Chapter 31

1. Dong-Sun Lee/ CAT-Lab / SWU 2012-Fall version Chapter 31

2. Gas Chromatography (GC) Introduction Gas chromatography is a chromatographic technique that can be used to separate volatile organic compounds. Two types of GC are encountered: gas-solid chromatography(GSC) and gas-liquid chromatography(GLC). GLC is finds widespread use in all fields of science, where its name is usually shortened to GC. A gas chromatograph consists of a flowing mobile phase, an injection port, a separation column containing the stationary phase, and a detector. GSC is based on a solid stationary phase on which retention of analytes is the consequence of physical adsorption. GLC is based on partitioning behavior of the analyte between the mobile gas phase and the liquid stationary phase in the column.

3. Characteristics of GLC 1. Sensitivity : mg ~ pg (10–3 ~ 10–9 g) 2. Versatility : from rare gases to liquids and solids in solution with 800~1000 MW 3. Speed of analysis : typically 5 ~ 30 min , complex 3 ~ 30 mixture separation 4. Reproducibility : qualitative Accuracy : quantitative

4. HP 5890 gas chromatograph (Hewlett Packard) with an integrator GC-MS :GC-Q plus ion-trap GC-MSn ( Thermoquest - Finnigan ) Xcalibur software GC-14A gas chromatograph (Shimadzu) with an integrator.

5. Valve/ Gage  Fuel gas  Trap Make-up gas   Injector Detector  Regulator Electrometer Column Split vent Recorder Integrator Computer Column oven Fuel gas Carrier gas Schematic diagram of a gas chromatograph system. Basic components of Gas Chromatograph Carrier gas supply Sample introduction inlet Column and controlled-temperature oven Detector & oven Recorder

6. Carrier gas system in gas chromatograph The purpose of the carrier is to transport the sample through the column to the detector. The selection of the proper carrier gas is very important because it affects both column and detector performance. The detector that is employed usually dictates the carrier to be used. From a column performance point of view a gas having a small diffusion coefficient is desirable (high molecular weight, e.g., N2, CO2, Ar) for low carrier velocities while large diffusion coefficients (low molecular weight, e.g., H2, He) are best at high carrier velocities. The viscosity dictates the driving pressure. For high-speed analysis, the ratio of viscosity to diffusion coefficient should be as small as possible. H2 would be the best choice, followed by helium. Thepurity of the carrier should be at least 99.995% for best results. Impurities such as air or water can cause sample decomposition and column and detector deterioration. In temperature programmed runs, impurities in the carrier gas such as water can be retained at low temperatures but are then eluted at higher temperatures impairing the baseline. Many instrument problems have been traced to contaminated carrier gases. The carrier must also be inertto the components of the sample and the column.

7. Properties of common carrier gases Gas molecular weight Thermal conductivity Viscosity  × 105 at 100oC  × 10–6 at 100oC (g-cal/sec-cm- oC ) (P) Ar 39.95 5.087 270.2 * CO2 44.01 5.06 197.2 He 4.00 39.85 234.1 H2 2.016 49.94 104.6** N2 28.01 7.18 212.0 O2 32.00 7.427 248.5*** * at 99.6oC ** at 100.5oC **** at 99.74oC

8. Using the correct carrier and detector gases are an important factor in installing a new GC. The five gases commonly used as carrier gas and detector fuels in capillary gas chromatography are helium, hydrogen, nitrogen, argon-methane, and air. The types of gases necessary are partly determined by the detection system used. Factors to consider for each individual gas are discussed below. Carrier Gas Choice Carrier gases that exhibit a broad minimum on a van Deemter profile are essential in obtaining optimum performance. Volumetric flow through a capillary column is affected by temperature. When temperature programming from ambient to 300oC, the flow rate can decrease by 40 percent. A carrier gas that retains high efficiency over a wide range of flow rates and temperatures is essential in obtaining good resolution throughout a temperature programmed run. Figure 1 shows the van Deemter profile for hydrogen, helium, and nitrogen carrier gases.

9. Van Deemter curves for GC of n-C17H36 at 175oC, using N2, He, or H2 in a 0.25 mm diameter × 25 m long wall coated column with OV-101 stationary phase

10. Hydrogen is the fastest carrier gas (uopt), with an optimum linear velocity of 40cm/sec, and exhibits the flattest van Deemter profile. Helium is the next best choice, with an optimum linear velocity of uopt = 20cm/sec. Nitrogen's performance is inferior with capillary columns because of its slow linear velocity, uopt = 12cm/sec. Argon-methane has a slower optimum linear velocity than nitrogen and is not recommended for use as a carrier gas with capillary columns. Air is not recommended as a carrier gas because it can cause stationary phase oxidation.       With hydrogen and helium as carrier gases, the minimum H.E.T.P. values can be maintained over a broader range of linear velocities than with nitrogen, and high linear velocities can be used without sacrificing efficiency. Nitrogen is beneficial only when analyzing highly volatile gases under narrow temperature ranges where increasing stationary phase interaction is desirable. Otherwise, the use of N2 results in longer analysis times and a loss of resolution for compounds analyzed on a wide temperature range. http://www.restekcorp.com/gcsetup/gcsetup3.htm

11. Exert Caution when Using Hydrogen as a Carrier Gas • Hydrogen is explosive when concentrations exceed 4% in air. Proper safety precautions should be utilized to prevent an explosion within the column oven. Most gas chromatographs are designed with spring loaded doors, perforated or corrugated metal column ovens, and back pressure/flow controlled pneumatics to minimize the hazards when using hydrogen carrier gas. Additional precautions include: • Frequently checking for leaks using an electronic leak detector. • Using electronic sensors that shut down the carrier gas flow in the event of pressure loss. • Minimizing the amount of carrier gas that could be expelled in the column oven if a leak were to occur by installing a flow controller (needle valve) prior to the carrier inlet bulkhead fitting to throttle the flow of gas (for head pressure controlled systems only) as shown Fig. 2. • Fully open the flow controller (needle valve) and obtain the proper column head pressure, split vent flow, and septum purge flow rates. Decrease the needle valve flow rate until the head pressure gauge begins to drop (throttle point). Next, increase the flow controller (needle valve) setting so that the right amount of flow is available to the system. Should a leak occur, the flow controller will throttle the flow, preventing a large amount of hydrogen from entering the oven.

13. Make-up and Detector Fuel Gases Gas added to the stream after the column is called makeup gas. Choosing the correct make-up and detector gases will depend on both the detector and application. Most GC detectors operate best with a total gas flow of approximately 30ml/min. to ensure high sensitivity and excellent peak symmetry. Refer to your GC manual for optimum flow rates on different instruments. Carrier gas flows for capillary columns range from 0.5 to 10ml/min. which are well below the range where most detectors exhibit optimal performance. To minimize detector dead volume, make-up gas is often added at the exit end of the column to increase the total flow entering the detector. Make-up gas helps to efficiently sweep detector dead volume thereby enhancing detector sensitivity. Make-up gas can be added directly to the hydrogen flame gas for flame ionization detectors (FID), nitrogen phosphorous detectors (NPD), and flame photometric detectors (FPD) or added to the column effluent by an adaptor fitting. However, GCs such as Perkin-Elmer and Fisons do not require make-up gas.       Combustion type detectors (FID, NPD, FPD) use three gases: make-up, hydrogen (fuel gas), and air (combustion/oxidizing gas). For non-combustion detectors, such as the thermal conductivity detector (TCD), electron capture (ECD), and photo ionization detector (PID), only carrier and make-up gases are required. In the case of the electrolytic conductivity detector (ELCD), the make-up gas is hydrogen, as a reaction gas in the halogen and nitrogen mode or air in the sulfur mode. Table I shows recommended gases for various detectors.

14. Carrier gases and detector fuel gases for use with various GC detectors http://www.restekcorp.com/gcsetup/gcsetup3.htm

15. Effect of impurities - Impurities such as hydrocarbon, oxygen, water contribute to unwanted noise levels, excessive baseline drift. - Molecular sieve --- Moisture trap, Oxygen trap, Chemical filter Effect of water on column efficiency - Carrier gas dryness is very important !! (use anhydrous sodium sulfate) - Water can and usually does react with some portion of the column. This results in loss of resolution and tends to produce asymmetric or tailing peaks. Unwanted components or ghost peaks may also appear. Another effect is a net loss of sensitivity.

16. Gas purifiers The trap will remove any water vapor or oils that may have been introduced in the filling process since a number of gases are water pumped. The contaminants removed by the trap could otherwise interact with the column packing material to produce spurious peaks. In addition the contaminants can cause increased detector noise and drift. The traps should be reconditioned (about twice a year ) by heating to 300 oC for 4~8 hr with a stream of gas passing through it or in a vacuum oven.

17. Carrier gas purifiers 1 2 3 4 GC Gas cylinder 1. Hydrocarbon trap 2. Moisture trap 3. Oxygen trap 4. Indicating oxygen trap

18. Gas purifier recommendation for GC applications Capillary column GC Carrier Hydrocarbon, Moisture, Oxygen with any detector Make-up None - all detector but ECD moisture & oxygen Air for FID Hydrocarbon H2 for FID None ELCD reaction gas Hydrocarbon Packed column GC Carrier Hydrocarbon, Moisture, Oxygen with FID or TCD Packed column GC Carrier Hydrocarbon, Moisture, Oxygen with ECD, FPD, NPD, MSD

19. Flow requirements 1. Stable 2. Reproducible 3. Convenient The more constant the flow rates, the more precise and accurate the results. Flow controller ( Pressure controller ) --- to maintain precise and accurate flow rates

20. Effect of decreased flow rate or lower temperature • - All peaks have shifted to longer retention times • - Apparent loss of peak height • - The base of each peak is wider, • however, individual peak area remain constant. • Effect of increased flow rate • Sample components are squeezed toward • the injection point • Cause two components to elute together, • appearing as single peak

21. Regulations of carrier gas Carrier cylinder bottled at about 2500 psi(150-160 atm) Two stage pressure regulator : - first stage : high inlet pressure - second stage : low outlet pressure ( set at 40~100psi) Gas generators

22. Gas flow rate control A 1 % change in carrier gas flow rate will cause a 1% change in retention time. For all these reasons it is important to keep the flow of the carrier gas constant. 1. Control of carrier gas inlet pressure 2. Control of carrier gas flow rate In isothermal operation the means of regulation is immaterial because both means provide constant inlet pressure as well as constant flow rate. In temperature programmed runs, however, the situation is quite different. If one maintains the inlet pressure constant the flow rate will change. Therefore, with temperature programming of the column, the flow rate must be controlled. Pressure controllers 1. The second stage regulator on the cylinder 2. A pressure regulator mounted in the GC 3. A needle valve(variable restrictor) mounted in the GC 4. A fixed restrictor mounted in the GC

23. Flow measurement 1) Rotometer The column flow rate is typically indicated by a rotometer . ( Calibrate equilibrium position indicating the flow ) Rotometer is operated by the volume of gas passing a ball in a tapered cell. 2) Bubble meter 3) Electronic flow sensor

24. Relationships between inside diameter, column length, mesh size, and carrier gas flow for packed column Inside diameter Mesh size for Mesh size for Carrier flow mm length up to 3m length over 3m N2, ml/min He or H2, ml/min 2 100~120 80~100 8~15 15~30 3 100~120 80~100 15~30 30~60 4 80~100 60~80 30~60 60~100 John A. Dean, Analytical Chemistry Handbook, McGraw-Hill, 1995, p. 4.31 .

25. Comparison of 1/8-in(0.316 cm) packed, wide bore, and WCOT columns 1/8 in packed Wide bore WCOT Inside diameter, mm 2.2 0.53 0.25 Film thickness, m 5 1~5 0.25 Phase volume ratio() 15~30 130~250 250 Column length, m 1~2 15~30 15~60 Flow rate, ml/min 20 5 1 Effective plates(Neff) per meter 2000 1200 3000 Effective plate height (Heff),mm 0.5 0.6 0.3 Typical sample size 15 g 50 ng John A. Dean, Analytical Chemistry Handbook, McGraw-Hill, 1995, p. 4.31 .

26. Recommended ranges of gas flow rates Detector Gases Range Capillary Packed FID Carrier 2 ml/min 40 ml/min Hydrogen 30~50 ml/min 35 ml/min 40 ml/min Air 300~600 ml/min 350 ml/min 500 ml/min Make-up(N2) 10~60 ml/min 30 ml/min not used NPD Hydrogen 2~4 ml/min Air 40~80 ml/min Make-up(N2, He) 10~20 ml/min not necessary PID Make-up 5~10 ml/min Sheath 30~40 ml/min FPD Carrier 1~3 ml/min 30~50 ml/min Hydrogen 85~100 ml/min 100~120 ml/min Air 100~120 ml/min 110~135 ml/min

27. Inlet requirements 1. Temperature controlled 2. Low volume (total swept by carrier ) 3. Inert construction Column Overload If too large an sample were allowed to enter small bore(capillary) columns column overload and a loss of resolving power would like occur.

28. Liner • All liners help protect the vaporized sample form contacting the metal wall of the inlet as sample flows onto the column. Deactivated glass wool may be used as an aid for sample vaporization, to minimize discrimination based on boiling point, and to provide a surface on which non0volatiles can be trapped. The simplest liner is a straight tube, which gives all-around good performance at low cost. Single-taper liners improve on a straight tube by minimizing sample vapor contact with metal at the bottom of the injection port, although they are somewhat more expensive. Liners are deactivated borosilicate glass, except quartz where noted. Liners are guaranteed inert for phenols, organic acids and bases. • Why is Glass Wool Added to an Injector Liner? - General GC • The Glass wool serves three major purposes. • The Glass wool will prevent the small pieces of septa from reaching the column. • The presence of Glass wool will help the injected sample stay in the liner a little longer which will help the sample to vaporize and mix more thoroughly with the carrier gas. • If positioned properly it will wipe the outer surface of the syringe needle and improve the precision of the liquid injection.

29. Inlet configuration 1. Direct column inlet --- 1/8 " OD or larger column sampling syringe is actually inserted into the end of the column needle guide / cap / spring / septum mounting holes / carrier gas in / inlet body column Swagelock ferrules / Swagelock nut 2. Splitter inlet --- open tubular column or less than 1/8" OD column Because of the limited capacity for sample of these small bore columns and the difficulty of injecting extremely small volume samples, a large portion of the injected sample is vented to atmosphere by the inlet. septum / preheated carrier gas / mixing tube / restrictor buffer volume / tapered needle / gold gasket / column fitting

30. Injection port for split injection into an open tubular column. The glass liner is slowly contaminated by nonvolatile and decomposed samples and must be replaced periodically. For splitless injection, the glass liner is a atraight tube with no mixing chamber. For dirty samples, split injection is used and a packing material can be replaced inside the liner to adsorb undesirable components of the sample.

31. Common injection techniques 1) Hot flash vaporization Direct Cold-trap Split Splitless 2) Direct cold : on column Split or on-column Split 1) Simple 2) High column efficiency 3) Column may be protected On-column 1) Best accuracy 2) Thermolabile compounds 3) Trace analysis

32. Representative injection conditions for split, splitless, and on-column injection into an open tubular column.

33. Direct injector 1) Good sensitivity 2) Low column efficiency 3) Best for thick films, widebore column ( 0.53 mm ) Hot on-column injectors 1) Reduced column efficiency 2) Best with thick films, widebore columns 3) Nonvolatiles may damage column 4) Cold on-column injector may be used with 0.1 to 0.53 mm i.d. columns Advantage of on-column 1) Best reproducibility : Quantitative results 2) No split, no loss of high boilers 3) "Cold" on-column injection available

34. Advantage of splitless 1) High sensitivity ( 95 % of sample on column ) 2) Solvent effect produces narrow sample bands 3) Same hardware as split injection Disadvantage of splitless 1) Slow sample transfer to column 2) Must dilute sample with volatile solvent 3) Time consuming : must cool column 4) Poor for thermolabile compounds

35. Split and splitless injections of a solution containing 1 vol % methyl isobutyl ketone (bp 118 oC) and 1 vol % p-xylene (bp 138 oC) in dichloromethane (bp 40 oC) on a BP-10 moderately polar cyanopropyl phenyl ,ethyl silicone open tubular column(0.22 mm I.d., 10 m long, 0.25 m, column temperature =75 oC).

36. Common injection methods Syringe injection Valve injection Sampling Syringe 0 ~ 1 μL --- the sample is totally confined to the needle 0 ~ 5 / 0 ~ 10 μL needle / barrel / plunger Gas sampling valve Sample in Sample loop --- 1/4 or 10 mL loop size compatible with needs Sample vent Carrier gas to column

37. Solvent effect t-1 t-2 time t-1 --- just after injection, solvent and sample are condensed in a long plug at the front of the column. The column temperature must be cold enough to condense the solvent. time t-2 --- after some time, the column temperature has been raised, most of the solvent has evaporated, and the solvent effect has left the sample molecules concentrated in a narrow band. As the column is further heated, the remaining solvent and sample molecules are rapidly vaporized --- resulting in high column efficiency and narrow peak.

38. Syringe for solid phase microextraction. Sampling by SPME and desorption of analyte from the coated fiber into a gas chromatograph.

39. Purge and trap apparatus for extracting volatile substances from a liquid or solid by flowing gas.

40. GC column Parts of Column 1) Tubing material Stainless steel--- reactive ( steroids, amines, free acids ) Glass ------------ can be made inert, difficult handling Fused silica ---- flexible most inert most popular high resolution 2) Stationary phase Solid support --- carefully sized granular Liquid phase --- active portion of the column Porous polymers Adsorbents

41. Important column parameters 1) Inside diameter 2) Length 3) Film thickness 4) Stationary phase composition 5) Flow rate

42. Column diameter i.d. Resolution Speed Capacity Ease 100 micrometer +++ +++ + + (narrow bore ) 250, 320 ++ ++ ++ ++ (mid bore) 530 + ++ +++ +++ (wide bore) Column length N œ L R œ L1/2 tR œ L

43. 24-foot 1/8" packed column wound on 6" coil6-foot 1/4" packed column wound on 5" coil 60-meter 0.53mm metal wide bore capillary column wound on 3.5" coil  15-meter 0.53mm fused silica wide bore capillary column wound on 7" cage 30-meter .25mm metal narrow bore capillary column wound on 3.5" coil  http://www.srigc.com/catalog/columns.htm Column Glass wool --- both ends of the column 1- ½” (inlet side) 1/4” (detector side)

44. Fused silica surface made by the reaction of SiCl4 and water vapor in a flame - SiO2 contains 0.1 % –OH groups - Very inert - Uniform chemical surface Fused silica - High tensile strength - Flexible - Sheath of polyimide - Very inert

45. Fused Silica Capillary ColumnsA fused silica capillary column is comprised of three major parts (Figure 1). Polyimide is used to coat the exterior of fused silica tubing. The polyimide protects the fused silica tubing from breakage and imparts the amber-brown color of columns. The stationary phase is a polymer that is evenly coated onto the inner wall of the tubing. The predominant stationary phases are silicon based polymers (polysiloxanes), polyethylene glycols (PEG, Carbowax) and solid adsorbents. Figure 1. Capillary columns have to be properly installed to maximize their performance and lifetime. You can obtain enhanced column performance and lifetime by following these recommended installation guidelines. More detailed installation, operational and troubleshooting information can be found in the following references

46. WCOT= Wall Coated Open Tubular invented and patented by Dr Marcel Golay Tubing - Fused silica - Glass - Stainless steel Liquid phase coating WCOT - - - High resolution Film thickness : 0.5 to 5.0 micrometer i.d. : 0.10, 0.25, 0.32, 0.53 mm Length : 10 to 60 m Open tubular GC column

47. Operational guideline for open tubular GC columns WCOT narrow intermediate wide bore Column inner diameter, mm 0.25 0.32 0.53 Maximum sample volume, l 0.5 1 1 Maximum amount for one component, ng 2~50 3~75 5~100 Effective plates(Neff) per meter 3000~5000 2500~4000 1500~2500 Trennzahl(separation) number per 25 m 40 35 25 Optimum flow for N2, ml/min * 0.5~1 0.8~1.5 2~4 Optimum flow for He, ml/min ** 1~2 1~2.5 5~10 Optimum flow for H2, ml/min *** 2~4 3~7 8~15 * Optimum velocity is 10 to 15 cm/s for each column ** Optimum velocity is 25 cm/s for each column *** Optimum velocity is 35 cm/s for each column

48. Other types of capillary columns SCOT = Support Coated Open Tubular Solid support : Celite Liquid phase Not available fused silica tubing PLOT = Porous Layer Open Tubular Porous adsorbent : alumina or molecular sieve * Molecular sieve --- efficient for H2, Ne, Ar, O2, N2, CO, CH4. Porous carbon stationary phase ( 2 m thick) on inside wall of fused silica open tubular column.