Lec 7 Gas Chromatography PDF

Lecture 7 PHCM561-Biotech-WS24 Gas Chromatography and Supercritical Fluid Chromatography Dr. Heba ElNakib GC and SFC 1  Learning outcomes 1. Define supercritical fluid chromatography (SFC). 2. Compare between HPLC and SF...

Lecture 7 PHCM561-Biotech-WS24 Gas Chromatography and Supercritical Fluid Chromatography Dr. Heba ElNakib GC and SFC 1  Learning outcomes 1. Define supercritical fluid chromatography (SFC). 2. Compare between HPLC and SFC. 3. Describe Gas Chromatography (GC). 4. Compare between GSC and GLC. 5. Describe different types of columns and stationary phases employed in GC, and explain their use resp. 6. Compare between split, split-less, and on-column injection techniques. 7. Describe the function and usage of FID in GC. 8. Explain temperature / pressure programming. GC and SFC 2  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. GC GLC GSC Partition Adsorption Stationary phase is a non- The analyte is adsorbed volatile liquid coated on the directly on solid particles of inside of the column or on a fine solid support. stationary phase. GC and SFC 3  Set-Up of a Gas Chromatograph Carrier gases are: H2, He and N2. He is the most common. 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 the set up of a temperature gradient) flow through a detector whose response is displayed on a computer (chromatogram). The detector is maintained at higher temperature than the column so that all analytes will be gaseous. GC and SFC 4  Columns in GC 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 (Cu term is reduced ! good resolution) GC and SFC 5  Open Tubular Columns (OTC) The majority of OTCs are made of fused silica (SiO2), 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” O2 exposure at high temperature accelerates degradation. GC and SFC 6 Effect of decreased thickness of the stationary phase in GC Decreased retention times Decreasing thickness of Decreased sample capacity (less stationary phase thickness, less material) Increased resolution (less thickness, less material, so less diffusion). GC and SFC 7 Open Tubular vs. packed Columns (K’= tR-tM/tM) GC and SFC 8  CARRIER GASES H2 He give better resolution (smaller plate height) than N2 because solutes diffuse more rapidly through lighter gases (Cu term is reduced) Disadv.: Adv.: forms explosive mixtures most common gas because it is compatible with air when H2 > 4% vol. with all detectors catalytically react with unsaturated compounds on metal surfaces. Degradation of St. phase: High quality gases should be used & they should be passed through purifiers to remove O2 and H2O & traces of organic compounds prior to entering the column. GC and SFC 9  Types of injection 1. SPLIT INJECTION: Sample characteristics: used if analytes represent > 0.1% of sample content to avoid column overload. delivers only 0.2 – 2 % of the sample to the column. Process: < 1 µL is injected in < 1 s, at 350°C ! fast evaporation occurs. at split point, small fraction of vapour enters the column, but most passes to a waste vent. After sample has been flushed from the injection port, the valve is closed and the carrier gas is correspondingly reduced. Disadvantage: Quantitative analysis can be inaccurate because the split ratio is not reproducible from run to run. GC and SFC 10  Types of injection, cont. 2. SPLITLESS INJECTION: Sample characteristics: for trace analysis of analytes that are < 0.01% of the sample. approx. 80% of the sample is delivered to the column. ▪ Process: ▪ 2 µL 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) How to avoid band broadening due to slow injection? SOLVENT TRAPPING: column initial temperature is set lower than boiling point of solvent ! 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). ! Sharp narrow peaks GC and SFC 11  Types of injection, cont. 3. On-Column Injection: Sample characteristics: For samples that decompose above their boiling point, preferred for quantitative analysis. lowest possible temperature used for separation (! limited degradation of solutes). Process: ▪ solution is injected directly into the column, without going through a hot injector. ▪ low initial column temperature (! condensation of solutes in narrow zone). GC and SFC 12  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. Isothermal: 150°C Programmed T: 50°C- 250°C at 8°C/min GC and SFC 13  Detection Quantification of analytes: Peak area α Concentration. Qualitative analysis Based on comparison of retention times with standards, 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 area will increase. By Mass Spectrometric detection (GC-MS hyphenation): giving molecular weights of analytes and/or of their fragments GC and SFC 14 Detectors in Gas Chromatography GC and SFC 15 Flame Ionization Detector (FID) eluate is burned in a mixture of H2 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, then current (=movement of electrons) can be recorded. response is proportional to number of molecules – insensitive to non-hydrocarbons GC and SFC 16  Supercritical Fluid Chromatography Mobile phase: no gas nor liquid, but a supercritical fluid. V phase diagram of carbon dioxide pressur e 31,3 ºC, 73.9 bar -56.5 ºC, 5.1 bar temperature Density increases as pressure increases: The denser a mobile phase is ! the bigger its capacity for a solute ! the less distribution into stat. phase takes place ! the lower the retention of the analyte. GC and SFC 17  Use of Supercritical Fluids as mobile phase Speed and resolution better Properties (density) compared to liquid mobile phase between gas and liquid (faster diffusion inside the column cavity! faster equilibration “Cu term”) Supercritical fluids 1. possess lower surface tension than liquids (i.e. they spread faster over stat. phase). 2. dissolve non-volatile substances unlike gas mobile phase 3. evaporate upon pressure reduction after passing the column: analytes are in gaseous phase and thus easily detectable. mostly used SF: CO2 compatible with common detectors (FID, UV). low critical temperature. non-toxic (green analytical chemistry). GC and SFC 18  Effect Flow Rate in SFC ✓ best (smallest) plate HETP height at higher flow rate HPLC compared to HPLC SFC ✓ min. plate height only 1/3 compared to HPLC flow rate u What gradients do you know? In HPLC: usually solvent gradient In ion exchange: concentration or pH gradient In GC: usually temperature gradient In SFC: pressure gradient GC and SFC 19  5% presentation You are asked to prepare and present about Capillary Electrophoresis principles and applications in Biotechnology. You can work in groups of 3 to 6. The presentation is due in tutorial session#10 GC and SFC 20  References 1. “Principles of instrumental analysis, 5th ed. by Skoog, Holler, Nieman” Chapter 27. 2. “Quantitative Chemical Analysis, 7th ed. By Harris” Chapter 24. 3. Lecture of “Chromatography-III” by Dr. Raimund Niess, GUC, 2009. GC and SFC 21