Stationary

phase is liquid Mobile phase is gas called carrier gas Sample must me heated to vaporize (convert it to gas) Yields chromatograms

 Chromatogram

give Series of peaks each corresponding to one component of mixture Area of peak indicates relative abundance of that substance in the mixture (concentration) Retention time
Time for component to emerge from column By comparing to known retention times for substance can identify compound

 Function

Components
Common

uses Chromatographic resolution Sensitivity

 Separation

of volatile organic compounds Volatile when heated, VOCs undergo a phase transition into intact gas-phase species Separation occurs as a result of unique equilibria established between the solutes and the stationary phase (the GC column) An inert carrier gas carries the solutes through the column

 Carrier Injector Oven

Gas, N2 or He, 1-2 mL/min

Column Detector

GC syringe penetrates a septum to inject sample into the vaporization camber Instant vaporization of the sample, 280 C Carrier gas transports the sample into the head of the column Purge valve controls the fraction of sample that enters the column

 Programmable Isothermal-

run at one constant temperature Temperature programming - Start at low temperature and gradually ramp to higher temperature
More constant peak width Better sensitivity for components that are retained

longer Much better chromatographic resolution Peak refocusing at head of column

220C

160C

50C 0

Time (min)

60

Gas Chromatography GC
Gas chromatography is basically a separation technique in which the compounds of a vapourised sample are separated and fractionated as a result of partition between a gas and liquid or gas and solid. According to nature of S.P., GC may be divided into two classes --1. S.P. consist of solid material like granular silica, alumina or carbon. This type of GC is called as Gas Solid Chromatography (GSC). 2. S.P. consist of non volatile liquid held as a thin layer on a solid support. The most common support is diatomaceous earth or kieselgurh. This type of GC is called as Gas Liquid Chromatography (GLC).

Gas Solid Chromatography (GSC). GSC is quite limited in applicability because of following reasons --1. Difficulty in reproducing surface area. 2. Excessive retention of active gases on solid surface which reduces the available area. 3. Tailing of elution peaks caused by non-linear adsorption isotherms. 4. GSC is of greater value in the separation of permanent gases and low boiling hydrocarbons. Question: Why He gas is the most preferred M.P. in GC. He is preffered over N2, CO2, H2, Ne, Ar etc because of following reasons ---

1. One of the most useful detectors depends on the thermal conductivity of the gas. He has much greater thermal conductivity than other gases( almost equal to H2). The presence of even small quantity of organic compounds causes a relatively large decrease in thermal conductivity of the column effluent. As a result, the detector undergoes a marked increase in temperature. 2. He is having low density so greater flow rate can be employed (same is the H2 ). 3. No explosion danger with He like in case of H2. 4. It is extremely inert gas.

 Gas

chromatography (GC) is known by a variety of other names: Gas-liquid chromatography (GLC). In gas chromatography, the stationary phase is a high-boiling liquid and the mobile phase is an inert gas. Gas chromatography is generally used as an analytical tool rather than as a means of purification; however, like TLC, this technique can also be used to separate small quantities of compounds.

The process of gas chromatography is carried out in a specially designed instrument, called a gas chromatograph. About 1-10 L of liquid sample or solution is injected with a small hypodermic syringe into the gas chromatograph, through a rubber septum on the heated injection port of the instrument. The sample is vaporized and carried through a heated column by an inert carrier gas (usually helium or nitrogen). The adsorbent in the column is a high-boiling liquid suspended on a solid inert carrier. Because of differing interactions with the adsorbent and differing vapor pressures, the components of the sample move through the column at different rates. At the end of the column, each component passes through a detector, which is connected to a recorder. The recorder produces a tracing that shows when each component of a mixture passes the detector and also indicates the approximate relative quantity of each component.

GC is commonly used for:

1. Checking the purity of volatile-liquid samples, such as distillation fractions. 2. Checking the identity of a substance by comparison of its GC with that of a known. 3. Analyzing a mixture for the presence or absence of a known compound (such as alcohol in blood).

 In

most GC units, small quantities of sample are used and no attempt is made to collect the material that has been chromatographed. with preparative gas chromatographs, the separation and collection of samples are possible. Special techniques also allow the effluents from a gas chromatograph to be further analyzedfor example, by a mass spectrometer. As the vapor of a sample is carried through the column by the carrier gas, it continuously condenses and revapourizes. The amount of time the compound stays condensed depends on its volatility, and hence its boiling point.

 Once

a compound condenses, it can dissolve in the highboiling liquid. The time it remains dissolved depends on its solubility in the liquid phase (according to the rule "like dissolves like"). Thus, a nonpolar compound with a high vapor pressure moves along a non-polar column at a fairly rapid rate; a less volatile compound moves more slowly; and the compounds tend to concentrate in bands as they move through the column. Columns in a GC can be changed, although in practice they usually are not changed very frequently. It is important to report which type of column is used in an experiment so that the experiment can be duplicated in another laboratory.

1.

Efficient separation of compounds in GC is dependent on the compounds traveling through the column at different rates. The rate at which a compound travels through a particular GC system depends on all of the following factors: Volatility of compound. [Low-boiling (volatile) components

will travel faster through the column than will high-boiling components]. 2. Polarity of compound. [Polar compounds will move more slowly, especially if the column is polar]. 3. Temperature of the column. [Raising the column temperature speeds up all the compounds in a mixture]

4.

Polarity of the column packing material. [Usually, all

compounds will move more slowly on polar columns, but polar compounds will show a larger effect].

5.

Flow rate of the gas through the column. [Speeding up the

gas flow increases the speed with which all compounds move

through the column].

6.

Length of the column. [The longer the column, the longer it

will take all compounds to elute. Longer columns are employed

to obtain better separation].

1. A.

B.

The column : two type Capillary Column Packed Column

Capillary Column: CC is fabricated from capillary tubing and sometimes metal or organic polymers, the bore of which is coated with a very thin film of the liquid S.P. directly to the wall of column. These columns have very low sample capacity and used for partition. Size of the column is approx. 2.5 mm in dia and 100 mm in length. Advantage of CC is its increased power of separation.

 Packed Column The column of a typical gas Chromatograph is constructed of tubing, which is coiled so that a long column will fit into the small volume of the heated oven. The column is made of stainless steel or copper tube with either a solid substrate (GSC) or a liquid coating on an inert solid (GLC). The diameter of the metal is about 0.25 inch and length varies from 5-50 feet. In general, the longer length and smaller diameter columns give better resolution of components but have a lower capacity.

 The

column contains an inert but loosely packed solid (such as crushed firebrick or diatomaceous earth) that is coated with the liquid phase. The inert packing provides spacing and allows the carrier gas to flow through the column without the need for excessive pressure. The liquid phase is the active component in the column. In the instrument, the column is located in an insulated oven with adjustable temperature controls. The upper useful limit of temperature is determined by the vapor pressure of the liquid phase in the column.

 The

usual operating temperature is 100-200, but higher temperatures may be used with some types of columns. The operating temperature chosen depends on the boiling points of the compounds in the sample to be chromatographed.
A

typical liquid phase used in a GC column is

OV-

which is useful for a wide variety of compounds. For polar compounds, a more polar liquid phase, such as a Carbowax, may be used.

101,

 In

columns that have a nonpolar hydrocarbon as the liquid phase (a nonpolar column), the separation of compounds in GC is similar to their separation in distillation, because both processes depend primarily on relative vapor pressures. The low-boiling components of a mixture pass through the GC column first, followed by successively higher-boiling components. When the column contains a polar-liquid phase, the separation of the components in the mixture depends upon (1) their relative vapor pressures and (2) their relative interactions with the polarliquid phase. The greater the vapor pressure, the faster the compound will pass through the column. However, the greater the interaction, the slower the compound will pass through the column.

2. The detector The detector at the end of the column signals when a compound is being eluted from the column. A number of different types of detectors have been invented, but only two types are in common use: 1. Thermal conductivity (hot-wire) detectors 2. Flame ionization detectors 3. Electron capture detector

 In

a thermal conductivity detector, an electric current is passed through a wire located directly in the flow of gaseous effluent from the column. The electrical resistance of a hot wire varies with the heat conductivity of a gas passing over it. Helium, with a high heat conductivity, absorbs heat from the wire and keeps it relatively cool. Organic compounds have lower heat conductivities. Therefore, when the vapors of an organic compound pass over the wire, the wire becomes hotter; consequently, its resistance changes.

 The

hot wire is actually one arm (the sample arm) of a Wheatstone bridge circuit. The other arm (the reference arm) is a similar hot wire with pure helium (no sample) passing over it. When no sample is passing through the sample arm, the bridge is in balance and no message is sent to the recorder. The passage of a gaseous organic compound from the column through the sample arm of the detector changes the resistance of the sample arm. Thus, the bridge becomes unbalanced, and an electric current passes to the recorder.

 In

a flame ionization detector, part of the gaseous effluent from the column is mixed with a separate gas stream of hydrogen and oxygen, and this gaseous mixture is burned. When organic compounds are present in the effluent from the column, they are oxidized and converted to ions by the high temperature of the flame. The ions then pass through a metal ring, creating an electrical potential difference between the ring and the barrel of the burner. The small potential difference is amplified and then sent to the recorder.

Electron Capture Detector Introduction The ECD uses a radioactive beta emitter (electron, typically a metal foil holding 10 millicuries (370 MBq) of the radionuclide nickel-63.) to ionize some of the carrier gas and produce a current between a biased pair of electrodes. When organic molecules that contain electronegative functional groups, such as halogens, phosphorous, organometallic compounds and nitro groups pass by the detector, they capture some of the electrons and reduce the current measured between the electrodes.

The analyte concentration is thus proportional to the degree of electron capture. It is used for ultra-sensitive detection of halogen-containing species and Pesticide analysis.
Sensitivity: An ECD is 10-1000 times more sensitive than a flame ionization detector (FID), and one million times more sensitive than a thermal conductivity detector (TCD), but has a limited dynamic range and finds its greatest application in analysis of halogenated compounds. The detection limit for electron capture detectors is 5 femtograms per second (fg/s), and the detector commonly exhibits a 10,000-fold linear range.

This made it possible to detect halogenated compounds such as pesticides and CFCs, even at levels of only one part per trillion (ppt), thus revolutionizing our understanding of the atmosphere and pollutants.

The electrons are formed by collision with nitrogen because nitrogen exhibits a low excitation energy, so it is easy to remove an electron from a nitrogen molecule. The electron is then attracted to a positively charged anode, generating a steady current. Therefore, there is always a background signal present in the chromatogram. As the sample is carried into the detector by a stream of nitrogen or a 5% methane, 95% argon mixture, analyte molecules absorb the electrons and reduce the current between the collector anode and a cathode.

3. The chromatogram
The

recorder receives the electrical information from the detector and produces a graphthe chromatogramof the components passing through the detector. The time it takes for a particular compound to pass through the column is called the compound's retention time (RT). The retention time is a function of the physical properties of the compound, the rate of gas flow, the temperature, the liquid phase, and the length and diameter of the column.

The retention time is measured from the injection point ("start") to the top of the compound peak and is usually reported in minutes. If accuracy is desired, an inert compound such as a hydrocarbon can be added to the sample and relative retention times measured from this internal standard's peak instead of from the injection point. Another method of reporting position along the xaxis of the chromatogram is by retention volume, which is the product of the retention time and the carrier gas flow rate.

1.
2. 3.

There are several styles of GC recorders: Integrating recorders Chart recorders Computer interfaces. Integrating recorders : These instruments record the electronic input from the detector as peaks and they also integrate the area under each peak in the chromatogram. The printout reports the number of peaks, the retention time of each peak, and the area under each peak. Chart recorders : Older-style chart recorders feature a roll of paper that rolls under a recorder pen. When the detector sends an electronic message to the recorder pen, the pen moves, resulting in a "peak" on the chart paper. Computer interfaces: Retention times and areas under the peaks will be displayed on the screen and then printed out on a sheet of paper.

GC-MS

Principle GC/MS

1. 2. 3.

4.

The interface transports efficiently the effluent from the GC to MS. The analyte must not condense in the interface. The analyte must not decompose before entering the MS ion source. The gas load entering the ion source must be within pumping capacity of the MS.

 Molecular

weight Elemental composition (low MW with high resolution instrument) Structural info (hard ionization or CID)

 Gas-phase

ions are separated according to mass/charge ratio and sequentially detected

1.F

ood, Flavor, and Fragrance Analysis

A.New Crop Development B.Analysis of Beverages C.Halitosis D.Profiling in perfume industry E.Food analysis

Development of new crop speciesi.e. genetically modified to aid pest resistance requires tests of the new crops odor profile to ensure the flavor is enhanced, or at least comparable to previous species.

Forensic Mass Spectrometry

Analysis of Body Fluids for Drugs of Abuse Analysis of Hair in Drug Testing Sports Testing Analysis of Accelerants in Fire Debris Analysis of Explosives Use of Isotope Ratios

Essential Oil and Volatile Components GC-MS has been demonstrated to be a valuable analytical tool for the analysis of mainly nonpolar components and volatile natural products, e.g., mono- and sesquiterpenes described a method using direct vaporization GC-MS to determine approx 130 volatile constituents in several Chinese medicinal herbs. They reported an efficient GC-MS method with EI for the separation and structure determination of the constituents in ether-extracted volatile oils of Chinese crude drugs, Jilin Ginseng, Radix aucklandiae, and Citrus tangerina peels.

THANX