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4 Gas Chromatography/Gas Chromatography Mass Spectrometry

4 Gas Chromatography/Gas Chromatography Mass Spectrometry

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Chemical Extractions and Sample Preparation

Dry-Extraction Gas-Chromatography Modification

This method is used for raw samples and samples purified using thin-layer chromatography.

• Approximately 1 mg of sample is placed into a sample container using:

2-ml autosampler vial or

3 ì 50-mm culture tube

Approximately 1 ml of organic solvent is added to the sample container.

– The organic solvent used will depend on the solubility properties of the target compound.

– A 1-mg/ml sample concentration is an acceptable analytical standard.

(a) The actual concentration of components will be proportionally <1 mg/ml.

– Proper technique requires the use of an internal standard.

(a) An internal premix standard of 1 mg/ml concentration is recommended.

• The sample is agitated, and undissolved solids are allowed to settle.

– A filtration step may be required at this point.

• A sample of the solution is drawn from the vial and analyzed.



Gas-Chromatography Modification

The method is a slight modification of an acid/base extraction. It is typically used on impure samples and minimizes the

introduction of contaminates that could either adversely affect results or block the injection port of the gas


• Approximately 1 mg of sample is placed into a sample container using:

– 2-ml autosampler vial or

– 3 × 50-mm culture tube

• Approximately 1 ml of saturated sodium bicarbonate solution is added to the sample vial.

– Saturated sodium bicarbonate solutions have a pH = 8, which will create an environment conducive to the extraction of

acidic, neutral, and amphoteric (can be acidic or basic) compounds.

• Approximately 1 ml of organic solvent is added to the sample container.

– The organic solvent used will depend on the solubility properties of the target compounds.

(a) Chloroform extracts a wide range of compounds.

(b) Hexane is more selective.

– A 1-mg/ml sample concentration is an acceptable analytical standard.

(a) The actual concentration of the components will be proportionally <1 mg/ml.

– Proper technique requires the use of an internal standard.

(a) An internal premix standard of 1 mg/ml concentration is recommended.

• The sample is agitated and the phases are allowed to separate.

• A sample of the organic liquid is drawn from the vial and analyzed.


Infrared Spectroscopy

Infrared (IR) spectroscopy requires a highly purified sample for analysis. The slightest contamination can result in unexplained absorption bands or intensity shifts that complicate the identification process. Therefore, some compounds may

require chemical processing to be effectively identified using IR spectroscopy. For example, the free-base forms of many

phenethylamines are volatile, oily liquids that produce nondescript spectra. However, the salts of these same compounds

produce well-defined spectra with sharp, reproducible absorption bands.

The following methods are slight modifications of acid–base extraction and are required for IR-spectroscopy sample




Methanol Extraction


Acid–Base-Extraction Infrared-Modification-I

Acid–base-extraction IR-modification-I is used when the target compound is a solid at room temperature.

• Perform a complete acid–base extraction procedure for isolating an acid, base, or neutral compound depending on the

properties of the target compound.

• Evaporate the organic solvent to dryness.

• Analyze using IR spectroscopy after:

– Preparing a sample pellet using an IR-transparent material (dried potassium bromide, KBr). Grind 1 mg of sample with

80 mg of dried KBr using a mortar and pestle, or

– Dissolve and recrystallize a thin film of sample on IR-transparent material, or

– Place a drop of solution on IR-transparent material, typically salt plates.


Acid–Base-Extraction Infrared-Modification-II

Acid–base-extraction IR-modification-II is used when the target compound is either a basic liquid or a volatile oil at room


• Perform the complete acid–base extraction procedure for isolating a base.

• Bubble hydrogen chloride gas into the isolated organic layer containing the target compound.

– The HCl gas reacts with the free-base compound to produce a salt that is insoluble in organic solvents.

– HCl gas can be obtained by transferring the headspace air from a bottle of hydrochloric acid into the organic liquid

containing the free-base compound.

– HCl gas can also be generated through the reaction of sulfuric acid and sodium chloride.

• Evaporate the organic solvent to dryness.

• Analyze via IR spectroscopy after:

– Preparing a sample pellet using an IR-transparent material (dried potassium bromide, KBr). Grind 1 mg of sample with

80 mg of dried KBr using a mortar and pestle, or

– Dissolve and recrystallize a thin film of sample on IR-transparent window material, or

– Place a drop of solution on IR-transparent material, typically salt plates.


Methanol Extraction

Methanol is an excellent solvent for use in gas-chromatography mass-spectrometry (GCMS) examinations; however, it is seldom

used because it also dissolves a variety of impurities. Methanol extraction is preferred for the examination of residue only because

there is a high risk of sample loss during extraction. In residue analysis, the deposits are washed with approximately 2 ml of

methanol, and a sample of the resulting solution is run on the GCMS.

Most samples submitted to forensic laboratories for analysis require chemical processing to isolate the controlled substance. In some cases, separation can be accomplished using extraction techniques, while, in others, analytical instrumentation is the only viable alternative. Preliminary screening methods are often used to establish the direction of subsequent

analyses. Although extraction techniques are not technically classified as screening methods, the isolation of a compound

using extraction indicates the presence of fundamental properties that determine the appropriate confirmatory test.

It is important to recognize that no single extraction technique has universal applications in all instances. There is considerable variation among procedures, and the choice of particular technique will be based on the chemical and physical properties of the target compound. Notwithstanding these variations, product recovery is always greater when using multiple

extractions with smaller volumes as opposed to a single extraction with a larger volume. This is because the solubility limit

of the extracting solvent is often reached during the procedure. Also, the order of extraction steps may be changed with little,

if any, affect on the overall success of the technique. For example, is extraction more effective if you dissolve the sample and

extract the target, or dissolve the sample and extract impurities? The answer to this question will establish the order of extraction steps and is usually determined by availability of resources, time constraints on the analysis, and desired yield (quantity)

of the target compound. Regardless, extraction techniques represent some of the most reliable and cost-effective methods

used to isolate controlled substances.

















Chemical Extractions and Sample Preparation


Please describe chemical extraction to the jury.

Discuss the difference between solubility and affinity.

Describe the basic principles of liquid–liquid extraction.

What factors would be considered in the choice of liquid–liquid extraction over solid–liquid extraction?

Please explain to the jury the basic principles of acid–base extraction and cite any procedural differences when using this

technique to isolate an acid and a base.

Discuss a procedure used to isolate a neutral compound.

Briefly explain why IR spectroscopy requires highly purified samples.

Define the terms miscible and immiscible.

Provide two examples of immiscible solvent pairs.

Discuss why immiscible solvent pairs are required for extraction procedures.

Outline a common test used to verify phase identity in extraction procedures.

Discuss why multiple extractions maximize product yield.

In your opinion, would it be more efficient to perform a single extraction using a larger volume, or multiple extractions

using smaller volumes. Justify your choice.

Selected Reading

Bell, C. E. Jr.; Taber, D. F.; Clark, A. K. Organic Chemistry Laboratory with Qualitative Analysis, 3rd ed.; Harcourt College Publishers: New York,

2001; pp. 33–37.

Fieser, L. E.; Williamson, K. L. Organic Experiments, 8th ed.; Houghton Mifflin Company: Boston, 1998; pp. 104–110.

Pavia, D. L.; Lampman, G. M.; Kriz, G. S.; Engel, R. G. Introduction to Organic Laboratory Techniques, 3rd ed.; Saunders College Publishing:

New York, 1990; pp. 595–616.

Chromatography and Mass





A vast majority of samples submitted to forensic laboratories for identification are complex mixtures. Suspected drugs or

controlled substances recovered from crime scenes are often contaminated with various solvents or by-products from manufacturing and distribution. The identification process begins with the separation of the mixture into individual components.

The purified components can then be identified using various chemical or analytical techniques.

Chromatography (from Latin for color writing) is a general term used to describe any physical method of separation in

which the components to be separated are distributed between a mobile and stationary phase. Although several types of

chromatography exist, the underlying chemical principles are the same. The most common systems consist of a mobile phase

that is either a liquid or gas and a stationary phase that is a solid. The components of the analyte (mixture) will interact with

both phases with varying relative affinities. The physical process of separation will depend on these interactions as the

mobile phase pushes the components through the system. Generally, components with a higher affinity for the mobile phase

will move faster, while those with a higher affinity for the stationary phase will move slower. A common river provides a

good analogy to most chromatographic techniques. The rocks (analyte) in the riverbed are pushed down the river by the running water (mobile phase). The actual separation depends on how the rocks interact (affinity) with the riverbed (stationary

phase) and the running water. Normally, the smaller rocks move downstream faster, while the larger rocks move slower.

There are several different types of chromatography in common use and considerable variations exist. However, some are

more frequently used in forensic analysis than others. We have selectively chosen representative methods for discussion.


Chromatographic Techniques

10.2.1 Paper Chromatography

Paper chromatography is the simplest, and perhaps oldest, of all chromatographic techniques with references dating back to

the late 1800s. The stationary phase is a special paper that may or may not be pretreated. The inert (nonreactive) solid support

medium is cellulose (polymer of glucose), a major component in paper manufacturing. A small amount of water is adsorbed

from air at the hydroxyl groups of cellulose forming the stationary phase. Adsorption is different from absorption. Adsorption

refers to water molecules loosely bound at the surface of a molecule, while absorption refers to water molecules integrated

into the interior. A simple sponge illustrates these subtle differences quite effectively. Water molecules are absorbed into a

sponge when it is placed in water. Sand particles are adsorbed to the surface of a wet sponge once it is thrown into a sand


Paper chromatography is performed in a clear container called a developing chamber. Initially, the developing chamber

contains only the mobile phase, a suitable solvent often called the running or developing solvent. Covering the chamber

allows an equilibrium to be established between solvent molecules in the liquid phase and those evaporating from the surface. Thus, the environment in the developing chamber is saturated with solvent molecules.

J.I. Khan et al., Basic Principles of Forensic Chemistry, DOI 10.1007/978-1-59745-437-7_10,

© Springer Science+Business Media, LLC 2012




Chromatography and Mass Spectrometry

Fig. 10.1 Separation of samples using paper chromatography with a polar running solvent. Left-to-Right: Column 1 – a nonpolar component.

Column 2 – reference. Column 3 – mixture of polar components. Column 4 – a polar component. Column 5 – a component of intermediate polarity.

Note beginning reference line and solvent front (not drawn).

Small drops of sample and reference solutions are placed at regular intervals (spotted) at one end of the paper on a

horizontal line drawn in pencil. Samples are identified using numbers, labels, or codes commonly written directly on the

paper (in pencil) under each sample. The samples must be high enough to ensure that they will not be immersed in the

running solvent once the paper is placed in the developing chamber. The samples are allowed to dry and the paper is

placed in the developing chamber with the samples toward (but not immersed in) the solvent.

Capillary action draws the mobile phase up the paper and the eluting solvent contacts each sample positioned on the stationary phase. The components in each sample will interact with the mobile and stationary phases with varying affinities and

separation is accomplished using differential migration (different migration rates). In general, this is directly related to differences in polarity; for example, if a polar running solvent is used, polar components will travel more rapidly in the mobile

phase, while nonpolar components will move slowly or not all. A quick note, polarity can be equated to a bar magnet. The

characteristics and behavior of polar molecules are very similar to those of a magnet. When you encounter terms such as

polarity, dipole, polar, etc., simply think of a magnet to clarify the concept.

The procedure is complete when the running solvent stops eluting (moving) or when it approaches the end of the paper.

The running solvent should never elute to the end of the paper. The paper is removed from the developing chamber and a line

is immediately drawn in pencil across the solvent front. This must be done quickly because volatile solvents evaporate

quickly and the solvent front may soon be indistinguishable. The paper is allowed to dry and the separated components are

observed as spots. They are readily observed if they happen to be colored; if they are colorless, visual enhancement techniques will be required, i.e., oblique lighting with short- and long-wavelength UV (a black light) or iodine fuming. Iodine

fuming involves placing the paper in a covered beaker containing a few crystals of iodine. Iodine will readily sublime (convert from solid to gas) and the gaseous molecules adhere to the components coloring them light purple-violet (Fig. 10.1). The

color intensity of each component can be used to compare relative concentrations. More intensely colored components

(darker spots) are present in higher concentrations. In addition, an assessment of purity can be made using the results.

Samples containing a single spot are highly purified, while those containing many spots are contaminated. The paper, or a

clear sketch of the results, including visual enhancement method, should always be documented in the case file notes.

Paper chromatography produces both qualitative and quantitative results. Qualitative results are obtained by simply

observing the spots on the paper, i.e., purity, number of components, relative concentrations, degree of separation, characteristics of each component based on running solvent properties, etc. Quantitative results are obtained using retention factors

(Rf-factor). Rf-factors can be calculated for each component by dividing component travel by solvent travel. Component

travel is the measured distance from the line where the sample was initially spotted to the final location of the component on

the paper. Solvent travel is the measured distance from the same beginning line to the solvent front. Rf-factors can be used to

identify components in different samples separated under the same chromatographic conditions.

10.2 Chromatographic Techniques


There are several factors to consider when performing paper chromatography:

• The best resolution (observed separation) is accomplished using small, highly concentrated drops when spotting. The drops

will diffuse radially outward when they are applied to the paper. If care is not taken, the drops may contaminate one another.

• Never use ink when drawing reference lines on the paper. Ink will often separate and migrate in a fashion similar to the samples.

• Never allow the running solvent to elute to the end of paper. This affects overall resolution by clustering components at

the top of the paper.

• Never immerse the components in the running solvent. Components with characteristics similar to the solvent will simply

dissolve directly off the paper.

Paper chromatography is classified as a type of solid–liquid chromatography because separation is achieved using a liquid

mobile phase and a solid stationary phase.

10.2.2 Thin-Layer Chromatography

The procedure used to perform thin-layer chromatography (TLC) mirrors that used for paper chromatography with one

notable exception. The stationary phase is a thin layer of adsorbent material coated on a small sheet of glass or plastic. TLC

plates can be cut in any size or shape and most often resemble a coated microscope slide.

There are many types of adsorbents available but alumina (Al2O3) and silica (SiO2) are by far the most common. Anhydrous

alumina (no water adsorbed) adsorbs substances more strongly and is often used to separate nonpolar components such as hydrocarbons and alkyl halides, as well as molecules containing ether, aldehyde, and ketone functional groups. Silica is less active and

has common applications separating polar components containing alcohol, carboxylic acid, and amine functional groups.

Thin-layer chromatography is a sensitive, fast, and simple analytical technique; however, it gained significant popularity

with the development of cost-effective commercially available plates. The automated industrial application of adsorbent

material to plates creates a solid layer of uniform size and thickness. This produced a more stabile, even flow of solvent and

a smooth path for component travel. The consistency of separation using commercially available TLC plates was a dramatic

improvement over paper chromatography (Fig. 10.2).

Fig. 10.2 Component separation using a commercially available TLC plate. CTR is control, a reference sample containing the purified drug. All

samples test positive for the presence of the drug. Note the well-defined columns of separation, sample labels, and drawn reference lines. Color

intensity can be used to determine relative concentration. Comparison of spots midway through the each sample column indicates sample 13 has

the highest concentration of these components (darkest spots).



Chromatography and Mass Spectrometry

10.2.3 Column Chromatography

Column chromatography is a macroscopic version of TLC often used to separate large samples. In this method, the “column”

is a specially designed glass or plastic tube open at one end and fitted with a polyethylene frit and stopcock at the other. The

frit holds the stationary phase in the column while allowing the mobile phase to pass. The stopcock is used to control the flow

of the mobile phase.

The vertical column is filled with adsorbent material (stationary phase) in a process called packing. Like TLC, alumina

and silica are most often used, but hydroxapetite and cellulose are also quite common. Uniform packing of the column is

critical to the success of column chromatography. For this reason, the most efficient columns are rarely packed with powdered adsorbent, although this is an option. Instead, a slurry is prepared by mixing the adsorbent with the running solvent

(mobile phase) and poured into the column. The stopcock is opened and the slurry uniformly packs the column as gravity

pulls the solvent downward. The draining process continues until the solvent level is just above the top of the packed adsorbent. Care must be taken to ensure that the last portion of slurry forms a flat surface at the top of the column. Under no circumstances should the level of the solvent fall below the packing, and, if this occurs, the procedure must be repeated. The

size of the column can vary depending on the desired resolution (separation). In general, the height of a packed column

should be at least ten times the column’s diameter and the amount of adsorbent used should weigh at least 30 times the

amount of sample.

Highly concentrated samples work best; therefore, it is recommended that samples be dissolved in a minimum amount of

solvent. The sample is carefully added to the column without disturbing the top. The stopcock is opened and the solvent is

drained until the sample solution level is just above the packing. The sample must be added uniformly to the column in a

small, discrete band.

The first running solvent is added to the top of the column and the stopcock is opened. Gravity pulls the running solvent

(mobile phase) through the column (stationary phase) and soluble components are separated as they migrate (travel) down

the column at different rates (differential migration). Individual components arrive at the lower end of the column at different

times and are collected as they drain through the stopcock. The process is repeated with solvents of different polarity to

isolate the remaining components.

10.2.4 Ion-Exchange Chromatography

Ion-exchange chromatography (IEC) is very similar to column chromatography in that both use packed columns to achieve

separation. The two procedures differ in the column packing material and the method used to isolate individual components.

Ion-exchange chromatography relies on electrostatic forces of attraction (opposites attract) to bind charged atoms or polar

molecules to an oppositely charged resin packed in the column. The column is subsequently treated with an eluting solvent

of varying concentration to disrupt the attractive forces and recover the fixed molecules. Ion-exchange chromatography is

typically used in forensic analysis to isolated anions from aqueous solutions, i.e., chloride (Cl−), sulfate (SO42−), and nitrate


Ion-exchange chromatography is divided into two broad classes differing only in the composition of the stationary phase.

Cation-exchange chromatography isolates positively charged ions from a sample mixture using a negatively charge resin to

selectively bind the cations. Anion-exchange chromatography does the polar opposite (no pun intended!), using a positively

charged resin to isolate negatively charged ions. Regardless of which technique is used, ions are fixed to oppositely charged

resins as the sample passes down the column.

Once the components are bound, the column is treated with a low-ionic-strength solution (low salt concentration) to

establish an equilibrium between the resin and the solution. The fixed components are removed from the column using

a salt gradient applied to the running solvent, which gradually increases its ionic strength. The changing salt concentration will eventually reach an ionic strength that is capable of displacing the ions from the resin. Alternatively, the pH

of the running solvent can be changed to alter the fixed ions’ charge. The change in charge will disrupt the ion-resin

attractive forces and free the bound ions. In either case, the fixed ions are released from the resin and collected at the

bottom of the column (Fig. 10.3). Sometimes it may be more practical to select conditions that bind contaminants and

allow the target ions to flow through. In these cases, ion-exchange chromatography is virtually identical to column


10.2 Chromatographic Techniques


Fig. 10.3 A typical instrument

used to perform ion-exchange

chromatography. The automated

computer workstation is capable

of performing single-step

qualitative and quantitative

analysis using several variations

of ion exchange.

Fig. 10.4 Integrated HPLC workstations have dramatically advanced the practical applications of column chromatography. HPLC is a highly

sensitive technique that is capable of differentiating structural isomers. It is commonly used in forensic analysis to separate drugs and controlled

substances from complex mixtures with expanding applications in the isolation of designer drugs.

10.2.5 High-Performance Liquid Chromatography

High-performance liquid chromatography (HPLC) is a more controlled variant of column chromatography. Measurements

are performed using an automated instrument called a liquid chromatograph, which houses, among other things, a closed

system of pumps and the column (Fig. 10.4). In HPLC analysis, high pressures (up to 400 atmospheres!) force the liquid

phase (mobile phase) through a column containing densely packed, small-diameter microspheres (stationary phase). The

nature of the packing material dramatically increases the surface area for column adsorption, a significant improvement over

column chromatography. Separation is achieved in the same manner as column chromatography. The mobile phase pushes

the sample through the stationary phase and components travel at different rates, depending on their selective interactions

with both phases. The capacity to precisely regulate pressure provides direct control over solvent velocity, which enhances

resolution (degree of separation) and decreases the level of component diffusion onto the column. Several types of detectors

are used to differentiate components as they emerge from the column.

The detector records and displays component information on a chromatogram, a series of peaks on a graph of relative

abundance (y-axis) versus time (x-axis). The area under each peak is integrated by the instrument and is used to compare the



Chromatography and Mass Spectrometry

relative concentrations of each component. For example, if peak 1 has an integrated area twice that of peak 2, component 1

is present in twice the concentration of component 2. The actual concentrations however are not known. The instrument also

records retention times. Retention time is the amount of time it takes for each component to pass through the entire column.

These times can be used to identify the component because they are well defined for a variety of substances isolated under

specific chromatographic conditions. A subtle point of note – the use of high pressures in HPLC has somehow (erroneously)

infiltrated the name. Referring to HPLC as high-pressure liquid chromatography is incorrect and its use can sometimes call

into question your knowledge of the process.

Typical HPLC columns are stainless steel with a length of 10–30 cm and an internal diameter of 4–10 mm. The particle

size of column packing material can range in diameter from 1 to 10 mm (mm is micrometer, 1 mm = 10−6 m; 1 millionth of a

meter). The stationary phase is commonly treated with various chemical agents, depending on the nature of the molecules to

be separated.

HPLC is divided into two basic procedures: normal-phase HPLC and reverse-phase HPLC. The main difference is in the

composition of column packing material, which necessitates the use of different solvents. Normal phase was developed first,

but has since been replaced by the almost exclusive use of reverse phase. Therefore, any discussion of normal-phase HPLC

is more historical than practical.

Normal-phase HPLC uses a polar stationary phase and a nonpolar mobile phase to achieve separation. Columns are

packed with tiny particles of silica containing polar amino, diol, or cyano groups. The sample is dissolved in a nonpolar

solvent such as hexane (mobile phase) and pushed through the column. Retention times for polar components are longer than

those for nonpolar components in yet another example of “likes dissolve likes.” Essentially, the polar components adhere to

the modified silica particles (adsorbed) and are retained longer on the column. Nonpolar components proceed through the

column at different rates, depending on their relative interaction with the mobile and stationary phase.

Reverse-phase HPLC uses a nonpolar stationary phase and a polar mobile phase to achieve separation (the reverse of

normal phase). The column is packed with silica containing a bound hydrocarbon component. Alkyl groups (the hydrocarbon component) containing 8 or 18 carbons are preferentially used to make the silica particles nonpolar. A solution of water

and alcohol (methanol) is commonly used to create a moderately polar mobile phase. The sample is dissolved in the mobile

phase and pushed through the column. The nonpolar components are adsorbed on the surface of the modified silica particles

and are retained longer on the column (likes dissolve likes). The polar components travel through the column at different

rates and are separated. Retention times can easily be modified using reverse-phase HPLC. In general, adding more water to

the mobile phase will increase retention times of nonpolar components and decrease those of polar. Adding an organic solvent will decrease nonpolar retention times and increase those of polar components.

10.2.6 Gas Chromatography

Gas chromatography (GC), also called vapor-phase chromatography (VPC) and liquid-gas chromatography (LGC), is the

most popular method used to separate the individual components of a volatile mixture. It is, without question, the most frequently used chromatographic technique in modern forensic laboratories. Measurements are performed using a complex,

highly integrated instrument called a gas chromatograph (Fig. 10.5). The vital components of a gas chromatograph are,

among other things, the injection port, the column, detectors, and an oven. All components are integrated using a computerized workstation with highly programmable functions. The injection port is a heated chamber typically located on the top of

instrument. The column is contained in an insulated oven at the front of the instrument, which is easily accessible for maintenance, repair, or replacement. Detectors are generally located at the back and can be individually accessed through the

workstation. Injection port and column temperatures are independently regulated and are capable of reaching temperatures

in excess of 400°C.

In GC analysis, the mobile phase is an inert gas such as helium, argon, or nitrogen called the carrier gas. The stationary

phase is a high boiling liquid coated on the interior wall of the column (capillary GC) or on the surface of a solid support

medium (packed-column GC). The sample is vaporized and carried through the column by the mobile phase (carrier gas). A

detector at the end of the column records and displays component information on a chromatogram. GC chromatograms

closely resemble the appearance of those produced using HPLC. They provide the same information and are interpreted in a

similar manner.

The sample is introduced into the gas chromatograph using a glass microliter (mL) syringe. Typically, a 2–4-mL sample is

sufficient, but actual sample size may vary from 1 to 25 mL. The needle is pushed through a rubber septum at the injection

port. Care must be taken to avoid bending the syringe needle during placement. Also, the injection port is usually very hot and

10.2 Chromatographic Techniques


Fig. 10.5 Automated gas

chromatographs are commonly

used in forensic analysis to

separate volatile drugs and

controlled substances. The above

instrument is equipped with an

autosampler located on the top of

the instrument. This

configuration permits the

sequential examination of several

mixtures with minimal


direct contact should be avoided. The sample is vaporized in the injection port and mixed with a continuous flow of carrier

gas. Components with low boiling points will vaporize first, followed by those with higher boiling points. The sample–carrier

gas mixture runs through the column at a preset flow rate. The individual components are differentially adsorbed on the surface of the stationary phase and an equilibrium is established between molecules in the vapor and liquid phases. Easily

adsorbed components travel slowly through the column (high retention times), while those not readily adsorbed travel more

rapidly (low retention times). Flow rates must be carefully regulated to ensure good resolution. If the rate is too high, an equilibrium will not be established between molecules in the vapor and liquid phase. If it is too slow, the molecules will emerge

from the column over an extended period of time producing band (peak) broadening. Both conditions result in poor resolution


There are two types of columns used in GC analysis. The stationary phase is the same in both, but the column dimensions

and the method used to affix the stationary phase are different. The type of column is incorporated into the name of the technique and differentiates the two procedures.

Packed-column GC uses a column constructed of stainless steel or glass tubing typically 1.5–10 m (meters) long with an

internal diameter of 2–4 mm (millimeters). The stationary phase is a thin layer of high boiling liquid coated on the surface of a

solid support medium. The composition of the stationary phase can vary depending on the characteristics of the molecules to be

separated, hydrocarbon greases, silicone-based oils, and carbowaxes (derivatives of polyethylene glycols) are most common.

The solid support media is typically crushed firebrick, glass or nylon beads, silica, or alumina.

Capillary GC uses a wall-coated open-tubular (WCOT) column made of small-bore flexible tubing (often copper). WCOT

columns are generally 10–60 m long with an internal diameter of 0.2–0.5 mm. The tubing is wound around a solid support

to accommodate space limitations in the gas chromatograph (Fig. 10.6). The inner wall of the tube is coated with a stationary

phase of similar composition to that used in packed-column GC. Capillary GC produces more effective separation than techniques using larger packed columns because the small diameter and extreme length of WCOT columns increase the interactions between sample components and the stationary phase.

One of the clear advantages of GC analysis is the ability of gas chromatographs to accommodate a variety of detectors in

a single instrument. Switching detectors or using detectors in series is a relatively simple process, often requiring only a

sequence of keystrokes at the workstation. Although the type of detector used will depend heavily on the specific application,

flame-ionization detectors (FIDs) and thermal conductivity detectors (TCDs) are very common. Flame-ionization detection

is a destructive technique because the components are burned (ionized) as they elute off the column. The ions create (induce)

an electrical current that is measured and recorded as a peak on the chromatogram. Thermal conductivity detection is a nondestructive technique and would be preferred when sample quantities are limited. In this method, two separate filaments are

heated to a constant temperature using an electrical current. Both filaments are exposed to a continuous flow of carrier gas,



Chromatography and Mass Spectrometry

Fig. 10.6 A typical wall-coated open-tubular (WCOT) capillary

GC column. Notice the ends of the column on the right and left

of the center support. The small diameter and extreme length of

WCOT columns offer significant advantages over most

chromatographic techniques.

Fig. 10.7 Gas chromatography (GC) can be compared to the operation of coin-separating machines. This useful analogy can be used to clarify

GC analysis to members of a jury.

which cools the filaments. The final temperature of both filaments will be the same and represents the base line (starting

point). The eluting components of the mixture are directed over one filament and cause a temperature change specific to that

component. The current required to bring the filament back to base-line temperature is recorded as a peak on the


Flame ionization and thermal conductivity are versatile, highly sensitive techniques and both are commonly used to detect

a wide range of organic compounds. However, in cases requiring a higher degree of specificity, a more selective detector may

be desired. For example, electron capture detectors (ECDs) are often used exclusively to detect halogens (Group VII), organometallic compounds (contain metals), nitriles (contain CN group), and nitro-compounds (contain NO2). Also, nitrogen–

phosphorus detectors (NPDs) are frequently used to distinguish compounds containing nitrogen or phosphorus. Without

question, the most versatile and well-known GC detector is the mass selective detector (MSD). This detector is actually a

mass spectrometer connected directly to the gas chromatograph. This analytical instrument will be discussed in more detail

below. Gas Chromatography: A Simple Analogy

In many cases, the forensic chemist is called upon to describe complex chemical procedures to individuals that have a limited

understanding of scientific principles. Courtroom testimony is carefully prepared using common terminology and the presentation must be in a clear, simple manner that avoids confusion and misinterpretation. A gas chromatograph is a highly

integrated instrument used to separate a gaseous mixture into individual components. The complex details of the separation

process may easily confuse most people on a jury. Therefore, a description of how a gas chromatograph functions may contain a reference to coin-separating machines frequently found in local grocery stores. These machines separate the mixture

of coins based on size, and totals each pile based on weights. This analogy would illustrate how a gas chromatograph functions and may help members of a jury be more comfortable with testimony about this complex instrument (Fig. 10.7).

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4 Gas Chromatography/Gas Chromatography Mass Spectrometry

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