1. Chem. 230 – 10/14 Lecture

2. Announcements I Some Comments on Homework –glucose/levoglucosan separation: since R s = 2.3, resolution was more than optimal. So time could be shortened (at cost of lower R s ) –optimization equation (for two compounds separated on two GC columns) % change in R s not equal to % change in parameter

3. Announcements I – HW cont. For Past Example (N= 1015,  = 1.17, kB = 3.5) Note: % change in alpha and kB depend on initial conditions

4. Announcements II Change to Chem 294 Abstracts Deadline for Spring, 2015 (now due 12/1) Exam 2 today – first 40 min. Third Homework Set will be online soon Today’s Topics – Gas Chromatography

5. GC Instrumentation Sample Injection – Liquid Samples Split/Splitless Injectors –Injectors capable of running in two modes: split and splitless –Split injections used to avoid overloading columns Injection Process –Syringe pierces septum and depressing plunger deposits liquid –Analyte volatilizes –Part injected (usually smaller fraction) –Part passed to vent –Fraction vented depends on split valve outside Inside oven Septum Syringe port He in To Column Split vent Split valve liner

6. GC Instrumentation Sample Injection – Liquid Samples Split injection is used for: –Higher concentrations –Smaller diameter (OT) columns –More volatile components/less volatile solvents In split injection, solvent overload is less problematic Split ratios can vary from 3:1 (to vent: to column) to 1000:1 Splitless injection is used for trace analysis (~50% of injected sample put on column) Splitless injection usually requires temperature programming where solvent passes through cool column while analytes are trapped Split/Splitless injectors also used with SPME

7. GC Additional Information More Details on Split vs. Splitless Injection –Split liner contains baffles to help mix sample so that fraction to column is more consistent (but this fraction still varies to make split less quantitative) –Because of greater total flow in split mode, the liner is flushed out more quickly –In splitless injection, it takes time to purge vapors from liner, so peak would be wide without temperature ramping –In split injection, the split vent is closed later along with a decrease in He flow –In splitless injection, the split vent is opened later to flush out residual gases.

8. GC Instrumentation Sample Injection – Liquid Samples Other injectors: –Direct injection (liner to column) –On-column injection (needle goes into column) –First two types work with packed columns and wide-bore OT columns –Cold on-column (used with temperature programming on inlet)

9. GC Instrumentation Sample Injection – Syringes For gas samples, gas-tight syringes needed For liquid samples, manual injection results in variability in injected volume. An internal standard normally is required for quantitative work (to adjust for variation in amount injected). Use of autosamplers allows accurate injection of liquids.

10. GC Instrumentation Columns Stationary Phases discussed previously OT columns –Best resolution –More robust –High resolution (thin film 0.25 mm diameter) vs. higher capacity (thick film 0.53 mm diameter) –More expensive, difficult to make in lab Packed columns –More capacity –Better with less (mass) sensitive detectors (thermal conductivity detector)

11. GC Instrumentation Ovens/Heating The columns are housed in thermostated ovens Most GCs have temperature programming capability Most GC ovens have minimum temperatures of around 35 to 40°C The maximum temperature is usually based on avoiding damage to the column Operation at sub-ambient temperatures possible with cryogenic cooling (mostly for permanent gases/highly volatile liquids) Ramp and cool down rates are important for efficient use of GCs

12. GC Instrumentation Detectors - Classes Universal Detectors –Should detect wide range of compounds –Response should be based on mass or moles of analyte –Used for comprehensive understanding of samples Selective Detectors –Should detect only specific types of compounds –Best for selected analytes in complex samples Multi-dimensional Detectors (selective and universal)

13. GC Instrumentation Detectors Performance criteria –Minimum detectable amounts or concentrations (amount or concentration for peak with signal/noise = 3) –Linear range (or useful range for non-linear detectors) –Destructive vs. non-destructive –Concentration type vs. mass flow rate type –Other (cost, speed, size, precision, etc.) Most GC detectors perform well for cost vs. HPLC

14. GC Instrumentation Universal Detectors Thermal Conductivity Detector (TCD) –Principle: heat conducted by gas from heated filament Filament resistance is temperature dependent Heat conduction depends on gas MW –All gases (w/ MW ≠ carrier MW) detected, but variable sensitivity. –Simple, but not very sensitive

15. GC Instrumentation Universal Detectors Flame Ionization Detector (FID) –Principle GC effluent “ burned ” in hydrogen flame Ions produced in combustion and collected in electrode Current proportional to mass combusted –Good sensitivity –Hydrocarbons are detected; similar response for hydrocarbons, but decreased response with functional groups –Sample consumed in flame –One of the most common because quantitation is good and possible without standards

16. GC Instrumentation Selective Detectors Based on Gas Ionization –Electron Capture Detector (ECD) Uses β emitter to produce electrons that cause current Compounds with electronegative elements (e.g. halogens) “capture” electrons reducing current One of the most sensitive detectors available –Photoionization Detector (PID) Uses UV light to photoionize compounds (M + h ν → M + + e - ) Sensitive to unsaturated compounds

17. GC Instrumentation Selective Detectors Element Selective Detectors –Atomic plasma emission (expensive) –Specific detectors for S, N, P containing compounds based on ionization (NPD) or emission of light (flame photometric detector and sulfur chemiluminescence detector) Multiple detectors –Multiple dectors can be used provided 1 st is non- destructive if in series (e.g. PID/FID gives aromatic/unsaturated fraction of hydrocarbon samples)

18. GC Instrumentation Mass Spectrometer Detectors Very Popular Detector Both selective (single ion mode) and universal (total ion mode); plus allows structural information Will cover in more detail in separate section later

19. GC Instrumentation Some Questions 1.In response to high He prices, a lab director says that no more He can be purchased. Would you want to use Ne or N 2 ? (assuming reasonable prices for both of those gases)? What other change would be needed to get reasonable separations with Ne or N 2 carrier gases? 2.How is the retention of polar compounds affected by switching from He to H 2 as a carrier gas? 3.List two ways to inject gas samples on GCs set up for analysis of liquids. 4.For what type of columns can direct injection be used? 5.Why is temperature ramping more valuable with certain types of injections (e.g. splitless or on-column) vs. other types of injections (e.g. split) 6.A student is analyzing reaction products present in the parts per thousand level and is using a GC with a 0.25 mm OT column and a split/splitless injector. What type of injection should be used?

20. GC Instrumentation Some Questions 6.Which chromatogram to the right is from a split injection? Which is from a splitless injection? 7.A food sample is being analyzed by GC for P containing pesticides (about 10 in number). Even after extraction, there are hundreds of compounds. What injector, column (dimensions), and detector should be chosen?

21. GC Instrumentation Area of Discussion 1.With the advent of cheaper GC-MS instruments, is there still a need for other detectors? Answer: there is much less need for other detectors because GC-MS is: selective (by looking only at specific fragment ions) and universal (almost all gases give a MS response). Value in other instruments: 1) Selective detectors can offer even more selectivity for certain detectors (e.g. analysis of chlorinated pesticides by GC-ECD). Note: one can also get more selectivity in GC-MS by using GC-MS-MS instead (each fragment can get broken up a second time with analysis of secondary fragment mass to charge ratios) 2) Also, universal detectors often give more universal response (similar signal for similar number of C atoms in FID), which allows quantification without standards. MS ionization efficiency (and signal) depends on compound structure 3) Size/Power Requirements – many instruments without MS are small enough to be field portable and simpler to use

22. GC – Role of Temperature Temperature is the most common way of changing retention (high T, less retention) Relationship between temperature and distribution constant: Log K 1/T Plot for each compound Low T Homologous series Different selectivity possible for other compounds Isothermal = single T

23. GC – Role of Temperature For isothermal GC, peak gaps and widths tend to increase with increasing retention time (for constant logK) When wide separation for later eluting compounds, an increase in k is desired, but will result in unresolved early eluting peaks The solution is temperature programming where T increases with time so that k decreases with time Temperature is good for early eluting compounds Wide separation of late eluting compounds Later eluting compounds now elute faster Peak width stays narrower

24. GC – Role of Temperature Advantages of Gradient Separations –Compounds of a wide range of volatilities can be separated in a single run –Because compounds are “ trapped ” at the head of the column until the temperature is high enough that they are volatile, narrow peaks result (even when not injected as narrow plug) –Signal to noise is improved in late eluting peaks –Quicker and more efficient elution occurs –Less overlap of compounds with solvent peak

25. GC – Role of Temperature Disadvantages of Gradient Separations –Must include time for oven to cool and re-establish equilibrium in total analysis time (so decrease in time is not as large as chromatogram may indicate) –Baseline tends to be less stable (some detectors ’ response depends on T and increasing column bleed can be a problem for trace analysis)

26. GC – Other Topics Comprehensive 2 Dimensional GC (or GC x GC) –See Chapter 15, section 1 –Also materials from Crimi and Snow, LCGC North America (2008), 26, 62-70. –Another recent review (focused on applications) is Adahchour et al., J. Chromatogr. (2008), 1186, 67-108. –a simple TLC example of a 2 dimensional separation: Separation first in 1 dimension (e.g. hexane) Then separation in 2 nd dimension (using different solvent – say benzene) –GC x GC is most common multi- dimensional instrumental method (at least if excluding biochemistry) Resultant Plate High alkane affinity High aromatic affinity Inseparable in hexane In hexane In benzene 1 st Separation in 1 dimension Then Separation in 2nd dimension

27. GCxGC Effectiveness of Method –Useful when columns and samples chosen so 2 nd dimension gives additional separations –Not that valuable if 2 nd dimension does not help much (similar polarity for all compounds) –Separation principles in the two dimensional should be “orthoganol” (based on different principles) 1 st Dimension 2 nd Dimension Effective Use of 2 dimensions Less Effective Separation 2 nd Dimension 1 st Dimension

28. GC GC x GC GC x GC’s main advantages are much greater peak capacity and alternative selectivities GC x GC is useful for comprehensive analysis of very complicated samples (e.g. gasoline) in which one dimension does not have enough resolving power Also time requirement is not as great as high resolution 1-D run and relatively narrow peaks result Even greater resolution possible with GC x GC x MS

29. GC GC x GC Unlike the TLC example, in GC x GC the second degree of separation is in time, not in space The equipment needed consists of two GC columns (often in separate ovens), and a modulator (a way of stopping and injecting analytes into the second column) Column 1 modulator Column 2 (in oven) detector injector

30. GC GC x GC First column normally is non-polar in standard length in main oven Second column is normally polar and very short Main principle of separation is to have distinct separation mechanisms –In 1 st column, fastest eluting peaks are most volatile and most polar –In 2 nd, they are most volatile and least polar

31. GC GC x GC Modulation in GC x GC (one type) –Cooling column allows trapping of analytes exiting 1 st column (partly to allow completion of 2 nd column separation) –Fast heating allows small gas plug to flow to second column, allowing narrow peaks Flow in to modulator (from column 1) Cold jet holds up analytes Hot jet releases analytes 2 nd Cold jet holds up analytes Hot jet to inject analytes Cold jet stops more analytes from column 1

32. GC GC x GC It also is possible to modulate using valves First, effluent from column 1 goes to waste Then valve switches and effluent flows to column 2 briefly Valve switches back to waste This allows periodic separation of column 1 effluent on second column. This method is less efficient (much of sample is wasted). Switching Valve From column 1 To waste To column 2

33. GC GC x GC Speed related to 2 nd separation, modulation and detection must be fast (~ 50 ms detection, ~100 ms modulation, 3 s 2 nd separation) so that neither peak (first nor second dimension peak) is broadened (see example below) Data is response vs. time, but every ~3 to 5 s a new 2 nd dimension chromatogram appears. Software converts this to 3 dimensional chromatogram Second dimension is very short (so small N even if using efficient, low H column). The 2 nd Diminsion peak capacity is small, ~10, but this leads to ~10X original 1D peak capacity (e.g. 2000 vs 200)

34. GC GC x GC To achieve useful separations, 2 columns must be orthogonal (e.g. ideally 1 st dimension based on volatility, 2 nd based on polarity) Figure 3 (Crimi and Snow, 2008) shows poor orthogonality (2 nd column is only moderately polar)

35. GC GC x GC Better use of 2 nd dimension occurs with more polar 2 nd column (Fig. 5) 4 peaks resolved in 2 nd dimension off of 112 s peak: #3 (pentane, bp = 36.1ºC), #4 (MTBE, bp = 55.2ºC), #5 (2-propanol, bp = 82.3ºC), and #6 (acetonitrile, bp = 82ºC) Chromatogram shows complete separation of 57 solvents commonly tested for residue in pharmaceutical products 112 s pk (1 st Dim.)

36. GC GC x GC Some Applications of technology –Foods/Fragrances –Biological Samples –Organohalogen compounds –Environmental Samples –Petrochemicals

37. GC GC x GC Questions 1.Why is the second dimension in GC x GC limited in resolution? 2.For which column in GC x GC is it important to use columns of small diameter and thin films? 3.Why is overloading on narrow diameter 2 nd dimension columns less important than when using the same column with 1D GC? 4.How many dimensions does a GC x GC x MS chromatogram have? 5.Why does the peak capacity of a two dimensional technique somewhat over express how many compounds can be resolved? 6.Why are two hot/cold jets needed in the modulator? 7.For what type of samples is GC x GC particularly useful? a)Complex samples with compounds of similar polarity b)Complex samples with a variety of compound polarity c)Complex samples with a small number of analytes (e.g. pesticides in food) d)Related compounds with different sized alkyl groups R(CH 2 ) n CH 3

38. Supercritical Fluid Chromatography = SFC What is a supercritical fluid? –has properties intermediate between a liquid and a gas –Defined by region P – T phase diagram –Phase boundary between liquid and gas disappears at critical point –Demonstrated by heating two phase system –Fluid must operate above critical T and critical P Temperature Pressure solid liquid gas liquid + gas heat supercritical fluid critical point supercritical fluid initial state critical T critical P

39. Supercritical Fluid Chromatography Mobile Phase –Supercritical fluid –Most common fluids are: 100% CO 2 (31.3°C critical temp. + 71 bar critical temperature) Mixture of CO 2 and polar modifier (e.g. methanol), where modifier added to adjust retention –Properties vs. gases and liquids densities a little below liquids (solute – solvent interactions matter, unlike ideal gases) viscosities a little greater than gases – allowing minimal pressure drop vs. liquids diffusion coefficients closer to but greater than liquids, minimizing C-term broadening vs. liquids –Properties generally result in efficient separations that can be applied to a greater class of compounds than by GC

40. Supercritical Fluid Chromatography Stationary Phase –Since carbon dioxide is non-polar, polar stationary phase is most common –Both OT columns (smaller diameters since diffusion is slower than in GC) and packed columns can be used –Columns can be set up to take advantage of smaller pressure drops of supercritical fluids vs. liquids of using smaller particle sizes or longer column