Gas Chromatography

Gas Chromatograph

•Gas chromatography (GC), is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition.

Principle:• A method of analysis by which the analyte is

vaporized and introduced into a stream of carrier gas.

• It is conducted through a chromatographic column and separated into its constituents.

• These fractions pass through the column at characteristic rates, and are detected as they emerge in a time sequence.

• The detecting cell responses are recorded on a chart, from which the components can be identified both qualitatively and quantitatively.

•In GC, the moving phase (mobile phase) is a carrier gas, usually an inert gas such as helium or nitrogen.

•The stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside a piece of glass or metal tubing called a column.

•The instrument used to perform GC is called a gas chromatograph.

•The gaseous compounds being analyzed interact with the walls of the column, which is coated with different stationary phases. This causes each compound to elute at a different time, known as the retention time of the compound.

•The comparison of retention times is what gives GC its analytical usefulness.

Gas chromatography and column chromatography

• Gas chromatography is in principle similar to column chromatography, but has several notable differences: ▫ The process of separating the compounds in a mixture

is carried out between a liquid stationary phase and a gas moving phase, whereas in column chromatography the stationary phase is a solid and the moving phase is a liquid.

▫ The column through which the gas phase passes is located in an oven where the temperature of the gas can be controlled, whereas column chromatography has no such temperature control.

▫ The concentration of a compound in the gas phase is solely a function of the vapor pressure of the gas.

Typical uses of GC include:

• Testing the purity of a particular substance.

• Separating the different components of a mixture

(the relative amounts of such components can

also be determined).

• In some situations, GC may help in identifying a

compound.

• In preparative chromatography, GC can be used

to prepare pure compounds from a mixture.

GC Instrument

Schematic Diagram of Gas Chromatograph

Carrier gas:•The carrier gas must be chemically inert.

Commonly used gases include nitrogen, helium, argon, and carbon dioxide.

•The choice of carrier gas is often dependant upon the type of detector which is used.

•The carrier gas system also associated with pressure regulators and flow meters.

•In addition, it contains a molecular sieve to remove water and other impurities.

Flow control:•Flow rates are controlled by a 2 stage

pressure regulator:▫At the gas cylinder.▫Mounted in the chromatograph.

•Inlet pressure 10-50 psi → F = 25-150 ml/min with packed columnF = 1-25 ml/min with capillary column

The flow rate will be constant if the inlet pressure remains constant.

Sample injection port:

•The injector is a piece of hardware attached to the column head.

• It provides the means to introduce a sample into a continuous flow of carrier gas.

•For optimum column efficiency, the sample should not be too large, and should be introduced onto the column as vapor.

•Slow injection of large samples causes band broadening and loss of resolution.

Common injector types:

• Microflash vaporizer direct injector:

▫It involves the use of a microsyringe to inject the sample through a rubber septum into a flash vaporizer port located at the head of the column.

▫The temperature of the sample port is usually about 50°C higher than the boiling point of the least volatile component of the sample.

▫Used for packed columns, where the sample size vary from a few tenth of microliter to 20 ml.

Capillary columns require much smaller samples ( 10-3 ml), so a sample splitter system is used to deliver only a small fraction of the injected sample to the column head, with the rest going to waste.

Sample splitter (Split/Splitless) injector

▫ The injector can be used in one of two modes; split or splitless.

▫ a sample is introduced into a heated small chamber via a syringe through a septum (the heat facilitates volatilization of the sample).

▫ The vaporized sample/carrier gas mixture then either sweeps entirely (splitless mode) or as portion (split mode) into the column.

▫ In split mode, a part of the sample/carrier gas mixture in the injection chamber is exhausted through the split vent.

▫ Split injection is preferred when working with samples with high analyte concentrations (>0.1%).

▫ Splitless injection is best suited for trace analysis with low amount of analyte (<0.01%).

Column:•There are two general types of column:

▫Packed column ▫Capillary column (open tubular).

•All the GC studies in the early 1950s were carried out on packed column.

•In the late 1950s capillary column were constructed that was much superior in speed and column efficiency (≈ 300000 plates).

• Capillary columns did not gain widespread until the late 1970s due to several reasons:

▫Small sample capacity.▫Difficulties in coating the column.▫Tendencies of columns to clog.▫Short lifetimes of poorly prepared columns.▫Fragileness of columns.▫Mechanical problems in sample introduction

and connection to the detector.

Capillary column:

• Capillary columns have an internal diameter of a few tenths of a millimeter.

• They were constructed of stain-less steel, aluminum, copper, plastic, or glass.

• They can be one of two types; wall-coated open tubular (WCOT) or support-coated open tubular (SCOT).

• Wall-coated columns consist of a capillary tube whose walls are coated with liquid stationary phase.

• In support-coated columns, the inner wall of the capillary is lined with a thin layer of support material, onto which the stationary phase has been adsorbed.

• SCOT columns are generally less efficient than WCOT columns, but both types are more efficient than packed columns.

• In 1979, a new type of WCOT column appeared, the Fused Silica Open Tubular (FSOT) column. It was drawn from specially purified silica that contains metal oxides. These have much thinner walls than the glass capillary columns, and are given strength by an outside protective polyimide coating.

• These columns are flexible and can be bent into coils. They have the advantages of physical strength, flexibility and low reactivity.

Packed column:• Packed columns contain a

finely divided, inert, solid support material coated with a thin layer of liquid stationary phase.

• Most packed columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm.

• They are made from glass, metals, or Teflon.

Characteristics of solid support materials

• Hold the liquid stationary phase.• Consists of small, uniform, spherical particles

with good mechanical strength.• It should be inert at elevated temperature.• The most widely used is prepared from the

naturally occurring diatomaceous earth (skeleton of thousands of single-celled plants-diatoms- lives in lakes and seas).

• The efficiency of a GC column increases with decreasing particle size of the solid support.

Properties and characteristics of GC columns

Packed SCOT WCOT FSOT

1-6 10-100 10-100 10-100 Length, m

2-4 0.5 0.25-0.75 0.1-0.53 Inside diameter, mm

500-1000 600-1200 1000-4000 2000-4000 Efficiency, plates/m

10-106 10-1000 10-1000 10-75 Sample size, ng

high Low Low Low Relative back pressure

slow Fast Fast Fast Relative speed

Poorest → → Best Chemical inertness

No No No Yes Flexible

FSOT columns

•Coated with polymer, crosslinked▫Polydimethyl siloxane (non-polar)

▫Poly(phenylmethyldimethyl) siloxane (10% phenyl)

▫Poly(phenylmethyl) siloxane (50% phenyl)▫Polyethylene glycol (polar)

▫Poly(dicyanoallyldimethyl) siloxane▫Poly(trifluoropropyldimethyl) siloxane

Polar vs. nonpolar

•Separation is based on the vapor pressure and polarity of the components.

•Within a homologous series (alkanes, alcohol, olefins, fatty acids) retention time increases with chain length (or molecular weight).

•Polar columns retain polar compounds to a greater extent than non-polar .

Column temperature •For precise work, column temperature

must be controlled to within tenths of a degree. The optimum column temperature is dependant upon the boiling point of the sample. As a rule of thumb, a temperature slightly above the average boiling point of the sample results in an elution time of 2 - 30 minutes. Minimal temperatures give good resolution, but increase elution times. If a sample has a wide boiling range, then temperature programming can be useful. The column temperature is increased (either continuously or in steps) as

separation proceeds .

Oven•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

Typical Temperature Program

Time (min)0 60

50C

220C

160C

Detectors •There are many detectors which can be

used in gas chromatography. Different detectors will give different types of selectivity. A non-selective detector responds to all compounds except the carrier gas, a selective detector responds to a range of compounds with a common physical or chemical property and a specific detector responds to a single chemical compound. Detectors can also be grouped into concentration dependant detectors and mass flow dependant

detectors .

•The signal from a concentration dependant detector is related to the concentration of solute in the detector, and does not usually destroy the sample. Dilution of sample with make-up gas will lower the detectors response. Mass flow dependant detectors usually destroy the sample, and the signal is related to the rate at which solute molecules enter the detector. The response of a mass flow dependant detector is unaffected by make-up gas .

Detectors

•Flame Ionization Detectors (FID)•Electron Capture Detectors (ECD)

•Thermal Conductivity Detector (TCD)•Electron impact/chemical ionization

(EI/CI) -Mass spectrometry

FIDs

•Effluent exits column and enters an air/hydrogen flame. The gas-phase solute is pyrolized to form electrons and ions.

•All carbon species are reduced to CH2+

ions•These ions are collected at an electrode

held above the flame. The current reaching the electrode is amplified to give the signal.

The effluent from the column is mixed with hydrogen and air, and ignited. Organic compounds burning in the flame produce ions and electrons which can conduct electricity through the flame. A large electrical potential is applied at the burner tip, and a collector electrode is located above the flame. The current resulting from the pyrolysis of any organic compound is measured. FIDs are mass sensitive rather than concentration sensitive; this gives the advantage that changes in mobile phase flow rate do not affect the detector's response. The FID is a useful general detector for the analysis of organic compounds; it has high sensitivity, a large linear response range, and low noise. It is also robust and easy to use, but unfortunately, it

destroys the sample .

FID

•A general detector for organic compounds•Very sensitive (10-13 g/s)

•Linear response (107)•Disadvantage: specificity

ECD

•Ultra-sensitive detection of halogen-containing species

•Pesticide analysis•Other detectors besides MS

▫IR▫AE

Gas chromatography-Mass Spectrometry (GC-MS)

•Gas chromatography–mass spectrometry (GC-MS) is a method that combines the features of gas-liquid chromatography and mass spectrometry to identify different substances within a test sample. Applications of GC-MS include drug detection, fire investigation, environmental analysis, explosives investigation, and identification of unknown samples. GC-MS can also be used in airport security to detect substances in luggage or on human beings. Additionally, it can identify trace elements in materials that were previously thought to have disintegrated beyond identification.

•GC-MS has been widely heralded as a "gold standard" for forensic substance identification because it is used to perform a specific test. A specific test positively identifies the actual presence of a particular substance in a given sample. A non-specific test merely indicates that a substance falls into a category of substances. Although a non-specific test could statistically suggest the identity of the substance, this could lead to false positive identification.

Gas Chromatography Application In general, substances that vaporize below ca. 300 °C (and therefore are stable up to that temperature) can be measured quantitatively. The samples are also required to be salt-free; they should not contain ions. Very minute amounts of a substance can be measured, but it is often required that the sample must be measured in comparison to a sample containing the pure, suspected substance known as a reference standard.Various temperature programs can be used to make the readings more meaningful; for example to differentiate between substances that behave similarly during the GC process.Professionals working with GC analyze the content of a chemical product, for example in assuring the quality of products in the chemical industry; or measuring toxic substances in soil, air or water. GC is very accurate if used properly and can measure picomoles of a substance in a 1 ml liquid sample, or parts-per-billion concentrations in gaseous samples.

SEMI- QUANTITATIVE ANALYSIS OF FATTY ACIDS

C

C

CDetector Response

Retention Time

14

16

18

Peak Area (cm )

Sample Concentration (mg/ml)

2

4

6

8

10

0.5 1.0 1.5 2.0 2.5 3.0

2

The content % of C fatty acids =C

C + C + C

= the content % of C fatty acids14

14

TENTATIVE IDENTIFICATION OF UNKNOWN COMPOUNDS

Response

GC Retention Time on Carbowax-20 (min)

Mixture of known compounds

Hexane

OctaneDecane

1.6 min = RT

Response

Unknown compound may be Hexane

1.6 min = RT

Retention Time on Carbowax-20 (min)

GLC ADVANTAGES

1. Very good separation

2. Time (analysis is short)

3. Small sample is needed - ml

4. Good detection system

5. Quantitatively analyzed