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1. Introduction to GCMS
Gas chromatography (GC) being a very good method for separating components of a mixture and mass spectrometry ( MS) a powerful technique for identifying components in a mixture, combination of the two techniques (GCMS) may be considered as a natural marriage. GC and MS deal with samples in the gas phase and can handle very small quantities of material. A basic introduction will be provided here to explain how GC separate ans MS identify compounds. Gas chromatography relies on the fact that different compounds dissolve to different extents in a particular liquid. In the experimental practice a long thin tube with such a liquid adhering to the inner wall , which is heated and has a gas such as helium flowing through it may be used. As chromatographers call it, the tube is called a column or a capillary column and when a mixture is introduced into the hot column, a component that does not dissolve in the liquid would be vaporized by the heat and carried straight though the capillary column at the same speed as the helium gas which is called the carrier gas. A compound of the mixture that dissolves in the liquid called the stationary phase and has less interactions with the gas phase would remain near the start of the column and move through it with difficulty. Consequently, different compounds are separated within the column because they move through it at different rates, depending of the partition between the stationary phase and the mobile carrier gas. The experimental situation is depicted in figura 1.
Fig. 1 Separation of different molecule species in a capillary gas chromatographic column The compounds separated by GC are passed for identification into a mass spectrometer. The eluted molecules from the capillary column are introduced in the ion source of the mass spectrometer where neutral molecules are converted into charged particles. The gaseous charged particles move in electric and magnetic fields in a way that is dependent on their masses. Determining the molecular mass of a compound does not allow an unambiguous identification because acetic acic and urea have the same mass of 60 daltons to the nearest whole number. The molecular ions may be submitted to fragmentation processes characteristic of the substance and usually sufficient to identify the compouind. In the ionization process molecules are converted into ions which are caused to undergo fragmentation reactions. A record of all the ions produced in this process is a mass spectrum. In gas chromatography (GC) alone the time taken for a compound to pass through the column is called the retention time. which is the sole criterium for identifying the substance. In GC/MS analysis a second identifying parameter is available which is the mass of the compound. With this instrumental combination , the technique rarely fails to identify compounds under experimental investigation.
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Introduction la GCMS
New generation of Instruments for GC/TOFMS Analysis 1. Achieves stable, high-sensitivity spectrum analysis. 2. High-resolution measurement conditions are always possible. 3. Fast GC analysis which improves productivity. 4. Variety of optional attachments improves analytic capabilities.
2. Practical aspects of Gas Chromatography (GC)
Figure 4 illustrates a gas chromatograph coupled to a quadrupole mass spectrometer.
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2. Type of column and nature of the stationary phase 3. Column and injector temperature The column may be kept at a defined isothermal temperature or its temperature may be changed at a programmed linear rate. 2.1 Types of carrier gases Helium is most employed for capillary columns , nitrogen has been often used with packed columns but has a high viscosity and when used with narrow-bore capillary columns the elution of the compounds would be slowed down. Moreover traces of oxygen are to be eliminated and compared to helium , nitrogen may produce interferences in the lower mass range since nitrogen ha a mass of 28 daltons and helium a mass of 4. Hydrogen is the best gas to use with capillary columns , it is oxygen free, is inexpensive and has a low viscosity producing fast separations. Leaks must be avoided because of the formation of explosives mixtures with air. The use of hydrogen in GC/MS is not frequent. 2.2 Injection Samples are introduced into the analytical instrument in a vapor state and vaporizarion can take place during or after injection of the sample. Liquid samples may be introduced in the gas chromatograph using a microliter syringe and solid samples are to be dissolved before injection. The use of a hot injector system is the easiest method of vaporization. The critical step in gas chromatography is the introduction of the sample where reaction of the components of the sample may occur. Samples may be injected using : splitless injection, split injection, temperature-programmed injection on-column sample injection procedures.
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3. Columns
In GC-MS the minimization of the release of stationary phase called bleeding due to heating giving background in the mass spectrometer is an important factor in selecting a column.
Liquid phase: Stationary phases covalently bonded to the inner column surface provide greater stability so that the column tolerates high temperatures ,can be
washed with solvents and gives low bleeding. The general rule in phase selection is: separate apolar compounds on apolar liquid phases separate polar compounds on polar phases The table herunder gives some common stationary phases together with chemical details
Phase
OV-1 OV-101 OV-17 OV-225
Similar to
SE-30, BP-1, DB-1, CP-Sil5 SP-2100 HP-17
Structure
100 % dimethyl silicone 50 % phenyl 50 % methylsilicone cyanopropylmethyl-
Max temp
325C
Polarity
non non medium high high low low high non high
220 C
5 % phenylsilicone 95% methyl 325 C silicone 1 % vinyl silicone 5 % phenyl silicone 94 % methyl silicone 325 C 250 C 450 C 220 C
SP-1000, OV-351
polyethylene glycol 2-nitroterephtalic acid carboranemethyl silicone silicone with chiral center
OV-1 and OV-101 stationary phases of low polarity, stable to high temperatures tend to separate comopunds according to their boiling points and are frequently used in gas chromatography. The phenylsilicone more polar phases such as OV-17 show stronger retention of aromatic compounds and are used for the separation of TMS and TFA derivatives of steoids, sugars and drugs in the biomedical field. Cyanosilicone phases such as OV-225 used in the analysis of fatty acid methyl esters (FAME) show strong cis-trans selectivity with trans before cis elution. Carbowax 20M, sensitive to water and oxygen exposure, used for separation of essential oil components (hydrocarbons and terpenes) not at high temteratures provides excellent results. SE-52 and SE-54 are frequently used to separate polycyclic aromatic hydrocarbons (PAH). FFAP was developed for the separation of free fatty acids. Dexsil , a carboranemethyl silicone was developed for applications with high temperature operation. Chirasil-Val, used for the resolution of D- and L-amino acid derivatives and chiral drugs, is a silicone-based phase with an in-built chiral centre allowing the separation of enantiomers .
Film thickness
The thickness of the stationary phase film is 0.3-0.5 m. Sample capacity increases with film thickness and thicker films and/or longer columns are more suitable for lower molecular weight volatile compounds. Thin film columns (0.1-0.2 m) are used for high boiling compounds eluting near the upper temperature limit of the column.
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Flow rate
The flow rate of carrier gas can be controlled by injecting a small amount of an inert gas such as methane on to the column and by measuring the time for the signal to appear in the mass spectrometer. This time is generally 1.5 min for a 25 m x0.2 mm i.d. column. The linear velocity V is where L is the column length and tg gas retention time The volumetric flow rate F in ml/min is given by where r is the column radius
Capillary columns flow rates are set normally at 0.5-2.0 ml/min and actual flow rate can be measured with a bubble flow meter connected to the end of the column.
Compounds Retention
Compounds of different polarities move at different rates along the column and the retention time (tR) is a property of a specific compound and may be considered as an aid in its identification. Since the experimental operating conditons are changing with time (modification of column properties with time) retention times may be expressed relative to an internal standard and this is known as the relative retention index RRI. Relative retention index (RRI) where tRc is the retention of the compound and tRs is the retention time of the standard
Retention gap
A retention gap is a length of deactivated tubing placed between the injection port and the start of the capillary column and 1 m is recommended for every l os sample. It serves as a guard column and can help to prevent peak splitting and distortion often caused by differences in the boiling points and polarities of the solvents and the solutes and their interaction with the stationary phase.
Column care
Cutting: Columns should be cut with a diamond knife to ensure smooth edges and to avoid contamination from polyimide and increase in dead volume. Carrier gas: The carrier gas air content should be as low as possible because certain polar phases ( Carbowax 20 M) are very sensitive to the presence of oxygen as well as non polar phases such as SE-30 at elevated temperatures. Rinsing of the column : In order to effect regeneration of cross-linked chemically bonded columns solvent wash is possible as illustrated herunder A wash sequence in the opposite direction to the original carrier gas flow for an OV-1 column would be 5 ml of water, 5 ml of methanol, 5 ml of dichloromethane and finally 5 ml of hexane.
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With the introduction of open capillary columns for GC and GC-MS, an analyte enrichment interface is no longer required because the flow rate of such a column is compatible with the vacuum system of a benchtop GC-MS instrument. Two types of GC-MS coupling are now in use: the direct coupling and the open split coupling.
Direct coupling : Efficient pumps can cope with the reduced amount of carrier gas emanating from capillary columns and the helim flow rates of 0.5 to 2.0 ml/min
are handled by modern pumps. In priciple the best interface is no interface at all, just a flexible GC column introduced directly into the ion source. The main advantages of such a coupling are:: Simplicity, no interface dead volume degrading the chromatographic resolution, no sample loss because transfer efficiency is 100 % providing good analytical sensitivity. Moreover, the molecules only come into contact with the stationaty face until they reach the ion source and there are no other glass or metal surfaces on which molecules might decompose or adsorb. The drawbacks are: a risk of source detuning and contamination due to the solvent pulse, to flow-rate changes during temperature programming and sample contaminants. While in direct coupling the vacuum system must be switched off for changing the GC column and this operation is not required with the open split coupling.
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