Lecture 26- Nov 9 Chem 4101 – Fall 2011 Recent chromatographic developments 1- Chiral chromatography 2- GC x GC 3- LC x LC 4- Chromatography in microfluidic systems References Chiral chromatography: Hühnerfuss and Shah, J. Chrom. A, 1216 (2009) 481-502. GC x GC: Mitrevski et al., Anal. Bioanal. Chem. (2011) 401:2361-2371 LC x LC: Kivilompolo et al., LC•GC Europe, May 2011, 232-243 Microfluidics: Li et al., Anal. Chem., 2011, 83, 7863-7869 Suggested exercises: See the end of lecture notesLecture 26- Nov 9 Chem 4101 – Fall 2011 Chiral chromatography - analytes Stereoisomers made of the same atoms and bonds but with different 3D structures (configurations). When they are mirror images of each other, but are not superimposable they are called enantiomers. When they are not mirror images of each other, the possess different physical properties; there are called diastereomers Racemate or racemic mixture contains both stereoisomers in proportions 1:1. Stereogenic centerLecture 26- Nov 9 Chem 4101 – Fall 2011 Chiral chromatography - analytes Plane of symmetry No rotation Achiral No Plane of symmetry No rotation Chiral AtropoisomersLecture 26- Nov 9 Chem 4101 – Fall 2011 Chiral chromatography – Enantiomeric separations Chiral compound Chiral reagent Diastereomer products Chromatographic separation Chiral compound Chiral stationary phase Transient diastereomer complexes Chromatographic separation Chiral compound Chiral mobile phase Diastereomer complexes Chromatographic separation injectLecture 26- Nov 9 Chem 4101 – Fall 2011 Chiral chromatography – Ibuprofen S - ibuprofen R - ibuprofen Body transforms (R,S)-ibuprofen into active S-ibuprofen Detection of ibuprofen in river water Gas chromatography: Column: 6-TBDMS-2,3-Me-β-CD (505 in OV-1701) Temperature programming β -cyclodextrinLecture 26- Nov 9 Chem 4101 – Fall 2011 2D gas chromatography Tandem separations in two different columns that use different retention mechanisms (e.g. non-polar and polar stationary phases) Goal: increase peak capacity (n = TR/FWHM), resolve more components, improve S/N • Heart-cut technologies Collection of discrete elution volumes from the first column Injection into the second column Use microfluidic devices such as capillary flow systems, Swafer, etc. • GC x GC Periodic cryotrapping (refocusing) Remobilizing into second column by rapid temperature gradient The second separation must be fast (2-10 s) The detector must be fast (50 – 200 ms) • Detectors FID, TOFMS, nitrogen-phosphorous detectionLecture 26- Nov 9 Chem 4101 – Fall 2011 GC x GC of three ecstasy samplesLecture 26- Nov 9 Chem 4101 – Fall 2011 GC x GC: Column pairs Which par of columns is better? A, B, C or DLecture 26- Nov 9 Chem 4101 – Fall 2011 LC x LC Tandem separations in two different columns that use different retention mechanismsLecture 26- Nov 9 Chem 4101 – Fall 2011 LC x LC: Column selection Selection of Columns 1D Column i.d. = 1-2 mm length = 10-20 cm flow rate = 0.05-0.1 mL/min Eluent with low retention power in 2nd dimension 2D Column i.d. > i.d. of 1D column length = 2-10 cm flow rate = 0.5-5 mL/min Selection of sampling time (Modulation time) Minimize fraction volume Minimum of 2-3 fractions/peakLecture 26- Nov 9 Chem 4101 – Fall 2011 LC x LC Tandem separations in two different columns that use different retention mechanismsLecture 26- Nov 9 Chem 4101 – Fall 2011 LC x LC: Modulation time 3 fractions/peak in first dimension 1-2 fractions/peak in first dimension 3-4 fractions/peak in first dimensionLecture 26- Nov 9 Chem 4101 – Fall 2011 LC x LC: properly adjustedLecture 26- Nov 9 Chem 4101 – Fall 2011 Microfluidic affinity chromatography PDMS – glass device Cross-section of 3D geometryLecture 26- Nov 9 Chem 4101 – Fall 2011 Microfluidic affinity chromatography • PDMS – glass device • Antibodies immobilized on the walls Cross-section of 3D geometryLecture 26- Nov 9 Chem 4101 – Fall 2011 Anti-CD19 chip Target cells: Hut78 cells Impurity cells: Ramos cells Straight vrs. 3D geometriesLecture 26- Nov 9 Chem 4101 – Fall 2011 Questions 1. Are the analyte(s) of interest to your analytical problem chiral? Explain your answer. 2. If they are chiral, are they found as a racemic mixture in your sample? 3. If they are racemic, select chromatographic conditions to separate the stereoisomers. 4. Can the analyte(s) of interest to your analytical problem be analyzed by GC? Explain your answer. 5. If the answer to question 4 is YES, is it beneficial to use GC x GC? Explain your answer. 6. Do you have samples with complex matrices? Explain your answer 7. If the answer to question 5 is YES, is it beneficial to use LC x LC? Explain your answer. 8. If the answer to question 7 is YES, what pair of LC modes would you use? 9. How would you use a microfluidic device in your analytical problem? 10. Can you use afffinity chromatography in your analytical problem? Explain how you would modify the device created by Li, Gao, and Pappas to explore your analytical
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