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Sci.chem FAQ - Part 4 of 7
Section - 16. Laboratory Procedures

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16.1  What are the best drying agents for liquids and gases?

The Rubber Handbook lists the traditional information on drying agents
that involve on chemical action.  This lists phosphorus pentoxide and 
magnesium perchlorate as the most effective desiccants. However, later 
work by Burfield [1-9] has demonstrated that much of the traditional
information is misleading. He found that the efficiency of the desiccant 
is strongly dependent upon the solvent. He also found that Drierite 
( anhydrous calcium sulphate ) is only a moderately efficient desiccant for 
organic solvents [9], and that correctly prepared molecular sieves are 
often the preferred desiccant [2]. His publications are highly recommended.

16.2  What is the effect of oven drying on volumetric glassware?

Many older laboratory texts insist that volumetric glassware should not
be oven dried because of the danger of irreversible and unpredictable
volume changes. However most modern laboratory glassware is now made of
Pyrex, and work by D.R.Burfield has demonstrated that low temperature
drying does not significantly affect the calibration of volumetric
glassware [10]. He demonstrated that exposing volumetric flasks and
pipettes to 320C, either continuously or thermally cycled, resulted in no 
significant detectable change to the calibration. He concluded that
"oven temperatures in the range of 110-150C should provide efficient drying
of glassware with no risk of discernible volume changes, even after 
prolonged use, providing that Pyrex glass is the material of construction".  

16.3  What does the Karl Fischer titration measure?

In 1935 Karl Fischer used the reaction between iodine, sulfur dioxide, and 
water to produce a technique for quantifying water [11]. In aqueous solution, 
the reaction can be presented as I2 + SO2 + 2H2O <=> 2HI + H2SO4.
He used anhydrous methanol to dissolve the I2 and SO2, and added pyridine
to move the equilibrium to the right by reacting the acidic products. 

Fischer assumed his modifications did not change the reaction and one mole of 
iodine was equivalent to two moles of water. Smith et al.[12], demonstrated
that both the methanol and pyridine participate in the reaction and one mole
of iodine is equivalent to one mole of water. They suggested two steps:- 
(1)  SO2 + I2 + H2O + 3RN -> 2RN.HI + RN(SO2)O
(2)  RN(SO2)0 + CH3OH -> RN(SO4CH3)H    (where R = base = C5H5 for pyridine)

This was further investigated by E.Scholz [13], who proposed:
(1)  CH3OH + SO2 + RN -> (RNH)SO3CH3
(2)  H20 + I2 + (RNH)SO3CH3 + 2RN -> (RNH)SO4CH3 + 2(RNH)I   (where R = Base)

The advantage of the Karl Fischer titration is that it has few interferences
and can quantify water from < 1ppm to 100% in diverse samples, ranging from 
gases to polymers. It will measure all water that is made available to the
reagent. the endpoint is usually ascertained using a dead-stop endpoint,
and for low water levels coulometric techniques are used to quantitatively
produce the iodine by anodic oxidation of iodide. The procedures are 
described in detail in ASTM, AOAC etc.

16.4  What does the Dean and Stark distillation measure?

The Dean and Stark procedure can be used to measure the water content of
a diverse range of samples, and has been extensively used in industrial
laboratories to measure water in petroleum oils. The technique can measure
% levels of water, but is not as accurate as the Karl Fischer titration,
and is not applicable to samples where the water is not liberated by the
solvent. The sample is mixed with a solvent ( usually a toluene/xylene mix ) 
and refluxed under a condenser using a special receiver. There are two common 
designs of receivers, one for solvents that are heavier than water, and the 
more common one for solvents that are lighter than water - illustrations will
be shown in most laboratory glassware supplier catalogues. 

The water and solvent are refluxed, and as they condense the two phases 
separate as they run into the receiver. The water remains in the receiver 
while the solvent returns to the flask. The Dean and Stark technique is also 
useful for removing unwanted water from reactions, eg the synthesis of 
dibutyl ether by the elimination of water from two molecules of n-butanol 
using acidic conditions. An example of this is provided in the preparation 
of dibutyl ether described in Vogel, and detailed procedures for the 
determination of water using Dean and Stark are provided in ASTM and AOAC.
16.5  What does Kjeldahl nitrogen measure?

The Kjeldahl procedure is routinely used to measure the protein nitrogen 
content of organic compounds, especially natural foodstuffs. Contrary to 
popular belief, the procedure does not determine total nitrogen on all 
organic compounds, as it is not applicable to materials containing N-O or 
N-N linkages without modifications to the method. This discrepancy is 
becoming of more significance as automated nitrogen analysers using other 
techniques are producing different results because they measure the total 
nitrogen present.

The method usually involves high temperature ( 390C ) digestion of the 
sample using concentrated sulfuric acid, a catalyst ( Cu, Hg, or Se ), 
and a salt to elevate the acid boiling point. In some cases 30% hydrogen
peroxide is also used, making the digestion effectively a high-temperature 
piranha solution attack on the organic matter. After digestion, the sample
is made strongly alkaline and the ammonia is steam distilled into a boric 
acid solution, and aliquots are titrated against a standard acid using an
indicator solution endpoint.

Some organics compounds require aggressive digestion conditions to make 
all the organic nitrogen available, consequently Kjeldahl procedures should 
not normally be used on samples that may have N-O or N-N bonds. Details of 
procedures for foods are in the AOAC handbooks, and general Kjeldahl 
procedures are detailed in the ASTM volumes. 

16.6  What does a Soxhlet extractor do?

The soxhlet extractor enables solids to be extracted with fresh warm solvent 
that does not contain the extract. This can dramatically increase the
extraction rate, as the sample is contacting fresh warm solvent. The sample 
is placed inside a cellulose or ceramic thimble and placed in the extractor. 
The extractor is connected to a flask containing the extraction solvent, and 
a condenser is connected above the extractor. The solvent is boiled, and the
standard extractor has a bypass arm that the vapour passes through to reach 
the condenser, where it condenses and drips onto the sample in the thimble. 
Once the solvent reaches the top of the siphon arm, the solvent and extract 
are siphoned back into the lower flask. The solvent reboils, and the cycle
is repeated until the sample is completely extracted, and the extract is
in the lower flask.

There is an alternative design where the hot solvent vapour passes around 
the thimble, thus boiling the solvent in the thimble - this can be a problem 
if low-boiling azeotropes form. Procedures for using soxhlet extractors are 
described ( along with illustrations which might make the above description 
comprehensible :-) ), in Vogel and many other introductory organic laboratory

16.7  What is the best method for cleaning glassware?.

As scientific glassware can be used for a variety of purposes, from the
ultra-trace determination of sub-ppq levels of dioxin, to measuring % 
concentrations of inorganic elements, there is no single cleaning method that
is "best" for all circumstances. Difficult and intractable deposits often 
involve the use of hazardous and corrosive chemicals, and details of the 
necessary safety precautions for each cleaning solution should obtained 
before attempting to clean glassware. The use of heat and/or ultrasonic 
agitation can greatly improve the removal rate of many deposits, especially 
inorganic and crystalline deposits.

Whilst the semiconductor industry use piranha solution ( refer Section 
12.9 ), and several other reactive and toxic chemicals for cleaning, those
reagents can react dangerously with the residues found in laboratories, and 
their use is prohibited in some institutions. Such chemicals should only be 
used after extensive prior consultation with laboratory management and safety
staff - to either identify safer alternatives, or to ensure that appropriate 
protective and safety systems are in place.

If the probable composition of material deposited on the glassware is known,
then the most appropriate cleaning agent can be readily selected. There are
several safe aqueous cleaning solutions that are routinely used. If possible,
glassware should be washed or soaked immediately with an appropriate solvent 
for the residue. This will make subsequent cleaning easier, but all traces
organic solvents must be removed before using any cleaning solution.

The most common aqueous-based soaking solutions are commercial formulations 
that usually contain alkalis, chelating agents, and/or surfactants, and can 
be used either at ambient temperature, or temperatures up to boiling ( with 
ventilation - caustic fumes are noxious ). These are very effective for 
general grime, most labels, pyrogens, and many common chemical residues, and 
well known examples include RBS-35, Decon, Alconox, and Pyroneg. Their main
advantages are low toxicity and ease of disposal.

The next common strategy involves physical abrasion to remove deposits
inside flasks, usually with a bottle brush and an aqueous cleaning solvent
( like those above ) or a suitable organic solvent. A refinement is to add
sand, pumice, glass spheres, or walnut shell chips, along with some water 
or solvent, and shake vigorously. It's important that the sand should not 
have sharp edges - as it can scratch the glass. It has been suggested that 
table salt in solvent ( eg petroleum spirit, methylene chloride, acetone ) 
is superior, as it doesn't scratch the glass, can be easily removed by 
washing with water, and has minimal disposal problems [14].   
The traditional glassware cleaning solution is "chromic acid", and many
analytical chemistry texts detail the preparation [15,16]. Chromium (VI) is 
highly toxic ( mutagenic, carcinogenic ), and disposal is expensive, as all 
solutions containing more than 5 mg/l of chromium are considered hazardous 
waste in the USA. Disposal of chromic acid requires a two-stage process, 
involving bisulfite addition to reduce Cr(VI) to Cr(III), followed by 
neutralisation of the acid. There have also been several reports of 
spontaneous explosions of chromic acid cleaning solutions [17,18,19], 
consequently the use of chromic acid for cleaning glassware is declining,
and several alternative glassware cleaners have recently been evaluated [20].
Sodium dichromate dihydrate is usually used to prepare chromic acid, as 
potassium dichromate is less soluble in sulfuric acid. One technique is to 
dissolve 140g of technical grade sodium dichromate dihydrate in approximately
100 ml of water. Add two litres of technical grade 98% sulfuric acid to a 4-5
litre glass beaker that is sitting in a cold water bath in a fume cupboard. 
Carefully stir the acid gently and pour a few mls of the dichromate solution 
slowly into the acid. Keep repeating the addition every few seconds - after 
the previous dose has been dispersed. As long as the stirring is gentle and 
continuous, little or no splattering should occur, but the solution will 
become quite warm. Allow to cool before storing in a glass-stoppered reagent 
bottle. Always ensure that the stopper is sufficiently loose to release any 
gas pressure. Never use a screw-capped or similar types of sealed containers. 

If made correctly, the chromic acid solution should have no precipitate, will 
be a deep red colour, and will last for years in a glass-stoppered bottle. 
Ensure the glassware to be cleaned does not have any residual organic 
solvents. Chromic acid is very effective at around 80C, but an overnight soak
at ambient temperature is commonly used. If the solution develops a green 
hue, it is exhausted and should be disposed of, or regenerated, using 
appropriate procedures. Slowly pouring used acid down a drain with the cold 
water tap fully open is no longer considered appropriate. There is a recent
report of a technique to regenerate chromic acid cleaning solution ( by 
distillation of water and oleum ) that reduces disposal quantities [21]. 

The major problems with chromic acid are the multiple rinses, and perhaps 
even alkaline EDTA treatment [16], that are necessary to remove all the 
chromium from glassware - especially if it is required for cell culture or
trace analysis, and the increasing problems of safe and legal disposal of 
spent solutions.

An alternative to chromic acid is "Nochromix", which is commercial solid 
formulation that contains 90-95% of ammonium persulfate ( ((NH4)2)S208 )
along with surfactants and other additives. The powder is dissolved in 
water and mixed with 98% sulfuric acid. The solution is clear, but turns
orange as the oxidizer is consumed, and further additions of solid are
routinely required. It is available from Godax Laboratories, New York.

A similar bath that is reported to be very effective can be made by the 
addition of 19 grams of reagent grade ammonium persulfate to two litres of 
reagent grade 98% sulfuric acid [22]. Add more ammonium persulfate and acid 
every few weeks, as necessary.  

One popular replacement for chromic acid in organic laboratories has been 
alcoholic sodium hydroxide or potassium hydroxide solutions. These remove 
most deposits, with metals and hydrocarbons greases ( Apiezon ), as notable
exceptions. One advantage they have is that they will remove silicone grease
deposits from joints and stopcocks, especially if warmed to 65C, and the
glassware immersed for up to 10 minutes [23]. Prolonged immersion, even at 
ambient temperature, will damage ground-glass joints, dissolve glass sinters, 
and will leave glass surfaces translucent or opaque. The solution can be 
prepared by either adding two litres of 95% ethanol to 120 mls of water 
containing 120 grams of sodium hydroxide [16], or by dissolving 100 grams of 
potassium hydroxide in 50 ml of water and, after cooling, make up to one 
litre [15].  

Solutions based on hydrofluoric acid, usually containing 1-5% of HF, also 
rapidly attack glass, and destroy sinters, but are very effective for removal 
of carbonaceous and fine silica deposits. They also remove silicone greases,
but alcoholic caustic solutions are preferred [23,24,25]. Hydrofluoric acid 
is corrosive and extremely nasty if it comes in contact with humans. It 
requires extensive safety precautions before use. For most deposits, only a 
few minutes are required, and ultrasonic agitation often assists the removal
of deposits. Cleaned glassware usually remains transparent. Cleaners 
containing HF should not be used on volumetric glassware.  

Another acidic solution, comprising of a 3:1 mixture of concentrated sulfuric
acid and fuming nitric acid, is also extremely effective for removing grease 
and dirt, but also requires extensive safety precautions. The grease and dirt
can often be removed more safely using hot aqueous-based cleaners.

If you have intractable organic-based deposits in flasks without standard 
ground glass ( or clear glass ) joints, then some deposits can be carefully 
burned off in a glass annealing furnace. The glass needs to carefully 
follow a slow heating and cooling schedule to minimise thermal stresses and
distortion. My experience has been that standard joints do tend to freeze
more often after such treatment. Also note that glassblowers may not want 
to coat their annealing furnace with your rubbish, so they may prohibit 
the use of their furnace for such activity.

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