1. Lecture 9 Gas Chromatography Lecture 9 – Chromatography, 13-11-2012 Dr. Rasha Hanafi 1© Dr. Rasha Hanafi, GUC

2. Objectives 1.Define Gas Chromatography (GC) and distinguish between GSC and GLC. 2.Describe different types of columns and stationary phases employed in GC, and explain their use resp. 3.Distinguish between split, split-less, and on-column injection techniques. 4.Describe the function and usage of common detectors in GC. 5.Explain temperature / pressure programming. © Dr. Rasha Hanafi, GUC2 Lecture 9 – Chromatography, 13-11-2012

3. Gas Chromatography In Gas Chromatography (GC), gaseous analyte is transported through the column by a gaseous mobile phase, called the carrier gas. The stationary phase usually is a non-volatile liquid or a solid and the analytes are gases or volatile liquids. © Dr. Rasha Hanafi, GUC 3 Lecture 9 – Chromatography, 13-11-2012 GC GLC Partition Stationary phase is a non- volatile liquid coated on the inside of the column or on a fine solid support. GSC Adsorption The analyte is adsorbed directly on solid particles of stationary phase.

4. Columns Packed column. Typically used in HPLC, seldom in GC, they offer high capacity but poor resolution. Typically used in GC: rapid equilibration is accomplished by decreasing stationary phase thickness and reducing column diameter (C u term is reduced  good resolution) © Dr. Rasha Hanafi, GUC4 Lecture 9 – Chromatography, 13-11-2012

5. Open Tubular Columns (OTC) The majority of OTCs are made of fused silica (SiO 2 ), coated with polyimide for support and protection from atmospheric moisture. The narrower the column, the higher the resolution, and the higher the required pressure, why? (ID: 0.10 to 0.53 mm and lengths: 15 to 100 m !!!!). Degradation of columns: Aging: stationary phase bakes off, silanol groups are exposed, tailing increases. The stationary phase could also leak from the column resulting in “ghost peaks in the chromatogram” O 2 exposure at high temperature accelerates degradation. © Dr. Rasha Hanafi, GUC5 Lecture 9 – Chromatography, 13-11-2012

6. Effect of decreased thickness of the stationary phase in GC Decreased retention times Decreasing thickness of stationary phase Decreased sample capacity (less thickness, less material) Increased resolution (less thickness, less material, so less diffusion). © Dr. Rasha Hanafi, GUC Lecture 9 – Chromatography, 13-11-2012 6

7. Open Tubular versus packed Columns In OTC we generally find: 1.higher resolution  greater length (up to 100 times) possible, and so more theoretical plates possible for OTC. 2.shorter analysis time  higher flow rates possible for OTC (less resistance). 3.lower sample capacity  not useful for preparative purposes when compared to packed columns. © Dr. Rasha Hanafi, GUC 7 Lecture 9 – Chromatography, 13-11-2012

8. conductivity Choosing the Column © Dr. Rasha Hanafi, GUC8 Lecture 9 – Chromatography, 13-11-2012

9. Packed Columns in GC They are used for preparative separation (greater amount of stationary phase allows greater sample size) or to separate gases (that are poorly retained by OTC). They have approx. ID 3 - 6 mm; length: 1- 5 m. The solid support material is usually diatomite (silica from algae that is silanized as shown in the reaction). packed column © Dr. Rasha Hanafi, GUC9 Lecture 9 – Chromatography, 13-11-2012

10. Set-Up of a Gas Chromatograph Volatile liquid or gaseous sample is injected by a syringe through a septum (=rubber disk) into a heated port, where it rapidly evaporates. The vapor is swept through the column by a carrier gas. The column must be hot enough to provide sufficient vapor pressure for analytes to be eluted in a reasonable time. Separated analytes (via a the set up of a temperature gradient) flow through a detector whose response is displayed on a computer. The detector is maintained at higher temperature than the column so that all analytes will be gaseous. © Dr. Rasha Hanafi, GUC10 Lecture 9 – Chromatography, 13-11-2012 Carrier gases are: H 2, He and N 2. He is the most common.

11. CARRIER GASES H 2, He give better resolution (smaller plate height) than N 2 at high flow rates because solutes diffuse more rapidly through H 2 and He than through N 2, so the C u term is reduced. He is the most common gas because it is compatible with all detectors. H 2 ‘s drawback is that it can catalytically react with unsaturated compounds on metal surfaces. It also forms explosive mixtures with air when H 2 > 4% vol. Impurities in the carrier gas degrade St. phase: High quality gases should be used & they should be passed through purifiers to remove O 2 and H 2 O & traces of organic compounds prior to entering the column. Steel or Copper tubing rather than plastic or rubber should be used because they do not release products in the gas stream. © Dr. Rasha Hanafi, GUC Lecture 9 – Chromatography, 13-11-2012 11

12. Types of injection 1. SPLIT INJECTION: routine method for introducing small sample volume into OTC. It delivers only 0.2 – 2 % of the injected sample amount to the column. It is used if analytes of interest are > 0.1% of sample because it is too much load for the column. small sample (< 1  L) is injected rapidly (< 1 s), at high temperature (350°C) fast evaporation occurs. flow or carrier gas sweeps sample through mixing chamber, where complete evaporation and good mixing occur, at split point, small fraction of vapour enters the column, but most passes to a waste vent. The proportion of the sample that does not reach column is called split ratio (usually 50:1 to 600:1). After sample has been flushed from the injection port, the valve is closed and the carrier gas is correspondingly reduced. Quantitative analysis can be inaccurate because the split ration is not reproducible from run to run. © Dr. Rasha Hanafi, GUC12 Lecture 9 – Chromatography, 13-11-2012

13. 2. SPLITLESS INJECTION: for trace analysis of analytes that are < 0.01% of the sample. approx. 80% of the sample is delivered to the column.  approx. 2  L of dilute sample solution in low-boiling solvent are injected slowly (2 s)  temperature is lower (220 °C) than at split injection to avoid decomposition (sample remains longer time in the injector – approx. 1 min- compared to split injection) SOLVENT TRAPPING : column initial temperature is below boiling point of solvent, therefore solvent condenses at the beginning of the column. As solutes slowly catch up with condensed plug of solvent, they are trapped (=concentrated in the solvent in a narrow band at the beginning of the column). Chromatography is initiated by raising the column temperature. This leads to sharp narrow peaks. © Dr. Rasha Hanafi, GUC13 Lecture 9 – Chromatography, 13-11-2012 Types of injection, cont.

14. 3.On-Column Injection: For samples that decompose above their boiling point, preferred for quantitative analysis.  solution is injected directly into the column, without going through a hot injector.  low initial column temperature (  condensation of solutes in narrow zone).  lowest possible temperature used for separation (  little loss of solutes). © Dr. Rasha Hanafi, GUC14 Lecture 9 – Chromatography, 13-11-2012 Types of injection, cont.

15. Split and splitless injections of a solution containing 1 % vol methyl isobutyl ketone and 1 % vol. p-xylene in dichloromethane. © Dr. Rasha Hanafi, GUC15 Lecture 9 – Chromatography, 13-11-2012 Effect of operating parameters in split and splitless injections

16. Detection  Quantification of analytes: Comparison of peak areas, using internal standard.  Qualitative analysis Can be direct or based on comparison of retention times with standards, or by "co-chromatography" where an authentic compound is added (spiked) to the unknown. If the unknown is identical with a component of the unknown, then the relative area will increase.  Detectors used for (direct) qualitative analysis: 1.Mass Spectrometer (GC-MS hyphenation): giving molecular weights of analytes and/or of their fragments 2.Fourier-Transform Infrared Spectrometer (GC-FTIR hyphenation): giving IR spectra of analytes that are compared to a library © Dr. Rasha Hanafi, GUC16 Lecture 9 – Chromatography, 13-11-2012

17. Detectors in Gas Chromatography © Dr. Rasha Hanafi, GUC17 Lecture 9 – Chromatography, 13-11-2012

18. Flame Ionization Detector (FID) eluate is burned in a mixture of H 2 and air. Only 1 out of 100,000 carbon atoms produce ions, but the number of ions is strictly proportional to the number of carbon entering the flame !. From carbon atoms (except C=O and COO) CH radicals are generated, that can be ionized in the detector flame: * CH + * O  CHO + + e – if voltage is applied between the electrodes, current (=movement of electrons) can be recorded. response is proportional to number of molecules + linear range is 7 orders of magnitude + generally sensitive to hydrocarbons – insensitive to non-hydrocarbons © Dr. Rasha Hanafi, GUC18 Lecture 9 – Chromatography, 13-11-2012

19. Temperature and Pressure Gradient  Temperature Gradient (="Temperature Programming"): Temperature of a column is raised during the separation to increase solute vapor pressure. as temperature increases, retention time decreases and peaks are sharpening  Pressure Gradient: Less common than temperature programming (+ used for analytes that can not tolerate high temperature and + avoids the long time wasted for a column to cool down when temperature programming is used). pressure of carrier gas is increased during the separation to increase flow, retention time decreases and peaks are sharpening. © Dr. Rasha Hanafi, GUC19 Lecture 9 – Chromatography, 13-11-2012 Isothermal: 150°C Programmed T: 50°C- 250°C at 8°C/min

20. References 1.“Principles of instrumental analysis, 5 th ed. by Skoog, Holler, Nieman” Chapter 27. 2.“Quantitative Chemical Analysis, 7 th ed. By Harris” Chapter 24. 3.Lecture of “Chromatography-III” by Dr. Raimund Niess, GUC, 2009. 20© Dr. Rasha Hanafi, GUC Lecture 9 – Chromatography, 13-11-2012