contains 1.0 x 10-2 M Sr2+ and Ca2+, how much SO42- can be added before Sr2+ starts to precipitate?, before Ca2+ starts to precipitate? What % of Sr can be isolated?

38. Extractions – Liquid to Solid • Temperature in precipitation processes • Example: Acetic Acid and Water Liquid “path” for cooling 50% acetic acid in water Eutectic Point Liquid solution T Ice + CH3CO2H (l) CH3CO2H(s) + H2O(l) Solid solution 0 100% X(CH3CO2H)

39. Some Questions From Last Lecture • What are advantages and disadvantages of Soxhlet extractions? • Suggest a way to isolate a polar organic compound from ionic compounds in urine. • It is desired to isolate CN- from CO32- by adding Ag+ and monitoring [Ag+] electrochemically. Assuming initial concentrations of [CN-] = 1.0 x 10-3 M and [CO32-] = 5.0 x 10-3 M and given Ksp values for AgCN and Ag2CO3 are 2.2 x 10-16 and 8.2 x 10-12, respectively, what would be the target [Ag+]? Will this separation be very efficient? • Can acetic acid be isolated from all solutions in water by freezing it out?

40. More Questions A 1.00 L sample of sea water is analyzed for phenols. The 1.00 L sample is passed through a solid phase extraction cartridge to trap the phenols. Then 25.0 mL of methanol is used to remove the phenols and then reagents are added that convert the phenols to methoxyphenols. The methoxyphenols are extracted by adding 25 mL of water to the methanol and extracting with two successive 25 mL portions of hexane. The hexane portions are combined, evaporated, and redissolved in 2.0 mL of hexane. An aliquot is then determined by GC and found to contain 22.1 mmol L-1 of a particular phenol. What was the original conc. of that phenol in sea water (in nmol L-1) if it is assumed that all transfers were 100% efficient? How could the sensitivity of the method be increased? The total NH3 (NH3 + NH4+) concentration of a water sample is determined by NH3 in the headspace above a sample. A water sample at a pH of 8.1 was found to have a headspace pressure of 2.4 x 10-7 atm. If KH = 1.6 x 10-5 atm m3/mol (at that T) and Ka(NH4+) = 5.6 x 10-10., calculate the total NH3 concentration in the sample

41. Liquid-Liquid Extractions One of more common simple separations Often used to introduce partition theory Equipment is simple (separation funnel or vials + syringes) Two liquids must be immiscible (form two distinct phases) Lower phase is more dense (usually water or chlorinated hydrocarbon) Most common with water (or aqueous buffer) and less polar organic liquids

42. Liquid-Liquid Extractions Partition Coefficient Kp = [X]raffinate/[X]extractant Kp depends on thermodynamics of dissolving X in two phases Most common rule for solubility is likes dissolve likes More polar compounds exist in greater concentration in water Koctanol-water values can be found in reference tables (octanol is assumed to be the raffinate) X(org) X(aq) If sample starts in aq phase, aq phase is raffinate, org is extractant

43. Next Time Will cover liquid – liquid extractions from a quantitative perspective in more detail