C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook [630682]
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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CHAPTER I
INTERNAL RULES OF LA BOUR HEALTH AND SAFE TY IN
CHEMISTRY AND CLINIC AL BIOCHEMISTRY LABORATORY
Laboratories from chemistry and biochemistry, and biochemistry and
molecular biology disciplines are for teaching, scientific and / or application
(clinical, medical, pharmaceutical, etc.) activities. In such laboratories are
carried a variety of jobs that require substances, glassware and utensils, and
equipment and facilities tailored to their goals.
The varied conditions for running some activities and the use of
chemically aggressive (flammable, explosive or toxic) substances require
taking various mea sures to avoid accidents. Accidents of any kind can be
avoided if the working conditions prescribed in working methods are strictly
respected. To this end there are general rules of a legislative nature referring
the safety and health at work, which should be well known to those working
in the profile laboratories.
Students work in the lab begins with a briefing on the norms on health
and safety at work. This training is consistent with Government Decision
no. 1425 from 11.10.2006 for approving the Methodol ogica l Norms for the
application of Law of labor safety and health, no. 319/2006 published in the
Official Gazette of Romania, Part I, no. 882 of 30.10.2006. The training is
followed by verification of knowledge in this area and the establishment of a
form of collective training where students will sign that they are aware of
legislative concepts submitted and undertakes to respect these general rules
and specific for laboratory, presented at the first meeting.
1.1. LABOUR HEALTH AND SAFETY
The rules for health and safety concerns the safety of people who
carries activities in sectors that may adversely affect health. These rules
include measures concerning the storage and handling of substances,
operations with glassware, utensils and laboratory equipment, hand ling and
preservation of biological products and, also of samples destinated for
analysis.
1.2. STORAGE AND HANDLING OF CHEMICALS IN
LABORATORY OF CHEMISTRY AND BIOCHEMISTRY
Storage and handling of chemicals concerns the nature and specificity of
these chemicals and rely on specific norms for clinical biochemistry and the
physic -chemical analysis laboratories.
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Corrosive chemicals (eg: alkali NaOH, KOH, concentrated acids: HNO 3,
H2SO4, HCl, CCl3COOH, HBr, etc. and other substances NH3, AgNO3,
N2O2, Br 2, etc.) and also volatile and flammable substances (eg: peroxides,
nitro compounds, ethers, etc.) are not kept inside the laboratory. For these
substances storage space with spec ially designed ventilation window are
provides. Beside is presented the European Union standard symbol used for
corrosive substances.
Liquefied gas cylinders (oxygen, acetylene, etc.) are also preserved in a
designated area, away from heat and with ventila tion possibilities. Also, to
anchor the wall by means of metal clamps is proceeded.
Chemicals with strong toxic action (eg: Hg, Pb, H2S, P, CO, CH, OH,
etc.) are kept in locked cupboards, and also corrosive and toxic substances are
handled with circumspect ion, wearing lab coat and protective equipment (lab
coat, goggles, gloves, etc.). Also, for these chemicals European Union
proposed a symbol by which to warn people that are coming into contact with
these substances on the degree of toxicity and hazard. Explosive substances
(eg: peroxides, nitro compounds, chlorine, perchlorates, etc.) will be handled
carefully avoiding strikes and proximity to heat sources. The symbol for
warning people about the explosive substances proposed by the European
Union is show n below.
Substances that produce gases or vapors in contact with air (eg: organic
solvents, etc.) will not be discharged into the canal. It will proceed to collect
them in appropriate containers and must be kept away from open fire sources
(gas burn er, electrical sparks, matches, etc.).
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Photosensitive substances are handled in appliances and cookware coated
with aluminum foil or paper in black or dark rooms. Keeping done in brown
bottles.
Filling large volumes of liquids in containers is done using glass traps,
baited with a rubber pear.
Solutions substances th at react violently with water are transferred only in
dry and heat resistant vessels. In the case of sulfuric acid (H 2SO 4) the dilution
of the concentrated acid is achieved by pouring it in to aqueous phase and
continuously cooling the flask externally wit h cold tap water. Do not pour
water into sulfuric acid because there is danger of sputter due to highly
exothermic and violent reaction.
For unknown substances it will be proceded to their identification, or
destruction. Avoid completely organoleptic contr ol (taste, smell).
1.3. OPERATIONS WITH GLASSWARE, REAGENTS AND
LABORATORY UTENSILS
In the laboratory of Chemistry and Biochemistry discipline reagents
(reagents), laboratory glassware and utensils are handled and employed by
their destination:
laboratory glassware must be washed and dried before use. If by ordinary
washing with water and detergent the glassware cannot be cleaned, then is
maintained in sulfur -chromic mixture. Glassware is dried on special holders,
in the oven or using a heating blower. Removal of organic substances (eg.
fats) from the glass vessel walls can be done with organic solvent (eg: ether,
benzene, alcohol etc.), the residue is collected in special containers for
recovery.
bottles containing liquid or solid chemical substances must be labeled
accordingly. Keep chemicals or reagents in labeled pots. Labels can be
covered with a thin layer of wax to protect information on the label. It is
mandatory that each label to mention the chemical formula of the substance,
called, data on purity (solid) or concentration (for liquids), factor solution – if
necessary. For some substances is necessary to specify the date on which the
reagent or solution was prepared. The bottles labeled sits on the shelf of
reagents for laboratory) near to laborator y table where are to be used.
Necessary reagents for experimental works are kept in special bottles with
glass stopper (grinded), and in the case of alkaline solutions with a rubber
stopper. Light sensitive reagents are kept in brown laboratory bottles kep t in
the dark. During lab work if more reagents are used, closing the bottles
immediately after use with its plug (do not change the substance or reagent
bottle caps) it has to be considered. Before working plug reagent bottles will
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sit on the lab table wi th the side in indirect contact with the substance, to
protect work surfaces.
run-over of reagents from bottles is done on the
opposite side of the label so the label will not be
damaged by the trickle of the bottle content. In case of
accidental release of a reagent it should be immediately
removed and that place cleaned (if necessary neutralized)
in order to clean all remaining traces of chemicals. After
use substance bottles are placed at their sits on the shelf
where they were found at the beginning of activity.
solid substances from original bottles are removed
only with clean spatulas or tools. Solution reagents are transferred with clean
pipettes or by pouring. It is envisaged that once they were transferred in other
containers or bottles of reagents are not to be reintroduced in the original
bottle. This helps prevent contamination and distortion of the original
composition of the substance or reagent.
clean pipettes are placed on special vertical racks pointing down, and the
used ones will be collec ted in special locations and containers.
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1.4. REGULATIONS CONCERNING HANDLING EQUIPMENT IN
LABORATORY OF CHEMISTRY AND BIOCHEMISTRY
Laboratory instruments and equipment is positioned so that it can be
easily used. At the beginning and at the end of determina tions the on/off
system will be check and they will be disconnected from the electricity system
if are not used for a long period of time.
Laboratory electrical equipment (centrifuges, ovens, thermostats, water
baths, heaters, etc.) as devices for analytic al work (spectrophotometers, pH
meters, etc.) will be connected to grounded outlets. Plugging and unplugging
will be made only with dry hands. When unplugging the drain body will be
kipped the protected part of the drain) and with the other hand the plug w ill be
hold, of course with dry hands.
If the execution of special works by using X -ray equipment or radioactive
isotopes special protective measures are taken. Their purpose is to prevent
irradiation of persons handling the device and those who come into contact
with the environment in question.
The electric motors will be provided with grounding. The same it will be
proceeded to annihilate the static electricity that occurs when operating
machinery with belts.
When using centrifuges prior the inserted te st tubes (or vessels) will be
balanced. Starting and stopping the centrifuge should be gradual to avoid
vibrations and loss of content from centrifuge tubes that undergo this
operation.
Avoid handling devices whose mode of operation and maintenance are
not fully known before being used.
Do not pull on the wires, do not use defective drains and plugs not to
cause short circuits, fire or electric shock.
1.5. HANDLING AND CONSERVATION OF BIOLOGICAL AND
ANALYSIS SAMPLE
Handling of organic products envisages their origin and the fact that is not
excluded that the collection of samples was made from animals that can
be "pathological cases" (unknown to the analyst). Any analysis that
involves the handling of biological samples will be made wearing
disposable gloves.
Volume measurement of biological samples will be done with automated
pipette or pipettes on which is necessarily to attach a pear special destined
for vacuuming liquids. This measure is taken to avoid aspiration of
biological samples directly into the mouth . This prevents the infection,
infestation or contamination by various viruses, bacteria and pathogens.
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It works carefully in order to avoid the overturn of biological samples
content on the work table, hands or clothes.
Laboratory glassware and utensils used in the biological products
operations at the end of work with will be cleaned of material debris with
warm water, detergent and wire brush. Glassware is washed with sulfur –
chromic mixture, tap water and finally with distilled water. Also, when
working with biological material from the unknown animal cases, utensils
and glassware are to be sterilized.
Conservation of biological degradable products, until biochemical
analysis is done by maintaining them at low temperatures (fridge,
freezer), as indicated by specific evidence protocol.
When handling or operating with biological products do not eat, drink or
smoke (valid measure also in the case of working with toxic substances).
After operating with biological samples the worktables are cleaned,
disinfecte d with solutions specially designed for this purpose and hands
will be washed thoroughly.
1.6. SAFETY AT WORK –
PREVENTION AND FIREF IGHTING
Knowing the rules concerning the prevention and firefighting (PSI) in
laboratories of didactic interest was be regulated by an order of ministry
concerned (Ord. 712/1975).
Their observance is the duty of all persons working in these
laboratories.
Fires can be triggered by mishandling of the gas network, fuel gas
cylinders, tubes or containers with flammable substances (eg ether, gasoline,
etc.), the electrical network or by using defective devices. To avoid fires is
required to be familiar with the provisions relating to the prevention and
firefighting.
1.7. FIRE PREVENTION AND LOCALIZATION MEASURES
In labor atories are displayed guiding plans comprising provided measures
for fire prevention and for handling such situations. Mentioned
inscriptions and signs are installed and the intervention team (from
laboratory personnel) and powers of its members are mentio ned.
The laboratory will be provided with fire extinguishers approved and
verified.
Avoid blocking of access and work spaces, doors and windows with
furniture, appliances, flower pots, billboards, posters, etc.
Smoking and flammable materials access in la boratory is forbidden.
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Work with flammable products will be executed in niches with good
ventilation, to prevent the formation of explosive mixtures and release
toxic fumes into the laboratory.
Compressed gas cylinders or tubes shall not be placed near hea t sources,
or near heaters.
Gas lamps are kept burning only during use as stated by the protocol. The
gas valves status is checked periodically by brushing with soap solution.
Avoid installing makeshift laboratory facilities or materials that can react
with used substances, thus creating explosives.
In case of accidental release of combustible or volatile liquids inside the
laboratory it will proceed to: extinguishing gas lamps and electrical
sources interruption; closing doors and opening windows; rags wiping of
surface on which the liquid is spilled; resume work after complete
removal of gas.
After starting/finishing the laboratory is required: disconnecting the
voltage electrical equipment (except refrigerators and thermostats
designed to store various laboratory materials); check shutting the gas
distribution network; clean debris of combustible materials from
workplace; light bulbs are shut off.
In the event of a fire it will proceed to locate and extinguish it by the team
designated for the purpose f rom laboratory staff helped by people in the
laboratory. The team designated for interventions, consisting of laboratory
staff, will act in accordance with the provided tasks, regarding the warning,
localization and fire extinguishing.
To extinguish the fire produced by inflammable substances, fire
extinguishers will be used and fire surface will be covered with sand.
Avoid using water because of its thermal dissociation oxygen and
hydrogen (fuel gas) is formed.
The actions will take pl ace dynamically, by circumscribing the fire site
and extinguish it. If it is determined that the fire spreads and the risk of
expansion or explosion is high, an immediate contact with emergency
services of the fire brigade by calling the 112 emergency services will be
made.
1.8. POSSIBLE INJURY –
SAFETY MEASURES AND PROTECTION
The possibility of accidents occurs due to ignorance and / or failure to
comply with work in the laboratory. Accidents can be grouped into the
following categories: poisoning, burns, trauma and electrocution.
Poisoning is caused by the penetration of certain toxic substances in
the body, followed by metabolic disorders and in the end of the onset of
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pathological processes. The penetration of substances in the body is achieved
in vari ous ways: the airway (breathing air by gases, vapors, dust);
gastrointestinal (with water and food); by tegument and / or mucosal path
(diffusing through the skin and mucous membranes to blood). Once in the
body, toxic substances can cause acute or chronic poisoning. Acute
intoxication is due to the penetration of substances in the body in a relatively
short period and in a quantity exceeding limits. Chronic poisoning is due to
penetration into a longer period of time, but in relatively small amounts,
which determines the cumulative processes.
Burns are injuries caused by physical agents (temperature: e.g. – hot
substances, ignition of volatile substances and radiation: egg – infrared) or
chemical (caustic substances: e.g. acids, bases, bromine, etc.). Afte r gravity of
burns different degrees are distinguished: I – with skin congestion followed by
edema and transient pigmentation; II – the occurrence of blisters with serocitrin
or bloody liquid (in case of deeper lesions); III – with extensive tissue destru ction
(epididymis, dermis, going deeper into muscles fascia and muscles) requiring
treatment in specialized medical services.
Injuries can be caused by blows, cuts or punctures due to wrong
handling of glassware, utensils, equipment and laboratory faciliti es.
Electrocution can be determined by body contact with ordinary
electricity network (120 -220 V) or high voltage. The causes of electrocution
may be due to faulty installation of electrical appliances, lack of grounding
network sockets defects, etc.
Prote ction measures aim at personal protection by proper handling of
chemicals, glassware, utensils, equipment and facilities. In this regard is
mentioned the need to respect the following rules:
Heating solutions in tubes is done with continuous stirring, avoi ding
"throwing" the liquid in the tube during boiling. The open end of the tube
does must not be kept towards the person making the operation nor for
neighbors.
In the case of operations with flammable substances is not allowed to be
carried near sources of heat (increased pressure in the storage vessel can
cause danger of explosion).
For evaporation, distillation, etc., operations, as heat sources electrically
heated water bath or infrared radiators are used.
In case of fire in the laboratory is envisaged that water -miscible
flammable liquids (eg ether, benzene) cannot be extinguished with water.
In these cases, using special extinguishers.
Handling acids and strong bases must be done with increased attention.
Operations with fuming acids and toxic gaseous substances are run under
niches. During these times the niche fan is functioning.
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If sampling and dosing of caustic or poisonous liquids, pipetting is done
by using a vacuum pump or a pipette with rubber pear. In chemistry and
clinical biochemistry testin g laboratories, automatic pipettes are used.
For reasons of hygiene and personal safety is forbidden to use glassware
for drinking and preserving foods. Inside the lab is not allowed to eat,
drink and smoke.
Tasting reagents is forbidden because most chem ical substances are toxic
or caustic. Smelling chemicals is not made by approaching to the nose,
but by proceeding to produce a stream of air containing vapors of
substances from the bottle neck by waiving palm, near nose.
After completion of laboratory se ssions (practical work or student’s
scientific research) proceed to hand washing as a measure of personal
hygiene.
By understanding the methods of analysis, by acquiring all the
knowledge on laboratory work and by respecting protection and safety rules,
laboratory work will cause students to achieve real knowledge gained in
practical activities.
1.9. GENERAL REGULATIONS IN LABORATORY
CHEMISTRY AND BIOCHE MISTRY
Each student is required to assimilate the theoretical knowledge prior to
each laboratory lesso n or theoretical problems related to applied Chemistry and
Biochemistry aspects, the method principle, reagents used and how to work and
also, notions regarding the use of laboratory equipment. During hours of
practical work in the lab students are require d to wear protective equipment,
namely white lab coat with long sleeves with cuff tightened. Also, students will
use the cloth specially designed for this purpose, to wipe bench.
The desktop is kept in order and the reagents are placed on shelf in
the orde r that were placed at the entrance to the lab. On the laboratory bench
is not allowed to place purses, handbags, food, drinks and clothes, except the
materials necessary to conduct practical work scheduled.
During the course of the scheduled work shifts wi ll be only performed
with laboratory activity purposes.
The practical work will be finished by writing the experimental
results, the calculations if they are presented in this paper and the conclusions
arrived at after respective analysis (test, applicatio n).
After completion of the laboratory lesson, reagents and equipment are
left entirely cleaned at the place they were at the entrance to the lab. Also,
glassware must be washed and placed on special laboratory devices in order to
dry and eventually, the s ink must be washed from precipitates reagents,
broken bottles.
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Laboratory work involves cleaning the place of work, so if the floor
laboratory was soiled as a result of work done in the lab, the students – that
people who worked in the lab – must collect, broom and even to wash the
floor – if this is required.
Leaving the lab will be only after the consent of the teacher and the
place will be leaved in order (including order on the work table and chair from
the laboratory bench).
On leaving the student mus t leave the laboratory where they performed
the work as they would like to find it at the arrival!
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CHAPTER II
GENERAL PRINCIPLES OF SAMPLING, TRANSPORT AND
STORAGE OF BIOLOGICAL SAMPLES
Sampling, handling, transport and storage of biological samples must
keep their quality. This involves the protection of the person making the
sampling, patient restrainers so as to prevent its aggressive reactions and used
instruments must be prepared acc ording to rules to prevent contamination.
Also, all procedures for handling biological samples until analyze must
preserve the samples characteristics at sampling .
2.1. COLLECTION OF BLOOD SAMPLES
The preferred site and the amount of blood taken from ani mals depend
on the purpose that is followed in the executed analysis.
Small amounts of blood are collected from the ear or nose by puncture
and larger quantities of blood are obtained by puncture in the veins or arteries.
From veins it can be taken directl y whit a needle or a syringe sterile
disposable in a special vessel. Thus, the collection of blood samples from
sheep, goat, dog – is made from the saphenous or jugular vein, from horse and
cattle – is made from the jugular vein and from pork is made from marginal
ear vein or tail vein.
The needle and syringe will be sterile, and the sampling container will
be adequate for biochemical purposes (biochemical analyzes that are needed).
The syringe, needle and sample collection container must be sterile and
preferable, disposable, because any traces of water or alcohol on their walls
can cause hemolysis or may give rise to errors resulting from variation of the
blood concentration.
For blood collected from fish, syringes and tubes used are made of
plastic, becau se the fish blood coagulates very quickly in contact with the
glass.
It is recommended that blood testing to be conducted in the morning;
tubes that are used for sampling must be fit for purpose, clean, dried and
sterilized. Sampling tubes and containers f or biological samples will be
suitable for biochemical tests that need to be executed. Currently, for the
biochemical blood sampling specific tubes are used for the biochemical
analysis, hematological analysis, for coagulation assays, for determining the
sedimentation rate of red blood cells, etc. After harvesting, tubes will be
appropriately labeled to identify the individual from whom the blood was
collected, the date of collection and analysis required by the laboratory.
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Blood analysis cannot be executed if prelevation was done improperly
or in inadequate containers (tubes).
Dosage of different biochemical constituents of blood is carried out
from whole blood, plasma or serum. In case the analysis is done from whole
blood or plasma, to prevent blood coag ulation, harvesting is done in tubes
containing anticoagulants.
Currently, glass or plastic vials that are sealed with rubber stoppers in
which there is vacuum (vacutainer), are used. By means of a needle exact
amount of blood (from 2 -20 ml) can be sample d. The vials may contain
substances that allow morphological and biochemical analyzes.
Depending on the used blood preservative, international standardization
of plugs provides the following colors:
• green – Heparin sodium or lithium is used for determina tions of plasma
• purple – contains potassium salt of EDTA and used for whole blood assays
• grey – contains fluoride or oxalate; Fluoride acts to inactivate enzymes on
substrates such that the latter they will not be consumed after storage of
samples
• blue – contains citrate and is used for determinations that require further
clotting.
• dark blue – contains heparin sodium but may contain EDTA as an additive –
for analysis of trace elements in the blood
• pink – similar with purple vacutainers, contains EDTA and are used for the
storage of blood
• red (glass) – contains no additives and is used in particular for determining
serum biochemical parameters, serologic tests, and the determination of
antibodies and the presence of drugs
• yellow – containing ACD (acid – citrate – dextrose) and are used for
phenotype studies, paternity tests, etc. ("BD Vacutainer ® venous Blood
Collection – Tube Guide", 2007 -05-30)
Containers for blood sampling will be chosen in respect to the
parameters to be deter mined.
Blood clotting is a complex physical -chemical process, which is due to
a relatively large number of factors: when hemorrhage occurs, an enzyme
called prothrombin or thrombogen ( inactive in the bloodstream) is activated,
and produces coagulation.
Inactive prothrombin is converted to active thrombin under the
influence of calcium ions. Thrombin acts on fibrinogen, convert it to insoluble
fibrin. Inside blood vessels, as blood does not clot because does not produce
thrombin, prothrombin activation is in hibited by antithrombin. When injured,
from the damaged tissues and blood platelets, thrombokinase is released
which will combine with antithrombin.
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Schematically process occurs as follows:
Prothrombin + Thrombokinase + Calcium = Thrombin
Thrombin + Fib rinogen = Fibrin
In the blood coagulation process a clot is formed which then is
immediately retracted, releasing a yellowish liquid that is serum. This
phenomenon is called syneresis. The clot is formed of a network of fibrils
containing erythrocytes, leu kocytes and platelets. The fibrils are made up of
fibrin resulting from the passage of plasma fibrinogen from soluble state
(hydrosol) in insoluble state (hydrogel).
Plasma contains fibrinogen and can coagulate to form a fibrin clot. By
centrifuging blood treated with anticoagulant, the figurative elements are
sedimented, and the resulting solution is plasma .
Anticoagulants are substances which prevent the coagulation of blood,
through their intervention, in different phases of the coagulation process.
Thus , for the inactivation of thrombokinase, hirudin, heparin or germanina
may be employed.
For the precipitation of calcium ions, potassium oxalate, sodium
fluoride or sodium citrate are used.
To prevent coagulation of fibrinogen, glass beads are stirred in t he
collection container.
Type of anticoagulant differs regarding the type of required analysis.
The EDTA (disodium salt of ethylene diaminetetraacetic acid) complex
with calcium ions. In this sense, in the sampling and collection tube is used
1mg EDTA / 1c m3 of collected blood. It is used in particular for determining
blood count.
Sodium fluoride is primarily used in collection of blood for glucose and
urea determination using a rate of 10 mg for 1cm3 of blood. Sodium fluoride
precipitates calcium and inhibits the formation of white blood cells, blocking
the coagulation and also glycolysis.
Potassium oxalate is used for collection of blood for measurement of
pH, alkali reserve and the dosage of plasma and globular chlorine at a ratio of
20mg to 10cm3 of blood.
Potassium oxalate was purified by recrystallization. The recrystallized
salt was sprayed thoroughly and is prepared a 20% solution which is
neutralized to pH = 7.4 ± 0.2 with potassium hydroxide solution or oxalic acid
in the presence of 0.02% phenol red as indicator.
From the neutralized solution 0,1cm3 (20mg) are inserted in collection
tubes and are evaporated in a drying oven at 105 -110șC. Potassium oxalate
remained in the tube is sufficient to yield 10cm3of blood.
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Sodium citrate : It acts in a similar manner as potassium oxalate. Use
5mg of citrate are used for of 1cm3 collected blood, in particular for
determining the erythrocyte sedimentation rate (ESR).
Heparin and hirudin are ideal anticoagulants because they do not alter
the phy siological state of the blood. They are used in an amount of 75 units /
1cm3 of blood (heparin 0.5cm3 / 1cm3 blood), especially in the collection of
blood for the determination of inorganic constituents, in order to not introduce
minerals that may alter th e distribution of inorganic elements between the
figurative elements and plasma. The heparin solution is evaporated at 100 ° C
directly into collection tubes.
The blood serum is deprived of fibrinogen and figurative elements.
The separation of serum . To ge t 5cm3, 10cm3 of blood serum are
required. In winter clot retraction is slowed. To obtain the serum, the blood is
maintained immediately after collection at a temperature of 35 -37șC until the
formation of the clot. Upper clot adhere to the sampling vessel walls, from
which detaches with a glass rod and then insert the thermostat at 37șC where
clot retraction and separation of a pale yellow liquid occurs, that is serum.
After clot retraction, the liquid is decanted into a centrifuge tube, which
is equilibrat ed in a balance and then centrifuged for 5 minutes with 1500 rot /
minute. Centrifuge must be started gradually to avoid red blood cell hemolysis
(which inevitably accompany serum) when spin speed is too high, and to
avoid centrifuge fret.
Obtained serum m ust be yellow, unhemolyzed red blood cell
suspension is sedimented by centrifugation. Under certain conditions serum
aspect is different, namely:
Opalescent – if it contains fat particles that normally are becoming
from a diet too rich in fats. Opalescence occurs in pathological cases and
nephrosis lipoidica. In this case, the serum can be divided by stirring with a
mixture of alcohol and ether.
Yellow -brown color may be due normally to a vegetarian diet, being
accompanied by skin pigmentation, and the path ological condition that this
color indicates is that the serum contains excess of bilirubin. Exposed to air
the serum receives a green color due to the fact that the bilirubin is converted
to biliverdin.
Pink to deep red color is due to hemolysis. Serum hemolysis usually
occurs a technical mistake in blood collection (using wet a syringe needle and
sampling vessels). The osmotic pressure of plasma by dilution is lower than of
the erythrocytes, those absorb water, become turgid and release hemoglobin, a
phenomenon known as hemolysis.
The separation of the plasma . The above stated amount of blood
thinner chosen is placed in a test tube, according to the purpose of analysis
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that is performed, over anticoagulant blood is collected a nd stirred for 30
seconds. This non -coagulated blood by is centrifuged for 5 -10 minutes at
1500 rot / minute. The figurative elements are sedimented on the bottom of
the tube and so the plasma is obtained.
Plasma is a yellow viscous liquid with an alkaline reaction with protein
content of 78 -80%. Such separated plasma is kept from 7 -10șC temperature
for 10 -12 days.
To prevent coagulation of fibrinogen, glass beads are used, which are
stirred together with the blood in the collection container thereby obtain ing
defibrinated blood.
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2.2. COLLECTION OF URINE SAMPLES
Urine collection is done in perfectly clean and tight glass or plastic
vessels. If a bacteriological test is necessary, glassware and utensils should be
sterilized.
Complete urinalysis is to be pe rformed on urine within 24 hours from
collection. Urine collection will be performed in the morning in disposable,
sterile containers, designated for this purpose. Urine collected is kept cool
without freezing. If it is necessary to be kept for longer time , urine is
preserved by the addition of preservatives.
Thus, formaldehyde (preservative used in the determination of urinary
sediment) is added in the amount of 10cm3 to 100cm3 of urine or toluene,
xylene or chloroform may be added but only at the surface of the urine.
Before urinalysis, toluene, xylene or chloroform, are removed using a
separating funnel. Chloroform is not used in preserving urine in the case of
carbohydrates and acetone determinations.
In the case of steroid hormones dosage of the urine sample 10n of HCl
solution (50cm3 HCl in urine sample within 24 hours) is added.
Full examination of the urine comprises physical, chemical and
bacteriological determinations. Currently a brief examination of the urine is
performed.
Brief examination of urine means to analyze the general physic –
chemical characteristics, identification and analysis of pathological
components of urine sediment.
2.3. COLLECTION OF MILK SAMPLES
2.3.1. Collection of raw milk sample
For sampling raw milk, samples are co llected from tanks and ponds,
minimum 500 ml each, after a preliminary homogenization. Collecting from
cans involve the formation of an average sample collected randomly from
10% of cans in a batch.
From the well homogenized average sample 500 ml is collec ted for
laboratory examination.
2.3.2. Sampling of drinking milk
2-3 packaging unit are collected from each lot. Samples must arrive at
the laboratory in maximum 4 hours from collection , the transport being
made in refrigerating conditions.
After collec tion, each sample will be individualized by labeling and
packaged separately so that is not damaged during transport. Samples must
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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be transported to the laboratory in useful time, so that they retain integrity
parameters unchanged.
2.4. COLLECTION OF SPER M SAMPLES
Depending on the species, collection of sperm is achieved through
various techniques: electro -ejaculation (ram, bull, dogs, birds), abdominal
massage (birds), massage of deferens vas ampoules and seminal glands (the
bull) and masturbation (in boa r and dog).
The sampling is carried out in a sterile and graduated glass flask with
double walls, being able to maintain a constant temperature at 37 ° C using
warm water. Immediately after collection the flask sits on a thermostatic area
so the optimum t emperature is ensured throughout semen handling.
Seminal plasma separation is achieved by centrifugation using a spin
speed that will not harm sperm membranes, which would lead to the release
of intracellular compounds. Transport and storage at the laborat ory until
analysis is performed in chilled or frozen pellets placed in liquid nitrogen
containers.
2.4.1. Conservation of sperm samples
The need of long term conservation of sperm outside the body – after
sampling, appeared with the practice of artific ial insemination of animals,
practice that is successfully realized in most species.
Sperm preservation methods are based on the following principles:
1) The gradual decline in sperm temperature to near 0 ° C (between 0o
and 4o) and creating an anaerobic environment for sperm. Low temperatures
retard spermatozoids metabolism and prevents their exhaustion; also prevents
the development of bacterial flora, which can lead to decomposition and
shortens sperm preservation. Conservation at this temperature maint ains
sperm in a somehow apparently dead – anabiosis – which is reversible, in case
of changing these conditions spermatozoids are regaining their viability.
2) Decreasing sperm density by using diluted substances must meet the
following conditions:
– not be toxic to spermatozoids
– to neutralize glands secretions that are abundant in some species (stallions,
boars, which contain large amounts of electrolytes: KCl, NaCl).
– to neutralize also the products of metabolism of the spermatozoids (carbon
dioxid e, lactic acid, etc.).
– to correct the natural environment of sperm by adding nutrients: glucose,
fructose, etc.
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– to increase the sperm resistance to heat shock by their content of lipoprotein
substances.
3) pH of diluted medium should be conducive to ma intaining sperm
viability. Diluted medium for ram and bull semen must have a pH of 6.2 to
6.8 and for the stallion and boar semen from 7 to 7.2, as much as possible
close to pH of respective sperm. The high acidity and alkalinity lead to rapid
death of spe rmatozoids.
4) Diluted medium should be isotherm with the sperm of respective
species.
5) Diluted medium must be easily to be prepared and economic.
In conclusion, sperm storage is based on two methods: sperm dilution and
cooling. In order to obtain the be st possible preservation the two methods
are combined. Both the sperm dilution and cooling must be carried out
within half an hour after harvest.
For sperm conservation most used diluents are: egg yolk and sodium
citrate and sterilized and non-cream milk. Thinner yolk – citrate has the
advantage that ensures good preservation of sperm. This is due to the egg
lecithin. By adding sodium citrate egg yolk acidity is neutralized and the
vitelus is separate in egg yolk, allowing the examination of sperm in good
conditions.
Tabelul 1.1 Sperm conservation by using some diluents
Diluent composition bull ram
Sodium citrate 1,5 g 2,8 g
Anhydrous glucose 3 g 0,8 g
Egg yolk 20 cm3 20 cm3
Distillated water 100 cm3 100 cm3
Diluents preparation . Dissolve the chemicals (citrate, glucose) in the
proper amount of distilled water – in a Berzelius glass after the table. In a
second glass the egg yolk is introduced and stirred with a glass rod upon that
the solution prepared above is pour ed in small portions (with continuous
stirring) that must have a pH ~ 6.8.
The diluents must have the room temperature (18 -25șC). The diluted
solution prepared by this method is added to the sperm to be maintained in a
ratio of 1: 3, 1: 5 or 1: 20. It is stored in a refrigerator. In case when the
diluent is cow's milk, this, whether is freshly milked, has to be filtered
through filter paper or sterilized gauze and then boiled, cooled and filtered
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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again. Dilution with milk brought to room temperature, will be made in vials,
in the ratio of 1: 1, 1: 3, 1: 7 and 1: 15. For preservation is kept in a
refrigerator, performing a slow cooling of the sample.
Observation:
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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CHAPTER III
CONCEPTS OF CHEMICAL AND BIOCHEMICAL ANALISYS
3.1. DEFINITION AND CLASSIFICATION OF THE METHODS
FOR CHEMICAL AND BIOCHEMICAL SEPARATION
Chemical analysis aims to study the chemical composition of
substances (natural substances, row materials, intermediate or finished
products in various t echnological processes, etc.). Biochemical analysis deals
with determining the composition of living organisms , animals, plants,
microorganisms and animal or vegetable products for consumption or other
practical uses .
In human and veterinary medicine the biochemical analysis helps
complete the clinical diagnosis and treatment , and because of this the term of
clinical analysis is also used.
The study of the chemical composition of substances, forms a special
branch of chemistry, named analytical chemistry .
After purpose, analytical chemistry is divided into:
1) Qualitative analytical chemistry – identify the elements or groups of
chemical elements that forms a substance.
2) Cuantitative analytical chemistry – establish the quantitative ration
of the element s from a chemical substace.
The methods used to determine the exact quantity of an element of a
substance or of a substances in a mixture , are chemical methods (volumetric
or gravimetric ) and physico -chemical methods (instrumental techniques ).
In biochemical analysis are used both gravimetric and volumetric
analysis methods, and also physico -chemical methods. Physico -chemical
methods have become research methods specific for biochemical analysis (for
examples: paper electrophoresis, radiochemical methods , electrochemical
etc).
3.1.1. Volumetric chemical analysis – general concepts
The name of volumetry comes from the fact that this type of analysis is
based on measuring the volume of the solution with the Burette . The main
operation used in volumetry is the titration , this method is called also
titrimetry .
Volumetric analysis is based on accurate measurement of the volume of
the solution of reagents, with a precise and known concentration , required for
a quantitative analyse .
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Chemical reactio ns are very important in volumetric analysis and must
fulfill the following conditions :
to be stoichiometric – that means to be expressed by a precise chemical
equation, respecting the law of conservation of mass ;
to be total reactions , meaning that the balance is moved to the
formation of thefinal products of the reaction ;
to be fast ;
the end of the reaction to be marked by a sudden change of one of the
chemical properties of the system.
The types and reactions used in volumetric analysis are:
1. Ionic r eactions with double ion exchange , which can be :
a) neutralizing reaction, of the proton exchange;
b) precipitation reactions (resulting in the formation of insoluble
reaction products in the used solvent ).
2. Oxidation – reduction reactions or electrons exchange.
Depending on the type of reaction used methods can be divided into :
• Neutralization method – based on the neutralizing reaction .
a) Acidimetry – use strong acid solution, with known concentration,
for determination of strong bases , weak bases , and salts with
alkaline hydrolysis .
b) Alkalimetry – use strong base solutions, with known concentration,
for dosing of strong acids, weak acids, and salts with weak acid
hydrolysis .
• Precipitation method – based on precipitation reactions .
• Oxidation -reducti on methods – based on oxidation -reduction
reactions :
Manganometry – use as oxidant the potassium permanganate solution
(KMnO 4).
Iodometry – use as oxidant the iodine solution (I 2).
Volumetric analysis is important in sensing the moment
corresponding to total reaction, moment called equivalence point . The
achievement of the equivalence point can be observed by adding substances
known indicators in the reaction medium, which allowed to see the presence
of the reactiv excess (by changing the color ).
The In dicators
Indicators are substances added to the reaction that indicates the end of
the reaction (see Appendix 1).
After the types of reactions that for the base of the titration methods ,
the indicators are as follows:
neutralizing indicators
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redox indicators
precipitation indicators
complexometric indicators
In some cases, the occurrence of colors is due to the excess of
reagents that is itself a colored substance (as in the case of titration with
potassium permanganate ), and the addition of the indicator is no longer
necessary .
Universal indicators are mixed indicators that change the coloure for
different pH values .
The indicators for the neutralization reactions are chosen so that the
equivalent point of the reaction to be included in the turn of indicator area.
3.1.2. Equipment and utensils used in volumetric chemical analysis
(volumetry)
For chemical analysis typically we used utensils like: tubes , conical
flasks (Erlenmeyer flasks), cylindrical glasses (Berzelius flasks), funnels ,
clamps, porcelain capsules, tripods , triangles porcelain , metal mesh with
asbestos , Bunsen burner etc.
Quantitative analysis requires in addition to those tools, other flasks and
glassware used to accurately measure gas volumes or solutions , technical
balances and analytical balances , densimeters for measuring the density of
liquids , centrifuges etc.
As unit volumes used ml (1ml = 0.001 l) or cm3 (1ml = 1cm3) and as
a unit of mass , the gram (g) (1 mg = 10-3g, g1 = 10-9g).
The main glass utensils used to measure liquid volumes are graduated
cylinders , flasks , pipettes , burettes (see Annex 2).
Graduated cylinders – the measure units are in “ml”, and are used in
approximate volume measurements . Cylinder grades range from 5 –
2000cm3).
Volumetric flasks – are glases with flat bottom and elongated neck ,
which includes a circular mark (graduate), which indicates the fill of them.
Flasks are each provided with a polished sttoper for sealing . Theie
capacity ranging from 10 to 5000cm3 and is used in the preparation of titrated
solutions .
Pipettes – depending of calibration type can be: pipette with bulla and
graduated pipettes . Pipettes with volumes below 1cm3 are called
micropipettes .
Pipettes with bulla – have marked only one sign or two signs . On the
bubble is engraved the capacity and temperature calibration . With these
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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pipettes we can measure only the amount for which they were calibrated .
Their capacity can be between 1 -100cm3 . ……………………………….
Graduate p ipette – are cylindrical , with marked volume and is
divided into ml and tenths of ml. They have capacities between 1-25cm3.
Micropipettes – were divided in hundredths of cm3 volume and
capacities varies from 0.01 to 0.02 cm3.
Automatic pipettes – works on the principle of suction piston pumps ,
which are calibrated for a fixed or adjustable volume .
Pipettes are used for accurate measurement of small volumes of
liquid . For accurate measurement of a volume of solution we proceed as
follows: insert the pipette tip into the solution and aspirate the volume, until
the liquid level goes just over the mark , and then close pipette thumb . Remove
the pipette from the liquid, remove the pipette tip and then gently lifting your
finger to drain the liquid until it is tangential to the graduation fixed mark ,
then passes into the reaction vessel volume set.
Burettes are graduated glass tubes, provided at the bottom with
devices for stopping or regulating leakage (rubber or tube clamp Mohr ).
Burettes have volumes between 10 -100cm3 , with graduations of 0.02 cm3 to
0.2 cm3.
In biochemistry often use burettes capacity 1-5cm3 and graduations
0,01cm3 called microburette .
Burettes with glass tap are used to tap the glass when working with
solutions of acids and oxidizers , and the biuret with rubber tube and the clamp
when using alkali . After using the burettes has to be washed with tap water
and then with distilled water , and – to avoid dust deposition – it can cover the
upper part with alluminum folia or plastic.
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C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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3.2. SOLUTIONS
3.2.1. Measurements used for solution preparation
Relative atomic mass is a number showing how many times the mass
of the atom of an element is greater than the 12th of the mass of the carbon
atom, taken as a unit and which has been accorded as 1 Dalton .
Gram atom is the quantity in grams of a chemical element,
numerically equal to the atomic weight of the element.
The molecular weight of an element or combination of elements , is a
relatively small number which shows how many times the mass of the
molecule that item or substance is greater than that of the mass 12 of carbon
atom , taken as a unit . The molecular weight was also expressed in Daltons .
Gram molecule (mol ) is the amount of substance in grams
corresponding to the molecular weight .
Chemical equivalent (equivalent mass) is a number that indicates
how many parts by weight of an element or chemical compound are
combined, replace or release 1.008 parts of hydrogen or eight parts of oxygen.
The equivalent gram (Eq) or equivalent weight of a substance is
the amount , in grams, that can replace or can chemically combined with 1.008
g of hydrogen or 8 g of oxygen . Or we can say that the equivalent weight is
the mass of one equivalent, where the mass is represented by the mass of a
substance t hat can combine or displace directly / indirectly 1.008 parts by
mass of H, or 8 parts of O, or 35.5. parts of chlorine; or can react or can
supply one mole of H+ in acid -base reaction; or can react or can supply with
one mole of electrons in the case of a redox reaction.
The equivalent is calculated as:
For elements:
valenceatom gramweight Equivalent
Examples:
Equivalent gram of oxygen = 16 / 2 = 8 g O
Equivalent gram of carbon = 12.01 / 4 = 3.0025 g C
The equivalent weight is calculated differently depending on the
substance used .
In calculating the equivalent weight of composed substances must
take into account the chemical reaction :
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Acids
hydrogens ionizedof numberM acidof gram moleculegram Equivalent)(
Or, in other words, the equivalent weight (Eq) for acids can be
calculated as the ratio of the molecular weight of the acid and the number of
transferred protons (the number of H+ in the molecular formula of the acid).
Examples:
Eq HCl = 36.465 / 1 = 36.465 g
Eq H 2SO 4 = 98.076 / 2 = 49.038 g
Eq CH 3COOH ═ 60.03 / 1 ═ 60.03 g
5,361HCl
HClMEq
49298
242
42 SOH
SOHMEq
22366
3316153
34 3
4 3 POH
POHMEq
Bases – equivalent weight = gram molecule of the base / the number of
hydroxyl groups (-OH) in the molecule taking part in that reaction .
hydroxylsof numberM baseof gram moleculegram Equivalent)(
Examples:
Eq NaOH = 40.005 / 1 = 40.005 g
Eq Ca(OH) 2 = 74 / 2 = 37 g
In other words , the base or equivalent weight (eq) is calculated as the
ratio of the molecular weight and the valence of the metal base (or the number
of OH in the molecular formula of the base ).
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401NaOH
NaOHMEq
27254
22) (
2) ( OHCa
OHCaMEq
33,21364
3)116(313
33) (
3) ( OHAl
OHAlMEq
Salts – equivalent gram = molecule gram / number of hydrogen
replaced by metal in the formula of refered acid.
metalsby replaced hydrogensof numberM saltof gram moleculegram Equivalent)(
Example:
Eq MgCO 3 = 84.3 / 2 = 42.15g
Eq NaCl = 58.55 / 1 = 58.55 g
In other words , the equivalent weight for the salts (Eq) is calculated as
the ratio of the molecular weight of the salt and the product of the valence of
the metal and the number of metal atoms .
5,5815,3523
11NaCl
NaClMEq
1
11
1ClNa
712142
2141632223
2142
42 SONa
SONaMEq
2
141
2 ) ( SO Na
33,52696326
6)41632(3213
233)4(2
3)4(2 SOAl
SOAlMEq
2
343
2 ) ( SO Al
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In redox reactions , chemical equivalent (Eq) of obtained salts is
calculating by the ratio of molecule gram of the salt and the number of
electrons receive d or donated by the substance, in its formation reaction .
Example:
2KMn+7O4 +10Fe+2SO 4 +8H 2SO 4 →
→ 2Mn+2SO 4 + 5Fe 2+3(SO 4)3+K 2SO 4 + 8H 2O
So, the changed of electrons is as follow s:
Mn7+ + 5e – Mn2+(reduction) / x 2
2Fe+2 + 2e- 2Fe+3 (oxydation) / x 5
The equivalent of KMnO 4 will be the ratio of the molecular weight
(158.03), and number of changed electrons (5)
EqKMnO4 = 158.03 / 5 = 31.606 g
In other words , the equivalent weight (Eq) for substances participating
in redox processes (oxidation -reduction) is calculated as the ratio of the
molecular weight of the substance and the number of transferred electrons .
772154
241632229
2142
42 SOCu
SOCuMEq
2
141
2 ) ( SO Cu
5,622125
24163229
124
4 CuSO
CuSOMEq
2
142
1 ) ( SO Cu
Chemical milliequivalent (mEq ) is a thousandth part of the chemical
equivalent.
mEq = Eq / 1000
Milliequivalent gram represents chemical milliequivalent expressed
in grams.
The solution is a homogeneous liquid mixture, consisting of a
substance dispersed molecular or ionic (called solvit) in a dispersion medium
(called solvent).
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Preparation of the solutions
To prepare standardized solutions we have to use volumetric flasks . If
we weighting of a standard substance to prepare a solution in a flask at 20oC
and the temperature is less than 20oC, the solution will be more diluted,
because at 20oC the volume would be higher ; when working at a temperature
higher than 20oC, the solution will be more concentrated , as by loweri ng the
temperature at 20oC, the solution volume will be reduced. Thus, for a different
operating at different temperature of 20oC, a correction must be made for the
volume.
In preparing a solution we have to weight very precise the amount of
dry substance or we have to use a well determined volume for a liquid when
the substance which is prepared is a liquid solution . We have to placed in a
volumetric flask the substance and dissolved substance in suitable solvent ; the
solvent is gradually added while stirr ing the contents of the flask until the
circular mark on the neck of the flask is tangential to the meniscus of the
liquid. If the determination is made at a different temperature to 20oC, then is
necessary to carry out the correction volume depending on the working
temperature .
Mix the solution by inverting a few times the ball , closed with a
polished cap before handling .
3.2.2. Expresion of the solutions concentration
Concentration of a solution is the amount of a solute (disolved
substance) in a give n volume of solution. Concentration is the ratio of the
solute to the solvent. Depending on the choosen units, the concentration can
be expressed in many ways :
The percent concentration – represents grams of disolved solution in
100 grams of solution (%). Such s olutions are percent solutions .
Example: 1 % of oxalic acid contains 1 g of oxalic acid disolved in 100
g of soluție.
Exercise – percent concentration ( C%)
Calculate the percent concentration of 200g of solution of sodium
chloride (NaCl ), where we know that for solution preparation has been
dissolved an amount of 10g of pure NaCl .
We know that to establish the percent cocentration (C%) of a solution
we have to calculate the amount in grams (g) of pure substance dissolved in
100g solution.
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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Thus,
200g NaCl solution C % ……………………………..10g NaCl pure
100g NaCl solution C % ……………………………… x
pure NaClg C 520010 100
%
So, in conclussion, the percentage NaCl solution is 5%.
The molar concentration – represents moles of substance dissolved in
1000 cm3 solution. The s olutions which is expressed by the molar
concentrations are called molar solutions .
These solutions are noted by M or m. It can be used multiples or sub-
multiples and in this case the symbol M (or m) will accompany the number
representing multiples or submultiples of moles, as follows: solutions molar
(M), bimolare solutions (2M), decimolare (0.1 M), millimolar (0,001M), etc.
Exemple: 2M oxalic acid solution contains 2 moles of oxalic acid in
1000cm3 solution, so 2 x 126.068 = 252.135 g of oxalic acid
A 0.1M HCl solution contains 0.1 moles of HCl, thus, molecular mass
is 36.5g, therefore in 1000cm3 solution we will have 3.65 g of HCl.
Exercise – molar concentration ( CM)
Calculate what molar concentration (CM) has a volume of 500 ml of
hydrochloric acid (HCl ) that contains 35 g pure HCl .
1 cm3 = 1 ml
1 L = 1000 ml = 1000 cm3
500 ml HCl = 0.5 L HCl
0.5 L HCl solution C M …………………………… 35g HCl pure
1 L HCl solution C M ……………………………… x
1000 x 35
CM = –––––– = 70g HCl pure
500
MHCl = 1 + 35.5 = 36.5
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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1 mole HCl …………………………………….36.5g HCl pure
x moles HCl …………………………………… 70g HCl pure
solutionLin pure HCl moles x 1 92.15.36701
CM = nr of moles disolved in 1 L of solution
CM of a s olution of HCl = 1.92
Normal or equivalent concentration – is expressed as the number of
equivalents gram of a solute in 1000 cm3 solution . A normal solution contains
an equivalent weight of substance in 1000cm3 solution.
Whose solutions of its concentration is expressed by normal solutions
are called normal solution or equivalent solutions . It is noted with N (or n)
accompanying by the number indica ting the number of multiple equivalents of
solution , that is exactly why we called normal or equivalent solution .
Knowing the equivalent weight of a substance we can prepare any normal
solutions .
Example: For the preparation of 1000 cm3 of 1N NaOH solution first is
calculated the equivalent weight of NaOH .
ENaOH = 40.005 / 1 = 40.005g.
The corresponding quantity of equivalents wheight is dissolved in a
volume of water, so that together they will form a volume of 1000cm3
solution .
0.3N of NaOH solution will contain in 1000cm3 solution, 0.3 x
40.005g , so 0.3 equivalents weight of NaOH .
4N of NaOH solution will contain 4 x 40.005 g NaOH .
The volume of solution required but may be greater or less than 1000
cm3.
To calculate the amount of substance required to prepare a given
volume of the solution ( V), of a particular normality (N) the equivalent weight
is multiplied by the normality and the volume ( in liters).
Weight (g) = Eq x N x v
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Example: to prepare 300 cm3 of 0.5N H2SO 4 solution we will first
calculate the equivalent weight of H2SO 4, then apply the above rule :
N = 0.5
V = 0.300 l
Weigth (g) = 49.04 x 0.3 x 0.5 = 7.256 g
Exercise – normal concentration ( CN)
Calculate the amount of the substance necessary for preparation of
500cm3 solution of MgCl 2 with 0.2N concentration . Explain how to prepare
the solution .
1 cm3 = 1 ml
1 l = 1000 ml = 1000 cm3
500 ml MgCl 2 = 0.5 l MgCl 2
1 L MgCl 2 solution 0.2N …………………… 0.2 Eq MgCl2 pure
0.5 L MgCl 2 solution 0.2N ………………… x Eq MgCl2 pure
2 1.012.05.0MgClEq x
MMg = 12; M Cl = 35.5
MMgCl2 = 12 + 35.5 x 2 = 83
5.41283
22
2 MgCl
MgClMEq
1 Eq MgCl2 …………………………………….41.5g MgCl 2 pure
0.1 Eq MgCl2 ………………………………….. x g MgCl 2 pure
pure MgClg x2 15.415.411.0
To prepare 500 ml of 0.2 N MgCl 2 solution we have to weight 4.15g
of pure MgCl 2 which is transferred quantitatively to a 500 ml volumetric
flask , then add about 150 ml distilled water at room temperature ; the solution
it have to be stirred for dissolving the solid; then fill up to the volume (500
ml) with distilled water. The solution was stirred so the undissolved solid does
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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not exist . Keep in bottles to prevent contamination and evaporation of the
solution .
Notes:
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3.2.3. Titrated solution . Factor and normality
The titre of a solution is the amount of the substance in grams of a
solution from 1 cm3 of solution. It is noted with T. Whose solutions are called
standard solution .
Examples: The titer of 0.1N NaOH solution is exactly
0.0040005g/cm3 (equivalent gram of NaOH is 40.005 ).
The titre of exactly normal H2SO 4 solution is 0.049g/cm3 (H2SO 4
equivalent weight is 49 g).
Theoretical titer of a solution can be calculated by the ratio:
Tt = equivalent weight x normality / 1000
The titre of a solution is expressed with 4 semnificative numbers.
The substances which , by simple weighing and dissolved in a
volumetric flask to known volume, can be obtained with the known titre , are
known standard substances or titred substances .
From these substances it can be prepared exact normality solution ,
and the real titre of this solution is equal to the theoretical titre (Tt).
Standards must meet the following conditions:
to be sufficiently pure ;
to have well-established formula and stable working conditions ;
to can be obtained easily in the required purity.
Examples of titred substances:
oxalic acid – used in alcalimetrie and manganometrie ;
anhydrous sodium carbonate – in acidimetry ;
sodium chloride – in the argentometry ;
sodium thiosulfate – to iodometry ;
calcium carbonate and magnesium carbonate or magnesium sulphate
– in Complexometry .
Since few substances meet those criteria, in practice we
prepared solutions with approximate normality , by weighing a quantity of
substance close to that theoretically required. The titer of these solutions is
called real titer (Tr).
In this case, it is necessary to know the normal factor for the passage
from a certain volume of the approximately normal solution to the solution of
the exact normality .
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For titrated solution with a given norm ality between the theoretical
titer and the real titre there is the following relationship :
where: Tr = real titre of solution; Tt = theoretical titre; F = normality factor.
The normality factor indicates how many times the approximate
normal solution is more concentrated or diluted than normal exact solution .
Exact normality solutions will have Tt = Tr, so F = 1.
The approximate normality solutions have approximately F = 1; If the
factor is less than 1 solutions are more diluted and if normality factor is
greater than 1, then the solutions are more concentrated .
The limits of F variation allowed in practice are:
0.9 < F < 1.1
Normality factor allows the conversion of approximately normal
volume solution (Vr) in exact volume solution (Ve).
V e= V r x F
The normal solutions are used in volumetry because meet the property
called the law of same normality solutions equivalence which states as
follows: equal volumes of different substances solutions, but with the same
normality, are equivalent to each other .
On this property is based the calculation of the unknown
concentration of solution, by measurement of the volume of the reaction
solution, with the same normality , whose concentration is known .
F1xV 1 =V 2xF2
where:
F1, F2 = normality factors corresponding to the used solutions for
titration;
V1,V2 = the volumes used to the titration.
3.2.4. Mixing rule (Rectangle rule )
For the preparation of solutions of certain concentrations is used the
rule of mixtures.
TtTrF
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
38
To prepare from two solutions of different concentrations of a% and
b% (a > b) an intermediar concentration solution c% (a > c > b), we proceed
as follows:
In the top corners of a rectangle we put the number representing
concentrations (percentage by weight) of initial solutions (a% and b%) in the
middle of the rectangle – at the intersection of two diagonal – we write the
concentration c% that want to reach .
In the bottom corners note the difference between the initial solution
concentration a% and b%, and concentration c %.
The difference is the volume of solution obtained , in gra ms, to be
mixed and to obtain the desired concentration solution .
Example :
To prepare a solution of H2SO 4 with a density of 1.24 to 32.28 % from
two solutions of H 2SO 4, one with a density of 1.84 that is 95.6% and the other
having a density of 1.15 that is 20.91 %.
For the calculation it applies the mixing rule:
Whei gt parts 11.37 H 2SO 4 95.60 % 63.32 H 2SO 4 20.91 %
So,
%60.95 179.684.137.11
4 2SOH volumeof parts
%91.20 60.5515.132.63
4 2SOH volumeof parts
total volume parts 61.239
For 1000 cm3 solution we calculate:
61.239 volume parts ……….6.179 volume parts H 2SO 4 95.60 %
1000………………………….x
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
39
%6.95 9.100239.611000 179.6
4 23SOHcm x
61.239 volume parts …………..66.060 volume parts H 2SO 4 20.91 %
1000…………………………….y
%91.20 1.899239.611000 060.55
4 23SOHcm y
So: 100.9 cm3 H2SO 4 95.6 % + 899.1 cm3 H2SO 4 forms 1000 cm3
H2SO 4 solution of 32.28%
Notes: :
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C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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3.3. VOLUMETRIC METHODS BASED OF
NEUTRALIZING REACTIONS
Volumetric analysis methods based on protolitic reaction
(neutralization) or proton exchange, include alkalimetry and acidimetry –
which use as a standard solution a base solution or an acid solution.
Alkalimetry – is part of titrimetry comprising methods for
determination of amount of bases or alkali in solutions, based on chemical
reaction, using titration of their solutions with a titration solution of acid.
Acidimetry – are methods of quantitative analysis of strong acids or
substances with acid reaction, by titration of their solutions with a titration
alkali solution .
Alkalimetry and acidimetry together include p rotolitice titrimetry or
neutralizing reactions, based of the following general reaction :
3.3.1. Alkalimetry
3.3.1.1. Determination of the titer and the factor of NaOH solution
0.1N with oxalic acid solution with exact 0.1N (titred
solution)
Principle of method:
The basis of this method is neutralization reaction of oxalic acid and
sodium hydroxide :
Reagents
Hydroxid of sodium NaOH p.a.
Oxalic acid H 2C2O4 x H 2O p.a.
Phenolphthalein (alcoholic solution 1%)
Modul de lucru
pipette 10 cm3 H2C2O4 ~ 0.1 N , and place it in an Erlenmeyer flask ;
wash the flask walls with distilled water ;
H+ + HO- H2O
COOH
COOHCOONa
COONaNaOH H2O + 2 + 2
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
43
add 2 -3 drops of phenolphthalein ;
titrate with NaOH solution 0.1 N , adding this solution drop by drop in
the Erlenmeyer flask and stirring continuously , until the pink color that
occurs when adding each drop will not go away, and stay coloured in light
pink for aproximately 30 seconds .
heate the solution up to 70-80oC (for removal the CO 2 from solution );
the solution from flask will discolour ;
continue the titration until the light pink color appear again , which
will last about 30 seconds;
note with V cm3 the volume of NaOH used for titration ;
to obtain accurate results, run 2-3 parallel samples .
Calculation:
Considering the properties of the solutions with the same normality, we
can write:
V NaOH ~ 0,1n cm3 x F NaOH ~ 0,1n = 10 cm3 H2C2O4 x F H2C2O4~ 0.1 n
H2C2O4 is a tritred solution, so F H2C2O4~ 0.1 n = 1.000
rezultă că
thus:
10 cm3 H2C2O4
F NaOH ~ 0,1n =
V NaOH ~ 0,1n cm3
From F = T r / Tt results:
T real NaOH ~ 0,1n = T theoretical NaOH ~ 0,1n x F NaOH ~ 0,1n
Notes: :
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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3.3.2. Acidimetry
3.3.2.1. Determination of the titer and the factor of H2SO 4 solution ~ 0.1N
with NaOH solution 0.1N with known factor (titred solution)
Principle of method:
The method is based on the neutralization reaction between the H2SO 4
solution of ~ 0.1N and NaOH solution 0.1 N with known normality (known
factor):
H2SO 4 + 2 NaOH = Na 2SO 4 + H 2O
Reagents:
Sodium hydroxide NaOH ~ 0.1N with known factor;
Sulfuric acid H2SO 4 ~ 0.1N (in a flask of 1.000 cm3, is placed approx.
400 cm3 of distilled water are added carefully over, 2.8 cm3 concentrated
H2SO 4 with density
= 1.84 g/cm3 measured with the graduated cylinder .
Stir the contents of the flask and fill up the flask to the marked with distilled
water, after cooling the solution );
Methyl Red Indicator (0.1g methyl red in 100 cm3 distilled water) .
Procedure:
pipette 10cm3 of 0.1 N of NaOH – with known factor, and introduce it
into a conical flask ;
wash the flask’s wall with distilled water, so that any droplets which
could be found on the walls to be brought into solution ;
add 2 -3 drops of methyl red (indicator);
titrate with H2SO 4 ~ 0.1 N to turn the indicator in orange (methyl red
in basic medium is yellow , red in acidic , so equivalence orange );
heat for aproximately 2 minutes to remove CO 2;
cool, and then titration is continued until the transition from yellow to
orange color ;
record the final volume V of reagent consumption (H 2SO 4 ~ 0.1 N) .
Calculation:
V H2SO4 ~ 0,1 n ml x F H2SO4~ 0,1n = 10 cm3 NaOH ~ 0,1 n x F NaOH ~ 0,1 n
F H2SO4 ~ 0,1 n = 10 cm3 NaOH ~ 0,1 n x F NaOH ~ 0,1n /V H2SO4 ~ 0,1n cm3
Treal H2SO4~ 0,1n = T theoretical H2SO4~ 0,1n x F H2SO4~ 0,1n
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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Notes:
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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3.3.2.2. Dosage of acetic acid
Principle of method:
The dosing is based on the neutralization reaction of acetic acid
with a base (NaOH):
CH 3COOH +NaOH → CH 3 COONa + H 2O
Reagents:
Acetic acid CH 3COOH
Phenolphthalein – sol 1%
Sodium Hydroxide NaOH sol ~0,1N with known factor
Procedure:
pipette 10 cm3 of acetic acid solution with un -known concentration,
and was placed in a conical flask;
wash the flask’s wall with distilled water, so that any droplets which
could be found on the walls to be brought into solution ;
add 2 -3 drops of phenolphthalein (indicator)
titrate with NaOH 0.1N solution with known factor, until to the
appearance of orange color which remains 30 seconds
noted by "a" the volume in cm3 NaOH ~ 0.1N used in titrat ion
Calculation:
Knowing that 1Eq of NaOH (40 g) neutralize 1 Eq CH 3COOH (60 g)
we can calculated the quantity of acetic acid – neutralized by 1cm3 NaOH 0.1
N of exact normality (with know the factor), which contains an amount of
NaOH equal to the theoretical titre (0.004 g).
40 g NaOH neutralize …………..60 g CH 3COOH
0.004 g NaOH ……………………….X
X = 0.006 g CH 3COOH
To calculate how many grams of CH 3COOH were neutralized by the
volume of NaOH ~ 0.1N solution used for titration, it is necessary first to
calculate the corresponding volume of NaOH with exact normality by
multiplying by a factor of normality.
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
47
Thus:
a cm3 NaOH ~ 0.1n x F NaOH~ 0,1n mean the volume (cm3) of exact
normality.
If 1 cm3 of NaOH with exact normality neutralize 0.006g of
CH 3COOH, the volume used for titration will neutralize:
a cm3 NaOH ~ 0.1n x F NaOH ~ 0.1n x 0.006 g CH 3COOH
The amount of acetic acid which was neutr alized, was found in 10cm3
volume of acetic acid solution.
In order to express the quantity of acetic acid as a percentage (%), we
should reportethis to 100cm3 .
10 cm3CH 3COOH……a cm3NaOH~0.1x F NaOH ~ 0.1n x 0.006g CH 3COOH
100 cm3 CH 3COOH …………x
a x F x 0.006 x 100
X =
10
% CH 3COOH = a cm3 NaOH~ 0.1n x F NaOH ~ 0.1n x 0.006×10
Notes: :
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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3.4. VOLUMETRIC METHODS BASED ON REDOX REACTIONS
Oxido -reduction methods are volumetric methods based on reactions
that occur after electron transfer between substances which react each other .
Intimate mechanism of redox reactions is therefore based on the loss or
capture of electrons.
The loss of electrons is called oxidation, while the reduction is meaning
gain on electrons .
The dosage based on oxido -reduction reactions are carried out with the
titrated solution by oxidizing or reducing substances.
Depending on the type of oxidant, we can define different oxidimetry
methods .
3.4.1. Manganometry
Dosage method is called manga nometry, because as the oxidant it used
the potassium permanganate . Potassium permanganate is a strong oxidizing
substance for acid and neutral or basic solutions .
Volumetric technique for dosage of KMnO 4 uses the oxidation reaction
in strong acidic medium . Manganometric based reaction is:
2 KMnO 4 + 3 H 2SO 4 = 2 MnSO 4 + K 2SO 4 + 3 H 2O + 5 O
From this equation it is noted that manganese is reduced from the
valence +7 to +2 , accepting 5 e-, so equivalent weight of KMnO 4 is calculated
as molecular mass reporting to 5.
The oxygen released in this reaction is used for the oxidation
substances which can react with it .
The oxidation reactions with KMnO4 , do no use any indicator , and the
end of reaction is detected because of the color excess of permanganate
solution in excess.
To ensure a strong acidic medium to the solution is added a strong
mineral acid (H2SO 4 4N).
3.4.1.1. Determination of the titer and the factor of a solution of
KMnO 4~0.1N with oxalic acid solution 0.1N
Principle of method:
The reaction underlying the assay, it may be expressed by the equation:
5 H 2C2O4 + 2 KMnO 4+ 3H 2SO 4 = K 2SO 4 + 2 MnSO 4+ 10 CO 2 + 8H 2O
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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In the reaction of potassium permanganate with sulfuric acid is released
oxygen .
2 KMnO 4 + 3 H 2SO 4 = 2 MnSO 4 + KSO 4 + 3 H 2O + 5 O
This will react with oxalic acid according to the equation :
(Oxalic acid = HOOC – COOH)
5 (COOH) 2 + 5 O = 10 CO 2 + 5 H 2O
Electron change is:
Mn +7 + 5 e- Mn2+ (reducere) / x2
COO-
– 2 e- 2 CO 2 (oxidare) / x 5
COO-
Reactivi
solution of potassium permanganate – KMnO 4 ~ 0.1 N (3.2 g KMnO 4
is dissolved in distilled water and fill up to a volume of 1.000 cm3; after 8-
10 days is filtered andwe can determining the factor )
oxalic acid – H2C2O4 x 2 H 2O crystals ;
sulfuric acid – H2SO 4 4 N (112 cm3 conc . D = 1.84 is added gradually
in about 400 cm3 of distilled water and, after cooling to 1000 cm3.
Procedure
pipette 10 cm3 of oxalic acid 0.1 N (Titrofix) in a conical flask ;
washed with distilled water the inner wall of the flask ;
add 15 cm3 of sulfuric acid 4N ;
heat the content of the flask up to 70 – 800 C
titrated with KMnO 4 solution ~ 0.1N until it appears a weak pink
coloration which remains stable for 1 minute
note the consumed volume of KMnO 4 ~ 0.1 N (V) for titration .
NOTE: If we used more diluted solutions (0.01N), then the procedure
is:
pipette 10 cm3 of oxalic acid 0.01 N (Titrofix) in a conical flask ;
washed with distilled water the inner wall of the flask ;
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
50
add 5 cm3 of sulfuric acid 4N ;
heat the content of the flask up to 70 – 800 C
titrated with KMnO 4 solution ~ 0.01N until it appears a weak pink
coloration which remains stable for 1 minute
note the consumed volume of KMnO 4 ~ 0.01 N (V) for titration .
And the calculati on will be the same as for 0,0N solutions
Calculation:
10 cm3 solution H 2C2O4 , 0.1N contains 0.063 g of substance. One
equivalent gram of H 2C2H4 (63.034), reacts with one equivalent gram of
KMnO 4 (31.606).
IF 63.034 g H 2C2O4 x H 2O ……………31.606 g KMnO 4
THEN a g H 2C2O4 x H 2O ………………… V cm3 KMnO 4 x Treal
VaTKMnO034.63606.31
4
theoreticreal
KMnOTTF4
where a = grams of oxalic acid pure (wheight).
Notes: :
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C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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3.4.2. Iodometry
The volumetric method based on a redox titration where iodine is
involved is known as iodometry , or iodometric titration .
Iodometry analysis is base don the floowed oxido -reduction reaction:
(1) I2 + 2 e- 2 I-
(2) 2I- – 2e- I2
In the first reaction, the iodine
In the first reaction , iodine (I2) act as an oxidant and in the latter one ,
yhe iodide ion (I-) acts as a reducing agent .
Iodometric dosing methods can use two reagents density: I2 solution or
solution of KI.
Determination of iodine (I2), in the case of using the first solution , or
ion (I-) in the case of using the second solution is performed by titration with
sodium thiosulphate solution . As indicator in iodometry is used starch
solutions – that forms an absorption compound in the presence of iodine
intensely colored in blue.
3.4.2.1. Determination of titer and factor of a solution of iodine ~0.1 N
with a solution of sodium thiosulfate ~ 0.1 with known factor
Principle of method:
2Na 2S2O3 + I 2 = 2NaI + Na 2S4O6
The reaction product is sodium tetrathionate . From this reaction is
observed that 2 molecules of sodium thiosulfate reacts with the iodine
molecule .
The equivalent of sodium thiosulphate is equal with the molecular
weight (248.19g) and of iodine with the atomic weight (126.92) because:
2S2O32 – – 2e_- = S 4O62-
2 I0 + 2e- = 2I-
The progress of the reaction depends on the pH of the environment . In
the alkaline environment the reaction occurs with the formation of hypoiodite
and iodine, and the oxidation of sodium thiosulphate to sulphate :
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
53
Na 2S2O3 + 4I 2 + 10 NaOH = 2 Na 2SO 4 + 8NaI + 5 H 2O
So, the reaction will be carried out in an acidic medium .
The starch is added during titration with thiosulfate when the brown
solution of iodine reached the yellow coloure , because otherwise some of the
iodine will be retained by starch in a stable blue color which requires more
time for reduction with sodium thiosulfate , therefore, consumption of the
reagent will be higher.
Reagents
iodine solution ~ 0.1N . Weigh 12.692 g resublimat iodine with the
analytical balance , place in 1000 c m3 volumetric flask . It has to be dissolved
in a solution of KI (25 g KI in 35 cm3 distilled water) . After dissolution is
filled up to the mark with distilled water ;
sodium thiosulfate solution Na 2S2O3 0.1N . Accurately weigh 24.82 g
sodium thiosulfate , place in 1 dm3 volumetric flask . Dissolve in distilled
water and fill up to the mark with distilled water;
starch solution 0.2%. Combine 2 g of soluble starch with 10 mg HgI 2
and add a little distilled water stirring to obtain a homogeneous mixture.
Bring to a volume of 1.000 cm3 with distilled water.
Procedure
pipette 10 cm3 of solution of iodine 0.1N (V1) which is diluted with
about 25 cm3 of distilled water ;
titrate with Na 2S2O3 solution ~ 0.1N with known factor (F2) until to
light yellow ;
add 1 cm3 of starch solution 0.2%, with role of an indicator ;
continue titration until the solution becomes discolored (blue coloure
disappears, but it could show like green coloure because blue with yellow
forms green)
note the volume V2 of sodium thiosulfate solution 0.1N in cm3 used in
the titration
Calculation:
Apply the property of equivalency solutions with the same
normality :
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
54
V1 x F I2~ 0,1 n = V 2 x F Na2S2O3~ 0,1n
11.0422 2
1.02VFVFN OSNa
N I
TrI2~ 0,1n = F I2~ 0,1n x T t = F I2~ 0,1n x 0.012692
Notes:
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3.5. VOLUMETRIC METH ODS BASED ON COMPLEX
FORMATION
(COMPLEXOMETRY or CHELATOMETRY)
Underlying these methods is volumetric reaction forming internal
complexes called chelates, so the complexometric determination are named
chelatometric methods .
Internal complecs are coordinative combinations with a special
structure .
Polycarboxylic amino acids have a great power for complexing and
therefore were called " complexons".
Complexone III (the disodium salt of ethylene diaminetetra -acetic
acid, or EDTA ) is most commonly used as a reagent in complexometric
reactions .
Complexon III notes as Na2H2Y and has the following formula :
Equivalence point is determined by indicators of complexometry .
Complexometry indicators are complexons , dyes, which have
complexing capacity less than complexons using for titration .
In the presence of free metal ions they form colored compounds. and in
titration release metal ions, so the solution changes its coloure , indicating the
end of titration.
Complexometry main indicators are: murexide , Eriochrome black T,
sulfosalicylic acid, titron , catechol violet , phtaleine Complexon etc.
Complexometry indicators change their coloure also with the pH variations .
3.5.1. Determining titer and factor of a solution of complexone III
Principle of method:
Establishing the titer of a complexone solution using as titrosubstances
– salts of metal ions, there with the complexone gives a stable compound. The
most used salts of metal ions are : CaCO 3, MgSO 4 x 7 H2O, ZnO, Cu, Zn, Bi,
etc.
Because in the reaction results hydrogen ions , leading to a reduced
complexon compound stability and a influence on the indicator by changing
pH, the titrations has to be performed in buffers.
CH2CH2 N NCH2
CH2CH2
CH2COOH
COONaHOOC
NaOOC
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
57
The basic reaction is as follows :
Reagents
complexone III solution. Weigh 9.34 g N2H2Y x 2 H 2O, introduce it in
1000 cm3 volumetric flask , dissolve in distilled water and fill up to the mark
with distilled water ;
solution of NaOH 1%;
buffer solution of ammonium chloride and ammonia – NH 4 + NH 3.
Weigh 70 g NH 4Cl, introduce in 1000 cm3 volumetric flask , add 570cm3 of
NH3 solution d = 0.90g/cm3 and fill up to the mark with distilled water ;
mixture Eriochrome black T p.a. with NaCl p.a. (in the ratio 1 :500);
weighed 6.162 g of magnesium sulfate heptahydrate MgSO 4 x 7 H2O
(before use, store in desiccator for 24 hours) , introduce it in a flask of
1.000cm3, dissolve in distilled water and fill up to the mark with distilled
water ;
Procedure
pipette 10 cm3 of MgSO 4 solution 0.05N to a conical flask ;
add NaOH solution until pH = 7;
add buffer solution NH 4Cl + NH 3 to pH 10 . The pH is controlled by
indicator paper ;
add a spatula tip of eriochrome black T + NaCl mixture (in a ratio of
1:500 );
titrate with complexone III solution 0.05 N to turn from red to blue ;
note the volume of complexone III solution used for titration with V2.
CH2CH2 N NCH2
CH2CH2
CH2COONa
CNaOOC
C O O
O-O-+ Me +
CH2CH2 N NCH2
CH2CH2
CH2COONa
CNaOOC
C O O
O O2
2Me +
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
58
Calculation:
Given the same normality property of the solutions we can write :
V 2 cm3 x F complexon ~ 0,05n = 10 cm3 MgSO 4 0.05n x F MgSO4 0.05n
MgSO 4 is a titrosubstance, so F MgSO4 0.05n = 1.000
Results that:
3
205.04310
cmVMgSOcmFN
complexone
From relation F = T r / Tt results
T real complexon ~ 0.05N = T theoretic complexon ~ 0.05N x F complexon ~ 0.05N
1 Eg complexon III = 186.2g
T theoretic complexon ~ 0.05N = 186.2 x 0.05 = 0.009325 g/cm3
T real complexon ~ 0.05n = 0.009325g/cm3 x F complexon ~ 0.05N
Notes: :
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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CHAPTER IV
PHYSICO – CHEMICAL METHODS OF BIOCHEMICAL
ANALYSIS
4.1. pH metry – GEN ERAL INFORMATIONS
pH metry is a physico -chemical analysis which is characterized by a
reaction (in acidic, basic or neutral medium ) and represents the number of
hydrogen ions.
The hydrogen ion concentration from a solution can vary within the
limits 1-10-14, being correlated with the concentration of hydroxyl ions,
according to the equation:
1410 OH H
Since the limits of variation of these concentrations are very wide , we
use the term pH, introduced by Sorens en, representing the negative logarithm
of hydrogen ion concentration, that is the exponent to which the number must
be 10 (basis of the decimal logarithm ), to express the value of the equivalent
concentration of hydrogen ions .
HH pH1lg lg
The connection between pH and [H+] is presented as:
pHpHH10110
The pH can vary within the limits 0-14.
Solutions with pH = 7 are neutral , the solutions with pH < 7 are acidic
and those with pH> 7 are alkaline .
The pH of the biological fluids is relatively constant , but can vary in
small limits , for example:
in blood the pH = 7.3 – 7.5, the average being 7.36
in stomach the pH = 1.5 – 2.5
in the intenstin the pH = 7.8 – 8.7
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In pathological cases it can cause changes in the normal values of pH.
Thus, when blood pH decrease below the normal range is called acidosis (in
the case of diabetes , the accumulation of ketones or lactic acid) and when
blood pH increase above the normal range is called alkalosis .
Determination of pH can be performed by two methods: colorimetric
and potentiometric.
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4.1.1. Determination of pH with indicator paper
Principle of method:
Determination of the pH is based on the property of indicators to
change their color depending on the concentration of hydrogen ions . The pH
indicator paper is impregnated with different indicators (universal indicators ).
It is a colorimetric method .
The pH indicator paper is presented either as books or leaflets, either in
the form of discs enclosed in a plastic box whose lid contains shows t he colors
corresponding to different values of pH.
Fig.1. Indicator paper for pH (presentation mode)
Procedure:
From a strip of pH indicator paper cut several small paper bands . With
tweezers take a band and moistening into the solution whose pH want to
determine . The paper b and will be coloured and then compare the color with
the color band on the standard color scale . Identity means the identity of the
pH color .
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4.1.2. Potentiometric method for the determination of pH
Principle of method:
Determination of pH with the potentiometric method is an
electrochemical method and is based on measuring the difference of electrical
potential between two electrodes inserted into the sample solution . One of the
electrodes , the negative electrode, is the indicator electrode, and the most
widely used is glass or measuring electrode (Figure 2).
Fig.2. Glas electrod Fig. 3. pH -metry
The second electrode , the positive one, is a reference electrode (calomel
electrode or AgCl electrode ) whose potential is always constant .
The two electrodes bind to the + and – terminals of a device called
potentiometer or pH -meter, which is an electronic millivolt . The value of the
measured electric intensity is very low, preventing the discharge of the
galvanic cells consist of two electrodes, during the measurement .
Procedure
electrodes are washed with distilled water in a Berzelius flask, and then
are rinsed with buffer calibration with known pH.
electrodes are placed in a buffer solution of known pH. With a
thermometer it is measure the temperature of the solution. The
compensator temperature adjustment wheel (button) is moved to the
operating temperature value.
the calomel electrode is connected to the positive terminal (+) and the
glass electrode terminal to the negative terminal ( -) of the device.
check if the device is suitable voltage stabilizer power socket.
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
63
connect the stabilizer after 5 minutes to make one swi tch in the operating
position. The red indicator should light up.
After 5 -10 minutes (during heating of the device), the switch is brought to
zero position, required working position (on the pH range of 0 -7 or range
to 7-14).
The indicator needle of the me asuring instrument is brought by turning
the pH button (wheel) at pH of the buffer solution.
Ensure proper functioning of the device with at least one standard buffer
solution. The device should indicate the pH of the solution. In this way
the device is re ady for work
introduce the electrodes in the solution of which pH we want to determine
and adjust the wheel to pH 0 -7. If the indicator needle is stopped in this
interval, the pH of the solution is in the acidic range and can be read from
the scale device. If the indicator needle exceeds 7, switch the wheel on the
7-14 pH. Requested value is read on the scale of the device which in this
case is in the alkaline range.
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Notes:
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4.2. Buffer systems
Buffer systems are mixtures of solutions which are able to maintain
aproximately constant the pH in the case of small amounts addition of acids or
bases.
Buffer solutions may consist of :
1) Weak acids and their salts with strong bases ;
2) weak bases and their salts with strong acids ;
3) primary and secondary salts of polybasic acids .
The pH of the buffer system is given by Henderson -Hasselbach
equation :
AHApK pH
log
One example of the first buffer solution class is the mixture of sodium
acetate (CH 3COONa ) and acetic acid (CH 3COOH). The mechanism of action
of this buffer system the addition of strong acids is as follows :
CH 3COO-+ Na+ + CH 3-COOH + H+ Cl- = Na+ Cl – + 2CH 3COOH
Instead each equivalent weight of hydrochloric acid will form an
equivalent weight of acetic acid that is a weak acid , and almost did not
dissociate, but the pH will change very little because of the blocking the
hydrogen ion into the solution by forming weak acid , undissociated. .
The mechanism of action of the same buffer system adding a strong
bases in this case will be :
CH 3-COO- + Na+ + CH 3COOH + Na+ + OH- = 2 CH 3COO- + 2Na+ +H 2O
Hydroxyl ions coming to the solution with strong base will lead to
dissociation of acetic acid to form a water molecule, also practically
undissociated . The pH of the solution will not change in this case, because no
hydroxyl ions will remain free in solution.
Described buffer system will have buffering action, and will have
maximum pH values at pH = p Ka, where the buffering range will be 1.7.
To obtain a buffering effect in a higher range of pH, we can use
universal buffer systems which contain polybasic acids and their primary and
secondary salts .
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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4.2.1. Preparation of universal buffering system
Procedure :
Se iau 9 eprubete curate și uscate numerotate de la 1 până la 9, și se
introduce cu ajutorul unei pipete volume de amestec de acid citric 0,1 M și
acid boric 0,1 M în raport de 1/1(25 ml acid boric 0,1M + 25 ml acid citric
0,1M) și soluție de NaOH 0,1 N î n cantitățile prevăzute în tabelul nr.1.
Nine clean and dry tubes are used, numbered from 1 to 9. Add with a
pipette volume of a mixture of citric acid 0.1M and boric acid 0.1 M in a ratio
of 1 / 1 (25ml boric acid 0.1M + 25ml citric acid 0.1M) and NaOH 0.1N
solution in volumes presented in table 1 .
In each tube is also inserted, using a pipette dropping, 6 drops of
universal indicator and shake the content. The obtained solutions with pH
values from 3 to 11 , and the color of the solution after the introd uction of
universal indicator ranges from red to violet. Buffering system will buffering
throughout the mentioned pH range, correlated to the acid constant of the used
acids (citric acid and boric acid) – which will act as independent buffers in the
range of pKa 1.7.
Table 1. Universal buffering system
Tube Nr. 1 2 3 4 5 6 7 8 9
Acid
mixture 7,7 6,1 4,8 3,9 3,7 3,55 3,3 2,9 2,1
NaOH
0.1M 2,3
3,9 5,2 6,1 6,3 6,45 6,7 7,1 7,9
pH 3 4 5 6 7 8 9 10 11
Coloure
of
indicator red Red-
orange orange yellow Green –
yellow Green Blue Viole
t Violet –
reddish
The structure of citric acid and boric acid are presented here:
Buffering range of Buffering range of
citric acid boric acid
pKa 1 = 3.06 1.36 – 4.76 pKa 1 = 9.14 7.44 – 10.84
pKa 2 = 4.74 3.04 – 6.44 pKa 2 = 11.74 10.04 – 13.4
pKa 3 = 5.40 3.70 – 7.10 pKa 3 = 13.80 12.10 – 14
CH
CH
CH2HO COOH
COOH
COOHBOH
OH
OH
Acid citric Acid boric
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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4.2.2. Highlighting the buffering action of the
buffer system
Test 1
One of the buffer system solution is divided into 3 tubes, adding into
two tubes about 1/3 of the contents of the test tube no. 5 which has a pH = 7.
In one of the tubes add 2-3 drops of HCl 0.1 N , and in the other 2-3
drops of NaOH 0.1 N . Compare the coloure of the solutions from the two
tubes with the coloure of the tube where was not added neither acid nor base.
It appears that the coloure of the solution from both tube has not changed
which means that the pH of the solutions remained constant due to the
buffering action .
Test 2
Take 3 clean tubes and add in each 10cm3 distilled water (or deionized)
and 6 drops of universal indicator.
Add in the first tube 2 -3 drops of HCl 0.1 N and in the second tube 2 -3
drops of NaOH 0.1 N. Compare the color with the third tube, where was not
inserted any acid or base.
It is observed that in the first tube the indicator is coloured to red, and
in the second tube the color turns to blue, which means that the pH of the
distilled water was influenced by the addition of H+ and the HO-, due to the
absence of systems for tamping.
Test 3
Take 3 clean and dry tubes and add in each 10 cm3 of tap water and 6
drops of universal indicator.
Into the first tube add 2 -3 drops of HCl 0.1 N and in the second tube 2 –
3 drops of NaOH 0.1 N. Compare the colors of the first two tubes with the
third tube, ob serving that for the first two test tubes the pH will change very
little due to the buffer systems from the tap water due to the presence of
carbonic and phosphoric acid and their salts.
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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4.2.3. Measuring the buffering system of blood and urine
Buffering power is the amount of acid or base needed to change the
pH by one unit.
Principle of method:
For the determination of blood serum or urine buffering power it is
necessary to determine in advance their pH. Then add a small volume of HCl
0.1 N solution and determine again the pH. The ratio between the number of
cm3 of acid added and the pH variation that occurred, is the buffering power
of the tested biological fluid.
Reagents :
citric acid 0.1M solution (21.014g C 6H8O7 x H 2O dissolved in boiling
deionized water and fill up to 1 dm3).
boric acid 0.1M solution (6.184g H 3BO 3 dissolved in boiling deionized
water and fill up to 1 dm3).
universal indicator. The mixture was dissolved in 100 cm3: 60 cm3 of
ethanol and 40 cm3 of deionized water and the fol lowing amounts of 0.02 g
of methyl red indicator, 0.04 g Bromothymol blue, 0.04 g Thymol blue, and
0.02 g of phenolphthalein.
Procedure :
Determine the pH of blood serum or urine pH meter.
Place in a test tube 2 cm3 blood serum or urine and add 0.2 cm3 of HCl
0.1N. Determine again the pH of the solution thus obtained.
Calculation:
Calculate the difference between the two values of pH and then
calculate the ratio of the number of cm3 HCl 0.1N added and this difference.
Example of calculation, con sidere the initial pH of the alkaline urine
is equal to 8, and after the addition of HCl 0.1 N the pH was equal to 7.8. The
difference between the two measurements is 0.2. The buffering power will be
0.2 / 0.2 = 1.
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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Buffer systems of animal organisms
Blood buffer systems are:
1) carbonic acid – sodium hydrogen carbonate in ration to 1/20.
– in plasma H 2CO 3 : NaHCO 3 (sodium bicarbonate)
2) primary sodium phosphate – secondary sodium phosphate ratio 1/5
– NaH 2PO 4 : Na 2HPO 4
3) Plasmatic Protein – Sodium Protein compounds
4) oxyhemoglobin – potassium oxyhemoglobin
– in erythrocytes (HBO 2) H : (HBO 2) K
5) hemoglobin – potassium hemoglobin
– in erythrocytes (Hb) H : (Hb) K
Approximately 50% of the total buffering capacity of the blood is due
to the carbonic acid – bicarbonat, which is formed mainly from CO 2 from
metabolism.
Carbonic acid results from CO 2 under the influence of carbonic
anhydrase enzyme according to the reaction:
CO 2 + H 2O H2CO 3 H+ + HCO 3-
Negative logarithm of the first ionization constants of carbonic acid is
pK = 6.1 and with the equation Henderson – Hasselbach, the blood pH will be
given by:
HCO 3-
pH = pK + lg = 6.1 + log 20 = 7.4
H2CO 3
Buffers systems assure therefore, the regulation of acid -base balance
of the biological environment.
In urine regulating acid -base balance is determined mainly by the
ratio of primary phosphate NaH 2PO 4, and the secondary phosphate Na 2HPO 4,
whose report is 9/1, reverse much compared to the blood that is 1/5.
In the cell, the phosphate buffer system has a predominant role in the
maintenance of pH. Proteins and the amino acids due to their amphoteric
character, significant contributes also to the buffering action.
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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Notes :
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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CHAPTER V
QUALITATIVE ANALISIS OF MAIN BIOMOLECULES
GROUPS
5.1. PROTIDS
Protids are the primary components of living matter. They are organic
substances which contain the at least four elements: C, H, O and N, S and P
may also contain macromolecules are made up of amino acids linked together
by peptide bonds ( -CO-NH-), forming polypeptide chains.
Protids can be hydrolyzed under acidic, basic or enzymatic
environment. The products obtained from hydro lysis can be classified as:
Aminoacids
Protids Peptides oligopeptids
polypeptids
Proteids - holoproteids (proteins)
heteroproteids
Proteins give characteristic color and precipitation reactions.
5.1.1. Color reactions
Protein color reactions are due to the presence of amino acids that react
specifically with differ ent reagents.
Biuret Reaction
Principle of method:
Biuret reaction is due to the presence of peptide bonds in the protein
molecule CO – NH -. Biuret reaction is positive for all substances in their
structure at least two peptide bonds. Protids react with copper salts in alkaline
medium, to produce a violet color.
This reaction is called biuret reaction, as biuret (the substance obtained
by the elimination of a molecule of ammonia from two molecules of urea) and
giving it the reaction, presenting two p eptide bonds.
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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The biuret reaction is also utilized for proteids dosage.
Reagents
solution of proteids (serum, egg white diluted in distilled water)
copper sulfate 5% solution
sodium hydroxide 20 -30% solution
Work method
Place in a test tube 1 cm3 (protein) of research solution, add an equal
volume of sodium hydroxide 20 -30% and then 3 -5 drops of 5% copper
sulfate. Observe the appearing of a violet blue coloration.
Ninhydrin reaction
Principle of the method :
Amino acids, peptides and proteids give through treatment with a hot
solution of ninhydrin a violet -blue color that is due to the free – NH 2 groups.
NH2
O C
NH
O C
NH2
Biuret
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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Reagents
solution of glycocol 1%
solution of proteids
solution of ninhydrin 0,1% in ethanol.
Work method
In a test tube are placed 2 cm3 of glycocol and in another 2 cm3 of
proteids solution (serum, albumin). In both test tubes is added 0.5 cm3
solution of ninhydrin and heated to boiling. Observe the apparition of the blue
violet color.
Ninhydrin reaction is used to highlight the amino acids separated by
paper chromatography.
Xantoproteic reaction
Principle of method:
The reaction is due to the presence of the protein molecules with
aromatic rings of amino acids: phenylalanine, tyrosine and tryptophan.
By treating the protein with hot concentrated nitric acid a yellow
precipitate is obtained because of formed nitrated derivatives.
Reagents
proteids solution
concentrated HNO 3
diluted NH 4OH
Work method
1 cm3 of a protein solution is placed in a test tube
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
75
0.2-0.3 ml of concentrated HNO 3 is added, and a white precipitate
appears
the solution is heated to boiling, and the precipitate becomes yellow
cool the tube in running water
the medium is alkalinized (with 1 ml NH 4OH) – the orange color is
observed
Millon reaction
Principle of method:
The reaction is due to amino acids with phenolic structure – namely
tyrosine which treated with mercuric nitrate in nitric acid forms nitric
derivates – precipitated of dark red color.
Reagents
proteins solution
Million reagent (a part of metallic mercury is dissolved in 2 parts of
concentrated nitric acid, by heating the mixture. After the dissolution of
mercury a double volume of distilled water is added).
Work method
in a test tube are introduced 1 cm3 of proteids solution
add an equal volume of Millon reagent
gently heat the mixture without boiling
formatted precipitate is tighten, it becomes spongy and turned in dark
red color.
Sakaguchi reaction
Principle of method:
Guanidine derivatives (arginine) give in a alkaline environment, in the
presence of naphthol and sodium hypobromite, red colored combinations.
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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Reagents:
proteids solution
NaOH 10% solution
Mollisch reagent (0.1 g -naphthol was dissolved in 25 cm3 of alcohol
and diluted to 100 cm3 with distilled water).
solution of sodium hypobromite (12 drops of bromine in 6 cm3 of 30%
NaOH).
Work method
in a test tube are inserted 2 cm3 of proteids solution
is alkalinized with 2 cm3 of 10% NaOH solution
2cm3 of naphthol and 5 to 7 drops of sodium hypobromite are added
in the presence of arginine a red staining appears
The reaction with lead acetate
Principle of method:
Proteins containing tio -amino acids (cysteine, cystine, methionine) by
heating in a strong alkaline environment, release the sulfur as sulfate ions (S2-
).
R-SH+ 2NaOH Na 2S+ R -OH +H 2O
Sulfide anion S2- is emphasized by treatment with lead acetate, which
forms a black precipitate of lead sulfide (PbS), according to the reaction:
Na 2S + (CH 3-COO) 2Pb PbS +2CH 3COONa
Reagents
NaOH 10% solution
proteins solution
lead acetate 0,5% solution
Work method
2 cm3 of the protein solution are inserted in a test tube
add the same volume of 10% NaOH and boil for 5 minutes
add 2 -3 cm3 of 0.5% lead acetate and heat
a dark brown precipitate is obtained
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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5.1.2. Precipitation reactions
Protein precipitation reactions can be grouped into: reversible and
irreversible.
In reversible reactions proteins retains native molecular structure,
changes being only physical (moving from state of solute to the precipita ted
state). At the removal of the precipitation proteins revert to the original state.
Salification of the protein consists of partial dehydration of the protein
The polar groups of proteids molecules retain through hydrogen bonds or by
electrostatic attra ction, an amount of water. Light metal salts Na +, Mg 2+,
NH4 +, partially dehydrate the proteins due to pronounced tendency of cations
to hydrate, proteids molecules loose water, aggregate and thus precipitates.
In reversible reactions, protein denaturati on occurs due to structural
changes in molecules. Due to structural changes proteins no longer regain the
initial properties even at the removal of the precipitation agent.
5.1.2.1. Reversible precipitation reactions
The reaction of precipitation with ammonium sulfate
Principle of method:
Proteins from aqueous solutions can precipitate by the addition of salts
(sulfates) of sodium, magnesium and ammonium salts. Globulins, having
large molecule, precipitates at half -saturation (half -saturated solutio n),
albumin, having smaller molecule precipitates at saturation (saturated
solution).
Reagents
proteid solution (diluted serum)
saturated ammonium sulfate fresh solution
Work method
in a test tube are inserted 3 cm3 of protein solution (diluted serum)
an equal volume of saturated ammonium sulphate is added
the contents of test tube is shacked and the appearing of a precipitate
(globulins) is observed
the content is filtered
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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upon the obtained filtrate fine crystals of ammonium sulphate are
added up to saturation
observe the formation of albumin precipitate
at the addition of water the precipitate will dissolve
the method serves to separate albumin from globulin.
Protein precipitation with alcohol
Principle of method:
The proteins may be dehydrated w ith alcohol, because of dehydration
they will agglomerate and precipitate. By adding water to precipitate the
protein will dissolve.
The method is used to separate the different protein fractions, by
varying the concentration of alcohol, the pH and tempera ture. At the
prolonged action of alcohol on proteids, they denaturate.
Reagents
Protein solution
ethanol 95%
Work method:
1 cm3 of proteids solution is introduced in a test tube
add 1 cm3 of ethanol
the formation of a precipitate is observed
precipitate is divided into two tubes
immediately dissolve the precipitate in the first tube
observed that/if dissolves
in the second tube the precipitate is dissolved after 2 -3 hours
5.1.2.2. Irreversible precipitation reactions
Precipitation with min eral acids
Principle of method:
Concentrated mineral acids irreversibly precipitated proteins in aqueous
solution due to their denaturation.
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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Reagents
protein solution
concentrated HCl, concentrated HNO 3
Work method:
1cm3 of proteids solution are introduced into two test tubes
In the first tube add 1cm3 concentrated HCl
in the separation area of the two liquids a white ring is formed
(precipitated proteins)
by stirring, the ring disappears, is dissolved, due to the excess of HCl
in the second test tube, add 1 cm3 concentrated HNO 3
observe the formation of a white ring at the area of separation of the
two liquids
by stirring, the precipitate is not dissolved (ring is present)
Precipitation with organic acids
Principle of method:
Organic acids such as: trichloroacetic acid, sulfosalicylic acid, picric
and citric acid (reagent Essbach) precipitate the proteids. The excess of acid
does not dissolve the precipitate. Precipitation with trichloroacetic acid is used
for the de -proteinization of biological fluids. Precipitation with sulfosalicylic
acid is very sensitive and is used to identify the proteins.
ac. trichloroacetic ac. sulfosalicylic ac.citric ac.picric
Reagents
CCl3COOH CH2 COOH
CH COOH
HO CH COOHCOOH
OH
SO3HO2NOH
NO2
NO2
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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protein solution
trichloroacetic acid 5 % solution
sulfosalicylic acid 20% solution
Essbach reagent( 10 g of picric acid și 20 g of citric acid are
solubilized in distilled water up to 1 dm3 solution)
Work method:
in three test tubes are introduced 1 cm3 of proteids solution.
in the first tube 1 cm3 of trichloroacetic acid solution is added. It is
observed the formation of an abundant white precipitate.
in the second tube add a few drops of 20% sulfosalicylic acid. The
formation of a cloudlet of precipitate cam be observed
in the third test tube, add a few drops of Essbach reagent. Observe the
formation of a yellow precipitate.
The precipitation with th e salts of heavy metals
Principle of method:
Proteins are forming with heavy metals (Cu2+, Pb2+, Mg2+, Ag+) hard to
dissolve precipitates.
Reagents:
protein solution
Cu SO 4 3% solution
Pb (CH 3-COO) 2 0,5% solution
Work method
1 cm3 proteids solution is added in two test tubes
in the first test are added 0,5cm3 of CuSO 4 3%solution
in the second tube are added 0.5 cm3 0.5% solution of lead acetate
(CH 3 COO) 2 Pb
heavy precipitate apparition is observed
Precipitation of proteins b y heating
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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Principle of method:
Protein, under the action of heat, in an acid medium and in the presence
of electrolytes, precipitates due to a denaturation phenomenon. Precipitation is
greatest near the isoelectric point. Denaturing heat action can be canceled
completely in strongly acid or alkaline environment.
In such media, the stability of the proteids is maximum, as the pH is far
from the isoelectric point.
Reagents:
protein solution
CH 3COOH 0,1% solution
CH 3COOH 1 % solution
NaOH 1% solution
Work method
take 4 tubes which are numbered from 1 to 4 and insert in each 1cm3
proteids solution
tube 1 is heated to boiling and the precipitate is noted for most of
proteids
in the tube 2, 1 -2 drops of 0.1% acetic acid are added and the content
is heate d to boiling
precipitate amount is higher than the test tube 1, which shows that
the proteids are in an environment with a pH very close to the isoelectric
point
in the test tube 3, 1 -2 drops of 1% acetic acid are added and the
content is heated.
no precip itate is formed because the pH is far from the isoelectric
point
in the tube 4, 1 -2 drops of 1% NaOH solution is added and heated.
precipitate is not formed because of strongly basic environment (far
from the isoelectric point)
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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5.1.2. Determination of milk proteins by the Sörensen method
Principle of method:
Free acidity of the milk is neutralized by titration with 0.143 N NaOH
solution in the presence of phenolphthalein.
The formaldehyde, which blocks the amine groups ( -NH 2) of the amino
acids of the protein constituents, is added. The remaining free acid groups ( –
COOH), are titrated with 0.143 N NaOH. The color of the obtained titration is
compared with that obtained in the vial where in 50 cm3 milk, potassium
oxalate was a dded along with 1 cm3 of the cobalt sulfate solution.
Reagents:
sodium hydroxide – NaOH 0.143 solution free of CO 2 (in a dm3 of boiled
and cooled water 5.75 g of NaOH are dissolved. The titer of the solution
is determined by means of a solution of oxa lic acid 0.143 n = 9 g of oxalic
acid / dm3.)
alcoholic phenolphthalein solution 2%
solution of cobalt sulphate – 5% CoSO 4
neutral solution of potassium oxalate – K2C2O4 28%
37% formaldehyde solution (amount of formaldehyde required for daily
determination is neutralized with a sodium hydroxide solution 0.1 N).
Work method:
In two vials are introduced in each 50cm3 milk and 2cm3 of potassium
oxalate solution and then is shacked. In a vial constituting the sample 1 cm3 of the
cobalt sulfate solution is inserted and shacked.
A pink coloration appears. In the second conical vial are insert 1 cm3 of
phenolphthalein and sodium hydroxide dropwise is added from a burette to
neutralize the free acidity, till a pink color to th e same intensity as the control
sample is obtained. In the test sample, 10 cm3 of formaldehyde are added and
R CH COOH
NH2+ OC
HH R CH COOH
N CH2
H2O
protida baza Schiff
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
83
mixed. Is noted that the pink color disappears. Allow to stand 30 seconds,
shake and titrate again until the pink coloration of the same intensity with the
control sample appears.
Calculation:
Proteids titre = V x F / 2 where:
V= sodium hydroxide 0,143 n volume, used for the second titration
expressed in cm3.
5.1.3. Determination of the protein isoelectric point
At a certain pH value, protein molecules can dissociate so that the
amount of positive charges is equal to the amount of negative charges.
This pH value is called isoelectric point and is noted pH i. Dissociation
of anions or cations of amino acids occurs according to the reactions:
At pH values smaller than pH i, the proteins are cations, and at pH
values greater than pH i different proteins behave as anions. The isoelectric
point is a physical -chemical constants that is characterizing each type of
protein.
At isoelectric point som e physical properties of the protein change.
Thus, the protein solubility is minimal and stability is also minimal (they
precipitate easy), they do not migrate in an electric field because electrical
charges of opposite sign shall compensate each other, th e attraction of the two
poles become equal.
At the isoelectric point, positively charged groups and negatively
charged groups of the proteids molecules will compensate in such a way that
the electrical charge of the molecule is equal to zero and the molecu le is not
migrating in the electric field.
Determination of the isoelectric point of casein
H2N
RCOOH COO CH H-+ CH
RH2N+
aminoacid anion
H2O H3N HO
cation aminoacidRCH +-CH COO COOH
RH2N ++-
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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Principle of method:
Casein solution is stable only in a well defined range of pH when
colloidal particles have a specific electrical charge. As the pH approaches the
isoelectric point, the solution of casein becomes increasingly less stable and
coagulated. Maximum coagulation occurs at the isoelectric point.
Reagents:
Distillated water
acetic acid solution – CH 3COOH 0,01N
acetic acid solution – CH 3COOH 0,1N
casein solution in sodium acetate – CH 3COONa 0,1N
Work method:
Distilled water and acetic acid of 0,01N, 0.1 N and 1 N are introduced
in a series of nine tubes using a microburette or pipettes, in the amounts
specified in Table 10, where is indicated pH appropriate for the solution. In
each tube 1 cm3 casein solution in sodium acetate 0.1N is added.
The solutions are mixed and after 5 min a haze is observed in one of the
test tubes which is noted by +, and where the coagulation was produced is
noted by X. The most intense coagulation occurs at the isoelectric point.
Determine, from the table, at which pH value corresponds to the isoelectric
point of casein.
Tabelul 5.1. Determination of the isoelectric point of casein
Reagents [cm3] Test tube number
1 2 3 4 5 6 7 8 9
Distillated water 8,38 7,75 8,75 8,50 8,0 7,0 5,0 1,0 –
acetic acid 0,01N 0,62 1,25 – – – – – – –
acetic acid 0,1N – – 0,25 0,5 1,0 2,0 4,0 8,0 –
casein in
sodium acetate 1 1 1 1 1 1 1 1 1
pH 5,9 5,6 5,3 5,0 4,6 4,4 4,1 3,8 3,5
degree of
flocculation – – + +++ xxx xx ++ + –
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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Observations:
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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5.2. GLUCIDS
Carbohydrates are ternary components of living matter containing
carbon, hydrogen and oxygen. Their chemical properties are determined
by the presence of a carbonyl group in the molecule and a number of
hydroxyl functional groups (OH).
Carbohydrates are classified into two main groups: simple
carbohydrates and composed carbohydrates.
Carbohydrates: Simple glucids (ose): -monoglucids
Composite glucids (oside): – Oligosaccharides
– Polysaccharides
Monoglucidele are distinguished by the number of carbon atoms in
their molecule and by the carbonyl functionality, contained ther ein.
Composite glucids consist of simple glucids composite (monoglucids),
Oligosaccharides are composed by 2 -6 monoglucids and
polysaccharides of a large number of monoglucids.
5.2.1. Specific reactions for monoglucids
Monoglucids give the following types of reactions :
1) Solubility reactions 2) Color reactions
3) Redox reactions 4) Condensation reactions
5.2.1.1. Solubility reactions
Principle of method:
Monoglucids are soluble in water and insoluble in organic solvents.
Reagents:
glucose, fructose
alcohol – C2H5-OH, concentrated solution
distilled water
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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Work method
Into two tubes are introduced 0.2 g glucose and 0.2 g of fructose and
then to each vial are added 2 cm 3 of distilled water. Stir and observe their
solubilization.
Take another 2 tubes and insert into each 0.2 g and 0.2 g of fructose and
glucose and 2 cm3 alcohol, shake and found that both glucose and fructose
remain as unsolubilised.
5.2.1.2. Color reactions
Schiff Reactions.
Principle of methods:
Monoglucids, due to the presence in their molecule of masked aldehyde
or ketone group slowly give Schiff's reagent, as opposed to the free aldehyde
which recolors immediately the decolorized fuchsin.
Reagents
glucose 2%
formaldehyde 5%
Fuchsin sulphonic acid (Schiff's reagent)
Work method
In a test tube are introduced 1 cm3 of 2%, glucose solution and in
another test tube 1 cm3 of formaldehyde. To each tube 2 -3 drops of Schiff's
reagent (fuchsin sulphonic acid) is added. It is noted that in the tube wi th
glucose solution, red color appears much more slowly than in the vial
containing formaldehyde.
Selivanoff reaction
Principle of method:
Selivanoff reaction allows a differentiation between aldose and ketones.
Hexose sugars under the action of hydrochloric acid, is converted into
oximetilphurphurol. Ketones speed of crossing in oxymethyl phurphural is 20
times higher than aldoses. Formed ox imetilphurphurol reacted with resorcinol,
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
88
giving a red color. This reaction allows the identification of ketones, both free
and combined. Coloration is due to the chinoidic structure of oxidation
product obtained in the final stage.
CCHOH HC
C
OCCH H
C
OH OH CO
HOH2C CH2OH HOH2C HHO
H2O 3
fructozã hidroximetilfurfurol
Reagents:
fructose solution
2% glucose solution
2% sucrose solution
Selivanoff reagent (0.5 g resorcinol in 100 cm3 of 20% HCl)
Work method:
In three test tubes are introduced 1 cm3 of fructose, glucose and sucrose
solution, and then in each 2 cm3 of Selivanoff reagent freshly prepared. Place
the tubes in a boiling water bath for 2 minutes. In the tube in which fructose
and sucrose were introduced a red coloration is formed.
Molisch Reaction
Principle of method:
Monoglucides in concentrated sul furic acid environment are dehydrated
and converted into phurphural, in the case of pentoses and in oxymethyl
CCH HC
C
OHOH2C C O
H+OH OH
OH H CCH HC
C
O HC O
H OH HOH2C
OH OH – 2 H2O
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
89
phurphural in the case of hexoses. Oxymethyl furfural and furfural give a
purple characteristic coloration with α -naphthol. This reaction is given by
oligo and poly carbohydrates because under the action of mineral acids a
partial hydrolysis occurs till monoglucids.
Reagents:
2% glucose solution
concentrated sulfuric acid
Molisch reagent (alcohol solution of α -naphthol 0.5%)
Work method:
In a test tube are placed 1 cm3 of glucose and 2 -3 drops of alcoholic α –
naphthol solution (Molisch reagent) is then added 1 cm3conc H 2SO 4 in such a
way as to flow down on the tube. Two layers of liquid are formed. At their
contact surface a purple ring appears.
5.2.1.3. Redox reactions
Due to the presence of functional carbonyl groups in the molecule of
monoglucids they are of a reducing character. They reduce in the alkaline and
hot environment, heavy metal salts of Ag, Cu, Bi, etc. Reducing character is
more pronounced in monoglucids and decreases with increasing molecular
weight of carbohydrates.
Trommer reactions
Principle of method:
Copper hydroxide formed under the action of NaOH on CuSO 4 is
reduced by reducing carbohydrates first to cuprous hydroxide and then to red
copper oxide.
HC O
+ 2Cu(OH)2 (CHOH)4
CH2OH(CHOH)4 C O
+ 2 CuOH + H2O
CH2OH OH
2CuOH Cu2O + H2O
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
90
Reagents:
2% glucose solution
dilute solution of 1%copper sulfate
Sodium hydroxide 10% solution
Work method:
In to a tube is inserted the glucose solution with a few drops of diluted
CuSO 4 and NaOH and then is boiled. It is observed that the formation of a
yellow precipitate which then turns red.
Fehling reaction
Principle of method:
Reducing carbohydrates reduce cupro tartaric complex formed by
mixing Fehling's solutions I and II, to the red cuprous oxide and the aldehyde
group is oxidized to carboxyl.
Characteristic reactions of reducing sugars (eg. Glucose – aldo-
hexose) are shown below:
Reagents:
2% glucose solution
Fehling's solution I (3.5 g CuSO 4 was dissolved in 50 cm3 distill ed
water)
Fehling's solution II (17.3 g of Seignette salt and 6 g of sodium
hydroxide dissolved in 50 cm3 distilled water)
Work method:
In one test tube 1 cm3 of dilute solution of glucose are inserted and 1
cm3 Fehling's reagent I and 1 cm3 of Fehling's reagent II are also added. Heat
in the flame of a lamp for 3 min, it is observed the formation of a red
precipitate of cuprous oxide.
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
91
Benedict Reaction
Principle of method:
The method is based on the glucose reduction of copper from the
Benedict solution (cupric sulfate) to the cuprous oxide – which colors the
solution in red. The color obtained may vary from green to red depending on
the amount of cuprous oxide formed.
2 CuSO 4 x 5 H 2O + C 6H12O6 → C 6H12O7 + Cu 2O + 2 H 2SO 4 + 8 H 2O
(Cu2+) (Cu1+)
Reagents:
research solution
Benedict reagent.
173 g of anhydrous sodium citrate and 90 g of anhydrous sodium
carbonate are solub ilised in 600 cm 3 of hot distilled water. The solution is
filtered, and completed with distilled water to 850 cm3.
17.3 g of copper sulfate (CuSO4 x H 2 O) is solubilised in 100 cm3 of
distilled water and completed up to 150 cm3.
Solution "a" is poured un der stirring into the solution "b".
CuSO4 + 2NaOH = Cu(OH)2 + Na2SO4
HO CH COONa O CH COONa
Cu(OH)2 + Cu +2H2O
HO CH COOK O CH COOK
sare Seignette complex cupro tartric
CH2OHHC O
+
(CHOH)4COOH
(CHOH)4 O CH COONa
Cu +2H2O
O CH COOK
CH2OH HO CH COONa
2 +
HO CH COOK
Cu2O
hexozã complex cupro tartric sare Seignette acid aldonic
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
92
Work method
Place in a test tube 1.5 cm3 of Benedict reagent and add 3 drops of
research solution then leave for 2 minutes in boiling water bath and then cool.
It can get a green color or a green precipitate, orange or red. The color
depends on the amount of carbohydrates contained in the resear ch solution.
After the table below, we can appreciate the approximate concentration
of sugars, according to the color obtained.
Table 5.2. Assessment of concentration of sugars by the color obtained
from Benedict reaction
Color Notation Aproximate
concentration %
Unchanged – 0
Green free from the
precipiate 0,1-0,3
Green with precipiate + 0,5
Olive green ++ 1,0
orange +++ 1,5
Light red ++++ 2,0 and above
Silver salts reduction (Tollens Reaction)
Principle of mehtod:
Silver nitrate, by treatment with dilute ammonium hydroxide,
precipitate the silver oxide, which is dissolved in an excess of reagent to form
di-amonia -argentic hydroxide. This is reduced by reducing carbohydrates, t o
metallic silver, which is deposited on the walls of tube under mirror shape.
glucose gluconic acid
2AgNO3+2NH4OH=Ag2O+2NH4NO3+H2O
Ag2O+4NH4OH=2Ag(NH3)2OH+3H2O
HCO HOCO
(CHOH)4+2Ag(NH3)2OH= (CHOH)4+2Ag+4NH3+H2O
CH2OH CH2OH
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
93
Reagents:
2% glucose solution
1% solution of AgNO 3
Diluted solution of NH 4OH
Work mehod:
In a test tube are placed 1 cm3 of AgNO 3 and treated with diluted
NH 4OH to dissolve the precipitate initially formed (avoid adding an excess of
NH 4OH). The resulting solution is treated with an equal volume of glucose
solution. Heat the tube content s slightly and found the formation of mirrors on
the walls of the tube.
5.2.1.4. Condensation reactions
Reaction of osazones
Principle of mehtod:
Monoglucids, due to the presence of the carbonyl functional group in
the molecule give condensation reaction with phenylhydrazine in cold
forming the phenylhydrazone, which in excess of phenyl hydrazine in heat
pass into osazones.
Osazones are crystalline substances insoluble in water. The osazone of
each monoglucid presents a characteristic crystalline shape. Glucose and
fructose give the same osazone who viewed under a microscope appears as
colored yellow -green heads and the lactosazone appears as yellow crystals
radially disposed.
Reagents:
5% glucose solution
2% lactose solution
phenylhydrazine hydrochloride (pu re crystallized)
crystallized sodium acetate
Work method
Mix two parts of phenylhydrazine hydrochloride with 3 parts of sodium
nitrate and rub well in a mortar. 4 cm3 of the solution of glucose is added to 1
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
94
g of the mixture prepared above. Heat in a boiling water bath for 5 -10
minutes, stirring continuously. Repeat experience for lactose solution. When
the osazone yellow crystals start to appear leave the test tube in the rack to
cool large, crystals will form and examined on a microscope slide.
Fig.5.1. Osazone crystals
In Figure 5.1 are presented several crystal structures that can be
formed by the reaction of obtaining the osazones. Thus, to obtain crystals in
the form of yellow -green colored heads we have composed osdezones of
glucose and f ructose, and lactosazone appear as radially yellow crystals.
5.2.2. Diglucids specific reactions
Diglucids are formed by two monoglucid molecules bound by one
water molecule elimination.
Diglucids properties depend on how the water molecule removal
occurred, two types of diglucids exist:
a) non -reducing type called trehalose type , the elimination of water
molecule is made between the two glycosidic hydroxyl groups from
monoglucids molecules . This type of diglucid do not give reducing reactions
characteristic for the carbonyl group. Only after cleavage of the diglucid
molecule in components, which is performed by acid or enzymatic hydrolysis,
the reductive character is gained. Sucrose is an example of this type of
diglucides
b ) reducing type called maltose type , the elimination of water molecule
is between glycosidic hydroxil molecules of a monoglucide with any of the
other alcoholic hydroxyls from another monoglucide molecule. Diglucide
formed is reducing as it has one of t he groups free glycosidic hydroxil , which
retains the reducing character.
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
95
5.2.2.1. Non -reducing diglucids reactions
Sucrose is a diglucid formed by one molecule of – D- glucopyranose
and one – D – fructofuranose bounded 1 -2 glycosidic by a water molecule
elimination. In the formation of this bond participating in the glycosidic OH
groups at both molecule sucrose does not have reducing properties .
The reaction of formation of sucrose in the – D – glucopyranose and
– D – fructo – furanose is shown below :
Reagents
sucrose solution 2%
Fehling I și II solutions
Working method
Place in a test tube 1 cm3 sucrose solution and add 0.5 cm3 solution
Fehling I and II. The tube is heated to boiling. It appears that there is no
precipitate which indicates lack of functional groups with reducing properties.
5.2.2.2. Reducing diglucids reactions
Maltose is a diglucid made by 2 molecules of D – glucopyranose linked
by eliminating 1 -4 glycosid e molecules of water . In the second molecule of
glucose is present glycosidic hydroxyl with reducing properties, which prints
the whole maltose molecule a reducing character. Maltose -forming reaction
is as follows :
H
OHO
HHH
OHOH
HOO
OH
H OHH H
OHHO
H+
HO
OHH
H
HO H
HOH
HO H2CO
OOH CH2
HH2C HO
-D-glucopiranoza -D-fructofuranoza
(zaharoza)2-[D-glucopiranozil D-fructofuranoza- H2OHO CH 2
HO H2COH CH 2
H
HOH1 2 1 2
-D-glucopiranozaHOOH
OHH
H
HHO CH 2
O
OHH H
OHOCH 2 HO
HH
OHHO
-D-glucopiranoza+H OCH 2 HO
HH
OHHO
OHH
HHHO CH 2
O
OHH
O – H 2O
4-[-D-glucopiranozil]- -D-glucopiranoza
(maltoza)HOHHHH HHH
OH
H HOHHHH
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
96
Reagents
maltose solution 2 %.
Fehling I și II reagents
Working method
Pipete in a test tube 1 cm3 maltose solution and 0.5 cm3 solution
Fehling I and II. Heat to boiling and is found to form a red precipitate of
cuprous oxide
5.2.2.3.Sucrose hydrolysis
Method`s principle:
-D-Glucopiranosil -1→2 –
-D-fructofunarose) by boiling with
-D-
-D-fructofuranose), which give a reductive
character to the solution..
Sucrose
-D-Glucopiranose
-D-fructofuranose
Reagents
Sucrose solution 2%
Diluted HCl
Fehling I și II reagent
NaOH sol.
Working method
Place in a test tube 1 cm3 of sucrose and treated with diluted HCl 0.5
cm3. Heat to boiling contents of the tube and continue to boil another 5
minutes. After cooling alkalinized the solution with NaOH solution and add
2-[D-glucopiranozil D-fructofuranoza
(zaharoza)HO H2C
HCH2OH
OO
H2C HOOH
HH O
HHH
OHOH
HO
OHHCH 2OH
H2C HOCH 2 HO
-D-fructofuranoza -D-glucopiranoza+
HHO
H
OH HOHO
HOOH
OHH
H
HO
OHH
– H2OH H
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
97
0.5 cm3 of Fehling's I and II. By boiling a precipitate of cuprous oxide red is
formed.
5.2.2.4. Sucrose invertion
Sucrose is dextrorotatory , with a specific rotation of + 66.50, by
treatment with strong acids results in a equimolar mixture of glucose and
fructose , which is levorotatory . This is because glucose is the dextrorotatory
rotating the plane of polarized light clockwise by an angle of + 520 and
fructose is levorotatory rotating the plane of polarized light to the left at an
angle of – 920.
Reagents:
Sucrose solution 20 %
HCl 1n solution
Working method
Pipette 25 ml of sucrose solution in a 50 ml conical vial and placed in
the thermostat at 25 0C. In another conical vial place 30 ml of 1N HCl.
Before use conical vials must be thoroughly washed and dried. After
uniformity of temperature, remove with a pi pette 25 cm3 of acid and placed in
sucrose solution vial. Shake well and insert the vial contents in a polarimeter
tube whose temperature must be 250C. 5-6 will perform readings averaged
readings and note the time. The angle of rotation reading "0" initial ly the
angle of rotation. The subsequent measurements performed in 15 minutes 15
minutes and then at longer intervals. The solution is kept in the thermostat and
after 48 hours is measured final rotation.
It will be appreciated that the angle of rotation t o be taken with the
correct sign, at the right with plus and at the left with minus. The
measurement results will be entered in the table below:
T
(minutes) t 1 t2 t3 t4 t5 t…
Rotation
angle
5.2.3.Polysaccharides specific reactions
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
98
Poliglucids are made up of chains of monoglucids molecules. The most
important poliglucids are: starch, cellulose and glycogen . All three of them
are made up by molecules of glucose bounded by glycoside type bounds.
They are distinguished both by way of linkage and the size and shape of
catens . Polysaccharides have non – reducing character because all glycosidic
hydroxyl groups except those terminals were blocked. Starch and glycogen
are made up by D glucopyranose the base of structure being maltose .
Starch is composed by two components , amylose and amylopectin .
Amylose consists of long chain molecules -D glucopyranose bounded
1-4, maltose being the structural unit.
Amylopectin consists also of D glucose molecules are linked 1.6
1-4 gly cosidic bond to the side chain. Amylose gives blue with iodine.
Amylopectin gives a red color purple and glycogen gives a red – brown
coloration.
Glycogen has a similar structure as amylopectin , but more branched.
5.2.3.1. Starch reaction with iodine
Method`s principle:
Colloidal solution of starch with iodine give a blue color due to the
formation of an adsorption product.
Reagents
Starch sol. 1%( 20 g of soluble starch is mixed with cold distilated
water in a Berzelius beaker, than pour it in 100ml fla sk, add 10 mg HgI 2 for
preservation and boiled distilated water to the sign)
Glycogen sol.0,3 %.
Iodine in potassium iodide solution (2g iodine și 5g KI disolved in 100
cm3 distilated water).
Working method
CH2OH CH2OH
O O
OO O
O OCH2OH CH2OH
amilozan
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
99
Place in a test tube 1 cm3 of starch and added 2 -3 drops of the solution
of iodine in potassium iodide solution . Observe a blue coloration. Heat and
color disappears notes that reappears by cooling. In the case of reaction with
glycogen a red –brown color will be obtained.
5.2.3. 2 . Starch hydrolisys
Method principle:
By boiling the starch with strong mineral acids it gradually hydrolyse ,
resulting amilodextrine then eritrodextrine , acrodextrine , maltodextrin,
maltose and finally glucose. These intermediates give different staining
solution of iodine in potassium iodide .
Reagents:
Starch solution 2%(20 g of soluble starch is mixed with cold distilated
water in a Berzelius beaker, than pour it in 100ml flask, add 10 mg HgI 2 for
preservation and boiled distilated water to the sign)
HCl 10%
I2+ KI
Working method:
In a test tube, add 5 cm 3 of a 2% starch sol. and 0.5 cm3 solution of
HCl. Heat to boiling for assure the starch hydrolysis. During hydrolysis
remove from time to time using a pipette 0.5 cm3 of starch solution and put it
into other tubes. Add into each tube a drop of iodine solution. It appears that
early solution boiling colors blue then violet and red. After 10 minutes of
boiling solution gives no coloration w ith iodine.
Further, we give below in tabular form, how to track starch hydrolysis process
:
Table 5.3. Starch hydrolysis products
Time (min.) Hydrolysis product Color
0 min. starch blue
2 min. amilodextrins violet
8 min. erithrodextrins red
15 min. acrodextrins no colour
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
100
20 min. maltodextrins no colour
25 min. maltose no colour
30 min. glucose no colour
5.2.3.3. Glycogen reaction with iodine
Method principle:
Glycogen colloidal solution in the reaction with iodine has a red -brown
or blue color due to the formation of a product of absorption. The structure of
glycogen shows the presence of residues – D – glucopyranose linked by an
intermolecular 1 -4 and 1 -6 according to the type of molecule fragments
shown below:
Glycogen
Reagents
glycogen solution 0,3 %
iodine (I) in potasiu m iodide (KI) solution
Working method
Place in a test tube 1 ml solution of glycogen, add 2 -3 drops of iodine
solution in potassium iodide, and then a red – brown to blue staining is
observed . Heat and color disappears and its notes that reappears after cooling.
HO 34HCH2 HO
OOH
OHH
H
HO H
1
31H O
HHH
OHOHHO CH2
H
HO 34HCH2 HO
OOH
OHH
H
HO H
1
31H O
HHH
OHOHHO CH2
H
34HCH2
OOH
OHH
H
HO H
1
31H O
HHH
OHOHHO CH2
H
O O31H O
HHH
OHOHHO CH2
H4
OHO
Glicogen
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
101
5.2.3.4. Celulose hydrolisis reaction
Cellulose is a polysaccharide containing a large number of hydroxyl
groups which is reacted with various acids to form esters or with alcohols to
form ethers. Cellulose hydroxyl groups from the molecule participate in the
specific reactions to form the ester, ether , alcohol celulose treated with a
mixture of acetic acid and acetic anhydride can form mono – , di- or tri
celulose acetate fiber . By treatment with concentrated sodium hydroxide
solution, the cellulose forms alkaliceluloza ( alkoxide ) .
Molecular formula is ( C6H10O5 ) n, where 'n' is the number of -D-
glucopyranose and the value of " n" can be between 700 -3000 monoglucide
radical( -D- glucopyranosyl).
Method principle
As a result of hydrolysis reaction of cellulose simple sugars , such
as tri -, tetra – or hexa carbohydrates ( eg: glucose, cellobiose ) are obtained .
The reaction can be highlighted by Fehling reaction which can evidence
oligoglucids and glucose formation .
Celulose
Reagents
– finely divided filter paper
– sulphuric acid sol. 70%
– sodium hydroxide 30 – 40%.
Working method
Measure in a test tube, 1 ml of 70% sulfuric acid solution over
which add the finely divided filter paper ( cellulose -containing ) to obtain a
viscous solution. Immerse the tube in a water bath for heating the solution
HCH2 HO
OH
OHHH
HO H
1
31H O
HH
H
OHOHOHO CH2
H4
3O
n
-D-glucopiranozil-1 4- -D-glucopiranoza
Celuloza
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
102
until a brown stains appears . Cool the obtained solution and add a water
volume 5 times higher.
From the solution which is obtained is taken 0.5 ml and alkalinised
with 0.5 ml sodium hydroxide . Finally it highlights the presence of glucose
with Fehling reaction .
Observations:
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
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C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
104
5.3. LIPIDS
The lipids are esters of alcohols and fatty acids. These substances are
insoluble in aqueous environment and soluble in non -polar organic solvents.
The lipids are found in membranes of cells or cell organites composition, but
also in cytoplasm .
Lipids are classified into: – simple lipids (glycerides)
– complex lipids
5.3.1. Specific reactions for glycerides (acylglycerols)
The saponification reaction
Principle of method:
By alkali hydrolysis of the glycerides, glycerol and fatty acids are obtained
which, because of the alkaline medium are forming salts named soaps. This
reaction called saponification reaction takes place according to the equation:
Triacylglycerol glycerol soap
Reagents:
alcoholic solution of NaOH (20% NaOH in alcohol 60%)
vegetable oil
glacial acetic acid
saturated solution of calcium sulfate
saturated NaCl
Work method:
4 to 5 drops of vegetable oil are placed in a test tube
3-4 cm3 soda alcohol are added
The tube is heated in a boiling water bath until the disappearance of the
oil, which indicates complete saponification
approximately 10 cm3 water are added
stirr and observe the formation of a foam (detersive action)
triacilglicerolCH2OCOR
CH OCOR
CH2OCORCH2OH
CH OH+3R-COONa
CH2OH
glicerol sãpunNaOH +3
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
105
from the obtained soap solution, take in three tubes of equal amounts to
highlight some properties of soap
in a test tube solution is acidified with glacial acetic acid. The free fatty
acids are not soluble in aqueous medium and form a white precipitate.
in the second tube are added a saturated solution of CaSO 4. It is observed
the formation of a precipitate of insoluble calcium soap.
In the third tube is added an equal volume of saturated NaCl. It is
observed the formation of a flocculent precipitate of soap formed due to
the destabilization of colloidal thereof, at the addition of the electrolyte.
Obtaining of fatty acid
Principle of method:
The fatty acids can be separated from the soaps by acidulation of a soap
solution. For example, by acidification of sodium palmitate with sulfuric acid,
the palmitic acid is obtained, according to the equation:
2 CH 3- ( CH 2 )14 – COONa + H 2SO 4 → 2 CH 3-( CH 2)14-COOH + Na 2SO 4
sodium palmitate Palmitic acid
Reagents:
soap
1/5 N sulfuric acid solution
Congo red
Work method:
0.1 -0.2 g of soap are solubilized in distilled water;
is neutralized with sulfuric acid in the presence of Congo red;
free fatty acids can be observedat the surface.
The identification of unsaturated fatty acid from glycerides
Principle of method:
The presence of unsaturated fatty acids in some lipid molecules, can be
highlighted by the addition reaction of the halogens to the double bonds of the
fatty acids in the glycerides.
oleic acid 9,10 -diiod -stearic acid
+I2
HOOC-(CH2)7-CH-I HOOC-(CH2)7-CH CH3-(CH2)7-CH-I CH3-(CH2)7-CH
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
106
Reagents:
vegetal oil
chloroform solution of bromine
Work method:
tube is inserted in a few drops of vegetable oil;
add bromine water drop by drop;
bromine solution discolors due to its addition to the double bond.
Identification of the glycerides in the reaction to form acrolein
Principle of method:
By heating the glucids will decompose, releasing glycerol, which in the
presence of dehydrating (KHSO 4), loses 2 molecules of water, turning into
acrolein, a unsaturated substance with characteristic pungent odor.
CH 2-OH CH 2
-2H 2O
CH-OH CH
CH 2-OH H-C= O
glycerin acrolein
Reagents
vegetal oil
potassium hydrogen sulphate KHSO 4
Work method:
in a dry tube, put 2 -3 drops of vegetable oil;
Add 2 g of KHSO 4
heat in the flame;
fumes of a pungent odor are released;
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
107
keep at the topof the tube a filter paper soaked in silver nitrate ammonia
solution and notice the black coloring due acrolein vapor.
Highlighting the degree of rancidity of fats
Principle of method:
Kept long to air and light, the fats are decomposing into glycerol and
fatty acids. Fatty acids are oxidized in malodorous volatile compounds. The
degree of rancidity can be highlighted by color reactions.
Reagents:
fresh vegetal oil
concentrated hydrochloric acid
floroglucine solution (2% ether solution).
Work method:
2 cm3 of oil are introduced into a test tube;
2cm3 hydrochloric acid is added and stirred;
add 2cm3 fluoroglucine and shake again;
The degree of rancidity is found by red to purple coloration that
appears.
5.3.2. Specific reactions of sterids
Sterids are esters of the sterols with superior fatty acids. The most
widespread and important of animal sterols is cholesterol. Sterids can
recognize the color based on the reactions of sterols.
Salkowski reaction
Principle of method:
In chloroform solution, sterols in the presence of concentrated sulfuric
acid, are giving a red color.
Reagents:
concentrated sulfuric acid
chloroform
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
108
animal fat
Work method:
in a dry tube 2 -3 drops of oil are placed and solubilised with 1 -2 cm3
chloroform.
it is trickled on a side of the tube 1 cm3 of concentrated H 2SO 4. There is
the separation of the two layers, between them a red -orange ringis
obtained;
shake the tube. The chloroform layer will turn red while the sulfuric acid
will be a greenish fluorescence.
Lieberman – Burchard reaction
Principle of method:
Cholesterol with acetic anhydride, in the environment of sulfuric acid
will become of dark green color.
Reagents
acetic anhydride
concentrated sulfuric acid
animal fat
chloroform
Work method:
in a dry vial are placed 1 -2 drops of oil which is solubilized in 1 -2 cm3
of chloroform.
1 cm3 of acetic anhydride is added.
add 3 -4 drops of concentrated sulfuric acid and is stirred. At first a
bluish -green appears color that gradually goes to dark green.
5.3.4. Determining the quality indices of lipid
5.3.4.1. Determination of saponification indices
Saponification index is the amount in mg of potassium hydroxide
necessary for saponifying one gram of fat.
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
109
Saponification index value is directly proportional to the amount of
fatty acids contained, and i nversely proportional to their molecular weight, of
lipids respectively.
The table below exibits saponification index values for the main
vegetable oils:
Table 5.4. Saponification index values for the main vegetable oils
Nr.
crt. name of material Saponifi cation indices (Is)
1. cottonseed oil 191 – 198
2. sunflower oil 188 – 194
3. linseed oil 189 – 192
4. olive oil 189 – 196
5. corn oil 188 – 198
Principle of mehtod:
A known amount of glycerides, is saponified with potassium hydroxide
of known concentration, taken in excess.
Excess potassium hydroxide is titrated with a equivalent acid solution.
Reagents:
alcoholic solution of KOH 0.5 N (28 g KOH is solubilized in a small
amount of distilled water and supplemented to 1000 cm3 with 90% ethanol ).
0.5 N HCl solution (41 cm3 HCl concentration in 1 liter)
1% phenolphthalein alcohol solution
vegetable oil
Work method:
in an Erlenmeyer flask at which a reflux condenser can be
accommodate, is weighted with a analytical balance an amount of 0,5g
fat.
it is solubilized in 10 cm3 of benzene
add 20 cm3 alcoholic KOH 0.5 n solution measured exactly
in parallel a control test under the same conditions but without the fat is
prepared, using 20 cm3 0.5N alcoholic KOH
se titrează excesul de KOH cu acid clorhidric 0,5 n până la dispariția
culorii roșii.
fitt the refrigerens to the two samples.
heat in a water bath to boil for 30 minutes from the start
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
110
let for cooling and remove the refrigerant
add 1 -2 drops of phenolphthalein
excess of KOH is titrate d with 0.5 N hydrochloric acid until the red
color will disappear.
Exercise:
(V2- V1) x F HClx 28,052
Saponification index =
G
V2 = cm3 of HCl used for titration of control sample
V1 = cm3 soluție de HCl folosiți la titrarea probei cu grăsime cm3 of
HCl used for titration of sample with fat
G = weight in grams of sample with fat taken for determination
28,052 = mg of KOH that are f ound in one cm3 of KOH 0,5 n solution.
5.3.4.2. Determination of acidity index
The acidity index (I a) is the amount in milligrams of KOH required to
neutralize the free fatty acids in one gram of fat.
Principle of method:
A known amount of fat is titrated with a known concentration of the
KOH solution in the presence of an indicator.
CH 3-(CH 2)14-COOH+KOH CH 3-(CH 2)14-COOK+H 2O
palmitic acid potassium palmitate
Reagents:
mixture of ethyl alcohol and neutralized b enzene
solution of potassium hydroxide – KOH 0.1 N (5.6104 g / dm3)
animal fat
phenolphthalein, alcohol solution 1%
Work method:
weigh 2.5 g fat in an Erlenmeyer flask
add 20 cm3 of ethyl alcohol and benzene previously neutralized
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
111
titrate with 0.1 N KOH in the presence of phenolphthalein until the
appearance of pink color, persistent 30 seconds
if mixture becomes cloudy during titration, heat the vial a little in a
bowl with warm water.
Exercise:
5,6104x Vx F
Ia =
G
V=cm3 of KOH0,1 n used in titration
F=factor of KOH 0,1n solution
G=grams of analyzed substance
5,6104 = mg of KOH from one cm3 of KOH 0,1n solution
5.2.5. Extracting and dosing fat
from animal biological products by the Soxhlet method
Principle of method:
Extraction is carried out with a Soxhlet equipment, in a continuous
process and is based on the property of the fat in the body fluids and tissue to
be solubilized in organic solvents.
The solvent was removed from the extract and the amount of fat
containe d in the analyzed material is weighted.
The method provide the amount of crude fat, because other lipids are
partially extracted.
Reagents:
alcohol ethyl – ether mixture 1: 1
or petroleum ether
Work method:
the material to be analyzed is oven dried at 1050C, till a constant
weight.
weigh the quantity of dried material
triturate in a mortar with quartz sand
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
112
the material is inserted into a cartridge filter paper that closes with
plug of cotton wool
cartridge and plug of cotton are degreased in advance wi th the same
solvent to be used in the extraction.
Soxhlet equipment (fig. 5.2.) , used in extraction is composed of :
1. flask B – which collects the solvent and concentrates the fats
2. the extractor E – provided with a tube T, that communicate between the
top of the extractor and the bottom of the tube. Regular evacuation of
fluid from the balloon extractor is done by siphon S.
3. refrigerant with reflux
Functioning of Soxhlet equipment
the cartridge is inserted in extractor E with the material to be
analyzed
the extractor and reflux condenser are fitted
Fig. 5.2. Soxhlet equipment
through the inner tube of the condenser is introduced into the
extractor ether -alcohol (1: 1) solution until it’s rumples in the extraction
flask, then more solvent is added till 1/4 or 1/3 of the extractor
the boiling is adjusted so that of the solvent rumpling occurs within 3
minutes
extraction occurs in about 4 -6 hours
it is allowed to cool down
the solvent from the extraction flask, is passed through drainage, th e
cartridge is removed by disassemble the refrigerant
C. Tulcan, M. Ahmadi, O. Boldura Chemistry. – P. W. Handbook
113
facility is dismantled and the remaining liquid in the flask is
transferred into a Erlenmeyer vial previously weighed with analytical
balance
the flask is washed with small amounts of extraction solvent adding
the extract from Erlenmeyer flask
the solvent is evaporated on a water bath in a nish for chemical
substances
the remaining residue is crude fat
after drying for 30 minutes at 800C in the oven, is allowed to cool in
the desiccator
it is weighted with analytical balance
Soxhlet fat extraction method can be done indirectly. For this
purpose, the cartridge with the material from which the fat is extracted, is
weighted before and after extraction.
The difference between the two is weightings represent s the extracted
fat.
Exercise: G1 – G0
Crude fat % = x 100
G
G0= wieght of Erlenmayer flask
G1= wieght of Erlenmayer flask with fat after drying
G = wieght of analyzed product
Observatios:
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