Chem. 230 – 9/11 Lecture

1. Chem. 230 – 9/11 Lecture

2. Announcements I • HW Set 1 due • Additional Resources (show students site) • Application Paper (pass out)

3. Announcements II First Quiz First 30 minutes next Wednesday Will cover materials on Simple Extractions (in text and covered in lecture) Questions will be similar to those given as examples in lecture and in homework You should be familiar with equations needed, but constants will be provided Example Quiz + Solutions posted

4. Advanced Extraction TechniquesSPE Demonstration • Procedure: • Clean cartridge with removing solvent, then sample solvent • Apply sample; strongly retained compounds will remain on stationary phase, weakly retained/unretained compounds pass through • Rinse cartridge with sample solvent • Apply eluting solvent to remove strongly retained compounds • It is possible to increase solvent strength to remove compounds in several fractions

5. Advanced Extraction TechniquesSPE Demonstration Example: Mixture of methylene blue (organic cation) and I2 in 90% water 10% methanol Could in theory separate using either C18 or cation-exchange solid phase C18 used in example (and methylene blue is a “sticky” molecule – so sticks to many surfaces, though should pass through C18 column)

6. Advanced Extraction TechniquesSolid Phase Extraction • Solid Phase • Very Similar to HPLC packing particles • Smaller column • Larger particles (allowing low pressure elution) • Some Types • Silica Based (octadecyl or C18, phenyl, aminopropyl, etc.) • Ion Exchange (normally charged group on polymeric solid) • Others (e.g. graphitic carbon)

7. Advanced Extraction TechniquesSolid Phase Extraction Partitioning Strategies As before, both efficient phase transfer and good selectivity are desired To trap less polar compounds in polar solvents, hydrophobic stationary phases (also known as reversed-phase) are desired. (Example: pesticides in water) To trap more polar compounds in less polar solvents, hydrophillic stationary phases (also known as normal phase) are desired. (Example: sugars in acetonitrile, steroids in hexane) Trapping of polar compounds in polar solvents (or non-polar compounds in non-polar solvents) is difficult. “Breakthrough” often occurs. Larger analyte – solvent polarity difference allows better trapping but is limited by analyte solubility. To trap ionic compounds (usually in water), stationary phases with charged groups opposite in charge to analyte ions are used. It may be possible to produce several fractions by increasing solvent strength or changing pH.

8. Advanced Extraction TechniquesSolid Phase Extraction Reversed-Phase Groups C18 (most commonly used); best for trapping compounds with alkyl groups Phenyl: good for enhanced retention of aromatic compounds “Stronger” solvent is less polar Normal-Phase Groups Cyano (-CN) Amino (-NH2) Hydroxy (diol or SiOH) “Stronger” solvent is more polar

9. Advanced Extraction TechniquesSolid Phase Extraction Ion Exchange Stationary Phases Sulfonate groups common for cation exchange Ammonium groups –NR3+ common for anion exchange Trapping occurs in low ionic strength solvents; release occurs in high ionic strength Weak acids/bases need to be trapped in ion form but also can be released by pH adjustment

10. Advanced Extraction TechniquesSolid Phase Extraction Breakthrough and Release When SPE cartridges are used to trap and release compounds, losses can occur from incomplete trapping (breakthrough) or release of compounds. Breakthrough can occur because the partitioning equilibrium is not strong enough or due to capacity of cartridge is exceeded (sample overload) Breakthrough can be determined by measuring the concentration of solute passed through cartridge (either in whole sample or in intervals) Release can be determined by secondary rinses of SPE cartridge

11. Advanced Extraction TechniquesSPE - Questions It is desired to trap benzoic acid in an aqueous phase on an SPE cartridge and release it to an aqueous phase. Is this possible? Fish triglyerides are extracted in hexane. Describe a way to separate the triglyerides from more polar compounds (free fatty acids and steroids with OH groups). Trapping of trace amounts of phenols in water is attempted. To concentrate phenols, large volumes of water are used followed by small volumes of acetonitrile. What is a concern? Some of the phenols in water contain carboxylic acids. Suggest a way to trap both carboxylic acid-containing phenols and regular phenols while releasing them into two fractions for separate analysis. The pKa for carboxylic acids are about 4 and about 10 for phenols.

12. Advanced Extraction TechniquesSolid Phase Micro Extraction (SPME) First described in Arthur, C.; Pawlisyzn, J.; Solid phase microextraction with thermal desorption using fused silica optical fibers, Analytical Chemistry (1990) 62, 2145-2148. Can be used for subsequent analysis by GC or HPLC, but most common with GC Typically, non-exhaustive type sampling (meaning only a portion of analyte in sample is trapped). Quantitation is based on keeping exposure to samples the same (easier with autosampler). While quantitation is often difficult, sensitivity is enhanced relative to SPE because whole trapped sample is injected.

13. Advanced Extraction TechniquesSPME Procedure The needle pierces the septum to a sample (sample can be gas, liquid, or headspace) The sheath is removed allowing trapping of analytes on fiber Stirring helps the transfer The sheath goes back and the needle is withdrawn The needle pierces the septum to a GC, the sheath is withdrawn and the analyte is desorbed by the heated GC injector Fiber GC Inlet

14. Advanced Extraction TechniquesSolid Phase Micro Extraction (SPME) Sample Types (GC analysis) Liquid Samples (best when analyte concentrations are low) Headspace Sampling (avoids fiber fouling) Gas Samples In Fiber Derivatization (typically applied to polar organic compounds which often decompose on GC columns) Areas of Applications (reviews on these areas) Environmental Analysis (VOCs in air, pesticides in water, soil/sediment analysis, toxic metals) Biological Samples Food Analysis Natural Products

15. Advanced Extraction TechniquesSPME – Advantages/Disadvantages Advantages: Listed as “Solvent-less” technique (at least great reduction in solvent injected into GC) Less interference from solvent peak Reduced injection of non-volatiles Less sample handling (+ ability to automate) Can chose fibers for good selectivity Disadvantages: More difficult for quantitative results Limited lifetime of fibers Memory effects (slow desorption from fibers)

16. Advanced Extraction TechniquesOther Methods Emphasis toward microscale methods Liquid-Liquid Microextraction (drop scale liquid liquid extraction) Use of semi-permeable membranes (discussed in text) Stir-Bar Sorptive Extraction 1. Stir bar traps analytes 2. Stir bar transferred to GC inlet

17. Advanced Extraction TechniquesSome Questions A test for decomposition of a milk sample is made by measuring small aldehydes (e.g. butyraldehyde) by SPME through direct immersion in milk. A non-polar fiber is used and analysis is performed by GC with a non-polar stationary phase. Which of the following are advantages of using the SPME method: removal of interferents (other parts to milk) 2 dimensions of separation (on SPME fiber and on GC column) increase of concentrations by trapping on fiber avoiding need for more labor intensive methods (e.g. liquid – liquid extraction) If a fiber sits in a solution long enough, the peak area will reach a constant (be independent of time). Why is this? Is this exhaustive extraction? In SPME for HPLC, analytes are desorbed from the fiber into solvent that is injected into the HPLC column. Should the solvent be “stronger” or “weaker” than the sample solvent? In comparing direct headspace injections with SPME headspace injections, later eluting peaks (by GC)are larger in SPME. Explain why.

18. Chromatographic TheorySimple Separations vs. Chromatography Simple separations generally involve one to several process steps that lead to two to several fractions. Simple separations are limited to coarse fractionation of samples. Chromatographic separations are generally capable of isolating more than 5 compounds. Once the number of simple separation steps goes over a few (maybe 5 maximum), it becomes a labor inefficient way of performing a separation.

19. Chromatographic TheorySimple Separations vs. Chromatography Example of separation of two compounds by LLE. Compound X has Kp = 0.25 and Compound Y has Kp = 4. Extraction of X and Y using n washes with extractant phase (equal volumes and saving all extractant phase) To get efficient transfer of X means transferring a fair amount of Y also (poor selectivity)

20. Chromatographic TheorySimple Separations vs. Chromatography Continuation of example Better selectivity at same efficiency can be made by adjusting extract volume and increasing number of extractions In past example, using Vraf/Vext = 2.5/1 with 5 extractions results in 99% efficient transfer of X, while only transferring 38% Y Table shows dependence of %Y transferred on Kp values (assuming Kp(Y) = 1/Kp(X)) and 99% transfer of Y with 3 extractions (volumes adjusted to get ~99% transfer of X)

21. Chromatographic TheorySimple Separations vs. Chromatography Chromatography example Even column of poor efficiency can handle much more similar compounds Example: KY/KX = 1.25 (= a value) If we assume kX = 4, and resolution = 1.5 (minimum for “baseline”), a plate number of ~1000 would be needed (not very high)

22. Chromatographic TheorySimple Separations vs. Chromatography Conclusions to example: Unless order of magnitude differences in Kp values, simple separations have limited use (e.g. reduction of interfering substance). Simple separations are better for coarse separations Chromatographic separations can handle similar K values much better.

23. Chromatographic Theory Chromatography vs. Other Advanced Separation Techniques Chromatography is based on analyte partitioning between two phases Other methods use different mechanism for separation of analytes (e.g. electrolytic mobility in capillary zone electrophoresis) Some areas of overlap (e.g. Capillary electrochromatography and size exclusion chromatography)

24. Chromatographic TheoryPhase Definitions Mobile Phase (M subscript in later parameters) Fluid phase (gas, liquid or supercritical fluid) that moves through stationary phase Mobile phase defines the major classes of chromatography (GC, LC and SFC) Stationary Phase (S subscript) A non-moving phase (except in MEKC) to which compounds partition via absorption or adsorption Phase can be liquid (not very stable), liquid-like (most common), or solid (common for some applications) In past was second part of class name (for example GLC for gas-liquid chromatography)

25. Chromatographic TheoryMore on Stationary Phases Stationary phases come in several arrangements: in columns or on plates (used in thin layer chromatography) In columns, open tubular (coated walls), packed columns and monoliths are possible means of attaching stationary phase Packed columns contain packing material with the stationary phase either being the surface or being a coating on the surface Porous packing material is common Most common stationary phase is a liquid-like material chemically bonded to packing material or to wall (in open tubular chromatography).

26. Chromatographic TheoryMore on Stationary Phases Open Tubular (end on, cross section view) Packed column (side view) (e.g. Silica in normal phase HPLC) Column Wall Packing Material Stationary phase is outer surface (although influenced by adsorbed solvents) Mobile phase Stationary phase (wall coating) Expanded View Bonded phase (liquid-like) Stationary Phase Chemically bonded to packing material Packing Material Note: true representation should include micropores in sphere

27. Chromatographic TheoryDefinition Section Chromatograph = instrument Chromatogram = detection vs. time (vol.) plot Chromatograph Components Sample In Chromatographic Column Detector Flow/Pressure Control Mobile Phase Reservoir Waste or fraction collection Injector Chromatogram Signal to data recorder

28. Chromatographic TheoryDefinition Section – Flow – Volume Relation • Relationship between volume (used with gravity columns) and time (most common with more modern instruments): V = t·F V = volume passing through column part in time t at flow rate F Also, VR = tR·F where R refers to retention time/volume (time it takes component to go through column or volume of solvent needed to elute compound)

29. Chromatographic TheoryDefinition Section – More on Volume • Hold-up volume = VM = volume occupied by mobile phase in column • Stationary phase volume = VS • Calculation of VM: VM = Vcolumn – Vpacking material – VS VM = tM·F, where tM = time needed for unretained compounds to elute from column