PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Gas Chromatography1. Introduction2. Stationary phases3. Retention in Gas-Liquid Chromatography4. Capillary gas-liquid chromatography5. Sample preparation and inlets6. Detectors(Chapter 2 and 3 in The essence of chromatography)Sample preparation and inletA. Sample Preparation:1. The prerequisite in GC separation is that all solutes being separated must be: (a) fairly volatile, and (b) thermally stable. (c) Usually, the solute should be dissolved in a non-aqueous matrix (H2O changes column behevir ).2. Lack of volatility prevents the direct use of GC for many solute. One way to overcome this difficulty is to derivatize the solutes into more volatile forms.ClClOOHO2,4-dichlorophenoxyacetic acid(A cancer suspect agent).Silylation3. Derivatization of a solute can be used for any of the following reasons (a) To increase the volatility of the solute. (b) To increase the thermal stability of solute (c) To improve the response for the solute on certain detectors (e.g., incorporating halogen atoms into a solute so that it can be detected using an electron capture detector). (d) To improve the separation of the solute from other sample components (i.e., changing the structure of a solute will also affect its retention on the column)4. Most derivatization reactions can be classified into one of three group: (a) Silylation (b) Alkylation (c) AcylationMost of these reactions are performed using minimal amount of sample and reagents (i.e., 0.1~2.0 mL) are typical carried out at room temperature. Some, however, do require heating to moderate temperatures (60 ~ 100 OC).5. Silylation (a) This is the most common type of derivation techniques used in GC. (b) It involves replacing an active hydrogen on the solute (i.e. R-OH, RCOOH, R-NH2, etc.) with an alkylsilyl group (usually –SiMe3). The result of this reaction is that the solute is converted into a less polar, more volatile and more thermally stable form. (c) The most common reagent used in silylation is trimethylchlorosilane (TMS). Examples of its use are shown below:ClClOOHOClSiMe3ClClOSiMe3O+ClSiMe3R OHR OSiMe3++HClThe resulting Product of this reaction is usually just referred to as a TMS-derivative.(d) Besides trimethylchlorosilane, a number of other silylation reagents can also be used. These reagents have slightly different reactivity from trimethylchlorosilane. N, O-Bis(trimethylsilyl)acetamideBSA and BSTFA are highly stable TMS derivatives, with most organic functional groups, under mild reaction conditions. F3CNSiMe3OMe3SiR OHR OSiMe3F3CNSiMe3O++N,O-bis(Trimethylsilyl)trifluoroacetamideThe byproduct of BSTFA is highly Volatile.(e) Alylation i. Alkylation involves the addition of alkayl group to some active function group on the solute. A common example is esterification of a carboxylic acid, forming a volatile methyl ester. This is commonly done using borontrifluoride in methanol as the reagent. RCOOH + BF3/MeOH RCOOMe i. Acylation involves the conversion of a solute into an acylate derivates. This is often used to improve the volatility of alcohols, phenols, thiols and amine (e.g., -OH, -SH and -NH) containing compounds. As is true for other GC derivations, acylation can also be used to increase the response of a solute to a given detector (e.g., allowing the use of electron capture in solute’s detection by including fluorine atoms in the derivitizing agent.•methamphetamine,(f) Acylationmethamphetamineii. Trifluoroacetic anhydride (TFAA) is one common reagent used for acylation. OCOCF3COCF3N-CO-CF3NH++HOCOCF3Drug-of-abuse confirmation testing by GC/MSiii.Anther set of reagents used for solute with primary and secondary amines, as well as hydroxyl and thiol groups are N-Methyl-bis[trifluoroacetamide] (MBTFA). The reaction is under mild nonacidic conditions. MeNCF3OHByproduct is volatileSample preparation and InletsA. Sample Preparation:Hinj = σinj/L2σinj = Vinj/K22B. Sample Inlets:Sample inlet provide means by which the sample is vaporized and mixed with carrier gas.1. Direct Injectiona. Gaseous solutes can usually be directly injected onto a GC.b. Volatile liquid and solid solutes can also be applied directly to a GC system as long as they are dissolved in a solvent that does not interfere with solute peaks and does not contain other nonvolatile materials that may be deposited in the injector or on the column.2 Inlets for Packed column a. The solutes are injected by using a micro-syringe placed into a heated injection port. As a solute is injected into the port, it is quickly volatilized and taken by the carrier gas to the column.b. For thermally labile solute, the heated injected port may cause decomposition of the sample. For these solutes, direct injection of the sample onto the column is sometimes used, which allows a lower injection temperature.3. Inlets for open tubular (capillary) columnsa. Open tubular columns usually have a much smaller cross-section area than that of packed columns. This makes them more subject to extra-column band-broadening, requiring that special low volume injection techniques be used with them. b. Injection techniques used on open tubular columns include inlet splitters/splitless, cold on-column injectors and programmed temperature vaporizers. The aim of each is to apply a narrow plug of solutes to the column that is representative of the original sample.c. Inlet splitters are commonly used if the solute are reasonably volatile, thermally stable and each make up between 0.001 and 10% of the sample composition.Packed bedopen tubularplug of solutesVolume of injector(i) In this technique, the sample is first placed into the injection port and is vaporized.(ii) As the sample leaves the inject port, only a small portion of the vaporized samples is applied to the column (usually 1/20 to 1/200), with the remainder going to waste. This is splitting of the sample is used along with rapid injection, high carrier gas flow rate through the injectors, and high injector temperature to minimize the time that sample spends in the injector, which also minimizes extra-column band-broadening. (iii) The main difficulty with inlet splitters is that solute with different volatilities may not be