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Separating Complex Mixtures. Chapter 4. Introduction. Many commonly encountered types of physical evidence consist of complex mixtures of substances Examples include: Drugs are often mixed with other substances (sugars) Gasoline contains over 300 different substances
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Separating Complex Mixtures Chapter 4
Introduction • Many commonly encountered types of physical evidence consist of complex mixtures of substances • Examples include: • Drugs are often mixed with other substances (sugars) • Gasoline contains over 300 different substances • Piles of rubble from explosions are large, complex mixtures
Physical Separation of Solid Mixtures • Physical separations are used in cases where one or more components of the mixture must be recovered intact • Chemical treatment is not appropriate because the structure of the components may be altered • Example: If rubble from an explosion is treated with water to dissolve parts of the mixture, the explosives may also dissolve • A microscope and tweezers may be used to separate particles of cocaine from other substances
Chem. Separation of Solid Mixtures • Separating a mixture by solubility should only be done when: • The examiner knows what else is in the mixture • The crystal structure of the component is not an issue • Solubility is especially effective in cases where the particles are so small that it would be impossible to physically separate them or if only a small amount exists • For chemical separations of substances, chemists rely on pH and polarity
Chromatography • Chromatography means to analyze by color • All forms of chromatography contain two phases; • Stationary phase: The filling in a column or a thin coating of material • Mobile phase: Usually pumped under pressure and moves through the stationary phase • The analyte is the mixture that needs to be separated • Components of the analyte travel through the stationary phase at different speeds • Detectors electronically sense materials and display the results on a computer screen
Resolution • Resolution is a measure of the ability of a chromatographic technique to separate two similar substances • The goal is baseline resolution where each substance comes completely through the instrument before the next one • In other words, the peaks displayed on a computer are completely separated
Quantitative Analysis • All types of chromatography can be used for determining how many substances there are in a mixture and for separating them • Some types can also be used for quantitative analysis • Each peak is approximately triangular in shape • A peak formed from 1.0 mg of cocaine would be twice as tall as a peak formed by 0.5 mg • Quantitative analysis is done by running known samples (such as 1.0 mg of cocaine) and comparing the size of the peaks
Types of Chromatography • There are many types of chromatography used in chemical analysis • The following are important in forensic science: • Gas chromatography • Liquid chromatography • Thin-layer chromatography
Gas Chromatography (1) • Gas chromatography is used to separate very small amounts of gas or liquid • Can separate nanograms of material (10-9) • GC is used to separate drugs from cutting agents and to identify accelerants in fire residues • A detector sends a signal to a computer which displays a peak on a chart • The array of peaks (one for each component) is called a chromatogram
Gas Chromatography (2) • The most common detector used for GC is called the mass spectrometer (MS) • A mass spectrometer can identify each component of the analyte as it emerges from the GC column • Pyrolysis gas chromatography is used for materials with a higher vaporization temperatures (as much as 800oC) • These include paints, fibers, plastic, and rubber
Liquid Chromatography (HPLC) • In liquid chromatography, the mobile phase is pumped under pressure through the stationary phase • Detectors (mainly mass spectrometers) then analyze the substances • HPLC is used for a variety of evidence types • Drugs, soils, inks and dyes, and explosive residues
Thin-Layer Chromatography (1) • In TLC, there is no large instrument with columns, ovens, detectors, or computers • Instead, the stationary phase is typically a glass microscope slide coated with silica (known as the plate) • The plate is then immersed in a small amount of the mobile phase in a beaker or container • A spot of an unknown substance can be made next to known substances and compared
Thin-Layer Chromatography (2) • Capillary action causes the mobile phase to travel up through the stationary phase • Along the way, it carries with it the components of the analyte • Different components travel at different speeds • TLC is used for a wide variety of evidence types • Drugs, inks and dyes, explosive residues, cosmetics (lipstick, nail polish, etc.)
Electrophoresis (1) • Sometimes substances are so similar that no conventional chromatographic system can separate them • This is especially true for DNA fragments • For these separations, electrophoresis is used • Two types: Gel and capillary
Electrophoresis (2) • Gel electrophoresis is somewhat like TLC • The stationary phase is a slab of gelatin material (agarose or polyacrylamide) • DNA fragments are put in wells at one end of the gel • A strong electric current (mobile phase) causes the DNA to move • The lighter, smaller fragments travel faster and farther than the heavier larger ones
Electrophoresis (3) • Capillary electrophoresis uses a thin column of gel instead of a slab • As the analyte (DNA fragments) move through the column, they are detected by fluorescence • Capillary electrophoresis is more efficient at separating DNA fragments • The resulting electropherogram shows a series of peaks which gives quantitative data and the size of each fragment
