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7 Summary of Chemical Color Tests
Summary of Chemical Color Tests
Fig. 7.2 Results of color-screening tests on frequently encountered controlled substances.
Describe the use of color-screening methods in forensic chemistry.
Describe the chemistry of color formation.
List three ways colors are produced through chemical reactions.
List two limitations of chemical color tests.
Outline the documentation process for color tests.
Please explain to members of the jury why the Marquis’ test used in this case to test MDA produced two different
Why is it necessary to run a blank control when using Chen’s test?
Please explain to members of the jury what test you used to determine the sample was cannabis resin. Explain the procedure and results.
Describe two tests used to screen for heroin.
What test is commonly used to screen for LSD?
Why are blank controls always used in chemical color tests?
Identify the substance indicated from the following results: Marquis’ test, no color; tertiary amine test (neutral), no
color; tertiary amine test (acidified), blue.
Identify the substance indicated from the following results: Marquis’ test, orange-to-brown; secondary amine test, blue.
Identify the substance indicated from the following results: Marquis’ test, no color; tertiary amine test (neutral), no
color; tertiary amine test (acidified), no color; Chen’s test, no color; Janovsky’s test, no color; Dille–Koppanyi’s test, no
color; Van Urk’s test, purple.
Christian, D. R. Jr. Analysis of Controlled Substances. In Forensic Science: An Introduction to Scientific and Investigative Techniques, 3rd ed.;
James, S. H.; Nordby, J. J., Eds.; CRC Press: Boca Raton, FL, 2009.
Cole, M. D. The Analysis of Controlled Substances; John Wiley & Sons: New York, 2003; Appendix 1. Presumptive Color Tests.
Jeffery, W. Chapter 19. In Clarke’s Analysis of Drugs and Poisons 2004; Moffat, A. C.; Osselton, M. D.; Widdop, B., Eds.; Pharmaceutical Press:
Jones, H. S.; Wist, A. A.; Najam, A. R. Spot Tests: A Color Chart Reference for Forensic Chemists. J. Forensic Sci. 1979, 24, 631–649.
Jones, L.; Atkins, P. Chemisty: Molecules, Matter, and Change, 4th ed.; W.H. Freeman and Company: New York, 2000; p 267 & chapter 21.
King, L.; McDermott, S. Chapter 2. In Clarke’s Analysis of Drugs and Poisons 2004; Moffat, A. C.; Osselton, M. D.; Widdop, B., Eds.;
Pharmaceutical Press: London, 2004.
United Nations. Methods for Testing Barbiturate Derivatives Under International Control. A Manual for Use By National Narcotics Laboratories;
ST/NAR/18; United Nations: New York, 1989.
United Nations. Rapid Testing Methods of Drugs of Abuse. A Manual For Use by National Law Enforcement and Narcotics Laboratory Personnel;
ST/NAR/13/REV.1; United Nations Publication: New York, 1994.
United Nations. Recommended Methods for The Identification and Analysis of Amphetamine, Methamphetamine and Their Ring-Substituted
Analogs in Seized Materials. Manual for Use by National Drug Testing Laboratories; ST/NAR/34; United Nations Publication: New York,
Microcrystal test techniques are based on highly developed chemical-precipitation reactions in which a polarized microscope
is used to observe and distinguish the different types of crystals formed. Most of these tests were developed in the late nineteenth century for the identification of alkaloids. Over the years, they have been modified and are currently used in the identification of a majority of controlled substances. Despite the fact that they were developed more than 100 years ago,
microcrystal tests still have a role in modern forensic chemistry.
Microcrystal tests are confirmatory techniques often used to verify the results of preliminary screening methods. They are
fast, easy to perform, and can be highly specific. However, there is considerable debate on whether they are specific enough
to be used as a confirmatory test. The forensic community is divided on this issue into three main groups.
Traditionalists use microcrystal techniques as a confirmatory test in the forensic examination of controlled substances.
This older generation of chemists has used wet chemical techniques to identify compounds since the 1960s and 1970s.
Although not fully understood at the time, the chemistry of color formation is different from the chemistry of crystal formation. Traditionalists view microcrystal tests as independent tests and perform them in conjunction with color-screening
methods. Positive results obtained from two independent tests would represent definitive proof that the sample under investigation is a controlled substance.
The modern, younger generations of forensic chemists use microcrystal tests as a preliminary screening tool. They believe
the true chemistry behind microcrystal tests is unknown and that analytical examination (i.e., gas chromatography mass
spectrometry (GCMS) or Fourier transform infrared (FTIR) spectroscopy) is required before a positive identification can be
The clinical group does not use microcrystal tests and prefers more sophisticated instrumental analysis. This decision
appears to be based more on economic, rather than scientific, reasons. This group believes the automation of GCMS and the
chemical reliability of FTIR represent a more efficient and economical method of examination. They consider microcrystal
tests to be laborious techniques that require extensive training and skill. To address caseload requirements, the clinical group
prefers a single trained technician (to operate either the GCMS or FTIR instruments) over a group of scientists performing
Advantages of Microcrystal Techniques
Microcrystal tests are a low-cost alternative to GCMS and FTIR that are recognized by the scientific community. The
Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG) has established criteria for their use in the identification of controlled substances. Additionally, the American Society of Testing and Materials (ASTM) have established
methods for identifying cocaine and methamphetamine using microcrystal tests.
In general, microcrystal tests are safe and environmentally friendly. In most cases, the entire analysis can be performed
with as little as a single drop of reagent. This is in contrast to the volumes of chemicals required to prepare samples for
J.I. Khan et al., Basic Principles of Forensic Chemistry, DOI 10.1007/978-1-59745-437-7_8,
© Springer Science+Business Media, LLC 2012
instrumental analysis. For example, a mere 2 ml of organic solvent used in sample preparation for automated FTIR analysis
is roughly 40 times the volume required for a typical microcrystal test.
Purification of the controlled substance is not required for microcrystal testing. Diluents and impurities generally do not
interfere with the crystal features used in the identification process. Characteristic crystals can be observed at sample concentrations approaching 2% (by mass). This sensitivity can detect microgram quantities of controlled substance in a particular sample.
A clear advantage of microcrystal tests is their use in the detection of optical isomers (see Chap. 4; Chirality). The identification of enantiomers (optical isomers) is complicated by the fact that, except for optical rotation, they have the same chemical
and physical properties. Microcrystal tests can quickly and accurately differentiate optical isomers by forming different crystal
structures with each enantiomer. This can play an important role in the analysis of controlled substances when one isomer is
controlled and the other is not, or when one isomer can establish a particular method of drug production.
It should be noted that microcrystal formation does not affect the chemical and physical properties of the substance; thus,
it can be recovered for subsequent testing (i.e., instrumental analysis). This is clearly an advantage if sample quantity is
Disadvantages of Microcrystal Techniques
The principal disadvantage of microcrystal testing is that it is not applicable to all substances. Crystal-testing procedures do
not exist for a number of commonly encountered controlled substances and, given the current debate over their use, this is
not likely to change in the near future.
Microcrystal testing can produce more than one type of arrangement. The presence of additional crystal-forming agents
may interfere with the precipitation of the target compound. This interference may cause either distortions or variations in
the expected crystal form (polymorphism). This may complicate the identification process. In such cases, a purification procedure, such as thin-layer chromatography or extraction, is recommended before microcrystal analysis.
The formation of a solid crystal in solution begins when individual molecules or ions cluster together. This process of
nucleation continues until a visible particle appears. The speed of the nucleation process can influence the shape of the crystal. Crystals with definitive features are formed very slowly; while those formed rapidly can “mechanically trap” undesired
particles (i.e., solvent, impurities, dilutants, etc.). The trapped particles can distort the overall structure of the crystal and
complicate identification. Highly concentrated samples and reagents will develop crystals rapidly, resulting in polymorphism. Therefore, reagents and samples may require dilution to produce standard crystal forms for comparison and identification. Also, reference samples should be run with all reagents to verify reagent activity.
Microcrystal testing is a manual technique. The individual handling of samples and reagents requires care and consistency. The analyst’s results must be reproducible for definitive identification. This aspect is a barrier to automation and may
preclude its use in forensic laboratories with a high-volume caseload.
Microcrystal techniques lack the versatility of chromatographic methods offering single-step identification, quantization,
and documentation. Consequently, many would argue that they are not well suited for the production-oriented clinical environment of the modern forensic laboratory.
Identifying compounds using standard crystal features is not inherently subjective. Nonetheless, a degree of subjectivity is
always present in the interpretation of crystal test results. Characterizing shapes and structural features is influenced by the
experience and training of the analyst, concentration of sample and reagents, presence of interfering compounds, reagent age,
and crystal polymorphism. The effective use of microcrystal tests requires training and experience. The analyst must develop
recognition of unpredictable reagent behavior through practice. Unfortunately, this requires countless hours behind a
The results of microcrystal testing should be documented completely. Table 8.1 provides a list of terms and diagrams commonly used to describe crystals. In addition, comprehensive documentation would include: a complete description of
reagents (expiration dates, color, physical properties, photographs, etc.), a complete description of test substance (color,
physical properties, irregularities, notable markings, identifying characteristics, photographs, etc.), observations during
test performance (testing conditions, testing equipment, glassware, spot plate, colors, photographs, etc.), complete description
Table 8.1 Microcrystal descriptions
Cluster with the majority of the crystals lying in one direction
Rosette, which is so dense that only the tops of the needles show
Loose complex of crystals
Single cruciform crystal
Multibrachiate branching crystals
Small lenticular crystals
Long thin crystals with pointed ends
Crystals with the length and width that are of the same magnitude
Long thin crystals with square cut ends
Collection of crystals radiating from a single point
Small irregular rods and needles
Rosette with 4 or 6 components
Plates with appreciable thickness
Sector of a rosette
of results (crystal features, sketch, comparison to standard features, deviations, supporting evidence for conclusion, photographs, etc.).
Supporting documentation may or may not be required when microcrystal tests are used as a screening method. The documentation requirements are flexible on presumptive tests because the final opinion does not necessarily rely on the results.
Regardless, a physical description and diagram of the crystals must be documented for peer review.
Microcrystal tests used as a confirmatory method require documentation. It is recommended that a photomicrograph
(photograph) be taken of the crystals used for identification. Microcrystal results can easily be challenged as evidence, if the
examiner fails to provide documentation of performance and results.
Microcrystal Test Techniques
In microcrystal tests, the test sample is dissolved in a solution. A test reagent either is added to the solution or is already
present in the solvent. A reaction between the compound of interest and the test reagent forms a solid compound that is not
soluble in the test drop. The solid forms uniquely shaped crystals that can be observed with a microscope. Microcrystal-test
techniques are divided into two broad categories: aqueous techniques or volatility techniques.
Aqueous Test Technique
Despite considerable variation in testing reagents, the technique for aqueous testing remains unchanged. A reference standard is required and must be run concurrently:
• A small sample is placed on a microscope slide and dissolved in one drop of water or one drop of diluted acetic acid
• Place one drop of reagent next to the sample on the slide or place one drop of reagent directly into the test drop
• Mix the two drops (if side by side) (Fig. 8.3).
• Place the slide under a microscope and observe crystal formation.
– A slide cover is not required.
Fig. 8.1 Small sample placed on a microscope slide and
dissolved in one drop of water or one drop of diluted acetic
Fig. 8.2 Place one drop of reagent next to the sample on
the slide or place one drop of reagent directly into the test
Fig. 8.3 Mix the two drops (if side by side).
Aqueous Test Reagents
Volatility Test Technique
The volatility technique is used when the test substance is volatile (easily vaporized) or when a solvent is chosen that causes the
substance to be volatile. The sample vapors rise and react with a drop of reagent suspended on a slide over the substance.
Crystals form in the solution on the cover slide. A reference standard is required and must be run concurrently.
• The test sample is placed into the depression of either a clean spot plate or a volatility chamber.
• A drop of crystal reagent is placed onto a microscope slide.
– The reagent may contain a viscous material to aid in suspension.
• The microscope slide is inverted and placed over the depression containing the test sample. Align the reagent drop over
the test sample.
• The test sample is vaporized.
– It may require placing the spot plate onto a warm hot plate.
• Allow reagent drop and sample vapors to react.
• Place the slide under a microscope and observe crystals.
This technique is particularly useful in the detection of volatile poisons containing the aldehyde and ketone functional
groups. Controlled substances containing primary and secondary amines have been isolated using this technique. Also, it
may have possible applications in the identification of g-hydroxybutyric acid (GHB) and in the detection of explosive
Acid and Anionic Test Technique
This technique combines portions of both the aqueous and volatility techniques. A few crystals of sample are placed in the
cavity of a cavity slide, and one drop of alcohol solution (methanol or ethanol) is added. One drop of reagent is added immediately after the alcohol, and a cover slide is placed over the cavity to prevent evaporation. Crystal formation is observed by
placing the cavity slide (with cover) directly under a microscope. This method is often used to crystallize steroid hormones
and barbiturates, including phenobarbital.
Aqueous Test Reagents
The protocols for microcrystalline testing, including recommended procedures for the preparation of a vast number of
reagents and solvents, are readily available in a variety of scientific publications. However, only a few are used in forensic
investigation. The following tests are representative of those commonly used in most forensic laboratories, with only minor
variations to reagent preparation.
Gold Chloride Test
Reagent: 3-g gold(III) chloride (AuCl3) + 100-ml water + 0.25-ml concentrated HCl
Crystals: Combs and rosettes of needles
Gold Chloride in Phosphoric Acid Test
Reagent 1: 5-g gold(III) chloride (AuCl3) in 100-ml water
Reagent 2: Concentrated phosphoric acid (H3PO4)
Mix two drops Reagent 1 with one drop Reagent 2.
Crystals: Long needles and long barbs
Crystals: Long yellow rods and blades
Platinum Chloride Test
Reagent: 5-g platinum(IV) chloride (PtCl4) in 100 ml of 1 M hydrochloric acid (HCl)
Crystals: Combs of needles
Mercuric Iodide Test
Reagent: 10% hydrochloric acid (HCl) saturated with mercury(II) iodide (HgI2)
Crystals: Rosettes of dendrites
Note: The test is extremely sensitive, even with highly contaminated samples. Use caution when interpreting results;
product crystals are colorless and may be difficult to differentiate from undissolved reagent particles.
Mercuric Chloride Test
Reagent: 5-g mercury(II) chloride (HgCl2) in 100-ml water
Crystals: Small rosettes of rods
Potassium Permanganate Test
Reagent: 2-g potassium permanganate (KMnO4) + 100-ml water + 0.25-ml phosphoric acid (H3PO4)
Target: Phencyclidine (PCP)
Crystals: purple H-shaped plates
Note: The test is extremely sensitive, and the best results are obtained using very dilute samples. It is recommended to use
just enough sample to produce a light amorphous precipitate when reagent is added.
Sodium Acetate Test
Reagent: 10-g sodium acetate in 100-ml water
Crystals: Hexagonal plates
Crystals: Irregular logs
The interpretation of microcrystalline test results requires a great deal of care and attention. The subtle details of structural
features are often either lost or hidden by factors influencing crystal formation. The following list contains suggestions
designed to minimize the complexities associated with microcrystalline examinations.
• The best crystals form very slowly.
• Crystals get bigger with time, and larger crystals are more easily interpreted.
• Diluted test samples produce better crystals.
• Test solutions should never evaporate to dryness.
– The solid should separate almost immediately. Extended periods of time promote evaporation and increase the probability that undesired crystalline compounds will form, complicating the results.
• Crystals may be affected by changes in ambient temperature and humidity.
• Always run reference controls concurrently with samples.
• Reagent age can affect crystal formation.
The process of microcrystal formation in solution is not fully understood. Nonetheless, there appears to be no shortage
of theories that attempt to describe the procedure. Most have narrow applications and have trouble addressing even the
simplest of arguments. The dynamics of crystal formation is really quite simple; crystals will form in any solution when
the limit of solute solubility has been reached. But what factors determine a solvent’s capacity to dissolve solute? Alas,
this is the real question and, at present, that question is unanswered. There will be no theory that is universally applied to
precipitate formation in aqueous-solution chemistry. The complexities of crystal formation will ensure that.
Cite two reasons why microcrystal techniques would be used as a confirmatory test.
Describe the complete documentation of a microcrystalline-test result.
Cite two advantages of microcrystalline techniques.
Can you please explain to the jury how a basic microcrystal test is performed?
Please explain to the jury how you determined the substance was heroin using the sodium acetate test.
How would you test for amphetamine?
In your opinion, is this technique in question #6 a confirmatory or a screening examination? Explain.
Is it possible for two different substances to produce identical crystals? Explain.
Cite three disadvantages of microcrystalline techniques.
Discuss two critical factors that need to be considered when evaluating microcrystalline-test results.
What group considers microcrystalline tests obsolete?
Describe the crystals formed from cocaine and methadone.
Discuss an instance when complete documentation of testing results would not be required.
Compare and contrast the aqueous technique and the volatility technique.
California Department of Justice. Technical Procedures Manual for Controlled Substance Analysis; Sacramento, CA., 2006.
Chamot, E. M.; Mason, C. W. Handbook of Chemical Microscopy, Volume II: Chemical Methods and Inorganic Qualitative Analysis; McCrone
Research Institute: Chicago, 1989, chaps. 11–13.
De Forest, P. R.; Gaensslen, R. E.; Lee, H. C. Forensic Science: An Introduction to Criminalistics; McGraw-Hill: New York, 1983, chap. 5.
Evans, H. K. Drug and Microcrystal Tests for Forensic Drug identification. Microscope. 1999, 47, p.147.
Fulton, C. Modern Microcrystal Tests for Drugs; John Wiley & Sons. New York, 1969.
Julian, E. A. Microcrystalline Identification of Drugs of Abuse: The Psychedelic Amphetamines. J. Forensic Sci. 1990, 35, pp. 821–830.
Julian, E. A. Microcrystalline Identification of Drugs of Abuse: The White Cross Suite. J. Forensic Sci. 1981, 26, pp. 358–367.
Moorehead, W. A Brief Background and Justification for the Continued Use of Microcrystal Tests. CAC News, 2000, 3rd quarter, pp 11–15.
Chemical Extractions and Sample
Solutions are homogeneous mixtures (see Chap. 1) containing two or more substances. The component of a solution present
in the greatest amount is called the solvent and the dissolved substances are the solutes. It is possible to have more than one
solute in a particular solution; however, it is not possible to have more than one solvent. Solvents have a varying capacity to
dissolve particular solutes; for example; sodium chloride (NaCl) will readily dissolve in water, while silver chloride (AgCl)
will not. Solubility refers to the maximum amount of solute particles that can be dissolved in a specified volume of solvent
at a given temperature. Temperature affects the solubility properties of a solvent and, in general, solubility increases with
increasing temperature. A common example of this is illustrated using a simple cup of coffee. Have you ever wondered about
the dark sediment that mysteriously appears at the bottom of a cool cup of coffee? Stop blaming your dishwasher and the
coffee filters you purchased at the discount store; they are not the culprits. When hot water is added to solid coffee, only
specific components are dissolved (extracted) from the coffee grinds into water, that is, flavor, odor, caffeine, etc. The concentration of each component in the resulting solution is dependent on the temperature of water. If the water is very hot, a
“strong” cup of coffee results because the water can dissolve a higher concentration of the components (increased solubility).
As the coffee cools, the solubility decreases and the components precipitate out as dark sediment in the bottom of the cup.
The coffee/water system is an example of solid/liquid extraction because soluble components are transferred from a solid
phase (coffee) into a liquid phase (water).
Extraction is a general term used to describe a number of chemical techniques that separate the components of a mixture
using the solubility properties of various solute/solvent systems. In the context of extraction, solubility often refers only to a
solvent’s ability to dissolve a particular solute and not necessarily a quantitative maximum amount. Most extraction techniques are slight variations of three general procedures: solid–liquid extraction, liquid–liquid extraction, or acid–base
Solid–liquid extraction is most often used to extract a natural component from a solid natural source, such as a dried plant.
This technique is found in the isolation procedures of morphine from the opium poppy and cocaine from coca plants.
Although the basic concept is used in forensic analysis, a slight modification adds versatility to this technique. This method
relies heavily on the selective extraction (transfer) of soluble components from a solid phase into solution. Ideally, a carefully chosen solvent will dissolve only the target compound and no other components. Isolation is accomplished by filtering
out the insoluble contaminants and recovering the target compound from the filtrate (solution that passes through filter).
This method generally requires a single extraction, and an outline of the general procedure is below.
• Identify the components in the solid sample.
• Identify the solubility properties of each component.
• Select a suitable solvent, ideally one with a high solubility for the target compound and a low solubility for the remaining
J.I. Khan et al., Basic Principles of Forensic Chemistry, DOI 10.1007/978-1-59745-437-7_9,
© Springer Science+Business Media, LLC 2012