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Chapter 6 Separation Methods
Introduction to Forensic Science (CHE103)
Murdoch University
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CHAPTER 6 – SEPARATION METHODS
Since chemical evidence hardly occurs in a pure state, it is usually necessary to separate it from the matrix in which it is found at a crime scene. There are a wide variety of separation methods in analytical & forensic chemistry. They range from the very basic, such as gravity filtration to the complex, such as capillary electrophoresis. No best method. The separation method used in a material depends on what & how much evidence needs to be separated. 3 separation methods liquid phase extraction, solid phase extraction, & chromatography Analyte = describe the substance or substances being separated. Solvent = a liquid or liquid solution that is used to dissolve all or part of an analyte Solute = substance that is dissolved in a solvent.
LIQUID PHASE EXTRACTION:
Involves the separation of 2 or more substances in an analyte through a process where 2 solvents are employed. 2 solvents are chosen so that they do not mix with each other (immiscible). Eg: chloroform & water. Competition/distribution between 2 solvents for an analyte is called partitioning – involves analytes being distributed between 2 solvents according to certain chemical properties, mainly polarity & pH.
Polarity: The tendency of the compound to behave like a miniature magnet, with a positive side & a negative side. Not all compounds have this property. Most organic compounds tend to be nonpolar or slightly polar. Inorganic compounds can range from nonpolar to very polar. Non-polar compounds dissolve more readily & to a greater extend in non-polar solvent. Polar compound dissolves more readily & to a great extend in polar solvent. Compounds that are symmetric tend to be nonpolar. Polarity is caused by an excess of electron density on 1 side of a molecule & a deficiency on the other side. A simple rule about solubility & polarity is like dissolves like – means that polar compounds have a greater affinity for other polar compounds. If an analyte contains a mixture of substances where some are polar & others are nonpolar, they can be separated into 2 groups using a polar solvent & a nonpolar solvent. Cocaine is fairly nonpolar, it can be made more polar by reacting it with a dilute acidic, forming a salt. Salts are generally quite polar.
pH: Another property of certain chemical compounds is their acidity or alkalinity relative to water. Acids have pH values between 0 & 7. Alkaline pH is between 7 & 14. A neutral substance has a pH of 7. Drugs can be classified as acidic, basic, or neutral. Caffeine is a neutral drug, so are sugars and carbohydrates. Cocaine & heroin are basic. Liquid phase extractions are commonly used to separate mixtures of solids. If the mixture contains 2 substances of similar polarity & both are acidic or basic, such as cocaine & heroin, then a liquid phase extraction would likely fail to achieve a good separation. A relatively large amount of material, at least a few grams, is normally required for an effective liquid phase extraction. Separation of cocaine hydrochloride cut with fructose: 1. Dissolution in water 2. Addition of alkaline solution (ammonium hydroxide) to increase pH in separatory funnel. 3. Addition of non-polar solvent to separatory funnel 4. Separation of chloroform & water
SOLID PHASE EXTRACTION:
Does not involve a partitioning mechanism, it relies on adsorption, a process whereby a solid, liquid, or gaseous analyte is attracted to the surface of a solid adsorbing material. Adsoprtion = substance adheres to surface of solid phase, absorption = substance drawn into a solid phase.
If just 1 component of a mixture is adsorbed onto the surface of the adsorbent, it can be separated using this method. A number of chemical processes affect the tendency & tenacity of the adsorption including polarity. Example of a solid phase extraction is the "clean up" process of a biological sample containing a drug. A column of adsorbing material is inserted into a large tube & a blood or urine sample is then added. It flows through the tube under a vacuum & the drug is adsorbed on to the surface of the adsorbing material whereas the rest of the blood or urine passes through. What makes a good adsorbent? o High surface area, surface functionality/active sites eg with high polarity, reproducibility in degree of activation
Solid Phase Microextraction: In recent years, a modification of solid phase extraction has been developed – solid microextraction (SPME) – used for very small samples, a small wire is coated with an adsorbent & attached to a holder that can extend or withdraw the wire. The wire is extended into a vapor or liquid where adsorption takes place. Wire can be introduced into a gas chromatograph where the adsorbed materials are eluted & analysed.
CHROMATOGRAPHY:
Neither solid & liquid phase extraction techniques are quantitative. Chromatography (means "to write with colour"), developed by Tswett (1901). Separate complex mixtures of substances into their individual components & then display these components so that the analyst can get information about their number & chemical nature & sometimes concentration. Chromatography methods are more sensitive than extractions/separations All chromatography systems consist of: (1) stationary phase (doesn't move), (2) mobile phase (moves), (3) way of getting the mixture into the system, (4) way of telling if compounds in a mixture are coming out of the system.
How Chromatography Works: In all forms of chromatography, there are 2 phases present: 1. Stationary phase = finely divided solid material or a viscous liquid that is contained within a long column. The components of the analyte move quickly or slowly through the column, separating from other components along the way. 2. Mobile phase = a liquid (or liquid solution) or a gas under pressure. Moves through the stationary phase carrying the analyte mixture with it. Eg: Plant pigment separation – the column of silica is an example of a stationary phase. The plant pigment mixture is dissolved in a solvent that is then poured through the stationary phase. The solvent is the mobile phase. The stationary phase is designed to attract or repel certain members of the analyte, each one in a different way. Each type of chromatography has its own types of detectors that use various properties of the analytes to signal their presence, even eyes can be a detector. The relationship between the stationary phase & mobile phase is often described as normal or reverse phase. In a normal phase chromatography, the mobile phase is less polar than the stationary phase. This is always the situation with GC. In reverse phase chromatography, the opposite is true. Mobile phase is more polar than the stationary phase. In LC & thin-layer chromatography (TLC), either normal or reverse phase can be used.
GAS CHROMATOGRAPHY:
Stationary & Mobile Phases: Most versatile chromatography method is called gas chromatography (GC). Modern GC is called capillary GC because the stationary phase is contained within a very narrow, hollow tube that is made of glass or a synthetic polymer & is often coated with a plastic to add strength. The inside of the column contains the stationary phase, a thick, high boiling, viscous liquid, that has the consistency of molasses. The stationary phase forms a thin coating on the inside of the capillary. The columns can be up to 30m long. The mobile phase in GC is an inert gas. Most commonly used gas is helium. Best gas is hydrogen but it is more expensive & flammable. In a gas chromatography, the mobile phase is forced through the stationary phase under pressure. Analytes have to be in a vapor state. This results in one of the limitations of GC the analytes have to be heated until they
- Both would then be chromatographed & the areas under the cocaine & internal standard peaks for both the known cocaine & case exhibit are calculated. Through simple proportions, the weight of cocaine in the exhibit can be calculated & the % can be determined by knowing the weight of powder that was dissolved. This quantitative analysis helps determining how far up the distribution chain the seized drugs are. Eg: street cocaine can be 40% pure, but if the sample is 80% then we can assume it can from a distributor.
Pyrolysis Gas Chromatography: Substances that don’t vaporize at 350 degrees cannot be analysed by normal GC methods. These substances include fibers, paints, plastics, hairs, & other polymers. A modification of GC, pyrolysis gas chromatography (PGC) can make it possible for a gas chromatograph to analyse polymers. "Pyrolysis" means "to heat in the absence of air." If a polymer such as a fiber is heated to very high temperatures, up to 1000 degrees, in the absence of oxygen, it will not burn but instead will decompose into stable fragments, called pyrolyzates. In PGC, an apparatus that can hold a small fragment of polymer is inserted directly into the injector of the gas chromatograph. No oxygen is present so only pyrolysis & not combustion takes places. Pyrolyzer is heated to high temperatures (700-1000 degrees) & the polymer decomposes. Fragments are then separated. The resulting chromatogram is called a pyrogram.
Hyphenated Analytical Techniques: GC/MS GC-FTIR = fourier transform infrared spectrophotometer.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC):
Process can be sped up by having the mobile phase pushed through the stationary phase using pumps. Makes the experiment faster while keeping the high resolving power of the technique – high performance liquid chromatography (HPLC). Packed columns are routinely used & the stationary phase can be similar to those in packed column GC or can be very different. 1 of the most popular HPLC stationary phases used in forensic science is C hydrocarbon (octadecane) (like candle wax). Mobile phases can be either a single liquid or a solution containing 2 or more miscible liquids. HPLC has some significant advantages over GC. o In GC, the stationary phase is always more polar than the mobile phase. In HPLC, stationary phases & mobile phases can be designed so that the stationary phase is less polar than the mobile phase. Referred to as being reverse phase. o Another advantage of HPLC over GC is that the composition of the mobile phase can be altered during the run. This is called gradient chromatography – used when the analyte contains components of varying polarity. When the mobile phase stays constant during an HPLC run, it is called isocratic chromatography.
Parts of an HPLC: The Injector: o A liquid chromatograph has the same parts as a gas chromatograph. Differ in how they work because the mobile phase is a liquid or liquid solution. The injector consists of a sample loop. Mobile phase is pumped into the column containing the stationary phase. The Stationary Phase: o Typically columns are around 5mm in diameter. Stationary phase is either a solid or a viscous liquid coated onto spherical, solid particles. Columns are generally much shorter than GC columns (25cm). Detectors: o Detectors in an HPLC are different than GC detectors. GC detectors must detect solutions of gases, HPLC detectors must detect liquid solutions. o Detectors are designed to detect changes in the concentrations of substances in the mobile phase. The most popular detector for HPLC is a UV/visible spectrophotometer, usually a special type called a diode array detector (DAD) – measures the ultraviolent & visible spectrum of the solution as it flows through.
o A DAD simultaneously measures all of the wavelengths of UV & visible absorption of the analyte, so there are many ways that the data can be presented. o Other detectors that can be used in HPLC: - Fluorescence – detect only those substances that exhibit fluorescence, such as the illicit drug LSD. Extremely sensitive. Ultraviolet or visible light is used as the source. - Conductivity - Refractive index - Mass Spectrometry
Applications of HPLC: Has become increasingly popular. May be the method of choice for items that are sensitive to temperature, eg explosives. Some of more popular applications to evidence analysis drugs, soils, explosives, inks & dyes.
THIN-LAYER CHROMATOGRAPHY:
Paper chromatography is 1 of the oldest methods of separation. Paper acts as a stationary phase & the ink solvent is the mobile phase. Now called thin-layer chromatography. Stationary phase consists of a thin layer of solid material that is coated onto a small glass or plastic plate. TLC is similar to HPLC.
Stationary Phase: The stationary phase is a thin layer of a solid material combined with binders that are coated on to the surface of either a glass or plastic plate. When a non fluorescent sample is loaded onto the stationary phase, it will quench or blot out the fluorescence under the sample & it will be seen as a dark spot.
Mobile Phase: Mobile phase in TLC is an organic liquid (or water) or a solution of 2 or more liquids. Polarity ranges from the very nonpolar to very polar.
TLC Processes: Carried out in a chamber. Plate must fit so that it does not touch the sides of the chamber. Must also fit entirely within the chamber. Piece of filter paper is put inside the chamber up against the side. Top of the chamber is tightly covered. Analyte is dissolved in a small amount of a suitable solvent. Chloroform or methanol is often used. A spot of dissolved analyte is put onto the TLC plate. Spot is kept as tiny as possible. After the plate is loaded with samples, a small amount of the mobile phase is put in the chamber. When the mobile phase front as nearly reached the top of the plate, the process is stopped by removing & drying the plate.
Detection: Some of the spots may show up under UV light. Some may fluoresce. You could spray them with an aerosol. These reagents react with the analyte component to form a characteristic colour. Most nitrite (NO2) containing compounds, such as most explosives, will turn red when sprayed. Once the spots are visualized, their positions are measured as a retention factor (Rf) – distance that the sample component travels up the plate divided by the distance that the mobile phase traveled.
Applications of TLC: TLC can be used in all of the applications than HPLC & to some extent, GC are used for.
Advantages: Compared to GC & HPLC, TLC is much cheaper. No instrument is required. Multiple samples can be run simultaneously on TLC.
Disadvantages: Quantitative analysis can be easily performed using GC & HPLC, whereas this is much more difficult with TLC. Less sensitive – more sample is required. A method of visualization is needed for analyte components separated by TLC, whereas the other methods result in visible peaks on a chart.
Chapter 6 Separation Methods
Course: Introduction to Forensic Science (CHE103)
University: Murdoch University
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