SPOT TEST ANALYSIS 1

Spot Test Analysis areas of application in exactly the opposite direction. This
is the search for simple, compact and inexpensive analytical
devices for semiquantitative evaluation of certain elements
Ervin Jungreis or compounds where such approximate evaluation has
The Hebrew University of Jerusalem, Jerusalem, diagnostic value at least in the first stage of examination.
Israel Major manufacturers of chemical reagents concentrated
their efforts mainly in a number of applicative areas,
namely clinical analysis, forensic tests, air and water quality
control (QC) tests and soil and food QC tests.
1 Introduction 1 It must be emphasized that the applicability of spot test
2 Techniques and Equipment 2 methodology is limited and for the exact determination
2.1 Techniques 2 of chemical substances, complex analytical procedures are
2.2 Equipment 2 unavoidable. Although the use of spot and screening tests
3 Selected Tests in Inorganic Analysis 2 is marginal, the margin, however, is quite significant.
3.1 Examples of Preliminary Tests 2
3.2 Tests for Cations 3
3.3 Selected Tests for Anions 5 1 INTRODUCTION
4 Selected Tests in Organic Analysis 6
4.1 Preliminary Tests 6 Spot tests are defined as an analytical technique that
4.2 Selected Tests for Functional enables the analyst to accomplish satisfactory semimicro
Groups 7 and ultramicro tests with simple equipment in a minimum
4.3 Detection of Individual Organic time. The target is to reach the utmost sensitivity and
Compounds 9 selectivity with a minimum of physical and chemical
5 Selected Examples of Applications of operations, and the procedure must be as simple as
Spot Tests 11 possible.
5.1 Applications in Clinical Analysis 11 The modern systematic development of spot test
5.2 Forensic Application of Spot-test methods occupied Fritz Feigl.1,2/ for half a century in
Analysis 14 Vienna and Rio de Janeiro, but analytical chemists have
5.3 Selected Geochemical Applications long used single chemical tests on filter paper. These
of Spot Tests 15 are the indicator papers for the detection of an excess of
5.4 Rapid Selected Screening Tests for hydrogen or hydroxy ions, potassium iodide – starch paper
Soil and Plant Tissues 15 for chlorine detection, silver carbonate-impregnated filter
5.5 Selected Examples of Applications paper for uric acid detection in urine, and others.
of Spot Tests in Air and Water According to the writings of Pliny the Elder, the early
Quality Control 16 Romans detected iron in vinegar by means of a reagent
5.6 Food Quality Control Analysis by paper prepared by soaking papyrus in an extract obtained
Spot Test 17 from gall nuts.
Abbreviations and Acronyms 18 In parallel with the trend towards instrumental sophis-
Related Articles 18 tication using the phenomenal development of micropro-
cessors, a trend towards simplification was seen in some
References 18
marginal selected areas in the form of simple, rapid and
inexpensive spot and screening tests..3/ Commercial com-
panies sell vast numbers of compact spot-test systems in
Sensitive and selective qualitative tests based on chemical which the goal is the rapid establishment of the presence
reactions using small amounts of complex and reagents are or absence of certain materials. It would be wasteful to
referred to as ‘‘spot tests’’. The target is to reach the utmost devote resources to attaining precision or accuracy sev-
sensitivity and selectivity with a minimum of physical and eral magnitudes beyond that necessary in clinical urine
chemical operations. analysis, for example, when pathological changes in the
Rapid advances in spectral, chromatographic, nuclear, composition of urine are evidenced by a simple test. A new
mass spectroscopic and electroanalytical research have look was taken at the classical tests; sometimes they were
changed analytical chemistry spectacularly and developed refined and sometimes new ones were elaborated. In most
highly sophisticated analytical tools. At the same time of the tests the human eye was used as a detector; oth-
another development has taken place in certain distinct ers employed simple reflectometric instruments and UV

Encyclopedia of Analytical Chemistry, Online © 2006 John Wiley & Sons, Ltd.
This article is © 2006 John Wiley & Sons, Ltd.
This article was published in the Encyclopedia of Analytical Chemistry in 2006 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470027318.a8111
2 GENERAL ARTICLES
(ultraviolet) lamps. Most commercial companies nowa-
days concentrate their efforts in the following applicative
areas: clinical, forensic, soil and plant, air, water and food
QC tests, because of the vast number of screening tests
needed in these areas.
2 TECHNIQUES AND EQUIPMENT
2.1 Techniques
Spot-test procedures are the ultimate in simplicity.
Classical tests are ordinarily run by using one of the
following techniques:
ž by bringing together one drop each of the test solution
and reagent on porous or nonporous supporting
surfaces such as paper, glass or porcelain;
ž by placing a drop of the test solution on a medium
impregnated with appropriate reagents;
ž by subjecting a drop of reagent or a strip of reagent
paper to the action of liberated gases from a drop
of the test solution or from a minute quantity of the
solid specimen.
Commercial companies produce strips of absorbent
cellulose, one end of which is impregnated with the dry
reagent system. These strips are mainly used in clinical,
chemical and water analysis systems. The impregnated
reagent area is sometimes covered with a semipermeable
membrane to prevent staining by a colored matrix such as
blood. Another form of reagent system is a stable compact
reagent tablet, which can contain several compatible
agents such as buffers and sequestering agents. Test
strips for body-fluid analyses are prepared in a modern
technique by printing liquid-absorbing substances on a
support, drying the treated support and applying reagents
to the film formed on the support. Simple color comparing
devices may be part of commercial test systems. Some are
calibrated color charts printed on the container, others
are colored cubes molded of acrylic plastic and matched
to laboratory standards. A series of nonfading glass color
standards mounted on a circular disc may be used in a
comparator box or continuous color discs may be applied
for visual comparison.
For the semiquantitative testing of airborne contam-
inants in a workroom atmosphere, the detector tube
technique has gained ground. The reagent system placed
into the tube consists of supporting material impregnated
with the chromogen selective for the air contaminant.
The technique of separation of small water-soluble
molecules from high-molecular-weight proteins by a
semipermeable layer is utilized in the routine and simple
screening test for glucose and urea in blood.
Encyclopedia of Analytical Chemistry, Online © 2006 John Wiley & Sons, Ltd.
This article is © 2006 John Wiley & Sons, Ltd.
This article was published in the Encyclopedia of Analytical Chemistry in 2006 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470027318.a8111
2.2 Equipment
The amount and type of equipment needed is relatively
small. It is most important to have an ample supply
of reagents immediately available. Balances, UV lamps,
centrifuges, dryers, ovens, furnaces and a hood can be
considered to be essential in the spot-test laboratory.
Assorted sizes of beakers, volumetric flasks, Conway
cells, crystallizing and evaporating dishes, filter sticks,
separation funnels, extraction pipets, fritted glass cru-
cibles, graduated cylinders, vials, test tubes, centrifuge
tubes, cover glasses, white and black spot plates, porce-
lain crucibles and platinum dishes are standard for many
spot-test operations. Tweezers and spatulas, microburn-
ers, blow torches, sand and water bath are essential, too.
3 SELECTED TESTS IN
INORGANIC ANALYSIS
The sample should be subjected to preliminary tests
before beginning a systematic search, although it is often
possible to carry out conclusive tests with as little as one
drop of dilute sample solution, even though considerable
concentrations of other substances are present.
3.1 Examples of Preliminary Tests
A first test is carried out by heating the solid sample in
contact with air in a microcrucible. If the sample’s color
and structure remain unaltered, it shows the absence
of volatile compounds, e.g. absence of ammonium and
mercury salts, carbonates, organic compounds, water of
crystallization. If the sample melts, it is an indication of
the possible presence of nitrates, nitrites, carbonates,
formates, chlorates, silver chloride and bromide and
fusible organic compounds. If the sample chars, it is a
sign of the presence of organic materials, organometallic
compounds or metal salts of organic acids. If the sample
changes color without charring, it shows the presence of
certain heavy metal salts, such as cupric salts which are
oxidized to black cupric oxide, and cadmium salts which
are oxidized to dark-brown cadmium oxide. Lightening
of the color may indicate the decomposition of higher
oxides, e.g. lead dioxide to lead oxide, or oxidation of
sulfides, e.g. lead sulfide to lead sulfate.
A second test is carried out by heating the solid
sample in an ignition tube. If the sample gives off water,
the presence of salts containing water of crystallization,
metal hydroxides and oxyhydrates and ammonium salts
of organic materials are indicated. If litmus paper detects
acidic vapors, it shows the presence of acid salts or heavy-
metal sulfates or sulfites.
If alkaline vapors are detected by litmus paper,
ammonium salts of heat-stable nonoxidizing acids or
SPOT TEST ANALYSIS
complex metal amines might be present. If a white
sublimate appears, it indicates the presence of ammonium
salts of volatile materials, As2 O3 , Sb2 O3 , or mercury salts
of volatile acids or oxalic acid. The appearance of a
yellow sublimate is a sign of the presence of As2 O3 ,
As2 O5 , HgI2 or free sulfur. If a gray sublimate shows
up, it might be a sign of metallic mercury derived from
mercury compounds or of free arsenic. Yellow or brown
vapor might show bromine derived from bromide in the
presence of oxidizing agents, or nitrogen oxide derived
from nitrate or nitrite, or chromyl chloride provided by
dichromate and chloride. Colorless vapors show up when
CO2 derives from carbonates, CO derives from oxalate
and formate, HCN derives by thermal decomposition of
silver, mercuric or palladium cyanides, H2 S derives from
water-containing sulfide or thiosulfate or NH3 derives
from ammonium salts and so on.
3.2 Tests for Cations
A few selected examples are described here. The
selection is based on the simplicity of execution, on the
selectivity and on the sensitivity of the reagents. The short
description here might be enough to allow the tests to be
carried out, but Feigl and Anger.1/ should be consulted for
more detail and a deeper understanding of the reactions.
Aluminum ions form a lake stable to acetic acid
with quinalizarin. The test solution is placed on the
reagent paper (filter paper soaked in 10 mg reagent
dissolved in 2 mL pyridine C 20 mL acetone) and held
over concentrated NH3 until the initial blue color
disappears. Visible change: a remaining red-violet color.
Sensitivity: 0.005 µg aluminum. Interference: Be2C .
Ammonium ions liberate volatile NH3 by adding alkali
to them in gas-evolving test tubes. A strip of moist
red litmus paper is hung on the hook of the tube,
which is closed and warmed to about 40 ° C for 5 min.
Visible changes: blue color. Sensitivity: 0.01 µg ammonia.
Interference: cyanides.
Antimony ions reduce phosphomolybdic acid to molyb-
denum blue. A drop of the test solution is placed on filter
paper impregnated by 5% aqueous reagent and held over
steam. Visible change: blue coloration. Sensitivity: 0.2 µg
antimony. Interference: stannous ions.
Arsenic ions when oxidized to arsenate form an
insoluble compound with silver ions. The test solution is
warmed in a microcrucible with a few drops of ammonia
and 10% H2 O2 , then acidified with acetic acid and 1%
AgNO3 added. Visible change: red-brown precipitate.
Sensitivity: 6 µg arsenious acid. Interference: the test is
specific in the absence of chromates and ferricyanides.
Barium ions form an insoluble salt with sodium
rhodizonate solution. To a drop of the neutral or slightly
acid test solution on filter paper is added 0.2% aqueous
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This article is © 2006 John Wiley & Sons, Ltd.
This article was published in the Encyclopedia of Analytical Chemistry in 2006 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470027318.a8111
3
reagent (freshly prepared) dropwise. Visible change: red-
brown stain. Interference: bivalent heavy-metal salts must
be absent. Sensitivity: 0.25 µg barium.
Beryllium ions give red-violet color lake with
chromeazurole. One drop of the test solution is treated
with a drop of 2 N sodium acetate and one drop of
the alcoholic reagent solution. Visible change: red-violet
color. Sensitivity: 0.3 µg beryllium. Interference: Fe, Al,
Zn and copper salts must be masked.
Bismuth ions form insoluble double iodide with uni-
valent organic bases, among them cinchonine. Slightly
acidic test solution is dropped onto filter paper impreg-
nated with the reagent solution (1 g cinchonine dissolved
in 100 mL water containing a little nitric acid, by warm-
ing; after cooling, 2 g potassium iodide is added) Visible
change: orange-red stain. Interference: from lead, copper
and mercury can be avoided by zone precipitation.
Cadmium ions are precipitated with ferrous dipyridyl
iodide, a compound in which both the cation and the anion
are large complexes. A drop of the test solution is treated
with a drop of the reagent solution. Visible change: red
precipitate. Sensitivity: 0.05 µg cadmium. Interference:
mercury and nickel ions. The reagent solution is prepared
as follows: 0.25 g a,a0 -dipyridyl and 0.146 g FeSO4 Ð7H2 O
are dissolved in 50 mL water, then 10 g KI are added,
and after vigorous shaking any solids remaining are
filtered off.
Calcium ions form colored inner complex salts with
glyoxal-bis(2-hydroxyanil), which are stable against alkali
carbonate and extractable in chloroform. A drop of the
test solution is treated successively in a test tube with 3 – 4
drops of the reagent solution, one drop of 10% NaOH,
one drop of 10% Na2 CO3 and 3 – 4 drops of chloroform.
Visible change: red color in the chloroform layer. The test
is specific for calcium. Although barium and strontium
ion form a red complex with the reagent, they are not
extractable with chloroform. Sensitivity 0.05 µg calcium.
Chromium ions that are oxidized to chromate react
with diphenyl carbazide and form an inner complex salt
in which bivalent chromous ion participates. A drop of
the test solution is mixed on a spot plate with a drop of
saturated solution of potassium persulfate and a drop of
2% AgNO3 solution and allowed to stand for 2 – 3 min.
By adding a drop of 1% alcoholic diphenylcarbazide
solution, a violet to red color is formed. Sensitivity: 0.8 µg
chromium. Interference: Mo, Hg, V.
Cobalt ions form a series of cationic and anionic
complexes with thiocyanates in the presence of ethyl
alcohol or acetone. A drop of the test solution is
mixed with a few drops of saturated acetone solution
of ammonium thiocyanate on a spot plate. Visible
change: green to blue color. Sensitivity: 0.5 µg cobalt.
Interferences: Cu2C and Fe3C , which can be avoided by
removal of copper through precipitation in the form
4 GENERAL ARTICLES
of cuprous thiocyanate by adding reducing agents, and
complexing iron with fluoride, respectively.
Copper ions are precipitated with rubeanic acid (dithio-
oxamide). A drop of the neutral test solution is placed
on filter paper, held over ammonia and treated with a
drop of 1% alcoholic solution of rubeanic acid. Visible
change; black or olive-green color. Sensitivity: 0.06 µg.
copper Interferences: Ni2C , Co2C , Fe3C , AgC , Bi3C , Pt2C ,
Pt2C . All these interferences are avoided by complexing
them with 20% malonic acid and 10% ethylenediamine.
Gold ions (tervalent) are easily reduced to metallic gold
with strong reducing agents. Filter paper impregnated
with stannous chloride is dried at not more than 40 ° C
for 2 h. On adding a drop of a solution containing gold,
violet-colored metallic gold forms. Sensitivity: 1 µg gold.
Iron (bivalent) ions give a stable complex cation with
a,a0 -dipyridyl. A drop of the test solution is placed
on filter paper which has been impregnated with a
2% solution of the reagent in thioglycolic acid. The
latter reduces tervalent iron. Visible change: red color.
Interferences: considerable quantities of cobalt and nickel
ions. Sensitivity: 0.03 µg iron.
Lead ions are precipitated as chelates with dithizone. A
drop of the test solution is shaken in a test tube with the
reagent dissolved in carbon tetrachloride. Visual change:
red color. Sensitivity: 0.04 µg lead. Interferences: heavy
metal ions disturb, but complexing with alkali tartrate
and cyanide [CARE], makes the identification of lead
specific.
Magnesium ions give a blue lake with quinalizarin in
alkaline solutions. A drop of the test solution and a drop
of distilled water are placed in adjoining depressions in
a spot plate and mixed with 2 drops of an alcoholic
0.02% solution of the reagent. 2 N NaOH solution is
added drop by drop until a change to violet occurs. Visual
change: a blue precipitate in comparison with the violet
blank. Sensitivity: 0.25 µg magnesium. Interferences: La,
Be, Nd, Pr, Ce, Zr and Th.
Manganese ions are oxidized catalytically to violet
permanganates. A drop of the test solution is mixed with
a drop of concentrated sulfuric acid in a microcrucible.
A drop of 0.1% silver nitrate solution is stirred in,
followed by a few milligrams of ammonium persulfate.
The mixture is gently heated. Visual change: red-violet
color. Sensitivity: 0.1 µg manganese. Interference: halides
should be absent.
Mercuric ions form a red cuprous mercuric iodide
complex with a white suspension of cuprous iodide. The
acidic test solution (pH D 0) is treated on filter paper
with a drop of potassium iodide (5%) – sodium sulfite
solution (20%), followed by a drop of 5% CuSO4 solution
Visual change: red or orange color. Sensitivity: 0.003 µg
mercury. Interferences: AgC , Hg2 2C , Au3C , Fe3C , Ce4C .
All interferences might be avoided by a pretest treatment.
Encyclopedia of Analytical Chemistry, Online © 2006 John Wiley & Sons, Ltd.
This article is © 2006 John Wiley & Sons, Ltd.
This article was published in the Encyclopedia of Analytical Chemistry in 2006 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470027318.a8111
Molybdenum (tervalent) ions produce a red water-
soluble H3 [Mo(CNS)6 ] complex. A drop of the test
solution and a drop of 10% potassium thiocyanate
solution are placed on filter paper previously moistened
with 1 : 1 hydrochloric acid. In the presence of ferric
iron, a red stain appears which disappears when adding
5% stannous chloride in 3 N HCl. In the presence of
molybdate, a brick red Mo3C – thiocyanate complex forms.
Sensitivity: 0.1 µg molybdenum.
Nickel ions form a red chelate with dimethylglyoxime.
A drop of the test solution and one drop of 1% alcoholic
reagent solution are placed on filter paper and held
over ammonia. Sensitivity: 0.16 µg Ni. Interferences: large
amounts of oxidizing substances. Pd gives an analogous
yellow chelate.
Palladium(II) ions form an acid-insoluble yellow
chelate on the surface of red nickel dimethylglyoxime
paper. This layer protects the underlying red compound
against dissolution in acid. A drop of the test solution
is placed on the reagent paper and almost dried by
gentle warming. The paper is then bathed in dilute HCl.
The red compound dissolves except for the palladium
protected spot. Visual change: a red fleck remains.
Reagent paper preparation: filter paper is bathed in cold
saturated alcohol solution of dimethylglyoxime. After
drying, the paper is placed in 2 N Ni(NO3 )2 solution
made ammoniacal. The paper is washed with alcohol.
Sensitivity: 0.05 µg palladium.
Platinum(IV) salts, like those of gold, palladium and
osmium in acid solution, are reduced to the elementary
state by stannous chloride using the following procedure.
The platinum can be detected even in the presence of
these other noble metals. A drop of saturated thallium
nitrate solution is placed on filter paper, followed by a
drop of the test solution and another drop of TlNO3
solution. The paper is washed with ammonia. Tl2 [PtCl6 ]
remains on the paper only. A drop of stannous chloride
solution in strong HCl is added. Visual change: orange-red
stain. Sensitivity: 0.025 µg platinum.
Potassium ions are precipitated with sodium tetraphe-
nylboron. The test is particularly valuable if potassium
is to be detected in the presence of a lot of sodium.
A drop of the test solution is placed on a black spot
plate with a drop of a 2% water solution of the
reagent. Visual change: white precipitate. Sensitivity:
1 µg potassium. Interferences: NH4 C , CsC , RbC , heavy
metal ions.
Silver ions are precipitated with manganese salts and
alkali, as MnO2 and metallic silver. Both are black. Since
mercurous salts, noble metals and stannous salts react
analogously, AgCl should be isolated beforehand in order
to prevent such interferences. A drop of 0.1 N HCl is
placed on a filter paper followed by a drop of the test
solution and then a further drop of HCl. The fleck is
SPOT TEST ANALYSIS
blackened at the addition of a drop of 0.1 N manganese
nitrate and a drop of 0.1 N NaOH. Sensitivity: 2 µg silver.
Sodium ions impart a characteristic yellow color
in the flame. This flame test is extremely sensitive,
so even omnipresent sodium traces are detectable. A
sodium concentration of as little as 0.01 ppm yields a
very persistent and intense yellow coloration in the
flame.
Strontium ions react in neutral solution with sodium
rhodizonate and form a brown-red precipitate. To detect
Sr in the presence of Ba, the latter must be converted
first to insoluble sulfate. The solubility of SrSO4 allows
its detection with rhodizonate. A drop of the test solution
is placed on filter paper impregnated with saturated
sodium sulfate solution and the sulfates are formed.
After a minute, a drop of freshly prepared 0.2% reagent
solution is placed on the spot. Visible change: brown-red
precipitate.
Tin(II) ions reduce ammonium phosphomolybdate (in
contrast antimony salts reduce the free phosphomolybdic
acid only) to molybdenum blue. Filter paper impregnated
with an aqueous 5% phosphomolybdic acid solution is
held over ammonia until an insoluble yellow material
forms on the reagent paper. Quadrivalent tin must be
reduced with a tiny piece of zinc and a few drops of
concentrated hydrochloric acid. Visual change: deep blue
color. Sensitivity: 0.03 µg tin.
Tungsten(VI) ions are precipitated as a white amor-
phous product with 2,40 -diaminodiphenyl hydrochloride.
A drop of the test solution is mixed in a microtest tube
with a 1% solution of the reagent in 2 N HCl. Sensi-
tivity: 6 µg tungsten. Interference: molybdates disturb in
concentrations larger than 10%.
Uranium(VI) ions fluoresce in crystalline form, but
only slightly in solution. Sodium fluoride beads of uranyl
salts are most striking and are used to detect uranium.
Sodium fluoride is fused in a loop of platinum wire. A
neutral test solution is placed on the bead and evaporated
slowly. After fusing for a short time, the bead is cooled
and examined in UV light. Visual change: deep yellow
fluorescence. Sensitivity: 0.001 µg uranium. Interferences:
only niobium and beryllium give a much weaker similar
fluorescence.
Vanadium(V) as the vanadates forms a yellow pre-
cipitate in strongly acidic solution with a-benzoinoxime.
Analogous white compounds are formed with the same
reagent by molybdates and tungstates. Two drops of the
saturated reagent solution in ethyl alcohol are added to a
drop of the test solution followed by a drop of 3 N H2 SO4 .
Sensitivity: 1 µg vanadium. It is a specific reaction, only
the color of Fe3C might interfere. This can be avoided by
adding phosphate.
Zinc ions raise the oxidation potential of the
ferricyanide by removing ferrocyanide ions from the
Encyclopedia of Analytical Chemistry, Online © 2006 John Wiley & Sons, Ltd.
This article is © 2006 John Wiley & Sons, Ltd.
This article was published in the Encyclopedia of Analytical Chemistry in 2006 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470027318.a8111
5
redox equilibrium between ferricyanide and 3,30 -
dimethylnaphthidine. A drop of the reagent solution
(freshly prepared mixture of one part 5% solution of
potassium ferricyanide in water and two parts saturated
aqueous solution of 3,30 -dimethylnaphthidine chloride)
and a drop of weakly acid test solution are brought
together on a spot plate. Visual change: red-violet color.
Sensitivity: 0.1 µg zinc.
Zirconium(IV) ions with morin form a pale-yellow,
acid-resistant color in daylight and a yellow-green
fluorescing lake in UV light. A drop of the test solution
is mixed on the spot plate with a drop of 0.001% reagent
solution in ethyl alcohol and some drops of concentrated
HCl. Sensitivity: 0.1 µg zirconium. Interferences: it is a
specific reaction but cupric, ferric, vanadate, chromate
and noble-metal ions quench the fluorescence.
3.3 Selected Tests for Anions
If the original material does not consist solely of alkali
salts, the sample is boiled with strong sodium carbonate
solution or is fused with sodium potassium carbonate in
a platinum spoon. Heavy-metal salts are thus converted
in water-insoluble carbonates. The only soluble metal
carbonate, except for the alkali carbonates, is Tl2 CO3 .
Spot reactions to detect the various anions can be
successfully made on drops of the sodium carbonate
extract. Acetic acid is added to the carbonate extract
until no further carbon dioxide evolves.
Borates can liberate free boric acid which gives a deep
red compound when evaporated with tincture of curcuma
(turmeric). The boric acid changes the yellow curcumin
into the isomeric red-brown rosocyanine. A drop of the
test solution acidified with HCl is placed on curcuma
paper and dried at 100 ° C. Visual change: a red-brown
fleck, which turns blue to greenish-black on treatment
with 1% NaOH. Sensitivity: 0.02 µg boron. Interferences:
oxidizing substances (H2 O2 , CrO4 2 , NO2 , ClO3 and
iodides). They must be decomposed before carrying out
the reaction.
Chloride ions are detected in the presence of other
halide ions by selective oxidation of bromides and iodides
and halogenating 8-hydroxyquinoline with the halogen.
Chlorides are not oxidized and are precipitated with
AgNO3 solution. A drop of the test solution is warmed
with a drop of 2% reagent in 1 : 4 acetic acid and a drop
of H2 O2 solution (2 parts of 6% H2 O2 and 1 part dilute
CH3 COOH). A drop of 1% AgNO3 solution is added.
Visual change: white precipitation.
Cyanide ions can be converted to thiocyanate, which
can be identified by the sensitive iron reaction. A drop of
the test solution is stirred with a drop of yellow ammonium
sulfide on a watch glass and warmed until a rim of sulfur
is formed around the liquid. After the addition of some
6 GENERAL ARTICLES

dilute HCl, 1 – 2 drops of 1% FeCl3 are added. Visual
change: red color. Sensitivity: 1 µg cyanide. Interference:
in the presence of thiocyanates, the cyanide should be
first isolated as Zn(CN)2 .
Ferrocyanide, thiocyanate and thiosulfate anions are
detected simultaneously by capillary separation of their
respective ferric compounds. Filter paper is impregnated
with ferric chloride solution and dried. The paper is
spotted with dilute HCl and then with the test solution.
After a short time, individual colored areas are observed
due to capillary separation. From the center outward they
reveal: ferrocyanide, blue fleck; thiosulfate, white ring;
thiocyanate, red ring. The sensitivities of the detections
depend on the ratio of the three components but they are
lower than 1 µg each.
Iodide ions react with palladous chloride to form a
brown-black precipitate of PdI2 . The latter is insoluble in
mineral acids but soluble in ammonia, NaCl and MgCl2 .
A drop of the test solution is spotted on filter paper with
a drop of 1% reagent Sensitivity: 1 µg iodide.
Nitrate ions oxidize diphenylamine to blue quinoid
imonium dye. About 0.5 mL of the strongly acid diphenyl-
amine solution is placed on a spot plate, and in the middle
a drop of the test solution is added. Sensitivity: 0.5 µg
nitrate. Interferences: alkali halogenates, perchlorates,
periodates, permanganates, persulfates, peroxides and
nitrites. The interferences of the six anions can be
averted by drying the sample and heating the residue
to 400° – 500 ° C. All but the nitrate and nitrite are
decomposed. Sulfamic acid, on the other hand, destroys
the nitrite in advance.
Nitrite ions form diazonium cations with primary
aromatic amines in acid solution which couple with the
same (or with another) primary aromatic compound and
result in colored azo compounds. A drop of the test
solution is mixed on a spot plate with a drop each of 1%
sulfanilic acid in 30% acetic acid and a 0.03% solution
of a-naphthylamine in 30% acetic acid solution Visual
change: red color. Sensitivity: 0.01 µg nitrite.
Phosphate ions react with molybdates forming complex
phosphomolybdic acid. The latter has an enhanced
oxidizing power, oxidizing tetrabase to its blue quinoidal
compound. A drop of the acid test solution is placed on the
quantitative filter paper followed by a drop of ammonium
molybdate solution (5 g salt dissolved in 100 mL water
and poured into 35 mL HNO3 specific gravity 1.2) and a
drop of saturated tetrabase solution in 2 N CH3 COOH.
Sensitivity: 0.05 µg phosphate. Interferences: germanic,
arsenic and silicic acids behave analogously.
Silicate ions form the complex silicomolybdic acid
H2 SiO4 , 12MoO3 when treated with molybdate. It is
reduced by sodium stannite solution to molybdenum blue.
Three drops of ammonium molybdate solution is placed
on a watch glass followed by a drop of the test solution
and a drop of alkali stannite solution Visual change: blue
color. Sensitivity: 1 µg SiO2 . Interferences: phosphoric
and molybdic acid.
Sulfate ions decompose the red-brown precipitate of
barium rhodizonate by forming insoluble BaSO4 . A
drop of BaCl2 is placed on filter paper followed by
a drop of freshly prepared 0.2% solution of sodium
rhodizonate. The red fleck is treated with a drop of
the test solution Visual change: the red fleck disappears.
Sensitivity: 5 µg SO4 2 .
Sulfide ions form methylene blue with p-
aminodimethylaniline and FeCl3 . H2 S is liberated from
the test solution and absorbed by glass fiber paper
moistened with dilute NaOH. Add a mixture of a
drop each of concentrated HCl, 0.1 N FeCl3 and several
granules of p-aminodimethylaniline. Visual change: blue.
Sensitivity: 1 µg sulfide.
4 SELECTED TESTS IN ORGANIC ANALYSIS
The main field of qualitative organic analysis is in the
detection of certain groups of compounds, the detection
of characteristic types of compounds and, whenever
possible, the identification of individual compounds.
These goals are mainly attained through observation
of the results of the chemical reactions into which
the functional group enters. Such reactions include
chelate formation, catalyzed and induced reactions,
masking procedures, solid – solid reactions at elevated
temperatures, reactions in the gas phase through contact
with solid or dissolved reagents and reactions which yield
fluorescent or colored products or those which fluoresce.
These qualitative tests.2,4/ are instrumentally unso-
phisticated but their use has considerable practical
importance. Analytical problems seldom involve totally
unknown materials. The information available concern-
ing the origin, method of preparation, color of the sample
and so on, almost always gives valuable clues as to the
direction of the spot-test examination. The analytical
problem is not always to detect a particular organic com-
pound, but rather to find out whether members of the
class are present or absent.
4.1 Preliminary Tests
4.1.1 Sensory Tests (Color and Odor)
As most pure organic compounds are colorless and
odorless, the appearance of color and odor has some
limited diagnostic value. Yellow material can indicate
nitro, nitrozo and azo compounds, and a shift to a longer
wavelength might suggest the presence of conjugation,
chelation or dyestuffs. Fluorescence is indicative also,

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SPOT TEST ANALYSIS
but only in material carefully purified by sublimation or
recrystallization.
The detection of odoriferous materials such as men-
thol, phenol, pyridine, butyric acid and vanilin can
be extremely sensitive. Nitrobenzene and benzalde-
hyde have an almond odor, ethyl sulfide has a garlic
and long-chained fatty acids have characteristic rancid
odors.
4.1.2 Burning Tests
Different visual phenomena appear by ignition of various
classes of organic compounds under similar conditions.
Burning aromatic compounds and halogen compounds
furnish a smoky flame whereas the lower aliphatic
compounds burn with a smokeless flame. Compounds
containing a high percentage of halogen do not ignite
easily and oxygen-containing compounds show a bluish
flame. Proteins and carbohydrates burn with an odor
resembling burnt hair.
4.1.3 Pyrolysis
Rapid external heating generally leads to the forma-
tion of lower-molecular-weight compounds, which can
be detected in the gas phase. The latter are HCN,
(CN)2 , hydrogen halides, acetaldehyde, CO, SO2 , nitrous
oxide, phenols and so on. HCN appears in the gas
phase by pyrolysis of nitrogenous organic material.
The detection of (CN)2 is almost specific for uric acid
and purine derivatives by pyrolysis. Hydrogen halides
are released by aromatic and aliphatic halogen com-
pounds, and nitrogen– oxygen containing compounds
always split off nitrous oxide. Aromatic compounds
containing oxygen atoms release phenol on strong
pyrolysis.
4.1.4 Acid – Base and Redox Behavior
The basic or acidic behavior of a material can afford a
valuable clue in the detection of basic or acidic functional
groups. The derivatives of ammonia, hydroxylamine
and hydrazine are bases, whereas functional groups
which split hydrogen ions, such as carboxylic, sulfonic,
sulfinic, arsonic, nitroxylic, oximic, thioenolic, phenolic
and thiophenolic are detectable by acid character. For
water-soluble compounds, conventional indicators would
be useful, whereas for water-insoluble compounds, basic
groups are detected by shifting of the reaction equilibrium
of nickel with dimethylglyoxime. After removal of the
precipitate, the equilibrium solution which results may
react with the basic unknown and the consumption of
protons will lead to the appearance of the red precipitate.
On the other hand, extremely weak organic acids heated
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DOI: 10.1002/9780470027318.a8111
7
with prefused potassium iodate (which contains iodide)
leads to iodine liberation.
Organic reductants are indicated by a molybdenum
blue reaction, whereas a preliminary test with tetrabase
reagent paper might detect the few classes of oxidiz-
ing compounds, such as polyhalides of organic bases,
quinones and peroxoacids.
4.1.5 Elemental Analysis
The organic material is pyrolyzed with carbonates, strong
oxidizing acids, oxidants such as vanadium pentoxide or
heated in aluminum foil under a stream of oxygen. For
details, consult Feigl and Anger..1/
4.2 Selected Tests for Functional Groups
An organic molecule may include several of such groups
and their detection may largely characterize its chemical
and physical properties. The most common functional
groups are discussed here.
Aromatic compounds react with concentrated H2 SO4
containing formaldehyde and form red, green and violet
precipitates. The reaction occurs only when the para
position is free. This enables the formation of methylene
compounds, which are partly oxidized to p-quinoidal
compounds. A drop of the nonaqueous test solution or
a little of the solid is treated on a spot plate with a drop
of formaldehyde – sulfuric acid reagent (0.2 mL of 37%
formaldehyde in 10 mL concentrated H2 SO4 ). Sensitivity:
2 – 25 µg aromatics.
Primary, secondary and tertiary alcohols convert
[Ce(NO3 )6 ]2 into [Ce(OR)(NO3 )5 ]2 with a color
change from yellow to red. The reagent solution is 40%
[(NH4 )2 Ce(NO3 )6 ] in 2 N HNO3 . One milliliter of the
reagent solution diluted with 2 mL of dioxane is treated
with the test solution. Sensitivity: 400 µg alcohol. Inter-
ferences: aliphatic bases and oxidizable compounds.
Secondary alcohols fused with sulfur release H2 S on
heating. In a microtest tube, the test solution is treated
with a drop of 2% sulfur solution in carbon disulfide and
dried. The mouth of the tube is covered with a disc of lead
acetate paper and placed in a glycerol bath previously
heated to 150 ° C. Visual change: appearance of black
lead sulfide on the paper. Sensitivity: 10 – 200 µg alcohol.
Volatile phenols condense with 2,6-dichloroquinone-4-
chloroimine to give colored indophenols. A drop of the
test solution is dried in a test tube. The mouth of the tube
is covered with a disc of filter paper impregnated with
a saturated benzene solution of the reagent. The tube is
placed in a glycerol bath preheated to 150 ° C. The reagent
paper is held in NH3 vapors. Visual change: blue stain.
Sensitivity: 0.3 – 20 µg phenol.
Phenols react with nitrous acid containing mercuric
nitrate to form nitro compounds reacting with the phenol.
8 GENERAL ARTICLES
A drop of the test solution is mixed in a microcrucible
with a drop of the reagent solution (one part mercury
is dissolved in one part of fuming HNO3 and diluted
with two parts of water). Visual change: red color.
Sensitivity: 0.5 – 10 µg phenol. Note: di-o- and di-m-
substituted phenols do not react.
Aromatic and aliphatic aldehydes condense with pri-
mary aromatic amine forming colored Schiff bases.
Dianisidine is an especially suitable reagent for this. A
drop of the sample is mixed with 3 – 4 drops of the sat-
urated solution of the reagent in glacial acetic acid. The
light color is intensified on heating. Sensitivity: 0.05 – 50 µg
aldehyde. Interference: large amounts of certain ketones.
Aromatic aldehydes condense with N,N-dimethyl-p-
phenylenediamine to give an intensely red protonated
Schiff base. Several milligrams of the solid oxalate or
chloride of the reagent are added to a drop of the test
solution in a microtest tube and warmed in a preheated
water bath. Aliphatic aldehydes and ketones do not
interfere. Sensitivity: 0.3 – 0.5 µg aldehyde.
Aliphatic ketones react with m-dinitro compounds in
alkaline solution to yield violet products. The reaction
is selective for aliphatic monoketones. A drop of a 1%
solution of m-dinitrobenzene in alcohol is mixed in a
microtest tube with a saturated methanolic solution of
KOH and a drop of the test solution. The tube is warmed
to 70 – 80 ° C in a water bath for 2 – 3 min. Visual change:
violet-red color.
Carboxylic acids are converted via acid chloride into
hydroxamic acids. The latter react with ferric ions to
give a red or bluish-red chelate. A drop of the test
solution is evaporated to dryness in a microcrucible and
treated with two drops of thionyl chloride. The mixture
is evaporated almost to dryness. Two drops of saturated
alcoholic solution of hydroxylamine hydrochloride are
then added and made basic with alcoholic alkali. The
mixture is acidified with 0.5 N HCl and treated with
1% aqueous FeCl3 solution. Visual change: dark-violet.
Sensitivity: 10 – 100 µg carboxylic acids. Interferences:
citric and thioacetic acid.
Alkyl esters of carboxylic acids react with metallic
sodium to form acyloins. The latter is identified by its
reaction with 1,2-dinitrobenzene to produce the red acid
form of nitrosonitrobenzene. The test is conducted in
the depression of a spot plate. A small piece of metallic
sodium (CARE) is pressed into a disk with a glass rod.
A drop of a benzene solution of the test material is
added and then a drop of 2.5% benzene solution of 1,2-
dinitrobenzene. The system is stirred with a glass rod and
a drop of water is introduced. Visual change: deep-violet
color.
Ethers are oxidized at higher temperatures (230 ° C)
to etherperoxides which oxidize, via cupric peroxide,
tetrabase to tetrabase blue. A drop of the test solution
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is placed in a microtest tube. The tube is plunged into a
glycerol bath preheated to 230 ° C. The mouth of the test
tube is covered with a disc of filter paper moistened with
a mixture of 5% aqueous copper ethylacetoacetate and
5% chloroform solution of tetrabase. Sensitivity: 20 µg
diethylether.
Interferences: the test fails in the presence of benzene
and CS2 . However, it is also given by dioxan and
tetrahydrofuran; it seems that the CH2 O CH2 group
is responsible for the auto-oxidation.
Aromatic primary amines react with Na2 [Fe(CN)5 Ð
H2 O] and produce bluish-green Na2 [Fe(CN)5 ArNH2 ].
A drop of the test solution is mixed with a drop of 1%
reagent and a drop of 2% Na2 CO3 solution on a spot
plate. Sensitivity: 0.2 – 50 µg amines.
Primary and secondary aliphatic and aromatic amines
condense with p-dimethylaminocinnamaldehyde to yield
colored Schiff bases. A drop of saturated solution of
the reagent dissolved in methanol and trichloroacetic
acid is placed on filter paper and treated with a drop
of the test solution The filter paper is kept in an
oven at 100 ° C for about 3 min. Visual change: blue
for primary and purple color for secondary amines.
Sensitivity: 0.02 – 2.2 µg amines.
Primary and secondary aliphatic amines form dithio-
carbamates with carbon disulfide instantly at room
temperatures. The dithiocarbamates can be detected by
the catalytic enhancement of the sodium azide – iodine
reaction. A drop of the alcoholic test solution is mixed
in a microcrucible with a few drops of a 1 : 1 mixture of
alcohol : CS2 . The excess of CS2 is volatilized after about
5 min. A few drops of the reagent (3% NaN3 in 0.1 N
iodine solution) is added. Visual change: the color of
the iodide disappears and evolution of nitrogen bubbles
is observed. Sensitivity: 1 – 250 µg amines. Interferences:
mercaptans, thioketones and thiol compounds. Tertiary
aliphatic amines do not react.
Aliphatic secondary amines form blue-violet com-
pounds reacting with sodium nitroprusside and acetalde-
hyde in alkaline solution. Primary and tertiary amines
do not interfere. A drop of the test solution is mixed
with a drop of the reagent (freshly prepared 1% solution
of sodium nitroprusside to which 10% acetaldehyde is
added.) The mixture is made basic with a 2% solution of
sodium carbonate. Visual change: blue-violet color. Sen-
sitivity: 1 – 100 µg amine. Interference: diisopropylamine
does not react.
Nitro compounds added to molten tetrabase (melting
point 91 ° C) lead to yellow-orange-red melts, molecular
compounds are obtained between the two components.
One drop of the ethereal or benzene solution of the sam-
ple and one drop of 5% benzene solution of the reagent
are mixed in a microconical tube and dipped into boiling
water. Visual change: after evaporation of the solvent, a
SPOT TEST ANALYSIS
yellow-orange melt appears. Sensitivity: 0.3 – 50 µg nitro
compound. Interference: p-quinoid compounds.
Primary aliphatic nitro compounds are pyrolytically
reduced to nitrous acid when heated to 160 ° C with
benzoin. The benzene solution of the test material
is brought together with some solid benzoin in a
microtest tube. After solvent volatilization, the test tube
is immersed in a glycerol bath preheated to 130 ° C.
The mouth of the test tube is covered with a filter
paper moistened with Griess reagent (a mixture of 1%
solution of sulfanilic acid and 0.1 aqueous solution of
N-1-naphthylethylenediamine dihydrochloride) and the
temperature raised to 160 ° C. Visual change: red fleck on
the reagent paper. Sensitivity: 10 – 20 µg nitro compounds.
Aromatic nitro compounds can be reduced with metallic
zinc in weakly acidic solution to arylhydroxylamines.
The latter form violet complexes with pentacyanoamine
ferroate. A few milligrams of the sample are dissolved in
hot alcohol in a test tube and treated with a few drops
of 10% CaCl2 . About 50 mg of zinc dust is added and
the suspension is heated in a strongly boiling water bath.
The filtrate is cooled and 1 drop of 1% reagent solution
is added. Visual change: purple, blue or green color.
Sensitivity: 0.8 – 15 µg nitro compound.
Nitriles (cyanides) are converted to oxamide by heating
them with hydrated oxalic acids (pyrohydrolysis). The
resulting oxamide is detected by its sintering with thio-
barbituric acid. A drop of the test solution is dried in a
test tube, a few milligrams of solid thiobarbituric acid are
added and the mixture is heated to 140° – 160 ° C in a glyc-
erol bath. Visual change: orange product. Interferences:
ammonium salts, amides of acids, amino acids, ureides.
Interference is avoided by selective dissolution of nitriles
in benzene. Sensitivity: 2 – 20 µg nitriles.
Aliphatic nitriles release HCN pyrolytically, when
heated to 250 ° C (differentiation from aromatic nitriles).
A little of the solid test material is mixed with a few
centigrams of a 1 : 1 mixture of calcium carbonate and
oxide. The tube is placed in a glycerol bath preheated to
250 ° C and the mouth of the tube is covered with a paper
disc moistened with a mixture of 5% Cu ethylacetoacetate
and 5% tetrabase in chloroform. Visual change: blue stain
on the paper after 3 – 4 min. Sensitivity: 2 – 50 µg nitriles.
Thioketones and thiols accelerate the iodine – azide
reaction catalytically, in contrast to other organic sulfur
derivatives. A drop of a solution or the test substance in
water or organic solvent is mixed on a watch glass with a
drop of 3% NaN3 in 0.1 N iodine solution Visual change:
the brown color of the iodine disappears and bubbles of
nitrogen evolve. Sensitivity: 3 ð 10 4 – 0.6 µg.
Primary and secondary thiols when heated with concen-
trated NH3 at boiling water temperature release H2 S. A
small amount of the test material or a drop of the solution
is treated in a microtest tube with a drop of concentrated
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NH3 . The mouth of the tube is covered with wet lead
acetate paper and the tube is placed in a boiling water
bath. Visual change: black stain on the paper. Sensitivity:
1 – 200 µg. Tertiary thiols and thioketones do not interfere.
Sulfonic acids can be converted pyrohydrolytically to
sulfuric acid which is instantly reduced to sulfurous acid.
(R, Ar) SO3 H C H2 O ! (R, Ar) H C H2 SO4 . The
pyrohydrolysis is accomplished by phthalic acid at 230 ° C.
A little of the solid or a drop of its solution is mixed with a
few centigrams of phthalic acid and the solvent is removed
by evaporation. The test tube is placed in a glycerol bath
preheated to 230 ° C. A disc of filter paper moistened with
ferric ferricyanide solution (0.08% anhydrous FeCl3 and
0.1% of K3 Fe(CN)6 in water) is placed over the mouth
of the test tube. Visual change: blue stain on the paper.
Sensitivity: 1 – 20 µg sulfonic acids.
4.3 Detection of Individual Organic Compounds
The measurement of certain physical properties provides
a very reliable method for the identification of an organic
material. Determination of melting and boiling points,
density of compounds and optical qualities such as
refractive index, optical activity, UV and infrared (IR)
absorption are very useful, but the essential requirement
of isolating the compound in a state of a high purity is
very costly with regard to time and material. For this
reason rapid chemical identifications that do not require
expensive apparatus are important. The possibility of
finding a direct specific test for an individual compound is
highly improbable but the available information about the
origin, method of preparation and use of the material in
question provide valuable clues. In fact, the analytical goal
is often well defined by the question of whether particular
compounds are present or absent. The following section
contains descriptions of selected spot tests for technically
important individual compounds.
Acetone condenses with salicylaldehyde to form a
red conjugated compound. One drop of 10% alcoholic
solution of the reagent is mixed in a test tube with one
drop of 40% NaOH solution and a drop of the test
solution The tube is kept for 2 – 3 min in a water bath
at 70 – 80 ° C. Visual change: deep-red color. Sensitivity:
0.2 µg acetone.
Acetic acid forms a dark-blue adsorption compound
of iodine and basic lanthanum acetate when mixed with
iodine and ammonia. The interference of many anions
is avoided by the microdistillation of acetic acid in the
presence of concentrated H3 PO4 . In the depression of a
spot plate a drop of the solution is mixed with a drop of
5% solution of lanthanum nitrate, 0.01 N iodine solution
and a drop of 1 N NH3 . Visual change: blue color in a few
minutes. Sensitivity: 50 µg acetic acid.
Monochloroacetic acid heated with ammonium thio-
cyanate forms rhodamine, which subsequently, owing
10
to its reactive CH2 group, reacts with 1,2 naphtho-
quinone-4-sulfonate. A colored condensation product
results. A drop of the test solution is mixed with
some solid NH4 SCN and heated in boiling water for
2 min. The cooled mixture is treated with 1 – 2 drops
of 0.5% reagent and made alkaline with 1% NaOH
solution Visual change: blue color. Sensitivity: 5 µg
monochloroacetic acid.
Acetylene forms an orange precipitate with ammoniacal
Ag2 CrO4 . It is an acid-insoluble addition compound
Ag2 C2 ÐAg2 CrO4 which serves as a protective layer around
silver chromate. A drop of ammoniacal silver chromate
(Ag2 CrO4 dissolved in 1 : 5 NH3 and filtered) is placed in
each of two small porcelain crucibles. One drop of the
solution being tested for acetylene is added to one of
the suspensions, and a drop of water to the other. The
mixtures are stirred and treated with the same number
of drops of 1 : 10 HNO3 until the Ag2 CrO4 precipitate
in the blank disappears. Visual change: in the presence
of acetylene a residue of red-brown Ag2 CrO4 remains.
Sensitivity: 1 µg acetylene.
Anthracene evaporated with concentrated HNO3 is
oxidized to anthraquinone, which can be detected by
reductive conversion with solid sodium hydrosulfite
in alkaline solution to the red water soluble salt of
anthrohydroquinone. Sensitivity: 3 µg anthracene.
Ascorbic acid reduces ammonium phosphomolybdate
to molybdenum blue. A drop of the test solution is placed
on the reagent paper (filter paper immersed in a saturated
alcoholic solution of phosphomolybdic acid, dried in
cold air, bathed in concentrated Ag2 CrO4 solution and
dried again). Visible change: blue color. Sensitivity: 0.1 µg
ascorbic acid. Interference of uric acid or urate is avoided
by acidifying the sample.
Barbituric acid reacts instantaneously with nitrous acid
to give violuric acid. A drop of the test solution is mixed
in the depression of a spot plate with one drop each of
2 N CH3 COOH and saturated NaNO2 solution. Visual
change: red violet. Sensitivity: 10 µg barbituric acid.
Carbon disulfide forms dithiocarbamates with sec-
ondary aliphatic amines, which in turn produce chelates
with transition-metal ions. In a microtest tube a drop of
the test solution is treated with a drop of reagent solution
(9 mL 0.2% CuSO4 solution mixed with 1 mL concen-
trated NH3 and 0.5 g dimethylamine chloride.) The tube
is shaken with 2 drops of benzene. Visual change: yellow-
brown color in the benzene layer. Sensitivity: 3 µg carbon
disulfide.
Carbon tetrachloride vapor releases chlorine when
heated in the presence of quartz sand. It is most
likely that a disproportionation occurs on the surface
of the quartz. In a microtest tube a drop of the test
solution is mixed with several centigrams of quartz dust.
The tube is immersed in a glycerol bath preheated to
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GENERAL ARTICLES
150 ° C and the mouth of the tube is covered with a
disc of Congo Red paper moistened with 5% H2 O2
solution. Visual change: blue stain. Sensitivity: 5 µg
CCl4 . Interference: sym-dichloro(bromo)ethane and sym-
tetrachloro(bromo)ethane show the same behavior.
Citric acid is converted by alkali permanganate into
acetone dicarboxylic acid, which in the presence of
bromine precipitates as pentabromoacetone. A drop of
the test solution is mixed in a microtest tube with one drop
each of 0.01 N KMnO4 and a drop of saturated bromine
water. After cooling, the excess bromine and MnO2 are
destroyed with the addition of some solid sulfosalycilic
acid. Visual change: white precipitate. Sensitivity: 6 µg
citric acid.
Formaldehyde condenses with J-acid dissolved in
concentrated H2 SO4 to produce a yellow fluorescent xan-
thylium dyestuff. Add one drop of aqueous test solution
to a drop of a 0.1% solution of J acid (6 amino-1-naphthol-
3-sulfonic acid in concentrated H2 SO4 ) on a glass filter
paper. Visual change: yellow fluorescence under a UV
lamp after addition of a drop of water, the yellow solution
turns blue. Sensitivity: 0.01 µg formaldehyde.
Glycerol is dehydrated with concentrated H2 SO4
to acrolein, which according to the classical Skraup
synthesis forms 8-hydroxyquinoline with o-aminophenol.
The magnesium salt of 8-hydroxyquinoline is highly
fluorescent. Two drops of 2% alcoholic solution of o-
aminophenol are evaporated in a microtest tube. A
drop of the test solution and 4 drops 1% arsenic acid
in concentrated H2 SO4 are added. The tube is kept at
140 ° C for about 15 min, and after cooling 5 drops of
concentrated NaOH, 1 drop of 2 N MgSO4 and three
drops of concentrated NH3 are added. Visual change:
bluish-green fluorescence. Sensitivity: 0.5 µg glycerol.
Interference: crotonaldehyde.
Malonic acid fused with urea forms barbituric acid. The
latter is identified by its reaction with pyridylpyridinium
dichloride to give a pentamethine dye. A drop of the
test solution is treated with several drops of a saturated
methanolic solution of urea in a microtest tube. The tube
is heated for several minutes in a glycerol bath at 120 ° C.
A drop of 1% solution of pyridylpyridinium dichloride
in dimethylformamide (DMF) is added. The mixture
is heated to 120 ° C for several minutes. Visual change:
reddish-blue color develops on cooling, which shows a
red fluorescence red under UV. Sensitivity: 50 µg malonic
acid. The detection is specific.
Methanol is oxidized by acidic permanganate to
formaldehyde. The latter is detected specifically by
forming a bright violet p-quinoidal compound with
chromotropic acid. A drop of the test solution is mixed
with a drop of 5% H3 PO3 and a drop of 5% KMnO4 .
The mixture is decolorized by sodium bisulfate crystals
and 4 mL of 12 N H2 SO4 . A little finely powdered
SPOT TEST ANALYSIS
chromotropic acid is added and heated to 60 ° C for 10 min.
Visual change: violet color. Sensitivity: 3.5 µg methanol.
Naphthalene vapor reacts selectivity with chloranil to
give a brick-red addition compound. The solid unknown
or its ethereal solution is placed in a microtest tube and
the mouth of the tube is covered with a disc of filter
paper impregnated with a saturated ethereal solution of
chloranil. The tube is immersed in a water bath at 60 ° C.
Visual change: brick-red color on the paper. Sensitivity:
25 µg of naphthalene.
Oxalic acid decomposes pyrolitically at 250 ° C to give
formic acid, which forms aniline blue with diphenylamine.
In a microtest tube a drop of the sample is evaporated and
melted with a little diphenylamine over a free microflame.
After cooling, a drop of alcohol is added. Visual change:
blue coloration. Sensitivity: 5 µg oxalic acid.
Piperidine reacts through its reactive NH2 group with
1,2-naphthoquinone-4-sulfonate to produce intensely col-
ored p-quinoidal condensation products. In a microtest
tube a drop of the acidic test solution is mixed with several
centigrams of Na2 CO3 . The tube is immersed in a water
bath heated to 90 ° C and the mouth of the tube is covered
with a disc of filter paper moistened with a 2.5% aque-
ous solution of the reagent Visual change: red stain on
the yellow paper. Under the conditions described piper-
azine does not disturb the detection. Sensitivity: 20 µg
piperidine.
Pyridine reacts with trisodium pentacyanoamminofer-
rate, by replacement of the ammonia with pyridine in the
coordination sphere, forming a colored compound. On a
Whatman filter paper No.1, a drop of the test solution is
sprayed with one drop of 0.2% solution of the reagent in
buffer solution at pH 6.5. Visual change: yellow-orange
coloration. Sensitivity: 10 µg pyridine.
Quinones give colored condensation products with
rhodamine. A drop of the test solution is shaken with one
drop of a saturated aqueous solution of the reagent and
one drop of ammonia. Visual change: green or blue with
p-quinones and violet-red with o-quinones. Sensitivity:
0.2 – 5 µg quinones.
Salicylic acid is converted easily to fluorescent alkali
salicylate with dissolved alkali and alkaline-earth salts.
This acid can be separated adequately by sublimation on
heating the substance with concentrated H2 SO4 . A drop
of the test solution is evaporated in a microtest tube. A
drop of concentrated H2 SO4 is then added and the mouth
of the tube is covered with a glass-paper disc wetted with
3% KOH. The tube is heated in a glycerol bath at 130 ° C.
Visual change: violet fluorescence on the glass paper.
Sensitivity: 5 µg salicylic acid.
Tartaric acid heated with concentrated H2 SO4 contain-
ing b,b0 -dinaphthol produces green fluorescence. The test
solution is treated in a test tube with a few milliliters
of 0.05% concentrated H2 SO4 solution of the reagent
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and kept for 30 min in a water bath at 85 ° C. Visual
change: luminous green fluorescence. Sensitivity: 5 µg
tartaric acid.
Thiophene condenses with ninhydrin in concentrated
H2 SO4 to give a violet dyestuff. In a depression of a
spot plate one drop of freshly prepared 0.01% reagent
in concentrated H2 SO4 is covered with a drop of the
test solution Visual change: deep violet to pink color.
Sensitivity: 5 µg thiophene.
Urea is quickly hydrolyzed enzymatically to ammonia
even at room temperature. A drop of the neutral or
alkaline test solution is treated in the depression of a spot
plate with several milligrams of urease and after 2 – 5 min
one drop of Nessler solution (50 g KI dissolved in 35 mL
water is treated with saturated HgCl2 solution until a slight
precipitate persists. Then 400 mL of 9 N caustic alkali is
added. The solution is diluted to 1000 mL, allowed to
settle and decanted). Visual change: brown precipitate.
Sensitivity: 1 µg urea Interference: ammonium salts.
These can be eliminated by drying the test solution with
a drop of dilute alkali.
5 SELECTED EXAMPLES OF
APPLICATIONS OF SPOT TESTS
The classical spot tests have been successfully applied to
solving large-scale analytical problems in clinical analysis,
in crime laboratories, in judicial chemical studies, in geo-
chemical prospecting, in soil and plant tissue testing, and
in air and water QC. Commercial companies are market-
ing a great variety of compact test systems. Their extreme
simplicity, time and money-saving nature and ultimate
reliability make these tests very useful. These tests sys-
tems, marketed generally by commercial companies in
the form of reagent strip systems, prepared reagent pil-
lows or tablets constitute a significant proportion of the
tools used for solving the total spectrum of analytical
problems.
5.1 Applications in Clinical Analysis
Substantial changes in the concentrations of glucose,
protein, ketone bodies, bilirubin, urubilinogen nitrites,
leucocytes and erythrocytes, and so on in urine may have
significant diagnostic value..5/ A simple spot test can give
valuable quick information about many health anomalies
and provides the possibility of performing many rapid
bedside methods for some key materials in the human
blood. Selected examples only are presented here.
5.1.1 Tests in Urine
Glucose in urine is screened quickly by enzymatic tests
based on the activity of the enzyme glucose oxidase,
12
which uses dry-reagent chemical technology (Ames Co.,
Boehringer-Mannheim, Hoechst AG). A firm plastic
strip, to which a stiff absorbent cellulose area is affixed,
is impregnated with a buffered mixture of glucose
oxidase, peroxidase and o-tolidine. In the first stage
of the reaction glucose is oxidized by atmospheric
oxygen in the presence of glucose oxidase to gluconic
acid and hydrogen peroxide, while in the second stage
H2 O2 /peroxidase oxidizes o-tolidine to a blue quinoidal
compound. The detection is either visual or by simple
reflectance instruments, irradiating the dipstick with
polychromatic light and measuring the reflected radiation.
Protein in urine test is based ‘‘on the protein error’’.
The test exploits the fact that the aqueous solution of the
potassium salt of tetrabromophenol-phthalein ethyl ester
becomes yellow on addition of dilute acetic acid. If, how-
ever, a solution or suspension of native albumin is added
to a dilute blue solution, a great deal of acetic acid can be
introduced without causing the blue to change to yellow
(Ames Co., Boehringer-Mannheim). These standardized
tests for the estimation of protein in urine are composed
of a strip of absorbent cellulose, one end of which is
impregnated with the indicator and buffered at pH 3.
Ketone bodies in urine are quickly screened by a
dipstick utilizing the reaction of ketones with nitro-
prusside. The NO group of the nitroprusside gives
isonitrosoketone, which remains in the complex anion,
with the ketone whereas the Fe3C is reduced to Fe2C
(Ames Co., Boehringer-Mannheim, Hoechst AG). A
microprocessor-controlled benchtop reflectance monitor
(Kyoto) is designed to measure the change in the color of
the strips.
Bilirubin in urine can be screened with a tablet-
configuration reagent system involving a diazotization
procedure, which couples with the bilirubin to give a char-
acteristic purple color. It is a fiber mat test (Ames Co.)
using a tablet composed of p-nitrobenzenediazonium
p-toluenesulfonate, NH4 HCO3 , sulfosalycilic acid and
boric acid. The sensitivity of the reaction coincides
with the lower limit of accepted pathological signifi-
cance.
Urubilinogen in urine is tested by a dip-and-read solid
state reagent incorporated in a p-dimethylaminobenzal-
dehyde and an acid buffer-containing strip. Urubilinogen
forms a red Schiff base (Ames Co.)
Nitrite in urine tests with the dipstick technique are
based on the classical diazotization Griess method (Ames
Co., Boehringer-Mannheim, Hoechst AG, Merck Co.)
The tests reveal significant bacteriuria when the bacterial
count reaches 1 ð 107 per mL of urine.
Occult blood in urine is detected on the basis of
peroxidase-like activity of hemoglobin, myoglobin and
some of their degradation products. The pseudoperox-
idase activity of hemoglobin transfers an oxygen atom
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DOI: 10.1002/9780470027318.a8111
GENERAL ARTICLES
from the peroxide to the chromogen (o-tolidine, o-
dianisidine, 2,6-dichlorophenolindophenol.) Typical dip-
and-read spot tests for occult blood (Ames Co.,
Boehringer-Mannheim, Helena Laboratories, Finnpipet,
Smith Kline Diagnostics) consist of absorbent cellulose
impregnated with an organic peroxide, o-tolidine and
buffer system.
Hemoglobin in urine is detected with a test strip coated
with an organic hydroperoxide. The test strip can be used
for the detection of peroxidase-like substances.
Phenylketones in urine are detected by a stiff strip of
cellulose impregnated with iron(III) and magnesium ions.
The desired acidity (pH D 2.3) is provided by the presence
of cyclohexylsulfuric acid. The ferric ion forms colored
chelates with phenylpyruvic acid, whereas the magnesium
ions are masking agents to minimize interference in the
color development by urinary phosphate (Ames Co.)
Chloride in urine is estimated by discharging the red
color of silver dichromate by chloride ion. A solid-
reagent spot test for chloride is the Quantab chloride
titrator (Ames Co.) It is a chemically inert plastic strip
laminated within, which is a capillary column impregnated
with Ag2 Cr2 O7 . The reaction of Ag2 Cr2 O7 with chloride
produces a white color in the capillary column.
Ethanol in urine can be estimated by test strips
containing alcohol oxidase and horseradish peroxidase
with colorimetric reagents (3-methylbenzothiazolinone
hydrazone hydrochloride and 3-dimethylaminobenzoic
acid). The urine sample is placed in a centrifuge tube
and the test strip is suspended in the air space above the
sample. The stoppered tube is heated at 65 ° C for 5 min
and the color development is estimated from a color
chart.
Ascorbic acid in urine is screened by the reduc-
tive decolorization of 2,6-dichlorophenolindophenol. The
reagent paper set is prepared by impregnating and drying
filter paper in 0.1%, 0.05% and 0.0025% alcoholic reagent
solutions. They are suitable for detecting 0.4 – 1 mg mL 1 ,
0.2 – 0.44 mg mL 1 and 0.01 – 0.24 mg mL 1 ascorbic acid,
respectively.
Cysteine in urine can be tested semiquantitatively based
on the reaction between cysteine, sodium nitroprusside
and K2 CO3 to give a cherry-red complex. Limit of
detection: 0.003 – 0.12 mM cysteine.
Ionic strength and specific gravity in urine can be tested
using a strip prepared by soaking a filter paper in a
solution of sodium heparin and 1 mM methylene blue
and attaching the treated sample to a plastic holder.
The density of the developed color correlates positively
with the specific gravity and ionic strength of the urine
sample.
Edetates in urine screening is based on the complexing
action of edetate anion to remove ferric ion from
ferrioxamine B immobilized on a Dowex HRC(HC ) resin.
SPOT TEST ANALYSIS
Decomposition of the colored chelate leads to a gradual
fading of the deep brown-violet resin beads.
5.1.2 Tests in Blood
Glucose in blood screening is based on the specific
glucose – oxidase – peroxidase reaction. In a reaction cat-
alyzed by glucose oxidase, D-glucose is oxidized by
atmospheric oxygen to b-D-gluconolactone and H2 O2 .
The latter oxidizes the chromogenic system (Ames Co.,
Boehringer-Mannheim, Biodynamic, Inc.) The test con-
sists of a firm plastic strip to which an impregnated reagent
area is affixed. A semipermeable membrane serves as a
barrier that retains the high-molecular-weight and cellu-
lar components on its surface and can be wiped clean after
exactly 60 s. Although significantly hypoglycemic results
can be rapidly recognized with the visual test, small devi-
ations from normal values should be confirmed by simple
reflectance measurements.
Urea in blood screening is based on the principle
of catalyzed hydrolysis of the urea to carbon dioxide
and ammonium hydroxide, the concentration of which
is measured by the color change of the indicator (Ames
Co., Boehringer-Mannheim, E. Merck Co.). The test stick
is coated with a membrane which is freely permeable to
urea but prevents staining of the paper by blood pigments.
Lactate screening in whole blood is as follows. Apply
20 µL whole blood to a filter paper strip coated on its other
side with 3.65 µmol min 1 enzyme activity Pediococcus
lactase oxidase, 0.12 µmol 4-aminoantipyrine, 0.2 µmol 3-
(N-ethyl-m-toluidine)-2-hydroxypropane-10 -sulfonic acid
and 18.4 µmol min 1 enzyme activity horseradish perox-
idase and attached at one end of a supporting strip.
After 1 min the absorbance is measured at 560 nm by a
reflectometer.
Cholesterol in blood can be quickly analyzed by use of
the cassette strip system, which is based on enzyme action
on cholesterol to produce H2 O2 and determination of
the latter (Diagnostics, Inc.). The enzyme is cholesterol
oxidase and the strip contains horseradish peroxidase and
leuco dye.
5.1.3 Other Tests
Occult blood tests in feces depend on the estimation
of peroxidase activity as an indication of hemoglobin
content. Reagents used include guaiac, o-tolidine, o-
dianisidine and 2,6-dichlorophenolindophenol. The prod-
ucts of Smith Kline Diagnostics and Finnpipette are based
on the gauiacum resin, whereas other self-contained
systems contain o-tolidine which is oxidized by the
peroxidase-like reaction. The reagent tablets (Ames Co.
Stoke Pages) contain o-tolidine, strontium peroxide, tar-
taric acid and calcium acetate. In contact with water,
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DOI: 10.1002/9780470027318.a8111
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tartaric acid, calcium acetate and strontium peroxide
instantly produce H2 O2 , which in the presence of
hemoglobin oxidizes o-tolidine to a blue compound.
Spot tests are performed for urinary calculi, the most
common varieties of which are the calcium oxalate,
uric acid, ammonium urate and magnesium ammo-
niumphosphate calculi. The test element for oxalate
determination contains oxalate oxidase, peroxidase and
optional riboflavin (reaction promoter) and quinolines
(enhancer). A filter paper is soaked in the first solution
containing oxalate oxidase, peroxidase, riboflavin, citrate
buffer and sodium alginate and then in the second solution
containing tetramethylbenzidine (color indicator) and 3-
aminoquinoline. Uric acid and urates are identified by the
classical murexide test, heating the sample with a number
of drops of concentrated HNO3 . The resulting red murex-
ide indicates the presence of urates. Phosphates in calculi
are detected by heating the sample with concentrated
HNO3 and (NH4 )2 MoO4 to produce yellow ammonium
phosphomolybdate.
Urinary lactic acid screening is based on the transfor-
mation of the acid to acetaldehyde, which reacts with
sodium nitroprusside to produce a blue color. A sample
is added to a tablet, which contains 30 mg CaCl2 and
30 mg Ce2 SO4 in a test tube. The test tube is covered
with a piece of filter paper that has been impregnated
with a 2.5% aqueous sodium nitroprusside solution and a
drop of 10% aqueous morpholine solution. Lactic acid is
indicated by the appearance of a blue color.
Sweat test for cystic fibrosis (CF) is based on the
elevated concentration of chlorides characterizing this
genetic disease. An indicator system for CF consists of
three filter paper strips impregnated with 0.1 M K2 CrO7 .
The dry strips are soaked in 0.05 N, 0.066 N and 0.1 N
AgNO3 solution, respectively. The indicator system is
attached to a suitable part of the body of the child
for 15 min. The indicator strip is removed and read.
A positive result is signaled by the decoloration of
the originally red-brown papers. Zero or one indicator
showing a positive reaction means that the sweat chloride
ion concentration is 35 equivalents per liter and the
child is considered normal. Children showing chloride
concentration of 35 – 55 equivalents per liter (two of
three papers are reacting positively) need to be checked
more thoroughly and concentrations of more than 55
equivalents per liter chloride (all three indicators reacting
positively) indicate a high probability of CF.
Urine leucocyte reagent strip (Boehringer-Mannheim,
Shiojiri) detects leucocytes in urine. The detection is
based on the esterase activity of the leucocytes. Upon
contact between the reagent matrix and leucocytes in
urine, the indoxyl ester substrate is hydrolyzed by the
esterase to indoxyl, which then couples with a diazonium
salt to produce a purple azo dye.
14
5.2 Forensic Application of Spot-test Analysis
Inexpensive screening by spot-test analysis has consider-
able importance, among other disciplines, in a modern
police laboratory,.3,6 – 9/ mainly as quick first orientation.
Only selected examples are described here.
5.2.1 Firearm Discharge Residue Tests
These search mainly for the presence of antimony, barium
nitrates, nitrites and lead as residues after shooting.
The three elements can be identified by direct test
on the cloth used to remove the residues. The cotton
cloth is dried and a drop of 10% alcoholic solution of
triphenylmethylarsonium iodide is added. In the presence
of antimony an orange ring develops within 2 min. After
drying a drop of freshly prepared saturated aqueous
solution of sodium rhodizonate is added. A red color
within the orange ring indicates the presence of barium,
lead or a combination of both. After repeated drying
the red area is spotted with 1 : 20 HCl. The appearance
of a blue spot indicates lead. A reddish-brown spot
turning vivid red confirms barium. Nitrate and nitrite
detection on the hand is achieved by removing traces
from the fingers by hot filter paraffin. After solidification,
diphenylamine reagent (10 mL concentrated H2 SO4 C
2 mL H2 O C 0.05 g diphenylamine) is added to the cast.
A positive reaction is shown by the appearance of dark-
blue particles. Although the reaction is not specific, it is
accepted as corroborative evidence for the firing of a gun.
Since the color of the blood and the red azo dye
produced by the classical Griess reaction for nitrite
are similar, nitrite is detected in this case by fluor-
escence under UV radiation. A Whatman No.1 filter
paper is soaked in freshly prepared 5% solution of o-
phenylenediamine dihydrochloride and partially dried.
This paper is placed over the suspected area of the gunshot
on the clothing. The paper is then placed in a Petri dish
containing a 25% NH3 solution The paper is illuminated
with 254 nm radiation. The yellow fluorescence pattern
may be photographed as permanent evidence.
5.2.2 Some Quick Field Tests for Drug Detection
As the problem of drug abuse reaches frightening
proportions, the use of simple screening tests are gaining
considerable importance beside the enormously useful
and sophisticated instrumental methods. Only some
representative examples are described here.
Morphine can be detected by an internally referenced
test strip immunoassay. The test strips comprise two active
surfaces, each of which contains co-immobilized glucose
oxidase and antibody against morphine; the color that
develops is inhibited by the presence of morphine in the
sample. The reference surface contains antiperoxidase
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This article was published in the Encyclopedia of Analytical Chemistry in 2006 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470027318.a8111
GENERAL ARTICLES
and it is used to set the assay detection limit. To
determine morphine in urine, the test strip is immersed
for 1 min in the sample, then for 10 min in a ‘‘developer’’
solution containing morphine-bound peroxidase, glucose
and 4-chloro-1-naphthol as a chromogenic substrate for
peroxidase.
Barbiturates in blood and urine samples may be
screened by thin-layer chromatography (TLC) on Silica
Gel G with CHCl3 – acetone (9 : 1) or benzene – acetone
(17 : 3) as mobile phase. Visualization is effected by spray-
ing with diphenylcarbazone – HgCl2 reagent followed by
1% methanolic KOH.
LSD (lysergic acid diethylamide) is identified by its
reaction with Ehrlich reagent (also referred to as Van
Urk reagent) producing a purple to deep-blue color.
Amphetamines in urine are sensitively (>0.1 µg)
detected by a strip prepared using the Simon reagent.
A 2% sodium nitroprusside and 5% aqueous Na2 CO3
solution is dried for 3 h at 110 ° C. A strip is cut from
the plate to use in the test. Five milliliters of alkaline
urine is extracted with 2 mL of benzene and a portion
of the benzene layer is spotted onto the strip. When
exposed to acetaldehyde vapor an intense blue color
results.
Marijuana and coca leaf in illicit cigarette samples
can be revealed by shaking the suspected material
with a chloroform solution of Fast Bordeaux Gp. salts
(C.I. Azoic Diazo Component 1) and with 0.5 M NaOH
solution. Cannabinoids are indicated by the appearance
of red color in the chloroform layer. The test is applicable
to urine samples too.
Salicylates in overdoses are commonly detected in
urine by the classical violet iron(III)-salicylate chelate
formation. 0.3 mL of blood is mixed with 0.6 mL of
methanol and centrifuged. The supernatant solution is
filtered and 50 µL of 0.5% FeCl3 solution is added to
0.2 mL of the filtrate. A violet color at the diffuse interface
indicates the presence of salicylate.
5.2.3 Chemical Testing for Blood
Chemical testing is needed to provide physical evi-
dence in cases in which direct visual inspection is not
a decisive proof. Chemical spot tests can verify the
presence or absence of blood stains, when blood spots
on a dark background are not visible or a stain may
resemble a bloodstain. In most tests the blood is iden-
tified on the basis of the peroxidase-like activity of
the heme group of hemoglobin. Free hydroxy ions
liberated by this activity oxidize colorless phenolph-
thalein (reduced phenolphthalein with metallic zinc in
basic solution). Addition of a drop of 3% H2 O2 solu-
tion produces a pink color in the presence of blood
traces.
SPOT TEST ANALYSIS
5.2.4 Detection of Semen
Rapid identification of semen is necessitated by the high
incidence of sex crimes. Semen is generally identified by
its high acid phosphatase content, which hydrolyzes aro-
matic orthophosphoric acid esters; the phenol resulting
from the enzyme-catalyzed reaction can be detected with
the Folin – Ciocalteu reagent.
The suspected stain is cut and incubated for 30 min
at 37 ° C with two drops of an 0.1% aqueous solu-
tion of disodium phenylphosphate and two drops of
Folin – Ciocalteu reagent is added. Phenol forms molyb-
denum blue on reaction with the molybdophosphate
incorporated in the reagent.
5.2.5 Detection of Saliva
Saliva occurrence may be related to spittle, or may be
detected on objects in contact with the mouth. Two-
thirds of the solid content (0.6%) is salivary amylase. This
enzyme, also called ptyalin or diastase hydrolyzes starch
to sugar or insoluble cross-linked blue starch polymer
to soluble blue fragments. A cutting from the suspected
saliva stain is placed in a tube with 1 mL distilled water.
Some drops of slurry are made by suspending a reagent
tablet in 5 mL water which contains the cross-linked starch
polymer (Pharmacia Laboratories, Inc.). After 30 min
incubation at 37 ° C the enzyme action is stopped by
adding 1 mL of 0.5 M NaOH solution and the mixture
is centrifuged. The appearance of a blue color in the
supernatant fluid indicates the presence of saliva.
5.3 Selected Geochemical Applications of Spot Tests
The aim of geochemical prospecting is to find new
deposits of metals and nonmetals and to detect crude
oil and natural gas accumulation. Chemical tests may
indicate the extension of existing deposits. Most of the
analytical methods used in geochemistry are instrumental,
but because a precision of 10% at the 95% confidence
level is good enough for geochemical purposes, at least
in the initial stages of the project, simple colorimetric
screening tests are still applied, which can be carried
out in the field by relatively unskilled operators..10/ Very
low capital cost is associated with these methods and the
operators can be trained in a relatively short time. Many
tests applicable to geochemical exploration are described
in Feigl and Anger.1/ and Jungreis..2/
In semiquantitative field tests calibrated sampling
spoons and plastic standards, which are prepared by
adding suitable dyes to a transparent polyester resin,
are used. These standards are far more stable than
the conventional liquid standards. No time-consuming
preparation of a standard series in the field is necessary
and the hardened plastic endures rough handling.
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DOI: 10.1002/9780470027318.a8111
15
5.3.1 Some Examples in Geochemical Screening
Differentiation between calcite and dolomite is based on
the different behaviors of the two carbonates towards
weak acids. Calcite is attacked by dilute acid at room
temperature. Some crystals of the sample material are
placed in a depression of a spot plate. One drop of
0.1% alizarin S solution in saturated tartaric acid solution
is added. Calcite is indicated by the appearance of a
purple-red precipitate. Dolomite does not react.
A spot test for Beryllium in minerals and ores is
based on the conversion of all beryllium compounds
to the soluble alkali beryllium fluoride, which reacts
with ammoniacal quinalizarine solution and forms a
blue-violet complex. The test is specific. The sample is
fused in a platinum spoon with an excess of KHF2 . The
residue is put in some cold water and centrifuged. A
drop of the supernatant fluid is treated with a drop of
0.05% quinalizarine solution in 10% NH3 . A few drops
of bromine water destroys the violet color of the excess
reagent.
A test for chromium in rocks is accomplished by
grinding a tiny particle to a fine powder in an agate
mortar and mixing with a four-fold excess of 1 : 1 mixture
of K2 CO3 and Na2 O2 . A red-hot platinum wire is dipped
in the mixture to allow bead formation. The cold bead is
dissolved in 1 : 1 H2 SO4 in the depression of a spot plate
and a drop of 1% alcoholic diphenylcarbazide solution
is added. A violet color appears even when the rocks
contain Cr in concentrations as low as 0.04%.
Lead in ores and minerals is directly estimated even
in the most water-insoluble ores with the rhodizonate
reaction. In the presence of Ba or Sr the sample must
be fumed with H2 SO4 beforehand. In a depression in
a spot plate a drop of pH 2.8 solution and a drop of
freshly prepared 0.2% reagent solution is added. A red-
violet color appears on the surface of lead-containing
ores. Galena, cerussite, anglesite, crocoite and all water-
insoluble lead ores give positive results.
Molybdenum in ores is identified extremely sensitively
by the specific color reaction with potassium xanthate. A
few grains of the ore in a small crucible are digested with
concentrated NaOH and acidified with H3 PO4 . Add a few
crystals of the reagent. A dark-violet colored compound
MoO3 Ð2[SC(SH)(OC2 H5 )] forms in the presence of Mo.
5.4 Rapid Selected Screening Tests for
Soil and Plant Tissues.3/
The simplification of complex soil chemical analy-
ses became necessary to make them performable by
minimally trained personnel. The screening of soil quality
is required in conjunction with testing of plant sap.
A test of soil aeration shows the oxygen concentration.
The soil is sampled with an open-faced sampler and
16
a CaCO3 suspension is added dropwise to its surface.
The extent of penetration of the white chalk particles is
proportional to the degree of aeration.
A test of soil pH is accomplished with a mixed
indicator of bromocresol green, bromocresol purple and
bromocresol red (0.05%, 0.1% and 0.02%, respectively.)
covering a pH range of 4.0 – 7.5 (Hellige Inc.) Some dried
soil grains are dispersed on a white spot plate and some
drops of the indicator mixture are added. Purified barite
mineral masks the color of the soil.
A test for exchangeable potassium is necessary to
decide whether additional potassium fertilizer is needed.
A portable soil test laboratory (Hach Co.) estimates K
content using powder pillows containing tetraphenylb-
orate, which precipitates the ion. Because heavy-metal
ions interfere with the test a second powder pillow is
included that contains the appropriate complex-binding
agents.
A rapid test for nitrate is accomplished by
adding a noncorrosive stable reagent tablet to the
soil extract. The tablet contains sulfanilamide, N-
(1-naphthyl)ethylenediaminedihydrochloride, zinc dust,
sulfosalicylic acid, boric acid and magnesium stearate.
Rapid reduction of nitrate to nitrite is accomplished
by formation of the stable complex between zinc and
sulfosalycilic acid. The color formed is compared with a
standardized color chart.
Some tests of the sap of plant tissue are very useful
as guides to the interpretation of the relative amounts of
nutrients taken up by the plant.
A screening test for phosphorus in plant tissue sap
is based upon production of the complex phospho-
molybdic acid with sodium molybdate and the reduction
of the heteropolyacid to molybdenum blue with a
tin rod.
The plant tissue is sliced with a sharp knife and a
filter paper is wetted with a plant juice. A drop of 0.1%
NH4 MoO4 solution in 0.1 N HCl is added, and a tin rod
is pressed to the spot for ca. 10 s. If no color appears,
the plant is very deficient in phosphorus, a blue color
indicates a deficiency, a medium color indicates a slight
deficiency in phosphorus, and the appearance of dark
blue signals that the plant is adequately supplied with
phosphorus.
A screening test for potassium in plant tissue is based
on indirect testing for potassium deficiency which is
feasible through detection of iron in the nodes of corn
stalks. It has been shown that the iron concentration
is inversely related to the total potassium in the leaf,
nodal and internodal tissue. The stalk is cut with a
sharp knife and some drops of KSCN are placed on
the nodal area followed by a drop of 6 N HCl. K
deficiency is indicated by the appearance of a red
color.
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DOI: 10.1002/9780470027318.a8111
GENERAL ARTICLES
5.5 Selected Examples of Applications of Spot
Tests in Air and Water Quality Control
To protect the worker’s well-being the concentrations
of air pollutants in industrial areas must not exceed
certain admissible levels. The simple estimation of carbon
monoxide in ambient air is carried out by sucking the air
sample through a glass tube containing a mixture of
iodine pentoxide and fuming H2 SO4 . The extent of green
coloration is the indication of CO concentration.
Glass-indicating tubes containing solid reagents are
produced and marketed by Drägerwerke AG, Bacharach
Industrial Instruments Co., Davis Engineering Co., Union
Industrial Equipment Corp., Mine Safety Appliances
Co, Acme Protection Equipment Corp. and so on.
The simple operating procedure for the detecting tubes
is as follows: the two sealed ends of the tube are
broken and a recommended air volume is drawn through
the tube with a calibrated bellows pump. The color
change produced in the tube is compared visually
against a set of standard colors. The length over
which the color change has occurred in the tube is
an alternative indication of the amount of airborne
contaminant..11/
Arsine has a maximum allowable concentration (MAC)
of 0.05 ppm. A typical detector tube for it includes a
precleansing layer of cupric ions, which precipitate the
interfering reducing gases such as H2 S, H2 Se and mercap-
tans. The indicator layer is auric gold, which oxidizes
the arsine to violet-gray metallic gold (Drägerwerke
AG(DW); Mine Safety Appliances Co. (MSA); Union
Industrial Equipment Corp. (UIE)).
Carbon monoxide detector tubes utilize the selenium
dioxide-catalyzed reaction of I2 O5 by CO in the presence
of fuming H2 SO4 . The interfering materials are selectively
oxidized by a precleansing layer ((DW), Bacharach Ind.
Instruments (BII)), MSA, UIE).
Chlorine is estimated by an indicator tube, in which the
chromogen is o-tolidine. It is oxidized by chlorine into a
full-quinoidal yellow compound (DW, MSA, UIE).
Hydrocyanic acid is measured in the indicator tube by
its complexation with Hg2C ion, and liberation of HCl
indicated by methyl red (DW, UIE).
Sulfur dioxide is estimated in the tubes by reaction
with free iodine decolorizing the blue iodine – starch
complex. The interference of H2 S is screened out with
a cupric compound-containing precleansing layer (DW,
MSA, UIE).
5.5.1 Water Quality Screening
Since the Safe Drinking Water Act passed by the US
Congress in 1974 the Environmental Protection Agency
(EPA) proposed a list of materials that present potential
hazards when present in drinking water. Recent water
SPOT TEST ANALYSIS
pollution controls rely heavily on advanced instrumental
methods, but spot tests based on simple kits for quick
and inexpensive water testing still find wide application
and are approved by EPA (Hach Co., Merck Co.,
Gallard-Schlesinger Chem. Manufacturing, Motte Chem.
Products Co., Taylor Chem. Co., Hellige Inc., etc.). In
the present limited space only a few of them can be
mentioned..3,12,13/
The chlorine content in drinking water may be
estimated by a stable tablet test. It contains equal parts
by weight of KI, potassium bitartrate and soluble starch.
Although Br2 , I2 , ozone and other oxidants react similarly
to chlorine in this test, their presence is unlikely in water.
The EPA accepts the use of the visual comparison method
using color-step cubes (Hach Co.) or continuous color
discs (Hach Co., Hellige Inc., Merck).
Chromium ions are suspected carcinogens. Their semi-
quantitative detection is based on their oxidation in
alkaline solution to chromate and subsequent reaction
of the latter with diphenylcarbazide. On-the-spot esti-
mation kits use comparator blocks (Merck), calibrated
continuous color discs and color cubes (Hack Co., Hel-
lige Inc.).
Cyanide ions are estimated by the demasking of the red-
violet mercury(II) dimethylaminobenzylidenerhodamine
complex. An indicator stick based on this reaction is on
the commercial market (MINTEK, Council for Mineral
Technology, Randburg, South Africa).
Nitrate nitrogen in drinking water may be measured
semiquantitatively on test strips (Merck). The test is
based on diazotization with an aromatic amine of nitrous
acid produced in situ in the reaction zone by a reducing
agent and coupled with N(1-naphthyl) ethylenediamine.
Organophosphorus compounds are detected by a field
test in water based on an enzyme reaction, namely on
the inhibition of cholinesterase activity. Soman (GD)
(0.12 µg L 1 ), sarin (GB) (9.9 µg L 1 ) and tabun (GA)
(26 µg L 1 ) are detected, whereas the detection limit of
the pesticide dichlorvos is 50 µg L 1 .
Total hardness of water is evaluated by test strips
based on the complexing reaction between the Ca2C
and Mg2C and ethylenediaminetetraacetic acid (EDTA)
disodium salt. The test strip has four zones of gradually
increasing reagent content. It is dipped into the water
sample and the water hardness is given by the number of
zones changing color from green to red-violet (E. Merck,
Macherey-Nagel, Ames Co.).
5.6 Food Quality Control Analysis by Spot Test.3,14/
The partial substitution of one product for another
in which the substituted material is inferior to the
original product is defined as adulteration. Mineral oil
is sometimes substituted for vegetable oil and saccharin is
Encyclopedia of Analytical Chemistry, Online © 2006 John Wiley & Sons, Ltd.
This article is © 2006 John Wiley & Sons, Ltd.
This article was published in the Encyclopedia of Analytical Chemistry in 2006 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470027318.a8111
17
sometimes used instead of sugar. Starch is sometimes used
to extend powdered cocoa, and butter can be replaced by
margarine.
One of the most common food adulterants is water.
Some of the proven toxic materials can be detected by
simple screening tests, although most of the analytical
procedures used are of a complex nature. A few examples
of quick tests are described here.
Saccharin in food may be detected by conversion into
phenolsulfonphthalein. The aqueous extract of the food
is treated with a few drops of H2 SO4 and a few grains of
phenol and heated for 10 min at 180 ° C. After cooling, it is
neutralized with solid Na2 CO3 . The presence of saccharin
is indicated by the appearance of a red color.
Artificial sweeteners (saccharin, cyclamate, dulcin and
P-4000) can be separated and identified by TLC on silica
gel using a solvent system of butanol, ethanol, ammonia
and water. Saccharin is detected as a fluorescent spot
under shortwave UV radiation. Successive spraying with
solutions of 5% bromine in DMF– alcohol (1 : 1) and
2% N(1-naphthyl)ethylenediamine dihydrochloride in
alcohol reveals cyclamate as a bright pink spot, dulcin as
a brownish or blue spot and P-4000 as a brown-pink spot.
Objectionable food preservatives such as H2 O2 , salicylic
acid, boric acid, borates, formaldehyde and sulfites may
be screened using simple spot tests.
To test for hydrogen peroxide, equal volumes of milk
sample and 1% KI solution dissolved in 2% starch paste
are mixed. The presence of peroxides is indicated by the
appearance of a blue color.
To test for salicylic acid, 50 mL of the sample or a small
volume of the aqueous extract is acidified with 5 mL dilute
(1 : 3) HCl solution in a separation funnel and extracted
with diethyl ether. After evaporation of the ether, a drop
of 0.5% neutral FeCl3 solution is added. Violet ferric
salicylate indicates the presence of salicylic acid.
Boric acid produces a deep-red compound when
evaporated with tincture of curcuma (turmeric), changing
yellow diferuloylmethane into the isomeric red-brown
rosocyanin. It turns blue to greenish-black under alkaline
conditions. The test is specific for boric acid.
Formaldehyde is extracted from food samples with
water and after acidifying with phosphoric acid, the slurry
is distilled. One milliliter of the distillate is treated with
5 mL of 0.5% chromotropic acid dissolved in 72% H2 SO4 .
The tube is heated for 10 min in a boiling water bath. In the
presence of formaldehyde, a bright-violet color appears.
The presence of sulfur dioxide or sulfite as preservatives
is allowed in dehydrated fruits and vegetables, but their
addition is prohibited to meats. A test strip for rapid
identification of sulfite was prepared from a mixture
of orange I (C.I. Orange 20), brilliant green (C.I. Basic
Green 1), sodium bicarbonate and alumina. The black
strip in contact with the moist sample of food will turn
18 GENERAL ARTICLES

red within 15 s in the presence of ½5 µg sulfite and green QC Quality Control

in its absence. TLC Thin-layer Chromatography
UV Ultraviolet
5.6.1 Tests for Mammalian Feces and Urine
Residues in Food
Food-processing plants and warehouses are subject to RELATED ARTICLES
occasional invasion of rats and mice. The detection
of rodent-contaminated lots, urine-contaminated grains, Carbohydrate Analysis (Volume 1)
and mammalian feces in foods is a crucial task for law Disaccharide, Oligosaccharide and Polysaccharide Ana-
enforcement officials. lysis ž Monosaccharides and Sugar Alcohol Analysis
The spot test for mammalian feces is based on the
fact that the alkaline phosphate isoenzyme present in Chemical Weapons Chemicals Analysis (Volume 1)
the feces at certain pH splits phosphate radical from Detection and Screening of Chemicals Related to the
phenolphthalein diphosphate to produce the reddish free Chemical Weapons Convention
phenolphthalein. A pH 9.5 borate – carbonate reaction
buffer optimizes the response of the enzyme. Clinical Chemistry (Volume 2)
Mammalian-urine contamination in food is detected Clinical Chemistry: Introduction ž DNA Arrays: Prepa-
by strips (Warner-Chilcott) in which the lowest part is ration and Application ž Drugs of Abuse, Analysis of
impregnated with phosphate-buffered urease and the next ž Glucose Measurement ž Phosphorescence, Fluores-
band contains K2 CO3 . This area is bordered by a plastic cence, and Chemiluminescence in Clinical Chemistry ž
barrier. Above the barrier is the indicator area containing Urinalysis and Other Bodily Fluids
bromocresol green.
Adulteration of milk by addition of (NH4 )2 SO4 Environment: Water and Waste (Volume 4)
increases both the nonfat solid content and the nitrogen Soil Instrumental Methods
content as determined by the Kjeldahl procedure. One
milliliter of milk is treated with 0.5 mL each of 2% NaOH Food (Volume 5)
and 2% NaOCl and an aqueous 5% phenol solution. Food Analysis Techniques: Introduction ž Atomic Spec-
The mixture is heated in a boiling water bath for 20 s. A troscopy in Food Analysis ž Water Determination in
rapidly deepening blue color appears in the presence of Food
(NH4 )2 SO4 .
To test for differentiation of synthetic from natural Forensic Science (Volume 5)
vinegar, 10 mL of the sample is mixed with 1 mL Forensic Science: Introduction
concentrated H3 PO4 and 1 mL 3% KMnO4 solution.
After 5 min the KMnO4 color changes for natural but Pharmaceuticals and Drugs (Volume 8)
not for synthetic vinegar. The solution is then mixed with Alkaloids, Pharmaceutical Analysis of
1 mL saturated oxalic acid, 1 mL 10% H2 SO4 and 1 mL
Schiff’s reagent (0.1% magenta, 1% anhydrous Na2 SO3
in 0.1 N HCl). After 5 min natural vinegar gives a violet REFERENCES
color reaction, but synthetic vinegar does not.
1. F. Feigl, V. Anger, Spot Tests in Inorganic Analysis, 6th
edition, Elsevier, London, 1972.
2. F. Feigl, Spot Tests in Organic Analysis, 7th edition,
ABBREVIATIONS AND ACRONYMS
Elsevier, Amsterdam, 1966.
3. E. Jungreis, Spot Test Analysis, Clinical, Environmental,
CF Cystic Fibrosis Forensic and Geochemical Applications, 2nd edition, John
DMF Dimethylformamide Wiley & Sons, New York, 1997.
EDTA Ethylenediaminetetraacetic Acid 4. E. Jungreis, L. Ben-Dor: Organic Spot Test Analysis,
EPA Environmental Protection Agency Comprehensive Analytical Chemistry, ed. G. Svehla, Else-
GA Tabun vier, Amsterdam, Vol. X, Chap. 1, 1980.
GB Sarin 5. L. Gershenfeld, Urine and Urinalysis, 3rd edition, Saun-
GD Soman ders, Philadelphia, 1948.
IR Infrared 6. E. Jungreis, in Spot Test Analysis – Inorganic Ions, Ency-
LSD Lysergic Acid Diethylamide clopedia of Analytical Science, Academic Press, London,
MAC Maximum Allowable Concentration 4742 – 4748, 1995.

Encyclopedia of Analytical Chemistry, Online © 2006 John Wiley & Sons, Ltd.
This article is © 2006 John Wiley & Sons, Ltd.
This article was published in the Encyclopedia of Analytical Chemistry in 2006 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470027318.a8111
SPOT TEST ANALYSIS 19
7. E. Jungreis, ‘Organic Compounds’, in Encyclopedia of 11. Detector Tube Handbook: Air Investigations and Techni-
Analytical Science, Academic Press, London, 4748 – 4755, cal Gas Analysis, Dräger Tubes, 4th edition, A.G. Dräger-
1995. werk, Lübeck, Germany, 1979.
8. E. Jungreis, ‘Applications’, in Encyclopedia of Analytical 12. M.J. Suess, Examination of Water for Pollution Control,
Science, Academic Press, London, 4755 – 4760, 1995. Pergamon Press, New York, Vol. 2, 1982.
9. P.L Kirk, Crime Investigation, 2nd edition, Wiley, New 13. APHA Standard Methods for the Examination of Water
York, 1974. and Wastewater, 14th edition, American Public Health
10. R.E. Stanton, Rapid Methods of Trace Analysis for Association, Washington, DC, 1976.
Geochemical Applications, Edward Arnold, London, 14. F.L. Hart, H.J. Fisher, Modern Food Analysis, Springer
1966. Verlag, Berlin, 1971.

Encyclopedia of Analytical Chemistry, Online © 2006 John Wiley & Sons, Ltd.
This article is © 2006 John Wiley & Sons, Ltd.
This article was published in the Encyclopedia of Analytical Chemistry in 2006 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470027318.a8111