Southwestern University

College of Medical Technology

Analytical Chemistry
Compiled by:

JULIUS P. MARIO, MS
Asst. Professor
College of Medical Technology

INTRODUCTION TO THE LABORATORY

The following are the objectives of the laboratory work:
1. To provide opportunity for the study of the physical and chemical
properties of the more common compounds and their solutions.
2. To show how these properties and principles of chemistry can be
combined into a systematic scheme for analysis; and
3. To give the student experience in laboratory technique.
Before starting with any work in the laboratory, the student should know
the meaning of the different terms that are usually encountered in
qualitative and quantitative analysis. Below are the different terms and
their corresponding meanings:
1. Semimicro - scale between the macro and micro,
2. Macro - large scale.
3. Micro - small scale
4. Reaction - any process in which a new chemical substance is formed.
5. Reagent - a substance used to bring about a reaction
6. Precipitate - any insoluble substance produced in a solution.
7. Residue - that portion of a solid remaining after partial solution by a
reagent.
8. Clear solution — one that is not turbid or cloudy.
9. Colorless solution - one that is not colored. Do not use “clear” to
mean “colorless”, for example potassium chromate solution is clear
and colored (yellow).
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10. Filtration and Centrifugation - methods for separating a solid from a

liquid in which it is suspended.
11. Test - a laboratory operation designed to show the presence or
absence of some substances, usually some ionic species.
12. Positive test - may be indicated by (1) formation of a precipitate of
distinctive color or texture, (2) the dissolution of a precipitate, (3) the
appearance or disappearance of a color in a solution or in a flame, (4)
the evolution of a gas, or (5) the development of a characteristic odor
13. Sensitive test - the positive test given by the smallest amount or
concentration of a substance
14. Specific test - the test given by one and only one substance
15. Confirmatory test - When a test is nonspecific in character,
additional evidence is frequently sought as final proof of the presence
of the ion.
16. Blank - consist of all the reagents present and otherwise treated
exactly as the test. It does not contain the ion or substance tested.
17. Control – a known sample with the ion under consideration has
already been added.
18. Test solutions - contains the ion or substance for which tests are
made, that is, substances that may occur in the unknown. Tests
solutions are used in the preliminary experiments as a source of the
ions being studied.

Housekeeping
1. Clean up areas around the analytical balances and the reagent
weighing balances immediately after use.
2. Wipe up spills immediately.
3. You are responsible for the cleanliness of the whole lab as well as your
assigned working areas.
4. Certain aqueous solutions may be discarded down the drain, however,
common sense dictates that volatiles (e.g. HCI, ammonia, etc.) be
poured down drains ONLY IN THE HOODS!!
General Rules
1. No eating or drinking in the laboratory.
2. Those not taking the class have no business in the room. Conduct
personal business outside the laboratory.
3. Immediately report to the instructor reagents that are in short supply.
Safety Rules
1. Know the location and use of: fire extinguisher, fire blanket, fire exits,
safety shower, eyewash fountain, spill kits and first aid kit.
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2. Goggles or glasses must be worn in the lab at all times to protect your
eyes from chemicals. Contact lenses are not advisable.
3. Wear sensible clothing. No shorts, short skirts, open-toe shoes or
sandals allowed. Long hair should be tied back. Wear protective
clothing while handling dangerous substances.
4. Do not eat or drink in the laboratory.
5. Prevent cuts and burns. Discard broken or chipped glassware. Be
aware and keep your neighbors aware of hot objects. Heat materials
only in beakers, flasks and porcelain wares; never heat graduated
cylinders, burettes, pipettes or watch glasses.
6. Report immediately any accident, no matter how minor, to the lab
assistant or instructor.
7. Be cautious of others’ activities as well as your own.
8. Avoid flammable solvents near flames. Most solvents other than water
are flammable.
9. Never pipette by mouth. Use a pipetting bulb to pipette all liquids.
10. Do not conduct unauthorized experiments.
11. Do not work alone in the laboratory.
12. Follow all safety precautions given by instructor or lab assistants in
pre lab or during laboratory session. If you miss pre-lab, check with
an instructor before you start the experiment.
13. Keep all toxic and volatile materials in the fume hood.
14. Label all containers.
15. Clean up right after you finish your work.

Apparatus to Be Constructed and Assembled

The students may be asked to construct a few of the simpler pieces of
apparatus such as washbottles, stirring rods, droppers, and capillary
syringes.
Washbottle. The washbottle is used to store and dispense distilled water
for rinsing apparatus, diluting solutions, and washing and transferring
precipitates. A 500-ml (16-oz) bottle is the most popular. Many prefer a
bottle of half that capacity. The traditional breath-powered washbottle is
rapidly being replaced by the polyethylene washbottle that delivers liquid
when squeezed. The “squeeze bottle” has the advantages of being
unbreakable and easy to operate, but it cannot be heated. The figure
above shows a modified delivery tube that has advantages for certain
operations, particularly for transferring solids. The student may
construct a delivery tube of this kind; he will need a one-hole stopper
that fits the bottle, 6-mm glass tubing, and a short piece of rubber
tubing.
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To prepare a glass washbottle, obtain a 500-ml or 250-ml flask and fit it
with a two-hole stopper. Cut — lay the tubing flat on the bench, make a
single deep scratch with the edge of a triangular file, hold the glass with
the opposite scratch, and bend outward from the scratch — a length of
6-mm glass tubing somewhat longer than required for the mouthpiece,
another for the delivery tube, and a 6-in, length for nozzle. For making
the bends, heat an extended portion of the tubing about 2 in. in a
horizontal position in the flame of a burner equipped with wing-top.
Continue the heating with rotation between fingers until the glass is soft
enough to bend from its own weight when the support is taken from one
end. Remove the glass from the flame and bend it to the desired angle.
The interior angles should be about 450 for the delivery tube and 135° for
the mouthpiece, so that when assembled the upper end of the
mouthpiece is parallel to the portion of the delivery tube that carries the
nozzle. To make the nozzles, heat the six-inch length of glass tubing
near the middle in a Bunsen flame (without spreader) until quite soft,
remove it from the flame, and then draw it out. Trim all pieces of glass to
the desired length and then fire polish (heat to the softening point) the
ends of the tubes to round the sharp edges.
After the fire polished tubes have cooled, insert first the delivery tube and
then the mouthpiece through the stopper. Be careful. Lubricate the
inside of the stopper and the end of the tube to be inserted with water,
alcohol, soap solution, or other wetting agent. Grasp the tube near the
end that is to be inserted into the stopper. Push the tube through the
hole until it protrudes about two inches. Then grasp the end of the tube
and pull it into position. Attach the nozzle to the delivery tube with a
short length of rubber tubing.
Finally assemble the washbottle and fill it half-full of water. Test for leaks
by blowing into the mouthpiece and observing whether the water
maintains its position in the delivery tube when placed over the end of
the mouthpiece. When lull pressure is applied, the nozzle should deliver
a fine, even, unscattered stream of water.
Stirring rods. Stirring rods should be long enough to be used in hot
solutions without danger to the fingers, yet not long enough to make the
container top-heavy or easily knocked over. A rod that extends about 3 to
5 cm beyond the rim of the vessel in which it rests meets these
conditions. Make several 10-cm rods for use in small beakers, a number
of 14 cm rods for use in 13- x 100-mm test tubes, and an 18 cm rod for
use in large tubes, Make the 10-cm and 14-cm rod from 3-4-mm thick
glass rod and the 18-cm from 6-mm thick glass rod. Fire polish each end
in the flame. Cone-shaped and flattened stirring rods about 10 mm
across the side part are useful in 13- x 100-mm test tubes. Stirring rods
made from glass tubing by melting both ends shut are lighter than those
 5
made from glass rod. To close the end of a glass tube, hold the piece of
tubing nearly vertical with the tip in the edge of the flame while rotating
it between the fingers.
Droppers and capillary syringes. Droppers, or dropping pipets, may be
purchased or may be constructed from glass tubing. Uniform,
machine-made droppers, either 3 or 4 in. in length of glass may be
purchased in gross lots at nominal cost. The droppers with 4-in, glass
bodies are far superior to the usual 3-in. “medicine dropper” for they add
a full milliliter of solution without danger of getting the liquid into the
rubber bulb.
Dropping pipets of any size are easily constructed from glass tubing. Cut
lengths of 6-8 mm diameter soft glass tubing about twice the length of
the desired pipets. Heat the middle of the tube uniform in the hottest
part of the Bunsen flame. Hold the tubing both ends and rotate it slowly.
When the glass is soft, remove it from the flame and pull — slowly at first
and then more rapidly — as it cools. Trim the pipets to the desired length
by scratching with a file and breaking off the excess glass. Heat the
broad end until the glass softens, and then press the hot end down on an
asbestos pad, soapstone desk top, or other heat resistant surface to form
a flange to hold the rubber bulb.
Capillary syringes, useful for washing small quantifies of precipitate with
a fine stream of water, can be made by slightly modifying the procedure
used for constructing the dropping pipet. If a narrower band of glass is
heated somewhat hotter and is drawn out more quickly, the finer
opening characteristic of the capillary syringe is obtained.
To prepare a capillary syringe from a dropper, first heat — seal a short
length of glass tubing or rod to the tip. The extra piece of tubing or rod
serves as a handle. While rotating the dropper and handle with both
hands, heat the dropper side of the seal in the cooler part of the flame
until the glass just softens. Remove from the flame and pull to give a fine
point. Break the capillary to leave a small orifice. The tip should be so
small that about 15 sec are required to dispense an ml from the syringe
and the water is delivered in a very fine stream. If the orifice is too small
break off a short length to obtain the desired opening.
Since the laboratory procedures frequently call for the addition of 1 ml or
2 ml of water or of reagent, it is convenient to have several droppers
calibrated to contain 1 ml. To calibrate a dropper, fill the 10-mI
graduated cylinder to the 10-ml mark with water. Withdraw water into
the dropper until the water level in the cylinder falls to the mark. With
the edge of a triangular me make a scratch on the dropper at the bottom
of the meniscus.
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Calibration of test tubes. Test tubes calibrated at milliliter intervals to 5

ml are timesavers. To calibrate, add water 1 ml at a time from a
calibrated dropper or pipet and mark the glass at the bottom of the
meniscus alter each addition. Reasonably satisfactory marks can be
made with corner of a triangular file. A small carborundum disc in the
hand piece of a flexible shaft driven by an electric motor make fine lines.
A small aluminum oxide sphere on a shaft and rotated at above can be
used to number the lines. Ballpoint pens that use an enamel writing
fluid (“Marktech”) make semi-permanent lines and numbers without
weakening the glass by scratching it. After calibrating one test tube
carefully, hold a piece of heavy paper against the tube and mark the
paper at milliliter intervals. Then use the marked paper as a template to
mark additional tubes. Finally, add water in 1-ml portions to make
certain that the lines on the tubes are correctly spaced.
Water bath. For heating liquids in test tubes, a water bath is usually
superior to an open flame, where the heating is prolonged, as in
hydrolyzing thioacetamide, a water bath is a necessity. Either each
student may have his own small water bath or there may be a number of
larger baths distributed about the room. A 150-ml beaker half full of
water serves as a satisfactory individual water bath. A microburner will
keep this size bath boiling slowly. A few boiling stones in the bath will
prevent bumping. If a cover is desired to reduce loss of water by
evaporation, make one from a square of heavy aluminum foil, of thin
copper sheet, or even lead foil. With a cork borer, cut several holes to
insert test tubes. Then crimp the corners of the square over the rim of
the beaker.
A 600-ml beaker fitted with a stainless steel rack accommodating ten 13-
x 100-mm test tubes and heated on a small hot plate with variable
temperature control, makes an excellent bath for use by a group of four
to eight students. A few of these larger baths at strategic locations may
prove more convenient than individual water baths.
Wires for flame tests. Obtain two 10-cm lengths of heavy (#18) Chromel
wire. Also obtain two corks, preferably of the size that fit test tubes
suitable for storing the wires as shown below. At one end of each wire
bend a loop about 5-mm in diameter. The loop should be closed; any gap
prevents filling the loop with solution. The loop is most easily formed
using a pair of needle-nosed pliers, though any pliers can be used. Insert
the other end of the wire through the cork which serves as a handle
while the loop is the flame.
Other apparatus needed
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BALANCES
Top-pan balances are used to measure larger masses of substances
where exact masses are not necessary. For example if we need to add
1.00 gram of a chemical into a solution we should use a top-pan balance.
These balances usually measure to 0.01 gram. Weighing by addition is
the preferred technique when using a top-pan balance. In this technique
you weigh and record the mass of the container and then add the
chemical to the container. The difference in mass of the container with
chemical and the empty container (final weight - initial weight) gives the
mass of the substance. An option is to place the container on the top-pan
balance and press the tare button. This will give a mass of zero for the
container. When a substance is finally added the balance display will
then directly give the mass of the substance.
Analytical balances are used when an accurately known mass of
substance is required. For example, if we are preparing a standard
solution of an analyte for use in determining the concentration of that
analyte in a sample. We would want accurate results in our
determination and so we would need an accurate reference or standard
solution. This is what is known as calibration. Weighing by difference
is the technique of choice, In the technique of weighing by difference, a
person would weigh a container (weighing bottle) that contains the dry
substance. An amount of the sample in the weighing bottle would be
poured into a flask. The original weight of the weighing bottle and sample
minus the final weight would give the mass of sample transferred to the
flask. When doing this the amount of substance poured into the flask
must be estimated. It could take more than one transfer to get enough of
the substance into the flask. On the other hand, if too much is
transferred then either you throw out and clean the flask and start over
or you accept the mass of substance transferred. Usually when you
weigh by difference you are not looking to add an exact amount. We can
often accept some deviation from the amount the procedure requires, as
long as it not a large deviation.
There are a number of practical issues that you will need to keep in mind
when weighing chemical substances. They are as follows:
a. Adsorption of moisture from the atmosphere. Most samples are
dried in an oven at a given temperature and for a given amount of
time. The sample is placed in a weighing bottle. The weighing bottle
and lid are placed in a beaker covered by a watch glass. The watch
glass should be raised above the beaker by some means. The dried
samples are then stored in a desiccator to keep them dry. Weighing
bottles (with ground glass lids) are used to contain the sample during
drying and storage. The sample in the weighing bottle can then be
 8
removed for periods of time for weighing out the sample. The lid
should remain on the weighing bottle when not transferring sample.
Great care must be taken with some substances that are highly
hygroscopic (adsorb water quickly from the atmosphere).
b. Static electricity occurs on days where the humidity is very low. The
glass weighing bottle can become electrostatically charged and when
this happens it can be attracted to different metal parts of the balance
causing an inaccurate weighing. Wiping the weighing bottle with a
damp (not wet) chamois cloth can be helpful or possibly damping your
hands and drying them. Your slightly damp hands could reduce this
electrostatic charging. Be careful not to wet the weighing bottle.
c. Temperature differences can cause inaccurate weighing. Always allow
your sample to remain in your desiccator until the weighing bottle
and sample are at room temperature. If a warm object is placed on a
balance it will weigh too low. The reason for this is that the warm
object warms the air around it, which rises. The rising air causes a lift
to be felt by the balance pan. Why do blizzards and eagles find certain
places to circle in the sky? Warm air rising! Most objects heated to
110°C will take at least 30 minutes to cool to room temperature. This
time will depend upon how hot the oven is and how many hot objects
you have placed in the desiccator.
d. Fingerprints are another source of inaccurate weighing. A greasy
fingerprint can weigh as much as 0.5 mg. The best way to avoid
fingerprints is to hold the weighing bottle using finger cots (cloth
thimbles) or paper towel. You can form a paper loop using a Kim
Wipe. Fold a Kim Wipe lengthwise three times. You can then loop this
around your weighing bottle and holding only the paper lift of the
weighing bottle without touching it.
e. Buoyancy corrections will be mentioned when calibrating your
glassware in the first experiment.
f. Balance tables should be used with analytical balances. Our
analytical balances are placed on heavy marble tables, which tend to
dampen vibrations. You should not move around quickly in the
balance room or lean on the balance table.
VOLUMETRIC GLASSWARE
1. Volumetric flasks are containers that have been made to contain
(TC) an accurate volume of a liquid as long as it is filled to the mark
on the neck of the flask. These flasks have a number of markings on
them to indicate how they are used, what accuracy to expect, and
 9
what temperature is required for the most accurate work. On a
volumetric flask you will see a TC. This indicates that the flask is used
‘to contain its volume. You cannot fill it to its mark and then pour it
into another container and expect to transfer an accurate volume of
that liquid. Volumetric flasks are used to prepare solutions of
substances to accurate volumes. Either samples containing an
analyte or for the preparation of a standard solution of a reagent or
the analyte. Standard solutions can be used to determine
concentrations of the analyte in a sample. A standard solution is a
solution containing an analyte or reagent whose concentration is
accurately known. Each volumetric flask will also have a tolerance
value. The manufacturer expects each measurement to be within that
tolerance.
2. Transfer pipets are used to transfer accurately known volumes of a
solution. If 15.00 mL of a solution needs to be transferred to a
container then we would use a transfer pipet. Just as with volumetric
flasks, transfer pipets also have markings giving information about
how they are used and how accurate we expect them to be. Transfer
pipets have a to deliver (TD) design. This indicates, “to deliver”. They
each will also have a temperature and a tolerance value.
3. Burets are used in an analytical technique called a titration. In a
titration, a standard solution of a reagent (a solution with an
accurately known concentration of the reagent) is added slowly to a
sample containing the analyte until we have added a stoichiometric
amount of the reagent. How much reagent is necessary is determined
by the balanced chemical equation for the reaction that occurred. For
example HCI reacts with NaOH in a 1 is to 1 stoichiometric ratio, if we
were titrating the HCI with NaOH then we would slowly add the NaOH
to the HCI until we have an equal number of moles of each. Titrations
are a very common and accurate technique for determining
concentrations of analytes in samples.
4. Beakers, Erlenmeyer flasks, graduated cylinders and other
glasswares are not volumetric and should not be used when accurate
volumes and measurements are required. They might be used to
make up solutions whose concentrations only require one or two
significant figures.
5. Cleaning glasswares is a very important part of analytical chemistry.
Dirty glassware can give inaccurate results. Always clean glassware
before using it. A useful test of the cleanliness of a piece of glassware
is that water should drain evenly from the surface without the
formation of droplets. Droplets indicate dirty spots. When cleaning
glassware always begin with soapy water and scrub thoroughly. Rinse
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with tap water and then with de-ionized water. Before use, cleaned
glassware must be rinsed with deionized water at least three times. If
soapy water does not clean your glassware then we will try acid
cleaning solutions and if that does not work then we would use a
strong base solution. Check with your instructor before trying these
last two cleaning methods.
6. Proper use of volumetric glasswares such as pipettes and burettes
cannot begin without rinsing the glassware first with at least three
small portions of the solution being used. This removes any previous
rinsing solution and relieves us of having to dry our glasswares before
use.
7. Volumetric glasswares must be calibrated before use.

General Manipulative Procedures

Cleaning glassware. All reaction vessels should be thoroughly cleaned
before they are used. Even traces of contaminants in any test may give
rise to spurious results.
Detergent (Alconox, Sparkleen, or other laboratory preparation, or even
a kitchen detergent) solution applied with vigorous brushing and
followed by rinsing with tap and distilled water will clean most
glasswares. Pipet cleaners serve as brushes for loosening particles in
droppers and other small tubes.
Chemical reagents should be used for cleaning when brushing with a
detergent is ineffective or when the shape of the vessel does not permit
use of a brush. The chemical properties of the substance to be
removed should govern the selection of the solvent. Cadmium sulfide
dissolves in either 6 M HCI or 6 M HNO3. The HC1 is to be preferred for
HNO3 oxidizes the precipitate and converted to free sulfur which must be
removed further. Copper sulfide is soluble in HNO3 but not in HC1,
hence one must tolerate the precipitation of sulfur. Mercuric sulfide
requires a mixture of HC1 and HNO3 for solution. Silver chloride is not
soluble in any of the common acids but dissolves readily in aqueous
ammonia. Manganese dioxide is insoluble in nitric acid and only slowly
soluble in hydrochloric acid. However, the addition of a small amount of
hydrogen peroxide results in the rapid solution of the Mn02. Even sulfur
can be dissolved if the correct reagent — ammonium or sodium sulfide
solution — is used.
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Making Observation. Careful observation is a prerequisite to
trustworthy interpretation. Small white precipitates are occasionally
overlooked, particularly when the lighting is poor. A slight turbidity
in a solution is often ignored before a precipitating agent is added, yet
afterwards is called a precipitate. Obviously, a solution should be
examined carefully before the addition of a reagent as well as afterwards.
Overhead lighting is less satisfactory than a desk light for examining
solutions in test tubes. A 60-100W incandescent bulb with a milk-white
plastic or glass bowl for a shade mounted at eye level can help in
reducing observational errors. A white precipitate or turbidity is most
easily observed when the tube touches the shade just above the level of
the liquid in the tube. A colored precipitate shows up strikingly when the
tube is held against the white bowl. Tube positions for optimum lighting
are illustrated below.
A satisfactory lamp may be assembled from a ring stand or other vertical
rod, a utility clamp, an extension cord with a standard lamp locker, a
lamp, and an opalescent shade of either plastic or glass. Some plastic
shades screw onto the socket; others require a brass adapter. As long as
the bulb is at approximately eye level almost any wall lamp, gooseneck
study lamp, or table lamp fitted with a plastic, glass, or even white
parchment opalescent shade can be used to advantage.
Measurement of reagents. In semimicro analysis the volume of liquids
are measured in milliliters (ml) or in drops. The milliliter is
one-thousandth part of a liter, the defined unit of volume. Use of the
term cubic centimeter (cc) as a unit of volume is discouraged by the
National Bureau of Standards. A standard drop is 0.05 ml; hence,
there are 20 standard drops to a milliliter. In practice the volume of
a drop varies with the size of the tip of the dropper, with the angle at
which the barrel is held, and with certain properties of the liquid being
delivered. To get drops of more uniform size and to prevent the
chemical solution from getting into the rubber bulb, always hold a
dropper in a vertical or near vertical position. Liquids with high
specific gravity and/or low surface tension form smaller than average
drops. Except where the quantity of reagent is critical, variations in
drops size are usually ignored.
The quantity of solids is measured by weighing. A triple beam balance
sensitive to 0.01 g (10 mg) is usually satisfactory in qualitative work.
Except when an exact quantity of solid is important, the weight may be
estimated. A nickel weighs 5 grams and a dime weighs 2.5 grams. A
grain of rice weighs about 20 mg. Water weighs 1 gram per milliliter or
0.05 gram (50 mg) per drop. Since the density of most solids is two to
 12
three times that of water, an amount of solid the size of a standard drop
of water weighs 0.1 gram.
Identification of vessels. When several test tubes or other vessels are
used in the same experiment, or when solutions are to be saved from one
laboratory period to the next, each should carry a label or other
identification. Do not try to carry in your head information that is
more reliably retained in writing on the container. Gummed labels
are easy to attach but are a nuisance to soak off. Wax pencils make
satisfactory marks on glass provided the pencil is not too old and the
glass is clean and dry. The enamel markings of the “Marktech” pen are
long lasting but require organic solvent (ethyl acetate or acetone) for
removal. Many vessels have sandblasted or enameled spot which is
readily marked with a soft lead pencil. Even test tubes are now available
with the white enamel marking spot.
Mixing solutions. Because of the narrow bore of test tubes used in
semimicro methods of analysis, the mixing of a reagent with a solution is
a tedious operation. Nevertheless thorough mixing should precede the
drawing of any conclusions. Ideally each drop of reagent added should be
mixed with the solution before the next drop is added. The student
should observe the effect of each drop of reagent as it is added and
mixed into the solution. Attention has already been called to the need
for careful examination of the solution both before and after addition of
a reagent.
When a test tube is less than half full, mixing can usually be
accomplished by shaking or flicking the tube. If the tube is over half full
it may be necessary to pour the liquid into a second vessel and then
return it to the test tube or to suck up a portion of the solution in a
dropper and expel it for several cycles. As the liquid becomes deeper
in a test tube stirring with a glass rod becomes less efficient. A
combination of circular and vertical motion with the rod gives more rapid
stirring than circular motion alone. Shaking a tube closed with a cork or
finger results in thorough mixing of the contents, but danger of
contamination of the solution form the cork and of chemical injury to the
finger makes this technique impractical.
Heating solutions. All solutions have to be heated carefully to avoid
bumping and spattering. The solution ejected from the tube by bumping
may give rise to painful burns. The loss of liquid may impair the
analysis.
Solutions contained in a 10-ml (13-x 100-mm) test tube and occupying
one-third of the capacity of the tube or less may be heated directly in the
flame of a micro-burner or in a small flame of a Bunsen burner. The test
 13
tube may be held in the fingers (unless boiling is to be prolonged), with a
yoke made from a strip of paper, or with a wire test tube holder. Point
the mouth of the tube away from yourself or any other person near
by. Bumping may be avoided by slow, careful heating along the side of
the tube near the top of the liquid provided that alter each few seconds of
heating the tube is withdrawn from the flame and the contents mixed by
shaking the tube. Alternately, the heating may be carefully done by
repeatedly flicking the end of the test tube into the flame, that is, by not
allowing the test tube to remain in the flame more than a fraction of a
second at a time. The flicking operation keeps the solution mixed.
Quickly withdraw the tube from the flame as soon as there is any sign of
boiling and proceed very gently using only the edge of the flame.
Solutions contained in very small test tube (less than 13-mm diameter)
should be heated in a bath of boiling water. This technique also applies
to 10- ml test tubes that are more than one-third full of solution and for
all solutions that required prolonged heating, for example, the hydrolysis
of thioacetamide.
Evaporation of solutions. The analytical procedures may specify
evaporation of a solution to dryness. At other points the procedures
may specify evaporation to a smaller, definite volume that is more
convenient for use in subsequent operations.
Evaporations should be carried out in a small porcelain casserole. When
evaporation is accompanied by the evolution of corrosive fumes or
unpleasant odors, it should be carried out in the hood. The casserole
should be heated over a small flame, preferably a micro-burner. It should
be rotated slowly with a motion that allows the liquid to come into
contact with the hot walls, thereby facilitating evaporation. Avoid any
jiggling motion that might throw the liquid from the vessel. At the first
sign of boiling move the casserole to a cooler part of the flame since
boiling is always accompanied by spattering, evaporation should be
conducted without boiling the solution. Speed of evaporation should be
attained by keeping a large area of the casserole walls hot and wet not by
boiling the solution.
In evaporating to dryness, remove the casserole from the flame while
there is still a drop or two of liquid left. The heat in the vessel walls is
usually sufficient to complete the operation. If additional heat is
necessary, warm the casserole very gently to avoid igniting (dry heating)
the residue.
Where time permits, or where loss of small amounts of solution form
spattering must be avoided smooth evaporation may be accomplished by
 14
heating on a water bath or by suspending an infrared heat lamp just
above the casserole.
Regulation and testing of acidity. The formation or dissolution of a
precipitate, the stability of a reagent, or the color of a solution frequently
depends on the acidity (or the alkalinity) of the solution is an important
step in nearly all procedures. Failure to attain the proper acidity is one of
the more common causes of spurious results.
To determine the acidity of a solution indicators or indicator papers
(prepared by impregnating filter paper with a solution of indicator and
then drying the paper) are used. Indicators are substances that change
color with a slight change of acidity. Litmus is red in acidic solution and
blue in basic solution. Methyl violet is green at a hydrogen ion
concentration of 0.3 M, yellow in more acidic solution, and blue in less
acidic solution. Hence, methyl violet paper is used to check the acidity of
the solution during the precipitation of the copper-arsenic group of ions
where the hydrogen ion concentration must be maintained at 0.3 M.
Wide range papers have been impregnated with a mixture of the
solution changed.
In many neutralizations — perhaps a majority — some physical change
occurring within the solution serves as a rough indicator. When acidic
solution of cupric ion is made alkaline with ammonia, the solution
changes from a pale blue to a deep blue with the first excess of ammonia.
When alkali is added to an acidic solution of the acid and has been there
a permanent precipitate in the funnel is an indication that most of the
acid has been neutralized. Hence, during the neutralizing procedure the
solution should be held before a bright light while the reagent is added
and watched carefully for the formation of a precipitate or color in the
vicinity of the added drops. The change occurring around the added
drop becomes more persistent as the neutral point is approached.
Observation of these changes can cut down on the use of indicator paper
and reduced the time required for adjusting acidity.
Whenever possible, anticipate the volume of acid or base that will be
required for neutralization. Add about two-thirds of the calculated
volume rapidly and then continue adding the reagent slowly until the
solution becomes neutral. For example, if the solution being neutralized
contained eight drops of 6 M NH4OH, then about 16 drops of 6 M NH4OH
would be required for neutralization. About 10 drops of ammonia could
be added rapidly, then additional base cautiously added until the
solution showed a neutral reaction to indicator.
To test the acidity or alkalinity of a solution, dip the end of a
stirring rod into the solution and apply it to the indicator paper. In
 15
this way several tests may be made with a single strip of indicator paper.
The indicator paper may be placed on a watch glass or on a clean
piece of paper, or may be held between two fingers, but it should
never be placed directly on the desk top.
1 2 3 4 5 6 7 8 9 10 11 12 13 14
pH
Methyl violet (0-2) Yellow-------violet
Thymol blue (1-3) Red---------------yellow-----------------------blue
Methyl orange (3-5) Red----------------yellow
Bromcresol green (4-6) Yellow---------------------blue
Methyl red (4-6) Red------------------------yellow
Litmus (4-8) Red---------------------------------blue
Bromcresol purple (5-7) Yellow-------------------------purple
Bromthymol blue (6-8) Yellow----------------blue
Phenolphthalein (8-10) Colorless--------------------------------pink
Thymolphthalein (9-11) Colorless--------------------------------blue
Alizarin yellow R (10-12) Yellow--------------------------------------purple
Trinitrobenzene (12-14) Colorless-----------------------------------orange
The following technique is recommended for neutralizing a solution in a
test tube, Hold the tube in the left hand, a stirring rod between the
forefinger and the second finger, and a strip of indicator paper between
the second and third fingers. Add about two-thirds as much acid or base
as you think will be required to neutralize the solution. If a physical
change within the solution can be used as an indicator, continue
alternately adding reagent and mixing the solution by shaking the tube
until the neutral point is approached. If no physical change that will
serve as indicator occurs, stir the solution with the rod and touch the
rod with the indicator paper. Continue, alternately adding reagent and
testing the acidity, until the solution is neutral.
When acid or base is added to a solution in a test tube, some of the
reagent may get on the glass above the solution. If the stirring rod, as it
is withdrawn, touches the side of the tube and picks up some of the
reagent, the test with the indicator paper is unreliable. Also, unless
mixing is thorough, the top portion of the solution is richer in the last
reagent added than that near the bottom of the tube. A rod withdrawn
from an unmixed solution does not transfer a representative portion of
the solution to the indicator paper and again the test is unreliable.
Therefore, when the solution appears to have changed in its reaction
with the indicator, always tilt the tube and turn it to wash the inside
surface free of reagent, stir the solution again, and make a confirmatory
test with the indicator paper.
Indicator papers should not be dropped into a solution or even dipped
into it. The dye may leach out of the paper and discolor the solution.
Also, fibers from the paper contaminate the solution. Occasionally,
 16
exception to this rule, it is convenient to use a ¼ - in. strip of litmus
paper in the solution during neutralization and then fish the paper out
with a stirring rod and discard it. Litmus is nearly insoluble in water.
Flame test. Two wires should always be used for a flame test: one for the
unknown solution and the other for a solution known to contain the ion
in question at a concentration of about that expected in the unknown.
Never rely on your memory in judging the characteristics of a flame.
Alternately apply the two wires to the flame, one carrying the unknown
and the other carrying the known solution.
Nitrate and sulfate salts do not give as bright a flame as the
corresponding chlorides. In the flame many nitrates and sulfates break
down into the corresponding oxides which do not volatilize readily.
Therefore a few drops of 6 M HC1 solution should be added to any
solution before making a flame test. To make a flame test on a solid,
make the powder into a thin paste with 6 M HCI and transfer the paste
to the flame on the wire loop.
To get the solution in the loop, either dip the loop into the solution or,
with the loop perpendicular to the plane of the table, feed a drop of
solution into it with a dropper. The rounded drop placed there with the
dropper has about five times the volume as the film form dipping.
Obviously, if the ion sought gives a weak flame test or if the
concentration of the ion is low, the larger drop in the loop is to be
preferred.
Make the test using a small blue flame. The background should be
dark, preferably black. Do not use the hottest part of the flame but
some flame colors last a relatively long time; some are very ephemeral,
disappearing quickly.
Procedures When Centrifugation Operations Are Used
The separation of a solid from a liquid may be accomplished either by
centrifugation or by filtration. Centrifugation is the more rapid and
leaves the solid in a test tube where it may be treated with the next
reagent. Filtration leaves the precipitate spread over a circle of paper
from which it must be transferred to some vessel, an operation that may
involve considerable loss. On the other hand the supernatant liquid
decanted following centrifugation is more likely to contain a little
suspended solid than is the filtrate. When small amounts of the solid
being removed from the suspension interfere with subsequent tests the
procedures recommend that the supernatant liquid be decanted through
 17
a filter. If the first wash solution is to be added to the supernatant liquid
it is decanted through the same filter.
The centrifuge and its operation. There are two general types of
centrifuges used for analytical purposes – the hand-driven and the
motor- driven types. The hand-driven are not recommended but can be
used. A typical motor-driven centrifuge is shown below.
The motor-driven consists of a driving motor with an extended shaft that
carries the head, or rotor. Openings in the head are fitted with cylindrical
shield in which the tubes that are to be spun are place. When the rotor is
spinning a centrifugal force is developed which, like gravity, brings about
the settling of the precipitate. The difference in the time required for
settling and centrifugation results form the centrifugal force being much
greater than the gravitational force at the surface of the earth. There is a
simple formula that contrasts centrifugal force with gravitational force.

Centrifugal force = ___f ___= radius in cm x (rpm)2

Gravitational force g 9 x 104

If the distance form the center of the centrifuge to the revolving particle
(radius) is 10 cm, and if the centrifuge operates at 2000 revolutions per
minute (rpm), the Relative Centrifugal Force (RCF) is equal to
10(2000)2 = 4 x 107 = 444 times that of gravity.
9x104 9x104
The rate of sedimentation or settling depends upon
(1) the centrifugal force;
(2) the size of the particle — the larger the particle the more rapidly it
settles;
(3) the difference in density between the solid and the solution — the
greater this difference the faster the settling; and
(4) the viscosity of the solution --- the greater the viscosity the slower
the settling. With these factors in mind the following considerations are
easily understood.
(a) The greater the density of the salt, the greater the sedimentation rate.
BaSO4 is very dense, as are the sulfides; hence, they settle quickly if the
particles are not too small. Sulfur has a much lower density than the
metal sulfides and because of this it is possible to make a partial
separation of sulfur from sulfides by controlling the speed of the rotor
and the time of spinning.
 18
(b) The greater the concentration of salts in the solution the slower is the
rate of sedimentation. The presence of salts increases the density of the
solution and decreases the difference in density between the precipitate
and the solution.
(c) The smaller the amount of solution in the test tube the faster the
sedimentation. With a larger amount of solution, some of the precipitate
is nearer the center of rotation where it is subjected to a smaller
centrifugal force.
(d) Colloids need a much longer spinning time. The small size and mass
of the particles results in slow sedimentation.
Because a particle travels down the side of a tube more rapidly than it
travels through a liquid, an angle head centrifuge is more efficient than
one in which the tubes swing perpendicular to the rotor shaft.
In operating a centrifuge always balance it symmetrically. See to it
that approximately equal amounts of liquid are in two opposite test
tubes. The tub should not be more than three-fourths full. An
unbalanced centrifuge vibrates. This vibration, in addition to wearing the
rotor bearings and occasionally breaking a tube, causes the centrifuge to
“walk” on the desk. If not stopped promptly the centrifuge may ‘walk” off
the desk and suffer permanent damage.
Before starting the centrifuge look for particles floating on the surface of
the liquid or adhering to the side of the tube. Surface tension effects
prevent the surface particles from settling Agitate the surface to sink the
floating particles and to dislodge any particles from the side nonionic
wetting agent to reduce the surface tension.
Under no circumstances use defective test tubes in a centrifuge. Discard
any tubes with cracks or chipped tips.
Two minutes of spinning time is usually ample, except for finer and
lighter precipitates. Do not usurp the centrifuge by spinning it an undue
length of time.

Do not attempt to retard the speed of the centrifuge with the hand until
the electric current has been turned off. The rotor of a “safety head”
centrifuge can be stopped in a few seconds. Since the force holding the
precipitate down decreases as the speed is decreased, the rotor should be
slowed rapidly at first by applying the palm of the hand to the smooth
outside surface of the head, and then decelerated slowly until the rotor
stops.
 19

The centrifuge should be well cared for at all times. Do not abuse the
mechanism in any way. Malfunction of the centrifuge, as with any other
laboratory instrument, should be promptly reported to the instructor.
Centrifuge tubes. Either round-bottomed test tubes (a) or special
tapered centrifuge tubes (b)) may be used in the centrifuge. Test tubes
are satisfactory if the supernatant liquid is to. be decanted and if there is
a moderate amount of precipitate. The tapered tube is preferable if the
supernatant liquid is to be removed with a capillary syringe or if an
unusually small amount of precipitate is to be collected. The tapered
tubes cost about ten times as much as test tubes and are more difficult
to clean with a brush.
Separation of the supernatant liquid. After centrifugation the
supernatant liquid is removed from the solid residue. The problem is to
remove the liquid as completely as possible while disturbing the solid as
little as possible. With tubes 13 mm in diameter and larger, the liquid is
usually decanted by slowly tipping the tube until the bottom end is
slightly above the mouth. With tubes of smaller diameter (10 mm or less)
where surface tensions effects make decantation difficult, the liquid may
be siphoned with a dropper. Before inserting the dropper into the
solution squeeze the bulb to expel the air. If there is danger that some of
the solid may be drawn into the dropper, wrap the tip with a tiny piece of
cotton which will serve as a filter.
Washing of precipitates. The precipitate retains some of the
supernatant liquid even alter the most careful removal of the mother
liquor. The surface of the particles may also have adsorbed ions from the
solution. To remove these impurities, the precipitate is washed.
The wash liquid may be distilled water, a dilute solution of the
precipitating agent, or a solution of an ammonium salt that will not
interfere in subsequent test. Lead sulfate is washed with distilled
water. Lead chloride is washed with dilute hydrochloric acid because of
its lower solubility in the presence of the chloride ion. Lead sulfide is
washed with dilute ammonium nitrate solution. If lead sulfide is washed
with water the adsorbed ions are removed and the aggregates of small
crystals become dispersed into colloid which cannot be easily
centrifuged. Ammonium chloride cannot be used in this wash solution
because the chloride ion interferes in the next step of the systematic
analysis.
The volume of wash liquid is governed by the amount of solid to be
washed. The volumes suggested in the procedures, usually 1 to 3 ml, are
for the maximum precipitate likely to be encountered. Obviously, if one
 20
has a very small amount of solid to be washed and the procedure calls
for 3 ml of wash liquid, this volume may be reduced to meet the
circumstance. Washing with two small portions is more efficient than
washing with a single portion of the same total volume.
Washing is accomplished by adding the required amount of distilled
water or wash solution to the solid, bringing the solid into suspension by
shaking the tube or by agitating it with a stirring rod, centrifuging, and
decanting the wash liquid. The first portion of wash solution is often
combined with the supernatant liquid. The second portion is usually
discarded.
Transferring precipitates. In semimicro procedures, it is seldom
necessary to transfer a precipitate from one test tube to another. It may
be necessary to transfer a precipitate from a test tube to a casserole or
from casserole to a test tube. Transferring precipitates with a spatula is
difficult — and unnecessary.
To transfer a precipitate from a test tube, first add the reagent with
which it is to be treated. Then bring the solid into suspension by shaking
or by agitating with stirring rod. Finally, pour the suspension into the
casserole. Rinse the test tube with another small portion of reagent and
add the rinsings to the casserole.
To transfer a precipitate from a casserole, pour the suspension into the
test tube, centrifuge, and decant the supernatant liquid. Add the first
portion of wash liquid to the casserole. Loosen any solid adhering to the
walls with a stirring rod or a “policeman”. Pour the wash liquid with the
suspended solid into the test tube with the main portion of solid,
Alternately, the supernatant may be returned to the casserole and used
to wash the remaining solid into the test tube. The supernatant liquid
can be returned to the casserole as often as necessary to transfer the
solid to the tube.
Procedures When Filtration Operations Are Used
A filter is any porous material through which a fluid is passed to cleanse
or strain it. Filters for liquids are most commonly made of paper. Filters
of unglazed porcelain, sintered glass, and even of porous platinum are
also available. Filter paper may be purchased in a wide range of
porosities, grades, and circle sizes as may be seen advertised in the
catalog of any laboratory supply house. In qualitative analysis, a paper
with moderately large pores, preferably semicrimped for more rapid
filtration will serve about ninety-five percent of the time. For filtering the
finer suspensions that are occasionally encountered, a more retentive
paper should be available.
 21

The choice between filtration and centrifugation as the means of

separating the precipitate from the solution should be based on several
factors. The time factor usually favors the centrifuge. Yet, while filtration
on the macro scale is one of the most time-consuming operations in the
laboratory, on the semimicro scale there is little difference in the time
required for filtering or centrifuging a suspension. Since it is nearly
impossible to decant the supernatant liquid without having a small
amount of solid accompanying it, the filtrate is usually cleaner than the
centrifugate. The major advantage in using the centrifuge is that the
solid remains in the test tube rather than spread about on a paper. There
is no easy way to transfer the solid from the paper into a test tube or
other vessel. The filter is superior under the condition that the solid may
be treated with the next reagent without removing it from the paper, and
that small amounts of solid decanted with the supernatant liquid
interfere with subsequent tests. In the laboratory procedures filtration is
recommended where it appears to have a distinct advantage over
centrifugation.
Filtration. For filtration on a semimicro scale, 55-mm (diameter) paper
in a 35-mm funnel is commonly used. The filter paper should be folded
to give a cone that seals tightly at the rim of the paper but fits loosely
near the apex The tight fit at the rim excludes air, the funnel stem fills
with liquid, and the weight of the liquid in the stem provides gentle
suction which speeds filtration. The loose fit at the apex allows the
filtrate to drain rapidly.
For funnels of modern design, 58° cone or 60° cone fluted near the stem
to promote rapid drainage. Fold the paper into exact halves and then into
exact quarters. Tear about half of one side parallel to the fold as
illustrated below. This tear seals the wet paper against the inflow of air
which would prevent the stem from filling with liquid. Insert the filter
into the funnel, moisten the paper with distilled water, and with the
fingers press the rim of the paper against the glass. Fill the cone with
water to test for proper operation. If the stem does not fill, again press
the glass where the bubbles of air leak in. Occasionally, a dirty stem
prevents the water column for forming. A pipe cleaner and detergent
solution is usually a quick cure from this difficulty.
For the older 600, inflated funnels, fold the paper into exact halves and
then not quite into quarters. Since the 58° funnel gives the most rapid
filtering with the 60° cone of exactly quartered paper, a 60° funnel filters
most rapidly when fitted with a 62° cone. Tear the tab from the smaller
fold and fit the paper into the funnel as previously directed.
 22
Allow the precipitate to settle. Decant the supernatant liquid down a
stirring rod. The rod should touch the lip of the test tube and direct the
solution to the apex of the filter as shown below.
If a portion of the precipitate remains in the precipitation vessel use
some of the filtrate to rinse it into the filter. A rubber “policeman” may be
used to detach the last traces of solid that adhere to the glass and the
loosened solid washed into the filter using a little of the filtrate as carrier.
When permissible heat the suspension and filter it while hot. Because of
its lower viscosity the hot liquid flows more rapidly through the filter.
Also, heat tends to coagulate many suspensions and the larger particles
are less likely to go through the paper or to clog its pores.

Washing the precipitate on the filter. Wash the paper and precipitate
with a jet of distilled water or wash solution from a capillary syringe.
Begin the washing at the upper edge of the paper. If possible, loosen the
precipitate from the paper with the jet and work it forward the apex.
Allow the filter to drain thoroughly before adding the next portion of
wash solution. As a rule the first portion of wash solution is caught with
the filtrate, subsequent portions are caught in a separate vessel and
discarded.
Transferring precipitates from the filter. Any of several procedures
may be used to remove a precipitate from filter paper. The properties of
the precipitates and the treatment to which it is to be subjected governs
the selection of method to be used. If the precipitate is soluble in the first
reagent with which it is to be treated, it may be dissolved by dropping the
reagent over the precipitate on the filter. A second procedure is that of
puncturing a small hole through the apex with a pointed glass rod and
then washing the precipitate into the receiving vessel with a fine stream
of water from a washbottle or a capillary syringe. Yet another technique
involves removing the filter from the funnel, unfolding and holding the
paper in an inclined position over a small casserole or other wide-mouth
container, and washing the precipitate into the receiving vessel.
If the precipitate is present in appreciable quantity and is allowed to dry,
the bulk of it may be scraped from the paper with a spatula and the
small amount retained on the paper may be washed of as previously
described.
If the amount of precipitate is small, tear off and discard those portions
of the filter that are free of precipitate. Then place the remaining paper
carrying the precipitate in a reagent that dissolves the precipitate. Finally
remove the paper by filtration or by fishing it out of the solution with a
 23
stirring rod, squeezing it between the rod and the side of the vessel, and
then discarding it.

Dispensing Reagents. The system used for the storage and dispensing
of reagents is dependent upon many local factors. Hence, some general
principles and suggestions are presented rather than a set of specific
directions of doubtful applicability. Under any system a major share of
the responsibility for keeping the reagents pure and their containers
clean and in orderly arrangement must be assume by the students.
All reagents and test solutions required for the preliminary experiments
and analytical procedures must be available. Ideally, each student has a
set of all reagents and test solutions for his exclusive use, stored at his
own desk. As the quantities of reagents needed by one student are small,
individual kits are often practical. In the least satisfactory arrangement,
all the students in a laboratory share a single set of reagents. Few
instructors will have to face this situation, but they may have to devise
some reasonable compromise. Certain desk and to use the same set of
reagents. Again, each student may have an individual kit containing the
most frequently used reagents while those less frequently used may be
available only at some central location.
Liquid reagents and solutions are conveniently dispensed from square
glass bottles with combined closure and dropper. The square shape
permits labeling and storage in trays or racks. For individual kits 15-ml
bottles with molded resin screw caps holding a rubber bulb and dropper
are most frequently used. If two or more students used the same set of
reagents, 30-ml bottles are convenient. These are available either with
the plastic cap assembly or with modified rubber stoppers that include
the bulb and glass pipet The rubber stoppered bottle is slightly more
convenient to use for less time is required to remove mid replace a
friction stopper than a screw cap.
Glass-stoppered bottles are required for concentrated nitric acid and
should be used for all strong acids at concentration of 6 M or greater.
Two forms of glass-stoppered dropping bottles provided with a channel to
regulate the rate of flow are illustrated below. Turning the stopper opens
and closes the bottle. Since the acids may be used in relatively large
amount 30 ml bottles are recommended for individual kits and 60-ml
bottles are preferred where several students share a reagent bottle.
Unless the bottles and stoppers carry the standard taper designation, ST,
each bottle and stopper form a noninterchangeable unit. Then identical
serial numbers should be scratched into the glass of each bottle and its
stopper before the bottles are put into use. Even in the best regulated
 24
laboratories unnumbered bottles and stoppers get mixed and the bottles
are then worthless.
Polyethylene dropping bottles are superior to glass as containers for
solutions of strong bases. However, the problem of fitting the off-sized
bottles into the kit may discourage their use. Glass bottles containing
strongly alkaline solutions become etched.
Vials with large neck openings are superior to those with constricted
necks for dispensing solids. The 4-dram (15 ml) size is recommended
since it fits into trays with either 15-ml or 30-ml square dropping bottles.
Vials may be purchased in many styles, some carry screw caps, others
friction plastic caps.
Some kind of storage rack or tray must be provided or the dropping
bottles soon become disarranged and scattered. The laboratory supply
houses, particularly those specializing in semimicro equipment, offer a
number of designs. Homemade racks, designed to meet local needs, are
often more useful.
Stock or supply bottles (250 to 1000 ml) kept at some central place
are required for filling the reagent bottles used at the desk. If only a few
sets of reagent bottles are in use, the instructor or his assistant may
assume responsibility for keeping them filled.