Laboratory techniques notes CT 105

A technique is an adaptation of a theory into a procedure, method or skill. In the chemistry

laboratory, there are three main categories of lab techniques

1. Isolative –this involves separation/isolation of actives from natural sources eg natural

chemical compounds from plants and animal materials, genetic actives, etc
2. Preparative – synthetic methods for fine chemicals, pharmaceuticals, catalysis
3. Analytical – done to identify and quantify actives/ analytes in material samples for drug
analysis, bioanalysis, radiochemical, pollution monitoring, certification of compounds,
etc

The embedded theory in a technique can be realized if;

-the analyst has the necessary skill
-adequate theoretical knowledge of the resources, equipment and reagents to be used
-analyst correctly follows the work instructions in the method

Techniques can further be classified according to the scale of the experiment. These are
Conventional / bench top/ semi macro techniques and Microscale techniques.

In conventional techniques, the quantities of stocks and equipment is sizeable and can fit on
benchtop work station, whilst in microscale, there are reduced dimensions and a micro station is
employed. Both techniques often involve batch processes. In industrialized/ commercial scaled-
up experiments stocks are large and equipment engineered to allow large production capacities.
The processes can be batch or continuous.

Scaling of experiments is determined by;

-cost of reagents

-occupational and environmental safety

-atomic economy efficient conversion methods

-work station space economy

-size of available equipment

- time for preparation and clear away

-waste at the source

Laboratory safety

1. Personal protective equipment

Full length acid proof labcoat, protective shoes (flat, leather), latex gloves, safety
googles. The afore-mentioned items are essential in ones PPE.
2. No eating/drinking in the lab. All chemicals should be treated as toxic and hence to
prevent accidental accidental ingestion, no food is to be eaten in the lab
3. Exercise caution when working with open flames. Do not smoke in the lab. Avoid direct
heating of organic solvents due to their flammability and high volatility.
4. Safety features such as eye wash facilities, shower
5. Exercise caution when working with organic solvents. They are flammable, toxic,
carcinogenic. Egs benzene, ether
6. Waste disposal- place the different types of waste generated in the lab in the appropriate
bins. There should be bins/ containers labelled organic, inorganic, broken glass, etc into
which specific waste goes.
7. Avoid pouring back reagents into stock feeds as this can cause reagent contamination.
8. Never carry out unsupervised/unauthorised experiments.
9. Know the position of the first aid kit and the procedures for dealing with minor cuts,burns
or accidental ingestion. Also know the position of the different types of fire extinguishers
needed to deal with the different types of fire.[ what are these?]
Cleaning glassware

Glassware should be cleaned as soon as you finish using it. As a general rule, soak it in soapy
water then scrub and rinse using distilled water/ double distilled/ deionised water. Organic
solvents can be used to remove organic dirt but in small amounts. Eg of appropriate solvents;
acetone, methylene - chloride, toluene. For troublesome stains use a mixture of HCI, HNO3 and
H2SO4. Increasing the temperature of the water can also help remove stains faster. Once the
stains are gone, wash with soap and rinse with distilled water.

Drying glassware

-allow glass to stand overnight

-place glassware upside-down on paper towels
-place in drying ovens at temperature ˂ 80 oC
-rinse with acetone for rapid drying
-blow a gentle stream of dry air

Conventional Laboratory techniques

1.Measurement of volume and weight

Accurate measurement of volume and weight is crucial in synthesis and analytical chemistry. It
is important to use the most appropriate apparatus and technique for a particular procedure.

Volumetric Apparatus- Examples of these types of glassware include graduated cylinders,

burettes, pipettes, and volumetric flasks. Ordinary beakers and flasks are not used for accurate
volume measurements; the markings on these pieces of glassware are generally only accurate to
within 5%. Each piece of volumetric glassware is marked with its total volume, the notation TD
or TC, and a temperature (usually 20°C).  The marked temperature indicates the temperature at
which the apparatus was calibrated.  Since density and volume change with temperature, the
volume markings are strictly correct only at the calibration temperature.  The notations TD and
TC stand for the phrases "to deliver" and "to contain."  The TD notation means that the apparatus
is calibrated to accurately deliver or transfer the stated volume to another container.  The TC
notation means that the markings give an accurate measure of the volume contained, but that
pouring the liquid into another container will not necessarily deliver the indicated volume.

a) Pipettes

A pipette is a laboratory tool commonly used to deliver (TD) a measured volume of liquid as
a media dispenser. Pipettes come in several designs for various purposes with differing levels
of accuracy and precision, from single piece glass pipettes to more complex adjustable or
electronic pipettes. Most pipette work by creating a partial vacuum above the liquid-holding
chamber and selectively releasing this vacuum to draw up and dispense liquid.  Pipettes that
dispense between 1 and 1000 μl are distinguished as micropipettes, while macropipettes dispense
greater volumes. Filling and dispensing is aided by a pump or pipette filler and should never be
by mouth and finger tips.

i) bulb pipette - allows the user to measure a volume of solution extremely accurately (accuracy
of 4 significant figures). These pipettes have a large bulb with a long narrow portion above with
a single graduation mark as it is calibrated for a single volume. Typical volumes are 10, 25, and
50 mL. Volumetric pipettes are commonly used to make laboratory solutions from a base stock
as well as prepare solutions for titration.

ii) Graduated pipettes - a macropipette consisting of a long tube with a series of graduations to
indicate different calibrated volumes. Can be blow out type or no blow out type. Blow out type
delivers its maximum volume when the last drop is blown out whilst the other should not be
blown and delivers its maximum volume when the meniscus is located on the last graduation
mark. Typical volumes for graduated pipettes are are 5, 10, 25 and 50 mL .

b) Burettes

A burette is a device used in analytical chemistry for the dispensing of variable, measured

amounts of a chemical solution. A traditional burette consists of glass tube of constant bore with
a graduation scale etched on it and a stopcock at the bottom. The barrel of the stopcock may be
made of glass or the plastic PTFE. Stopcocks with glass barrels need to be lubricated with a
specialized grease. Burettes are manufactured to specified tolerances, designated as class A or B
and this also is etched on the glass.

Burette accuracy /mL

Capacity, mL Class A Class B

c) The volumetric flask
10 0.02 0.04
The volumetric flask is calibrated to contain a
25 0.03 0.06 fixed amount of solution with high accuracy. 
The flask is used in two major ways.  In one
50 0.05 0.10 technique, a sample of known mass (the solute)
is placed in the flask and dissolved.  The
100 0.10 0.20
solution is then made up to the mark on the
flask by adding solvent, giving a solution of
precisely known volume containing a precisely known amount of solute. The second use of the
volumetric flask is to dilute a solution in a precisely known manner.  This technique involves
placing an aliquot of a solution of known molarity in the flask, then diluting to the mark with
solvent. Molarity is calculated using the eqn (1) below;

                               M1  x  V1  =  M2  x  V2                                                                              (1)

Measurement of mass/weight

1. Balances

Types: triple beams and top loadings

Triple beams are less accurate than top loading balances, with an error margin of 0.05g.
The balance was the first mass measuring instrument invented. In its traditional form, it consists
of a pivoted horizontal lever of equal length arms, called the beam, with a weighing pan,
suspended from each arm. The unknown mass is placed in one pan, and standard masses are
added to the other pan until the beam is as close to equilibrium as possible. In precision balances,
a slider mass is moved along a graduated scale. The slider position gives a fine correction to the
mass value.

Top loading and analytical balances are used for accurate weighing of solids and liquids (limiting
reagent). These have a balance pan, up right draught shields, balancing knobs at the back and an
internal reference standard for auto calibration. External recalibration should be done using
reference standards (5mg - 50g). The balances differ in sensitivities from 1 -5 decimal places, the
last being commonly known as the analytical balance.

Care of the balance involves;

 using a camel hair brush to brush off solids,

 avoid spillages, frequent dusting and wiping,

 placing the balance on a well –levelled, non vibrating surface in a draft proof room,
away from magnetic and electric fields

 One should never overload the balance beyond its maximum weight,

 Avoid corrosive chemicals and atmospheres

 Use the appropriate sample holders, forceps and spatulas

Weighing procedure

 Brush balance pan clean & check that it is well positioned

 Switch on & allow warm up (1 sec) for equilibration. If necessary, calibrate with
external standards or use balancing knobs
  Place object to be weighed at centre of pan, close draft shields and read displayed
weight

  the sample must be at room temperature to prevent natural convection from forming

air currents inside the enclosure from causing an error in reading.

Principle of operation - Electronic analytical scales measure the force needed to counter the
mass being measured rather than using actual masses. They use an electromagnet to generate a
force to counter the sample being measured and outputs the result by measuring the force needed
to achieve balance. Such measurement device is called electromagnetic force restoration sensor.
The force is triggered by a flow of current through the e/m compensating coil as the sample is
loaded. The micro processer converts the value of current into the digital display in grams.

Common errors in weighing

  Change in conditions of container or substances during weighing. This can be due to
absorption or loss of moisture; electrostatic effects; non equilibrated temperature; Mis-
calibration over time, due to drift in the circuit's accuracy, or temperature change

 Effects of buoyancy- erroneous reading of object in surrounding medium because of

differences in densities. Objects in air develop buoyancy force that is directly
proportional to the volume of air displaced. The difference in density of air due to
barometric pressure and temperature creates errors.

 Error in recording.

 Air gusts, even small ones, which push the scale up or down,

 Settling airborne dust contributing to the weight

 Mis-aligned mechanical components due to thermal expansion/contraction of components

 Chemical reactivity between air and the substance being weighed (or the balance itself, in
the form of corrosion)

 Condensation of atmospheric water on cold items

 Vibration and seismic disturbances

Heating and cooling methods

Most reactions need heating in order to complete reacting and cooling to stop the reaction. In the
chemlab, the following equipment & apparatus are used for heating

1.Water bath- This comes in a wide variety of shapes, sizes, and types, but traditionally is a
wide, cylindrical, usually metal container made of the following parts; an outer (or lower)
container that holds the water, an inner (or upper), smaller container that fits inside the outer one
and which holds the material to be heated, and sometimes a base underneath. Under the outer
container is an electronic element. Another heat source can be hot sand.

Typically the inner container is immersed about halfway into the water. The smaller container,
filled with the substance to be heated, fits inside the outer container, filled with the water, and the
whole is heated at, or below, the base, causing the temperature of the materials in both containers
to rise as needed. The insulating action of the water helps to keep contents of the inner pot from
boiling or scorching. Temp range normally 0-1000C.

2. Bunsen burner

The use of a naked flame is strictly limited in the chemistry laboratory because of the danger of
fire when handling highly flammable organic liquids. A flame should never be used to heat an
organic liquid in an open beaker or test tube, however it is useful if water is the solvent being
used or if a hot water bath is required. A Bunsen flame has the disadvantage of being a very
uneven source of heat. Use of a wire gauze spreads the flame to a large extent, however, the
bottom of the flask will always be considerably hotter than the rest of the flask.

3. Steam-baths

Steam-baths are usually located under the fume-hoods. These can be hooked up to the steam
outlets in the fume-hoods or on the benches. Steam from the heated water is used for heating.
The removable metal rings can be removed in order to accommodate flasks of different sizes.
The main disadvantage of a steam-bath is that it will generate only one temperature, a little
below 100 0C. This is sufficient to distil liquids with boiling points around 80 0C below. They are
useful in digestion of inorganic analytes and recrystallization

4. Electric heating Isomantle

An electric heating isomantle is a relatively safe form of heating. The mantles are hemispherical
and are lined with a ceramic covering to protect the heating element from mechanical and
chemical damage. The mantles are designed to accommodate a round bottomed flasks. This
device ensures uniform heating of the flask and its contents.

5. Electric Hot-Plate

These are usually placed in or near the fumehoods. Hot plates are used for boiling off solvents
excess water. It is not recommended for gentle heating eg digestion. Some hot plates are spark-
proofed, but those that are not are capable of igniting organic vapours, especially those denser
than air e.g. benzene, toluene, and ether.

6. Aluminium heating block

An aluminium block is fabricated into various shapes to hold vessels, flasks, vials. The Al block
is heated by electric elements then it transfers the heat to the vessels uniformly. Advantages
include high temps and high heating rate. Useful in distillation, reflux and extraction procedures.

7. Sand bath

A sand bath is a convenient source of heat for micro-scale reactions. A sand bath warms up much
more slowly than a steam bath. Unlike the hot water and steam baths, a sand bath can provide
temperatures that range from near room temperature at the surface up to 200 deg C and above,
deep in the sand.

8. Cold baths

For cooling a Cold bath is often used. This can be an ice bath or a dry ice bath. Cooling mixtures
to around 0oC is often done in an ice bath. Sodium chloride can be added to lower the temp to
below zero eg -10 oC. For temps around -78 oC, solid carbon dioxide mixed with isopropyl
alcohol can be applied. For -195 oC, liquid nitrogen can be employed.

Reaction Methods

1. Heating under reflux

Reflux is a distillation technique involving the condensation of vapours and the return of this
condensate to the system from which it originated. It is used in industrial and laboratory
distillations. The apparatus shown in the diagram represents a batch distillation. The liquid feed
mixture to be distilled is placed into the round-bottomed flask along with a few anti-bumping
granules, and the fractionating column/ water jacketed condenser is fitted into the top. As the
mixture is heated and boils, vapour rises up the column. The vapour condenses on the glass
platforms inside the column and runs back down into the liquid below, and the cycle begins. The
hottest tray is at the bottom of the column and the coolest tray is at the top. At steady state
conditions, the vapour and liquid on each tray is at equilibrium. The most volatile of the vapours
stays in gaseous form all the way to the top. The vapour at the top of the column then passes into
the condenser, where it cools until it condenses into a liquid. The separation can be enhanced
with the addition of more trays. The process continues until all the most volatile components in
the liquid feed boil out of the mixture. This point can be recognized by the rise in temperature
shown on the thermometer. For continuous distillation, the feed mixture enters in the middle of
the column.

Kuderna Danish concentration apparatus

Kuderna-Danish concentrator concentrates samples when testing for pesticides or other

pollutants prior to instrumental analysis. It makes use of refluxing as a method of concentrating
extracts. Concentration of 10ml to 1ml can be done in 24 to 72 hours.

Assemble a Kuderna-Danish (K-D) concentrator by attaching a concentrator tube to an

evaporation flask. Attach the solvent vapour recovery glassware (condenser and collection
device) to the Snyder column of the K-D apparatus. Collect the dried extract in a K-D
concentrator. Attach an erlenmeyer flask to the K-D column. Add boiling chips to the flask to
allow for smooth boiling, and attach a three-ball Snyder column. Place the K-D apparatus on a
hot water bath (20 0C above the boiling point of the solvent), so that the concentrator tube is
partially immersed in the hot water and the entire lower rounded surface of the flask is bathed
with hot vapour.

Stirring methods

A heated solution should be heated to avoid bumping ie the eruption of large bubbles from
solution causing loss of analyte and the possibility of fires. Stirring can be done manually using a
stirring rod or by magnetic stirrers.

Magnetic stirrers
A magnetic stirrer is a laboratory device that employs a rotating magnetic field to cause a stir bar
immersed in a liquid to spin very quickly, thus stirring it. The rotating field may be created either
by a rotating magnet or a set of stationary electromagnets, placed beneath the vessel with the
liquid. The small magnet (magnetic follower) placed inside the flask is often coated with Teflon
(an inert plastic) and can be found in a variety of shapes and sizes. For microscale analysis, a
triangular magnetic spin vane is used.

Since glass does not affect a magnetic field appreciably, magnetic stir bars work well in glass
vessels. On the other hand, the limited size of the bar means that magnetic stirrers can only be
used for relatively small experiments. They also have difficulty dealing with viscous liquids or
thick suspensions. Because of its small size, a stirring bar is more easily cleaned and sterilized
than other stirring devices. They do not require lubricants which could contaminate the reaction
vessel and the product. They can be used inside closed systems. Magnetic stirring systems can
have the rotating magnet driven by an electric motor embedded in a heating block. 

Addition of liquid reagents during distillation

1. Pastuer pipette : for open air reactions

2. A hypodermic syringe : the needle is inserted through a rubber septum and the liquid
added by the action of a plunger [anhydrous atmosphere, ]

3. Separatory funnel connected to the side arm of a 3 necked round bottom flask. Reagents
are leaked into the flask

Drying methods

Where substances are sufficiently stable, removal of solvents from recrystallized materials
presents no problems. The crystals, after filtering at the pump (and perhaps air-drying by
suction), are heated in an oven above the boiling point of the solvent (but below their melting
point), followed by cooling in a desiccator. In cases where heating above room temperature
cannot be used, drying must be carried out in a vacuum desiccator containing suitable
absorbents. For example, hydrocarbons, such as benzene, cyclohexane and petroleum ether,
can be removed by using shredded paraffin wax, and acetic acid and other acids are absorbed
by pellets of sodium hydroxide or potassium hydroxide. However, in general, solvent removal
is less of a problem than ensuring that the water content of solids and liquids is reduced below
an acceptable level.

Methods for removing water from solids depend on the thermal stability of the solids or the
time available. The safest method is to dry in a vacuum desiccator over drying agents eg;
phosphorus pentoxide, silica gel, calcium chloride. Where substances are stable in air and
melt above 100°C, drying in an air oven may be adequate.

Often, in drying inorganic salts, the final material that is required is a hydrate. In such cases,
the purified substance is left in a dessicator to equilibrate above an aqueous solution having a
suitable water-vapour pressure.

The choice of desiccants for drying liquids is more restricted because of the need to avoid all
substances likely to react with the liquids themselves. In some cases, direct distillation of an
organic liquid is a suitable method of drying both solids and liquids, especially if low-boiling
compounds are formed. Examples include acetone, aniline, benzene, chloroform, carbon
tetrachloride, ethylene dichloride, heptane, hexane, methanol, nitrobenzene, petroleum ether,
toluene and xylene. Addition of benzene can be used for drying ethanol by distillation. In
carrying out distillations intended to yield anhydrous products, the apparatus should be fitted
with guard-tubes containing calcium chloride or silica gel to prevent entry of moist air into
the system. Removal of water from gases may be by physical or chemical means, and is
commonly by adsorption on to a drying agent in a low temperature trap. The effectiveness of
drying agents depends on the vapour pressure of the hydrated compound - the lower the
vapour pressure the less the remaining moisture in the gas. Egs of drying agents;

P2O5, BaO, Mg(ClO4)2, CaO, MgO, KOH, conc H2SO4, CaSO4, Al2O3, silica gel, ZnCl2,
ZnBr2, CaCl2 , CuSO4,Na2SO4, K2CO3

Drying tubest- consists of a tube packed with drying agents placed between plugs of glass
wool. Used to prevent atmospheric moisture from entering reaction vessel.
Rotary evaporator

A rotary evaporator is a device used in chemical laboratories for the efficient and gentle removal
of solvents from samples by evaporation. The component liquids of interest in applications of
rotary evaporation are research solvents that one desires to remove from a sample after an
extraction, such as following a natural product isolation or a step in an organic synthesis. This
allows liquid solvents to be removed without excessive heating of what are often complex and
sensitive solvent-solute combinations.

The key advantages in use of a rotary evaporator are;

1. That the centrifugal force and the frictional force between the wall of the rotating flask
and the liquid sample result in the formation of a thin film of warm solvent being spread
over a large surface.

2. The forces created by the rotation suppress bumping. The combination of these

characteristics and the conveniences built into modern rotary evaporators allow for
quick, gentle evaporation of solvents from most samples, even in the hands of relatively
inexperienced users. Solvent remaining after rotary evaporation can be removed by
exposing the sample to even deeper vacuum, on a more tightly sealed vacuum system, at
ambient or higher temperature

3. Allows for efficient solvent recovery

Filtration
Filtration is the physical operation which is used for the separation of solids from fluids by
interposing a medium through which only the fluid can pass. The fluid that passes through is
called a filtrate. Oversize solids in the fluid are retained, but the separation is not complete;
solids will be contaminated with some fluid and filtrate will contain fine particles (depending on
the pore size and filter thickness).

The purpose of filtration is;

 remove solid impurities from a liquid

 collect a desired solid from a solution which it was precipitated or crystallised

Two main types of filter media are employed in any chemical laboratory— surface filter, a solid
sieve which traps the solid particles, with or without the aid of filter paper (e.g. Buchner funnel),
and a depth filter, a bed of granular material which retains the solid particles as it passes
(e.g. sand filter). The first type allows the solid particles, i.e. the residue, to be collected intact;
the second type does not permit this. However, the second type is less prone to clogging due to
the greater surface area where the particles can be trapped. Also, when the solid particles are
very fine, it is often cheaper and easier to discard the contaminated granules than to clean the
solid sieve.

Method Application

a) Gravity ; filter cones Vol ≤ 10cm3, solid saved

Fluted filters Vol ≥ 10cm3, solid impurity removed, desired

filtrate saved

Filtering pipettes Vol ≤ 10cm3, solid impurity removed

b) Vacuum ; hirsh funnel Collection of desired solid from small vol (1-
10ml)
 Buchner funnel Same as hirsh but bigger vol

c) Craig tubes Collection of desired solid from small vol

(~2ml)

d) Centrifugation Removal of suspended solids from liq

(1~25ml)

Fluids flow through a filter due to a difference in pressure — fluid flows from the high pressure
side to the low pressure side of the filter, leaving some material behind. The simplest method to
achieve this is by gravity. In the laboratory, pressure in the form of compressed air on the feed
side (or vacuum on the filtrate side) may be applied to make the filtration process faster, though
this may lead to clogging or the passage of fine particles. Alternatively, the liquid may flow
through the filter by the force exerted by a pump, a method commonly used in industry when a
reduced filtration time is important. In this case, the filter need not be mounted vertically.

Filter aid

Certain filter aids may be used to aid filtration. These are often incompressible diatomaceous
earth, silica, wood cellulose and other inert porous solids. These filter aids can be used as a
precoat before the slurry is filtered. This will prevent gelatinous-type solids from plugging the
filter medium and also give a clearer filtrate. They can also be added to the slurry before
filtration. This increases the porosity of the cake and reduces resistance of the cake during
filtration.

Centrifugation involves forcing the denser solid to the bottom, where it often forms a firm cake.
The liquid above can then be decanted. This method is especially useful for separating solids
which do not filter well, such as gelatinous or fine particles. These solids can clog or pass
through the filter, respectively.
Factors to consider when choosing filter paper

1. porosity – measure of the size of particles that can pass through the paper

2. retentivity – opposite of porosity

3. speed of filter paper – time taken to drain

Recrystallization

The most common method of purifying solid organic compounds is by recrystallization. In this
technique, an impure solid compound is dissolved in a solvent and then allowed to slowly
crystallize out as the solution cools. As the compound crystallizes from the solution, the
molecules of the other compounds dissolved in solution are excluded from the growing crystal
lattice, giving a pure solid.

Crystallization of a solid is not the same as precipitation of a solid. In crystallization, there is a

slow, selective formation of the crystal framework resulting in a pure compound. In
precipitation, there is a rapid formation of a solid from a solution that usually produces an
amorphous solid containing many trapped impurities within the solid's crystal framework. For
this reason, experimental procedures that produce a solid product by precipitation always include
a final recrystallization step to give the pure compound.

The process of recrystallization relies on the property that for most compounds, as the
temperature of a solvent increases, the solubility of the compound in that solvent also increases.
As the temperature of the solution decreases, the solubility also decreases, and the molecules will
begin to crystallize out of the solution.

The steps in recrystallization are;

 Find a suitable solvent for the recrystallization;

 Dissolve the impure solid in a minimum volume of hot solvent;

 Remove any insoluble impurities by gravity filtration;

 Slowly cool the hot solution to crystallize the desired compound from the solution;

 Vacuum filter the solution to isolate the purified solid compound.

Choosing a suitable solvent

There are four important properties that you should look for in a good solvent for
recrystallization.

1.The compound should be very soluble at the boiling point of the solvent and only sparingly
soluble in the solvent at room temperature. This difference in solubility at hot versus cold
temperatures is essential for the recrystallization process. If the compound is insoluble in the
chosen solvent at high temperatures, then it will not dissolve. If the compound is very soluble in
the solvent at room temperature, then getting the compound to crystallize in pure form from
solution is difficult. For example, water is an excellent solvent for the recrystallization of benzoic
acid. At 10°C only 2.1 g of benzoic acid dissolves in 1 liter of water, while at 95 °C the solubility
is 68 g/L.
2.The unwanted impurities should be either very soluble in the solvent at room temperature or
insoluble in the hot solvent. This way, after the impure solid is dissolved in the hot solvent, any
undissolved impurities can be removed by filtration. After the solution cools and the desired
compound crystallizes out, any remaining soluble impurities will remain dissolved in the solvent.

3.The solvent should not react with the compound being purified. The desired compound may be
lost during recrystallization if the solvent reacts with the compound.

4.The solvent should be volatile enough to be easily removed from the solvent after the
compound has crystallized. This allows for easy and rapid drying of the solid compound after it
has been isolated from the solution.

Dissolving the solid

Once a suitable solvent is selected, place the impure solid in an Erlenmeyer flask and add a small
volume of hot solvent to the flask. Erlenmeyer flasks are preferred over beakers for
recrystallization because the conical shape of an Erlenmeyer flask decreases the amount of
solvent lost to evaporation during heating, prevents the formation of a crust around the sides of
the glass, and makes it easier to swirl the hot solution while dissolving the solid without
splashing it out of the flask.

Keep the solution in the Erlenmeyer flask warm on a hot plate or in a water bath, and add small
volumes of hot solvent to the flask until all of the solid just dissolves. Swirl the solution between
additions of solvent and break up any lumps with a stirring rod or spatula. Occasionally there
will be impurities present in the solid that are insoluble in the chosen solvent even at high
temperature. If subsequent additions of solvent to the solution do not seem to dissolve any of the
remaining solid, stop adding solvent to the solution as this will decrease the percent recovery of
the desired compound. After the impure solid sample is dissolved in hot solvent, a small amount
of decolorizing /activated carbon is used to remove the coloured impurities from the sample.

Hot filtration
The solution is stirred and heated for a few minutes and then filtered whilst hot to remove the
decolorizing carbon and impurities using gravity filtration (fluted filter paper).

Recrystallisation

After the insoluble impurities have been removed, cover the flask containing the hot filtrate with
a watch glass and set it aside undisturbed to cool slowly to room temperature. As the solution
cools, the solubility of the dissolved compound will decrease and the solid will begin to
crystallize from the solution. After the flask has cooled to room temperature, it may be placed in
an ice bath to increase the yield of solid. Do not rapidly cool the hot solution by placing the flask
in an ice bath before it has cooled to room temperature-this will result in a rapid precipitation of
the solid in an impure form because of trapped impurities.

Sometimes the dissolved compound fails to crystallize from the solution on cooling. If this
happens, crystallization can be induced by various methods;

 by scratching the inner wall of the Erlenmeyer flask with a glass stirring rod. This is
believed to release very small particles of glass which act as nuclei for crystal
growth.

 add a small crystal of the desired compound, a seed crystal, to the solution. This seed
crystal acts as a template on which the dissolved solid will begin crystallizing.

 If neither of these two techniques results in crystallization, the compound was

probably dissolved in too much hot solvent. If so, reheat the solution to boiling, boil
off or distill some of the solvent, and then allow the solution to cool to room
temperature again to effect crystallization.

Collection of crystals via vacuum filtration

Once the compound has completely precipitated from the solution, it is separated from the
mother liquor by vacuum filtration using a Buchner funnel. Slowly pour the recrystallization
solution into the funnel and allow the suction to pull the mother liquor through. Rinse the
Erlenmeyer flask with a small volume of cold recrystallization solvent to remove any remaining
solid. Add this solvent to the funnel and then wash the solid in the funnel, called the filter cake or
residue, with a few millilitres of fresh, cold recrystallization solvent to remove any remaining
mother liquor and dissolved impurities.

Drying of crystals

The crystals can then be air dried, oven dried or vacuum dried in a dessicator depending on their
physical properties eg melting point.

Solvent extraction

Extraction is the transference of one or more solutes from one solvent into another that is
immiscible to the former, taking advantage of the difference in solubility’s in the two phases.

LLE- liquid –liquid extraction

It is an extraction of a substance from one liquid into another liquid phase. Liquid–liquid
extraction is a basic technique in chemical laboratories, where it is performed using a separatory
funnel. A separatory funnel is a piece of laboratory glassware used in liquid-liquid extractions to
separate / partition the components of a mixture into two immiscible solvent phases of different
densities. Typically, one of the phases will be aqueous, and the other a non- polar organic solvent
such as ether, dichloromethane, chloroform, or ethyl acetate. All of these solvents form a clear
delineation between the two liquids. 

The two layers formed are usually known as the organic and aqueous phases. Most organic
solvents float on top of an aqueous phase, though important exceptions are most halogenated
solvents. The organic solvent used for the extraction must not react with the substance to be
extracted or with water. It should also have a low boiling point so it can be easily removed from
the product.

To use a separatory funnel, the two phases and the mixture to be separated in solution are added
through the top with the stopcock at the bottom closed. The funnel is then closed and shaken
gently by inverting the funnel multiple times; if the two solutions are mixed together too
vigorously emulsions will form. The funnel is then inverted and the tap carefully opened to
release excess vapour pressure. The separating funnel is set aside to allow for the complete
separation of the phases. The top and the bottom tap are then opened and the two phases are
released by gravitation.

The organic solvent used for extraction must meet a few criteria:

1. Should readily dissolve substance to be extracted.

2. Should not react with the substance to be extracted.
3. Should not react with or be miscible with water (the usual second solvent).
4. Should have a low boiling point so it can be easily removed from the product.

Solid-liquid Extraction - SLE

A Soxhlet extractor is a piece of laboratory apparatus used for SLE. It was originally designed
for the extraction of a lipid from a solid material but is now used where the desired compound
has a limited solubility in a solvent, and the impurity is insoluble in that solvent.
Procedure

A solid material containing some of the desired compound is placed inside a thimble made from
thick filter paper, which is loaded into the main chamber of the Soxhlet extractor. The
extraction solvent to be used is placed in a distillation flask. The Soxhlet extractor is then placed
onto this flask as shown in the setup above. The upper end of the extractor is joined to
a condenser and the flask is placed in a heating isomantle.

When the solvent is heated, the solvent vapour travels up the distillation arm of the extractor,
condenses and flows into the chamber housing the thimble of solid. The chamber containing the
solid material is slowly filled with warm solvent. Some of the desired compound will
then dissolve in the warm solvent. When the Soxhlet chamber is almost full, the chamber is
automatically emptied by a siphon side arm, which runs the solvent back down to the distillation
flask. The thimble ensures that the rapid motion of the solvent does not transport any solid
material to the distillation flask. This cycle may be allowed to repeat many times, over hours or
days until all the desired analyte has been extracted.

The temperature of the isomantle is set such that it is high enough to vapourise the solvent but
not too high as to also vapourise the analyte. The solvent chosen for SLE should have a lower
boiling point than that of the analyte. The end result is that, after many cycles, the desired
compound is concentrated in the distillation flask due to the cycle of extractions with warm fresh
solvent. The advantage of this system is that instead of many portions of warm solvent being
passed through the sample, just one batch of solvent is recycled.

After extraction the solvent is removed by means of a rotary evaporator, yielding the extracted
compound. The non-soluble portion of the extracted solid remains in the thimble, and is usually
discarded.

Determination of physical constants

1. Melting point determination

The melting point of a substance is the temperature at which the material changes from a solid to
a liquid state. Pure crystalline substances have a clear, sharply defined melting point. During the
melting process, all of the energy added to a substance is consumed as heat of fusion, and the
temperature remains constant.

Capillary Melting Points

Capillary melting points, either in an oil bath or a melting-point apparatus, are most often used
for the determination of the melting point of a solid. A few crystals of the compound are placed
in a thin walled capillary tube 10-15 cm long, about 1 mm in inside diameter, and closed at one
end. The capillary, which contains the sample, and a thermometer are then suspended so they can
be heated slowly and evenly. The temperature range over which the sample is observed to melt is
taken as the melting point.

The thermometer and sample must be at the same temperature while the sample melts, so the rate
of heating must be slow as the melting point is approached (about 1 degree per minute).
Otherwise, the temperature of the thermometer bulb and the temperature of the crystals in the
capillary may not be the same. The transfer of heat energy by conduction takes place rather
slowly.

Filling a Capillary Tube - can be done by pressing the open end into a small heap of the crystals
of the substance, turning the capillary open end up, and vibrating it by drawing a file across the
side to rattle the crystals down into the bottom. The solid should be tightly packed to a depth of
2-3 mm.

A variety of oil baths can be used in a melting point determination, as well as in a boiling point
determination. The simplest use a burner flame and depend upon convection for mixing; the
more elaborate and accurate use an electric immersion heater and are stirred. It is easy to heat at
a low and steady rate with an electric heater, but almost impossible with a flame. When an oil
bath is used, the capillary can be fastened to the thermometer by means of a small slice of rubber
tubing used as a rubber band.

There is also a type of melting point apparatus in which the sample and thermometer are both
supported in an electrically heated metal block and the sample in the capillary can be observed
through a magnifying glass. Usually you can heat the block rapidly when the temperature is well
below the melting point, and slowly as the melting point is approached. If a compound begins to
decompose near the melting point, the capillary with the sample should be placed in the bath
after the temperature has been raised to within 6 or 10 degrees of the expected melting point, so
as to minimize the length of time that the sample is heated.

A pure substance melts at a precisely defined temperature, characteristic of every crystalline

substance and dependent only on pressure (though the pressure dependency is generally
considered insignificant).

Determining the MP is a simple and fast method used in many diverse areas of chemistry to
obtain a first impression of the purity of a substance. This is because even small quantities of
impurities change the melting point.

Melting point determination in a chemistry lab/ thiele tube

The Thiele tube is a glass tube designed to contain heating oil and a thermometer to which a
capillary tube containing the sample is attached. The shape of the Thiele tube allows for
formation of convection currents in the oil when it is heated. These currents maintain a fairly
uniform temperature distribution throughout the oil in the tube. The side arm of the tube is
designed to generate these convection currents and thus transfer the heat from the flame evenly
and rapidly throughout the heating oil. The sample, packed in a capillary tube is attached to the
thermometer, and held by means of a rubber band or a small slice of rubber tubing. It is
important that this rubber band be above the level of the oil (allowing for expansion of the oil on
heating). Otherwise, the oil softens the rubber and allows the capillary tubing to fall into the oil.
The Thiele tube is usually heated using a microburner with a small flame but a Bunsen burner
can also be used. When heating, the rate of temperature increase should be carefully controlled.
The rate of heating should be slow near the melting point (about 1-2oC per minute) to ensure that
the rate of temperature increase is not faster than the ability of the heat to be transferred to the
sample being observed. At the melting point it is necessary that the thermometer bulb and the
sample in the capillary tube be at thermal equilibrium.
Boiling point determination

When the vapour pressure of a liquid is equal to the atmospheric (or applied) pressure then
boiling occurs. The temperature, at which this occurs, for a given pressure, is the boiling point. It
should be noted, therefore, that the boiling point of a liquid decreases as the atmospheric
pressure decreases. The sample liquid is introduced by Pasteur pipette into a micro test tube (no
more than 0.5ml, which is about 10mm depth in the small test tube), and a piece of melting point
capillary tubing (sealed at one end) is dropped in with the open end down. The micro test tube
assembly is then attached to a thermometer with a rubber band or a thin slice of rubber tubing.
The whole unit is then placed in a Thiele tube.

Important points to note when placing the test tube / thermometer unit in the Thiele tube :

i. the micro test tube should be on the same side of the Thiele tube as the elbow

ii. Neither the test tube nor the thermometer bulb should be touching the glass walls of the Thiele
tube
iii. the rubber band should be placed well above the level of the oil in the Thiele tube. If the
rubber band enters the oil, the band may soften and break in the hot oil allowing the micro test
tube to fall into the oil.

Once the set up has been complete, the lower part of the side arm of the Thiele tube is carefully heated

with a small flame from the Bunsen burner moving the flame back and forth along the arm. During the

heating, there is an initial stream of bubbles as air is expelled and then, a little later, a rapid and

continuous stream of bubbles emerges from the inverted capillary tube. At this point stop heating. Soon

the stream of bubbles will slow down and stop. When they stop, the liquid sample will be drawn up in to

the capillary tube. The moment when the liquid enters the capillary corresponds to the boiling point of

the liquid, and the temperature should be recorded.

Distillation

Distillation is a method of physically separating mixtures based on differences in volatility of

components in a boiling liquid mixture. Commercially, distillation has a number of applications.
It is used to separate crude oil into more fractions for specific uses such as transport, power
generation and heating. Water is distilled to remove impurities, such as salt from seawater. Air is
distilled to separate its components—notably oxygen, nitrogen, and argon for industrial use.
Liquid chemicals for diverse uses are often distilled after synthesis to remove impurities and
unreacted starting materials.

Types of distillation

Simple distillation

In simple distillation, the vapour is immediately channelled into a condenser. Consequently, the
distillate is not pure but rather its composition is identical to the composition of the vapors at the
given temperature and pressure. As a result, simple distillation is effective only when the liquid
boiling points are very different or when separating liquids from non-volatile solids or oils. For
these cases, the vapour pressures of the components are usually sufficiently different that the
distillate may be sufficiently pure for its intended purpose.

Fractional distillation

For many cases, the boiling points of the components in the mixture will be sufficiently close.
Fractional distillation separates the components by repeated vaporization-condensation cycles
within a packed fractionating column. This separation, by successive distillations, is also referred
to as rectification.
As the solution to be purified is heated, its vapours rise to the fractionating column. As it rises, it
cools, condensing on the condenser walls and the surfaces of the packing material. Here, the
condensate continues to be heated by the rising hot vapours and it vaporizes once more. Each
vaporization-condensation cycle/ theoretical plate will yield a purer solution of the more volatile
component. The larger the number of theoretical plates the better the degree of separation.

A spinning band distillation system uses a spinning band of Teflon or metal to force the rising
vapours into close contact with the descending condensate, increasing the number of theoretical
plates. There are many types of fractionating columns that are used in fractional distillation.
They are all similar in that the surface area, which contacts the distilling vapor, is increased. The
larger the surface area contacted by the vapor, the more efficient the column is in separating the
components. There are columns which are open, columns with glass indentations called Vigruex
columns, and columns which are loosely packed with glass, metal or ceramic material. The
fractionating column is often insulated to keep the temperature of the column nearly constant. If
the temperature of the column fluctuates widely, it is difficult to maintain a slow, constant
distillation rate. Sophisticated fractionating columns have regulated heating coils built into them.

     

Types of fractionating columns:

(a)Vigreux
(b)Open
(c)Packed

Steam distillation

Steam distillation is a method for distilling compounds which are heat-sensitive. The temperature
of the steam is easier to control than the surface of a heating element, and allows a high rate of
heat transfer without heating at a very high temperature. This process involves bubbling steam
through a heated mixture of the raw material. By Raoult's law, some of the target compound will
vaporize (in accordance with its partial pressure). The vapour mixture is cooled and condensed,
usually yielding a layer of oil and a layer of water.

Applications of Steam distillation include various aromatic herbs and flowers. The distillates are
used as essential oils, perfumes, in aromatherapy, food processing and skin care.

Vacuum distillation

Some compounds have very high boiling points. To boil such compounds, it is often better to
lower the pressure at which such compounds are boiled instead of increasing the temperature.
Once the pressure is lowered to the vapour pressure of the compound (at the given temperature),
boiling and the rest of the distillation process can commence. This technique is also employed in
the rotary evaporator.

Vacuum distillation is also useful for compounds which boil beyond their decomposition
temperature at atmospheric pressure and which would therefore be decomposed by any attempt
to boil them under atmospheric pressure.

Column chromatography

Column chromatography is a method used to purify individual chemical compounds from

mixtures of compounds. It consists of a stationery phase (eg silicon dioxide, alumina) and a
mobile phase (solvent system). The stationary phases are usually finely ground powders or gels
and/or are microporous for an increased surface area for adsorption. The mobile phase / eluent is
either a pure solvent or a mixture of different solvents (multi solvent system). It is chosen so that
the retention factor value of the compound of interest is roughly around 0.2 - 0.3 in order to
minimize the time and the amount of eluent needed to run the chromatography. The eluent has
also been chosen so that the different compounds can be separated effectively. The eluent is
optimized in small scale pretests, often using thin layer chromatography with the same stationary
phase.
The chromatography column is a glass tube with a diameter ranging from 5 - 50 mm and a height
of 5 cm - 1 m with a tap at its base. At the bottom of the column, there is a filter (eg glass wool
plug) to prevent the loss of the stationary phase.

Two methods are generally used to prepare a column: the dry method, and the wet method.

 For the dry method, the column is first filled with dry stationary phase powder,
followed by the addition of mobile phase, which is flushed through the column until
it is completely wet, and from this point is never allowed to run dry.
 For the wet method, a slurry is prepared of the eluent (solvent system) with the
stationary phase powder and then carefully poured into the column being careful to
avoid air bubbles.

Procedure

A solution of the organic material is pipetted on top of the stationary phase. This layer is usually
topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic
layer from the velocity of newly added eluent. Eluent is slowly passed through the column to
advance the organic material. Often a spherical eluent reservoir or an eluent separating funnel is
put on top of the column.

The individual components are retained by the stationary phase differently and separate from
each other while they are running at different speeds through the column with the eluent due to
their different affinities for both the mobile and stationary phases. At the end of the column they
elute one at a time. During the entire chromatography process the eluent are collected in a series
of fractions. The composition of the eluent flow can be monitored and each fraction is analyzed
for dissolved compounds, e.g. by UV absorption. Coloured compounds can be seen through the
glass walled column as moving bands.
The main advantage of column chromatography is the relatively low cost and disposability of
the stationary phase used in the process. Disposal is necessary to prevent cross-contamination
and stationary phase degradation due to recycling.

TLC

Thin Layer Chromatography (TLC) is an extremely useful technique for monitoring reactions. It
is also used to determine the proper solvent system for performing separations using column
chromatography. TLC uses a stationary phase, usually alumina or silica, that is highly polar
(standard) or non-polar (reverse phase), and a mobile phase (solvent whose polarity you choose).

The procedure involves applying a reaction mixture in solution to the TLC plate then running the
plate by allowing a solvent (or combination of solvents) to move up the plate by capillary action.
Depending on the polarity of the components of the mixture, different compounds will travel
different distances up the plate. More polar compounds will "stick" to the polar silica gel and
travel short distances on the plate (higher retention), while non-polar substances will diffuse into
the solvent and travel large distances on the plate. The measure of the distance a compound
travels is called Rf. This number, between zero and one, is determined by measuring the distance
the compound moved from the baseline (where it was originally spotted) to where it stopped,
divided by the distance the solvent moved from the baseline to about ¾ up the plate at which
point the process would have been stopped(solvent front).
Steps for TLC:

1) Cut TLC plates into square or rectangular pieces of required size. Use a ruler and a pencil to
lightly mark baselines on the silica side of the plate, being careful not to remove any silica from
the plate.

2) Determine the solvent system. Your compounds will travel different distances up the plate
depending on the solvent you choose. In non-polar solvents like pentane and hexane, most polar
compounds will not move, while non-polar compounds will travel some distance up the plate and
vise-versa. A good solvent system is one that moves all components of your mixture off the
baseline, but does not put anything on the solvent front.

Standard solvents and their polarity

Very polar - Methanol > Ethanol > Isopropanol

Moderately polar additives - Acetonitrile > Ethyl Acetate > Chloroform > Dichloromethane >
Diethyl Ether > Toluene

Non-polar additives - Cyclohexane, Petroleum Ether, Hexane, Pentane

Common solvent combinations:

 Ethyl Acetate:Hexane - 0-30% ;

 Ether:Pentane - 0-40% ;
 Ethanol:Hexane/Pentane - 5-30%

3) Fill TLC chamber with 1-2 mL of the desired solvent system.

4) Spot the compound on the baseline of the TLC plate.

5) Run the TLC. Let the solvent go about 90% of the way up the plate.

6) Remove the plate from the chamber and mark the solvent front immediately with a pencil

7) Let the solvent dry off of the plate.

8) Visualize the TLC using non-destructive technique eg The UV lamp. Place your plate under
the UV lamp and circle any UV active spots with your pencil.
THE MICROSCALE LAB

Microanalysis is the chemical identification and quantitative analysis of very small amounts

of chemical substances (generally less than 10 mg or 1 ml) or very small surfaces of material
(generally less than 1 cm2).

Micro measurement of volume and weight

In micro reactions, you need to use small quantities of reagents and hence small pieces of
apparatus are employed.

Conical vial

The conical vial is used as a reaction vessel, for extractions, and as a storage container. Its flat
base allows it to stand upright on the laboratory bench. The interior of the vial tapers to a narrow
bottom, making it possible to withdraw liquids completely from the vial using a disposable
Pasteur pipette. The vial has a screw cap which tightens by means of threads cast into the top of
the vial. These threads also allow attachment of various other pieces such as a condenser or
distillation head, using the double-caps provided in the kit.
Since one rarely works with volumes larger than 2-3 mL, graduated cylinders are rarely used in
microscale experiments. Instead, one uses smaller scale volumetric devices such as syringes,
automatic pipettes, and calibrated disposable Pasteur pipettes.

Syringes are especially useful when anhydrous conditions must be maintained during an

experiment. The needle can be inserted through a rubber septum sealing the reaction vessel, and
the liquid added to the reaction mixture. To fill the syringe, insert the needle into the liquid and
draw in the required volume. Withdraw the syringe and pull the barrel back ever so slightly to
draw any liquid remaining in the needle into the syringe.

Disposable Pasteur pipets are used for dispensing small quantities of liquids, as filtration
devices, and as columns for small-scale column chromatography. Pasteur pipets may be
calibrated for use in operations where the volume does not need to be known precisely, such as
for measurement of solvents need for extraction and for washing a solid obtained following
crystallization.

A Filtering Pipet is used to remove solid impurities from a liquid with a volume less than 10-
mL. To prepare it, a small piece of cotton is inserted into the top of a Pasteur pipet and pushed
down to the beginning of the lower constriction in the pipet. It is important that enough cotton is
used to collect all the solid being filtered; however, the amount used should not be so large that
the flow rate throught the pipet is significantly restricted. The cotton plug can be pushed down
with a long thin object such as a glass stirring rod. In some cases, such as when filtering a
strongly acidic mixture or when performing a very rapid filtration, it may be better to use glass
wool in place of the cotton, as a filtering aid. To conduct a filtration, the filtering pipet is
clamped so that the filtrate will drain into an appropriate container. The mixture to be filtered is
transferred to the filtering pipet with another Pasteur pipet. If the volume of the mixture being
filtered is less than 1-2 mL, you should rinse the filter and plug with a small amount of solvent
after the last of the filtrate has passed through the filter. If desired, the rate of filtration can be
increased by gently applying pressure to the top of the pipet using a pipet bulb.

A Filter-tip Pipet is useful for transferring volatile solvents during extractions and in filtering
very small amounts of solid impurities from solutions. It is made by loosely shaping a tiny piece
of cotton into a ball, and pushing it to the bottom of the pipet using a wire with a diameter
slightly smaller than the inside diameter of the narrow end of the pipet. To use the filter-tip pipet,
simply draw the mixture to be filtered into the pipet using a pipet bulb and then expelling it.
With this procedure, small amounts of solid will be captured by the cotton.

Micro pipette

Micropipettes are lab apparatus are used by scientists in laboratories to precisely and accurately
deliver small volumes of a liquid substance. They come in different types and each is used to
administer a specific volume of fluid.

Consists of a handle that contains a spring- loaded plunger and a micrometer dial. The dial helps
in selecting the amount of liquid to be transferred. Delivers specific range eg 10-100 µL.
Operation- liquid should never be drawn into the pipette shaft. Attach a disposable plastic tip
when drawing out reagents. Only the tip should be filled. The plunger will stop at two different
positions when it is depressed. The first of these stopping points is the point of initial resistance
and will result in the desired volume of solution being transferred. The second stopping point is
when the plunger is depressed beyond the initial resistance until it is in contact with the body of
the pipettor. At this point, the plunger cannot be depressed further. This second stopping point is
only used for the complete discharging of solutions from the plastic tip
Regardless of the manufacturer, micropipettes operate on the same principle: a plunger is
depressed by the thumb and as it is released, liquid is drawn into a disposable plastic tip. When
the plunger is pressed again, the liquid is dispensed.

The tips are an important part of the micropipette and allow the same device to be used for
different samples (so long as you change your tip between samples) without washing. They come
in a number of different sizes and colours, depending on the micropipette they are used with, and
the volume to be dispensed.

The plunger can sit at any one of three positions: 

Position 1 is where Position 2 is reached by Position 3 is reached

the pipette is at rest pushing down on the by pushing down from
plunger until resistance is position 2
met

Microbalance

Microscale experiments involve quantities on the order of 200-300 mg at most, and it is thus
important to be able to weigh solid substances to the nearest milligram. This requires use of a
sensitive top-loading balance protected against drafts with a shield, or an analytical balance.

All weighings must be made into a previously weighed container. The weight is then subtracted
from the total weight of container plus sample to give the weight of the sample.
Solid samples are manipulated using microspatulas similar to those shown below. The larger
style is more useful when relatively large quantities of solid must be dispensed.

A Microbalance is an instrument capable of making precise measurements of weight of objects

of relatively small mass, µg. In comparison, a standard analytical balance is 100 times less
sensitive. It is the precision of the microbalance that distinguishes it from other weighing
devices. It also has very fast response times and a fully automatic draft shield.

Applications

 accurate weighing of toxic, hygroscopic, or heavily oxidizing samples

 Differential weighing (ashing or incineration, drying, measurement of coatings, checking
spillage quantities,
 Mass determination that allows for air buoyancy 
 Air density determination for metrological weight analyses

Microcrystallisation using a craig tube

Recrystallizations can be carried out using a conical reaction vial and conventional vacuum
filtration to collect the crystals on a small filter paper, or in a Craig tube, which is a device
designed specifically for recrystallization of very small quantities of materials.

In recrystallizations with a conical reaction vial, the conical vial simply takes the place of the
Erlenmeyer flask used for macroscale recrystallizations. The isolation of the crystals can be done
in a number of ways depending on their form:

(i) Once crystallization is complete, the mother liquors and crystals are filtered through a
small Pasteur pipet.
(ii) If the crystals adhere to the side of the flask, then filtration is unnecessary. Simply use
a filter-tip pipet to remove the mother liquors and transfer them to another flask.
Fresh, cold solvent is added to wash the crystals, and this is then removed with the
pipet in the same way. The crystals are then dried using a very light stream of air or
nitrogen, but care must be taken to ensure that the stream is light enough that the
crystals don't get blown out of the vial.

Craig tube Recrystallizations

Craig tubes are particularly useful for recrystallizing amounts of solid less than ~100 mg, the
main advantage being that it minimizes the number of transfers of solid material and thus
maximizes the yield of crystals. The separation of the crystals from the mother liquor with the
Craig tube is very efficient, and little time is required for drying the crystals. The steps involved
are fundamentally the same as those performed in macroscale crystallizations with an
Erlenmeyer flask and a Hirsch funnel:

Step 1. In crystallizations where a filtration step is not required in order to remove insoluble
impurities such as dirt or activated charcoal, this step can be done directly in the Craig tube. The
solid is placed in the Craig tube and the appropriate solvent is heated to boiling in a test tube
placed in a sand bath. Several drops of hot solvent are added to the Craig tube, which is then
heated in the sand bath while stirring continuously with a microspatula using a twirling motion.
This helps dissolve the solute and prevent the boiling liquid from bumping. Additional portions
of hot solvent are added until the solid is completely dissolved. Do not add too much solvent, in
order to maximize the yield.

Step 2. The hot solution is cooled slowly in the Craig tube to room temperature. This is done by
inserting the inner plug into the outer part of the Craig tube, and then placing the whole thing
into a 10-mL Erlenmeyer flask. This provides some insulation to slow the cooling rate. The
cooling rate can be slowed even further by first filling the Erlenmeyer flask with ~8 mL of hot
water at a temperature below the boiling point of the solvent. The Erlenmeyer flask containing
the Craig tube is then placed on a few layers of paper and left alone to cool to room temperature.
Once crystallization at room temperature is complete, the Craig tube is then placed in an ice-
water bath to maximize the yield.

Step 3. Once crystallization is complete, a 3" piece of copper wire is wrapped around the barrel
of the inner plug of the Craig tube, and a Centrifuge tube is placed over top of it. After bending
the copper wire back up the side of the centrifuge tube so that the Craig tube is held securely
inside it, the centrifuge tube is inverted. The solvent should seep out of the Craig tube, leaving
the crystals behind. The tube is then centrifuged for a few minutes to complete the separation of
the mother liquors from the crystals. Using a microspatula, the crystals are then scraped off the
end of the inner plug or from inside the Craig tube onto a watch glass or piece of paper. Minimal
drying will be necessary.     
Micro heating and refluxing

Heating is provided by a sandbath atop a magnetic stirrer/heater. A thermometer should be

clamped in contact with the sand so as to allow monitoring of the bath temperature. A typical
assembly for heating a reaction mixture under reflux is shown below. While an air condenser is
adequate for most applications, a water-jacketed condenser is also supplied, for cases where the
solvent is very volatile or where the ambient air temperature is very high. A "spin vane" might
also be included for magnetic stirring of the reaction mixture - this is a triangular device coated
with teflon, which is shaped to fit the bottom of the conical flask.

Note that the apparatus is clamped at the condenser rather than at the flask, as one would do for a
macroscale experiment using conventional ground-glass joint glassware. The apparatus can be
clamped in this way because of the screw-cap connection between the condenser and reaction
vial, which prevents the connection from falling apart.
Micro extractions

In microscale experiments, the conical reaction vial is the glassware item used for extractions.
The two immiscible liquid layers are placed in the vial, and the top is sealed with a cap and a
Teflon insert (with the Teflon side toward the inside of the vial). The vial is shaken to provide
thorough mixing between the two liquid phases. As the shaking continues, the vial is vented
periodically by loosening the cap and then tightening it again. After about 5-10 seconds of
shaking, the cap is loosened to vent the vial, retightened, and the vial is allowed to stand upright
in a beaker until the two liquid layers separate completely.

Two basic procedures are possible, depending on whether the solvent being used to extract the
desired product is heavier or lighter than water. Method A is employed for extractions where the
lower layer is a heavy solvent such as dichloromethane:

Method A - solvent heavier than water

Method B is employed for extraction with a solvent which is lighter than water, such as diethyl
ether. Note that in this technique, one draws both phases into the pipet and then returns the
heavy (aqueous) phase to the conical vial. Ether is so volatile that it is often difficult to hold it in
the pipet. Use of a filter-tip pipet for this procedure will help prevent the volatile organic layer
from squirting out in an uncontrolled way.

Method B - solvent lighter than water

 

Microdistillation

The key to successful microscale distillations is in avoiding long distillation paths, since this is
the main factor leading to loss of material during distillation. Short-path microscale distillations
are carried out using theHickman distillation head as the receiving device for the distilled
liquid. Two types of Hickman head, 'ported' and 'unported', are shown in the figure below. The
complete apparatus consists of a flask or vial containing the liquid and a magnetic spin vane or
boiling stone, attached to the bottom joint of the Hickman head. If desired, a condenser is
attached to the top joint. A thermometer can be suspended down the middle in order to record the
distilling temperature, with the bottom of the thermometer in the lower part of the Hickman head
just below the circular well. The vapours of the heated liquid rise upward and are cooled and
condensed on either the inside walls of the Hickman head or on the walls of the condenser. As
liquid drains downward, it collects in the circular well at the bottom of the still. The well can
contain as much as 2-mL of liquid.

 Collection of fractions is easiest with the ported Hickman head; the port is opened and the liquid
in the well removed with a Pasteur pipet (see 'C' below). With the unported head, the liquid is
drawn out from the top with a Pasteur pipet (see 'A' below). If a condenser or internal
thermometer is used, the distilling apparatus must be partially disassembled in order to do this. In
some stills the inner diameter of the head is so small that it is difficult to reach in at an angle with
the pipet and make contact with the liquid. This problem may be remedied by bending the tip of
the pipet slightly in a flame. Once removed, the liquid is transferred to a small vial and capped
with a Teflon-sealed cap.