(AOM) Chromatography Lesson

Chromatography

Chromatography is a technique used to separate the components of a chemical mixture by moving the mixture along a stationary material, such as gelatin. Different components of the mixture are caught by the material at different rates and form isolated bands that can then be analyzed. Forensic Scientists utilize several methods of chromatography including Thin Layer Chromatography, High Pressure Liquid Chromatography, Column Chromatography, and Gas Chromatography. It is important to note that there are many different types of chromatography, but the ones listed in this module are some of the most commonly used in Forensics. Chromatography is useful in separating compounds into component parts for both identification and further analysis. For example, brown paint appears as if it is only "brown" until it is broken down into the various colors used to produce it. The resulting bands of colors can then be used to identify specific samples of the same paint.

Thin Layer Chromatography

Thin Layer Chromatography is a form of chromatography in which components of a liquid mixture are separated by means of a thin layer of adsorbent material coated on a glass, plastic, or foil sheet. Adsorption is the process by which the molecules of a substance, such a liquid or gas, collect on the surface of another substance, such as a solid. The molecules are attracted to the surface but do not enter the solid's minute spaces as in absorption. This means that the substance being broken down using chromatography adsorbs to the paper in TLC.

Note that in picture A, the particles move past the barrier and into the solution on the other side of the barrier; this is absorption. In picture B, the particles are attracted to the surface of the solid barrier in a process known as adsorption, but do not move into the solution as in picture A.

Thin Layer Chromatography, or TLC, works by utilizing a liquid mobile phase across the sample and along a solid stationary phase such as Silica gel or cellulose. The sample is separated into the substances that make it up as they deposit at separate points along the stationary phase. The pattern made by the substance stopping points helps to identify the substance.

Retention Factor, or R_f, is a quantitative value for how far a sample solute or compound traveled in a specific solvent.

The R_f value is often used to tentatively identify similar samples, but not usually to produce a final confirmation of the identity of the substance. If the R_f values match in the TLC analysis, other confirmatory tests can be then be performed to more precisely pinpoint the identify of the substance.

Examine the photo below of a chromatogram of permanent black ink. Using TLC with a solvent of ethanol, the colors mixed in the black ink were separated in bands. The R_f value for each of the colors within the ink can be calculated. To calculate the R_f value of the orange ink, measurements need to be made of how far the orange ink traveled and of how far the solvent traveled. These distances are labeled D₁ and D₂ in the photo. For the purposes of this example, suppose that measurement of D₁ is 2 cm and D₂ is 4 cm.

R_f Values can be calculated by using the following formula:

R_f =     Distance solute moves (D₁)           

        Distance solvent front traveled (D₂)

Example:

D₁ = 2 cm, D₂ = 4 cm

R_f  = D₁ / D₂   

R_f  =  2   

R_f  = 0.5

        

TLC is often used in Forensic Science for various substances as a first step in trying to identify what type of substance a sample of evidence is or is not. Some of the common uses for TLC include:

• Analysis of the dye composition of fibers
• Separation of amino acids
• RNA fingerprinting
• Initial identification and comparison of drugs
• Analysis of explosives
• Analysis of ink and dye compositions

High Pressure Liquid Chromatography

High Pressure Liquid Chromatography, or HPLC, is a form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify the number of different compounds in a sample. HPLC utilizes a column that holds chromatographic packing material (the stationary phase), a pump that moves the mobile phase(s) through the column, and a detector that shows the retention times of the molecules. Retention time varies depending on the interactions between the stationary phase, molecules being analyzed, and the solvent(s) used. As the name implies, it utilizes high pressure to force a liquid through a column that is layered with the stationary material. The substances in the sample are attracted to the stationary material in varying degrees and deposited according to this. A graph is made of the position of each deposit and the amount of each substance in the sample is calculated.

 

HPLC is used in Forensics to:

1. Trace impurities in samples such as drugs or toxins (like pesticides). These impurities can be tracked and even linked to specific drug cartels with known manufacturing methods. Drugs are manufactured differently by the various drug cartels, and the types and amounts of impurities can sometimes identify the specific drug cartel.
2. Identify the components used to build a bomb.

Here is an HPFC chromatogram of J'Adore perfume:

Column Chromatography

Column Chromatography is chromatography that uses selective adsorption by a column of powders or adsorbent materials. Similar to Thin Layer Chromatography, the thin layer stationary phase materials such as silica gel are packed in a vertical glass column. In Column Chromatography, the sample is carefully "washed" in a solvent inside the column. As the solvent breaks down the compound, it adsorbs to the stationary phase packing in the column based on its polarity. The more polar the substance is, the slower it moves through the column because it is more strongly attracted/adsorbed to the stationary phase. This method of chromatography is also considered a form of "Elution". Elution is simply extracting one material from another, usually by washing it away with a solvent.

Column Chromatography is used in Forensics to test samples of:

1. Suspected illegal drugs
2. Toxins
3. Blood alcohol
4. Various narcotics

Gas Chromatography

Gas Chromatography, or GC, is defined as a technique for analyzing a mixture of volatile substances in which the mixture is carried by an inert gas through a column packed with a selective adsorbent. A detector records on a moving strip the conductivity of the gas leaving the tube. Peaks on the resulting graph indicate the presence of a particular component. Gas Chromatography is similar to HPLC, except that it is particularly useful in analyzing volatile liquids. This is because GC separates substances based on their boiling points and polarity.

To analyze a substance using Gas Chromatography, a sample is loaded into the stationary phase of the machine. The mobile phase is usually Helium gas, and it transports the sample through a glass column. The column is located in an oven to maintain a constant temperature. Substances with the lowest boiling points move through the column first and those with higher boiling points move slower. The time it takes for the sample to reach the detector is known as the retention time, and this is what is used to identify the substance. Records of known retention times are kept for comparison. Once the compounds reach the detector, a measurement of the concentration of the substances is graphed. The graph resembles triangular peaks along a horizontal axis (see right for sample). The graph can be compared to others graphs of known substances for identification.

Gas Chromatography is often used in Forensics in cases involving:

1. Arson; the accelerants can be differentiated and identified
2. Analysis of body fluid composition
3. Presence of illegal drugs/substances
4. Toxicology testing

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