Download Analitical chemistry 1

Structure of water
Water is a molecular compound
containing H2O molecules in which two
Hydrogen atoms are bound to the oxygen
atom forming an angle of 104.5°.
The OH distance (bond length) is 9.57 
1011 metres. Because an oxygen atom
has a greater electronegativity than a
hydrogen atom, the OH bonds in the
water molecule are polar, with the
oxygen bearing a partial negative charge
1
() and the hydrogens having a partial
positive charge (+).
The electron arrangement in the water
molecule can be represented as follows.
Each pair of dots represents a pair of
unshared electrons (i.e., the electrons
reside on only the oxygen atom).
2
This electronic structure leads to an
interaction between water molecules
that is called hydrogen bonding, in which
a positive hydrogen atom on one
molecule is attracted to the electron
density of one of the unshared electron
pairs on the oxygen atom of another
molecule.
3
4
Concentration based on volume
The majority of reaction studied by biochemists
occur in solution. Concentration based on the
amount of dissolved solute per unit volume are
the most widely used in biochemistry
laboratories.
Solution concentration:
Concentration is the amount of a solute in a
given amount of solution.
The molarity of a solute is the moles of solute
per liter of solution (the number of moles of
solute per liter of solution).
Molarity = Moles of solute/ Liters of solution
Units: 1 Molar (M) = 1mole/L
5
Molar con are usually given in square brackets.
Ex, [H]= molarity of H ion. To calculate M , we
need to know the weight of dissolved solute and
its molecular weight, MW.
Wtg/ MW = moles.
Dilute solutions are often expressed in terms of
millimolarity, Micromolarity, and so on ,
where:
1 mmole = 10-3 moles
1μmole = 10-6 moles
1nmole = 10-9 moles
Therefore:
1 mM= 10-3 M = 1mmole/ Liter = 1μmole/ml
1 μM = 10-6 M = 1μmole/Liter = 1 nmole/ ml
6
Normality (N)= the number of equivalents of
solute per liter of solution.
To calculate N we need to know the weight of
dissolved solute and its equivalent weight, EW.
Wtg/ EW= equivalents
One equivalent (EW) of an acid or base is the
weight that contains 1g-atom (1mole) of
replaceable hydrogen or 1g-atom (1mole) of
replaceable hydroxyl.
Ew= Mw/n
N= the number of replaceable H or OH per
molecule( for acids and bases)
7
The molarity and normality are related by:
N=nM
Ex, a 0.01M solution of H2SO4 is 0.02N.
Q: How many moles of NH3 are dissolved in 75
ml of 6.0 M NH3?
Q: How do you prepare 250 ml of 1.00 M
CuSo4 solution?
Mw of CuSo4= 249.7
8
Introduction
The branch of chemistry that deals with the
separation, identification and determination of
components in a sample. Thus, it is answers to
the basic questions:
 What is in this sample? and
 How much of it is there?
The first question is qualitative and it is related
to the detection and identification of the
components of a sample.
The second question is quantitative.
9
Applications of chemical analysis:
Applications of chemical analysis are found
everywhere in industry, universities,
hospitals…etc
The chemical analysis is playing an important
role in the following area:
1- Environmental:
Chemical analysis of air and water is required
for measuring pollutants or contamination.
2- Medical:
Measuring the levels of critical substances in
the blood stream of hospital patient. For
example, the presence of high amount of
bilirubin and concentration of transaminases
enzymes in patient s blood stream is assign of
an impaired liver function.
10
3-Nutrition:
Determination of nitrogen content of breakfast
cereals and other foods can be directly related
to their protein content and thus their
nutritional qualities.
3- Agricultural:
Chemical analysis of soil and plants is used to
determine the nutrients or fertilizers which
must be add to the soil to increase productivity.
4-Research
Quantitative analysis data are the backbone of
research activity in chemistry, biochemistry,
biology and other braches of sciences.
11
There are other areas in which chemical
analysis is playing an important role are
pharmacological and industry.
Analytical chemistry can be broken down in to
general areas of analysis:
1- Qualitative analysis deal with finding what
materials are present in an analytical sample.
It is not a method for determining the
concentration. If the chemical species
(materials) are known the concentration of
that species in the sample can be determined
by quantitative analysis. A variety of tools
are used for qualitative analysis such as
spectroscopic methods are quick and simple
12
ways to detect metals and organic
compounds.
Microscopic examination is also a powerful
qualitative tool. It may be necessary to do a
complete qualitative analysis on a sample before
discovering how to determine just one or two
components quantitatively. This is because
many elements are sufficiently similar in their
behavior that they interfere in quantitative
determination.
13
2- Quantitative analysis deals with the
determination of how much of a material is
present in a sample. The goal is to determine
the amount of each component in a sample.
Quantitative analysis may be classified based on
the size of sample which is available for
analysis. When a sample weighing more than
0.1g is available the analysis called as macro
analysis. Micro analysis deal with samples
weighing from 1 to 10mg.
14
Sampling:
The type of sample that must be taken for
analysis will depend on the information desired.
In the case of biological fluids, the conditions
under which the sample is collected can be
important. For example, whether a patient has
just eaten. Because the composition of blood
varies before and after meals and for many
analyses a sample is collected after the patient
has fasted for a number of hours. Preservatives
such as sodium fluoride for glucose
preservation and anti coagulants may be added
to blood samples when they are collected these
may affect a particular analysis. Blood samples
may be analyzed as whole blood or they may be
15
separated to yield plasma or serum according to
the requirement of the particular analysis. If
whole blood is collected and allowed to stand
for several minutes, the soluble protein
fibrinogen will be converted by a complex series
of chemical reactions (involving calcium ion)
into the insoluble protein fibrin, which form the
basis of a clot. The clotting process can be
prevented by adding a small amount of an
anticoagulant, such as heparin or a citrate salt.
Handling the sample:
Precautions should be taken in handling and
storing samples to prevent or minimize
contamination, loss. The sample may have to
protect from the atmosphere or from light.
16
Examples
1- Alkaline substance will react with carbon
dioxide in the air.
2-Blood samples analyzed for carbon dioxide
should be protected from the atmosphere.
3- Glucose is unstable and a preservative such
as sodium fluoride is added to blood samples.
4- Protein and enzymes tend to denature on
standing and should be analyzed without delay.
5- Addition 1 or 2 ml of acetic acid per 100 ml
of urine prevent precipitation (pH 4.5). Store
under refrigeration. Because urine samples are
unstable.
6-Whole blood, serum, plasma and tissue
samples can be frozen for prolonged storage.
17
Types of Methods
1- Volumetric analysis or titrimetric analysis.
Methods based on a measured volume
2- Gravimetry
Methods based on a measured weight
3- Electrochemical
Methods based on a measured of current,
charge and resistance.
4- Chromatography
Methods based on the interaction with two
different phases.
5- Chemometrics
18
The statistical treatment of data.
Q: What type of information do you need?
Complete analysis which is mean determines
the amount of each component in a sample.
Partial analysis:
Determining one or a limited number of species
in a sample. Ex, electrolyte levels in blood and
presence of lead in a water sample.
19
Titrimetric analysis (Volumetric analysis)
Titration slow addition of a standard solution to
a solution of analyte until the reaction between
the two is complete.
It is used for determining the concentration of a
solution. A known volume of a solution of
unknown concentration is reacting with a
known volume of a solution of known
concentration (standard).
The standard solution is delivered from a
burette so the volume added is known.
20
For example of titrimetric analysis is the
titration of a solution of an unknown con of
hydrochloric acid (HCl) with sodium hydroxide
(NaOH) solution of known conc. The end point
of titration is located with an acid-base
indicator. OH from NaOH combines with
Hydrogen ion HCl as the following:
H + OH
H2O
Generally, the burette is filled with the titrant,
while the titrate is in the conical flask. On every
titrant addition, the reaction consumes part of
the titrate. If all the titrate is consumed by the
21
last titrant addition, the equivalence point is
reached. Since both the concentration and the
volume of NaOH solution are known the
number of moles of OH which react with the
HCl can be calculated.
Equivalence point –reached when the amount
of added titrant is chemically equivalent to the
amount of analyte.
Standard Solution
Standard solution is a reagent whose
concentration is known exactly. Standard
solutions are used to perform titrations in
which the quantity of analyte in a solution is
22
determined from the volume of standard
solution that is consumes.
Prepared of standard solution
By dissolving a carefully weighed quantity of
the pure reagent and diluting to an exactly
known volume.
It is essential for the chemical standards to meet
the following:
1- High purity
2- Stability in air (not be attacked by
atmosphere)
3- Reasonable solubility in solvent
4- Availability at modest price
23
Classification of Volumetric methods
The are major general classes of volumetric
methods
1- Neutralization titrations
2- Oxidation- Reduction titration:
These redox titrations involve the titration of an
oxidizing agent with reducing agent. An
oxidizing agent gains electrons in a reaction
between them.
3- Precipitation titration:
In the case of precipitation, the titrant forms an
insoluble product with analyte. Ex, the titration
24
of chloride ion with silver nitrate solution. Also,
indicators can be used to detect the end-point.
Chemical Reactions in Solution:
A solution is a homogenous mixture.
The substance present in the greatest amount is
the solvent. The other substances are solutes.
In many reactions, one or more of the reactants
are in solution.
Solution concentration:
Concentration is the amount of a solute in a
given amount of solution.
25
The molarity of a solute is the moles of solute
per liter of solution (the number of moles of
solute per liter of solution).
Molarity = Moles of solute/ Liters of solution
Units: 1 Molar (M) = 1mole/L
Molarity is the conversion factor between moles
of solute and volume of solution.
Molar con are usually given in square brackets.
Ex, [H]= molarity of H ion. To calculate M , we
26
need to know the weight of dissolved solute and
its molecular weight, MW.
Wtg/ MW = moles.
Normality (N)= the number of equivalents of
solute per liter of solution.
To calculate N we need to know the weight of
dissolved solute and its equivalent weight, EW.
Wtg/ EW= equivalents
One equivalent (EW) of an acid or base is the
weight that contains 1g-atom (1mole) of
replaceable hydrogen or 1g-atom (1mole) of
replaceable hydroxyl.
27
Ew= Mw/n
N= the number of replaceable H or OH per
molecule( for acids and bases)
The molarity and normality are related by:
N=nM
Ex, a 0.01M solution of H2SO4 is 0.02N.
Q: How many moles of NH3 are dissolved in 75
ml of 6.0 M NH3?
28
Q: How do you prepare 250 ml of 1.00 M
CuSo4 solution?
Mw of CuSo4= 249.7
Atomic weight
An atom consists of protons, neutrons and
electrons. The protons and neutrons are in the
nucleus and electrons are somewhere around
the nucleus but at great distance in relation to
the size of nucleus.
Most of the weight of an atom is concentrated in
the nucleus. The mass of individual atoms is
around 10-24 gram. For example: one hydrogen
atom weighs 1.6738 × 10-24 g. Because atoms
29
have such small weights, scientists have agreed
a set of relative masses of atoms called atomic
weights
Because atomic weights represent relative
masses, one element must be selected as the
standard. Carbon isotope C12 was assigned
arbitrary value of 12 atomic weight units and
the weight of all other elements were related to
carbon- 12.
Note:
Isotopes are atoms of the same element which
differ only in atomic weight (as all atoms of an
element have the same number of protons,
30
isotopes differ in neutrons number) e.g. Carbon
has two isotopes 12 to 13.
Gram Atomic Weight: The gram atomic weight
of an element is the atomic weight in grams.
Atomic number is the number of protons in the
nucleus.
Ex; H = 1.0079
31
Molecular Weight
The molecular weight for a compound is the
sum of the atomic weights of each atom that
makes up the compound.
Example: the molecular (or formula) weight of
sulfuric acid H2SO4 is :
2× 1.01 = 2.02
1 × 32.06 = 32.06
4 × 64.00 = 64.00
Molecular weight = 98.08
Equivalent Weight
32
If a substance can react in more than one way,
it will have more than one equivalent weight.
Thus the definition of equivalent weight for a
substance is always based on its behavior in a
specific chemical reaction.
In neutralization reaction: the equivalent
weight of a substance is that weight which
either contributes or reacts with one mole of (H)
ion in the reaction.
For example the equivalent weight for
potassium hydroxide KOH and HCl must be
equal to their molecular weight because each
has only a single reactive hydrogen ion (H) or
Hydroxide ion.
Barium hydroxide , Ba (OH)2 is a strong base
containing two reactive hydroxide ions. This
base will necessarily react with two hydrogen
ions (H) in any acid-base reaction: thus its
33
equivalent weight will be one-have its molecular
weigh.
We define an equivalent of any substance as its
equivalent weight (aq. Wt) expressed in gram.
Thus, 1 equivalent of H2NO3
In an acid-base reaction, one equivalent of any
acid exactly neutralize one equivalent of any
base.
EW= MW/n
N= number of H or OH (per molecule for acids
and bases)
34
Electrolytes:
An electrolyte dissolves in water to form ions.
A non electrolyte does not form ions.
Strong Electrolytes:
Strong electrolytes are completely dissociated
into their ions, such as strong acids and strong
alkalis, also most inorganic salts are strong
electrolytes.
HCl is a good example as strong electrolytes.
Thus if 100 molecules of HCl are dissolved in
water, 100 H ions and 100 Cl ions are produced,
no HCl molecules exist in aqueous solution.
35
Another example of strong electrolyte is sodium
hydroxide NaOH which contain hydroxide ions
(OH) that completely dissociate when dissolved
in water.
Weak electrolytes:
They are substances that produce relatively few
ions when dissolved in water or other ionizing
solvents (poor conductors of electric current).
The most common weak electrolytes are weak
base and weak acids. Ex of weak electrolyte is
acetic acid when 100 molecules of CH3 COOH
are dissolved in water approx 99 molecules of
CH3 COOH remain intact and only H ion and
one CH3 COO- ion are produced (1%).
Non electrolyte:
36
Non electrolytes are substances that dissolve in
water but do not produce any ions.
Nonelectrolyt solution does not conduct electric
current.
Ex, ethanol and sucrose.
Sucrose is stable sugar which is very soluble in
water but produces no ions when it dissolves.
Acids and bases
Acid is a substance that donates protons
(hydrogen ions).
Base is a substance that accepts protons.
A strong acid is a substance that ionizes almost
100% in aqueous solution. Ex HCl in solution is
essentially 100% to H+ and Cl-. Other examples
of acids are H2 SO4 , HNO3,
A strong base is a substance that ionizes
extensively in solution to yield OH ions.
Potassium hydroxides are ex of strong inorganic
bases.
37
KOH in a solution is essentially 100% ionized to
K+ and -OH. Other ex. NaOH,
Buffers;
A buffer is a solution that resists change in pH
following the addition of acid or base. A buffer
solution consists of a mixture of a weak acid
(HA) and its salt (A-) or of a weak base and its
salt. A buffer can be created by mixing equal
concentration of a weak acid (HA) and its
conjugate base (A-).
The quantitative relation between the
concentration of a weak acid (HA) and its
conjugate base (A-) is described by the
Henderson equation.
38
The Henderson equation.
Weak acids are only slightly ionized in solution
and a equilibrium is established between the
acid and the conjugate base. If HA represents a
weak acid, then :
HA
H + + A-
Weak acid
Proton
salt form or conjugate base
The "salt" or conjugate base A- is the ionized
form of a weak acid.
39
The dissociation constant of the acid, Ka is
defined as:
[H+]
Ka =
[HA]
40
[A-]
What is gel electrophoresis?
Gel electrophoresis is a method that separates
macromolecules-either nucleic acids or proteins-on
the basis of size, electric charge, and other physical
properties.
Many important biological molecules such as amino
acids, peptides, proteins, nucleotides, and nucleic
acids, posses ionisable groups and, therefore, at any
given pH, exist in solution as electically charged
species either as cations (+) or anions (-). Depending
on the nature of the net charge, the charged particles
will migrate either to the cathode or to the anode.
Electrophoresis
The term electrophoresis describes the migration of
charged particle under the influence of an electric
field. Electro refers to the energy of electricity.
Phoresis, from the Greek verb phoros, means "to
carry across."
41
A GEL
A gel is a net. The suspended particles are single large
molecules or aggregates of molecules or ions ranging
in size from 1 to 1000 nanometers. Thus, gel
electrophoresis refers to the technique in which
molecules are forced across a span of gel, motivated
by an electrical current. Activated electrodes at either
end of the gel provide the driving force. A molecule's
properties determine how rapidly an electric field can
move the molecule through a gelatinous medium.
Agarose
There are two basic types of materials used to make
gels: agarose and polyacrylamide. Agarose is very
fragile and easily destroyed by handling. Agarose gels
have very large "pore" size and are used primarily to
separate very large molecules with a molecular mass
greater than 200 kdal. Agarose gels can be processed
faster than polyacrylamide gels, but their resolution is
inferior. That is, the bands formed in the agarose gels
42
are fuzzy and spread far apart. This is a result of pore
size and it cannot be controlled.
Agarose is a linear polysaccharide made up of the
basic repeat unit agarobiose. Agarose is usually used
at concentrations between 1% and 3%.
Preparation of Agarose
Agarose gels are formed by suspending dry
agarose in aqueous buffer, then boiling the
mixture until a clear solution forms. This is
poured and allowed to cool to room temperature
to form a rigid gel.
Polyacrylamide
polyacrylamide gel electrophoresis (PAGE)
involves separation of protein on the basis of
charge and molecular size.
The pore size of the gel may be varied to produce
different molecular sieving effects for separating
proteins of different sizes. In this way, the
43
percentage of polyacrylmide can be controlled in a
given gel. By controlling the percentage (from 3%
to 30%), precise pore sizes can be obtained, usually
from 5 to 2,000 Kdal. This is the ideal range for
gene sequencing, protein, polypeptide, and enzyme
analysis.
Gradient gels
It provides continuous decrease in pore size
from the top to the bottom of the gel, resulting
in thin bands. Polyacrylamide gels offer
greater flexibility and more sharply defined
banding than agarose gels.
Proteins
Proteins are important in the structure and
function of all living organisms. Some proteins
serve as structural components while others
function, defense, and cell regulation. Some
proteins serve as enzymes that act as biological
catalysts which control the biochemical events.
44
Amino Acids
The fundamental unit of protiens is the amino acid.
Each amino acid contains an amino group (-NH2)
and a carboxylic group (-COOH) attached to a
central carbon called the alpha carbon. A R-group
(or side chain) is also attached to the alpha carbon.
The R-group, or side chain, determines the nature
of different amino acids.
Twenty amino acids have been identified as
constituents of most proteins. These amino acids
differ from each other in the nature of the R-group
attached to the alpha carbon. The identity of the
particular amino acid depends on the nature of the
R group.
45
Classification of Amino Acids
Amino acids are classified according to properties
of the R groups. The first of these depends on the
polar or nonpolar nature of the side chain. The
second depends on the presence of an acidic or
basic group in the side chain. Another criteria that
is taken into account is the presence of functional
groups in the side chains and the nature of those
groups.
Electrophoresis of Proteins
Proteins can be separated and purified by
electrophoresis. Methods for separating
proteins take advantage of properties such as
charge, size, and solubility, which vary from
one protein to the next. Because many proteins
bind to other biomolecules, proteins can also
be separated on the basis of their binding
properties. The source of a protein is generally
46
tissue or microbial cells. The cell must be
broken open and the protein must be released
into a solution called a crude extract. If
necessary, differential centrifugation can be
used to prepare subcellular fractions or to
isolate organelles. Once the extract or
organelle preparation is ready, a variety are
available for separation of proteins. Ionexchange chromatography can be used to
separate proteins with different charges
(similar to the way amino acids are separated).
Other chromatographic methods take
advantage of differences in size, binding
affinity, and solubility. Nonchromatographic
methods include the selective precipitation of
proteins with salt, acid, or high temperatures.
In addition to chromatography, another
important set of methods is available for the
separation of proteins, based on the migration
of charged proteins in an electric field, a
47
process called (gel) electrophoresis. Gel
electrophoresis is especially useful as an
analytical method. Its advantage is that
proteins can be visualized as well as separated,
permitting a researcher to estimate quickly the
number of proteins in a mixture or the degree of
purity of a particular protein preparation. Also, gel
electrophoresis allows determination of crucial
properties of a protein such as its isoelectric point
and approximate molecular weight.
Amino acids differ not only in R-group
characteristics but also in molecular weight.
Different amino acids are linked together in a
linear chain by peptide bonds in various
combinations and sequences to form specific
proteins. The net charge of a protein will
depend on its amino acid composition. If it has
more positively charged amino acids such that
the sum of the positive charges exceeds the
sum of the negative charges, the protein will
have an overall positive charge and migrate to
48
the cathode (negatively charged electrode) in
an electrical field. Proteins even with a
variation of one amino acids will have a
different overall charge, and thus are
electrophoretically distinguishable.
polypeptides that make up complex proteins.
The break up of complex proteins into their
respective polypeptides allows us to study the
structure of proteins that result from the
interaction of several genes.
A gene is a discrete unit of hereditary
information that usually specifies a protein. A
single gene provides the genetic code for only
one polypeptide. Thus, a protein consisting of
four polypeptides requires the interaction of
four genes to synthesize that specific protein.
49
A molecular weight protein marker is used to
prepare a standard separation curve with which
various unknown proteins or polypeptide
fractions can be identified.
Nucleic Acids
The double helix structure originally
proposed by Watson and Crick is the most striking
feature of DNA structure. The two coiled strands
run in antiparallel directions and are held together
by hydrogen binds between complentary bases.
Adenine pairs with thymine and guanine with
cytosine. Eukaryotic DNA is complexed with
histones and other basic proteins, while prokaryotic
DNA occurs in "naked" form not complexed to
proteins.
50
Nucleic acids transmit hereditary information and
determine which proteins a cell manufactures.
There are two classes of nucleic acids found in
cells: ribonucleic acids (RNA) and
deoxyribonucleic acids (DNA). DNA comprises
the genes, the hereditary material of the cell and
contains instructions for making all the proteins
needed by the organism. RNA functions in the
process of protein synthesis. Nucleic acids are
large, complex molecules. Their name --nucleic
acid-- reflects that they are acidic and were first
identified in nuclei.
Nucleic acids are made of only four nucleotides in
a regular and an interesting arrangement. Beadle et
al experiments had shown that genes control the
production of enzymes, which are proteins.
51
Nucleic acids are polymers of nucleotides,
molecular units that consist of the following:
1. a five-carbon sugar, either ribose or
deoxyribose,
2. a phosphate group, and
3. a nitrogenous base, a ring compound
containing nitrogen.
The nitrogenous base may be either a double-ringed
purine or a single-ringed pyrimidine. DNA
commonly consists of


Purines
o adenine (A)
o guanine (G)
Pyrimidines
o cytosine (C)
o thymine (T)
with the sugar deoxyribose and phosphate. RNA
commonly consists of


52
Purines
o adenine (A)
o guanine (G)
Pyrimidines
o cytosine (C)
o uracil (U)
with the sugar ribose and phosphate.
The removal of the phosphate group from a
nucleotide yields a compound termed a nucleoside,
composed of a base and a sugar.
Nucleic acid molecules are made of linear chains of
nucleotides. The nucleotides are linked together by
covalent bonds to each other.
The specific information of the nucleic acid is
coded in the unique sequence of the four kinds of
nucleotides present in the chain. DNA is composed
of two nucleotide chains entwined around each
other in a double helix.
The base sequence of nucleic acids can be
determined in a manner similar to determination of
the amino acid sequence of protein, but it is more
efficient, particularly for DNA, to use specialized
techniques. Restriction endonucleases can be used
to cleave DNA molecules into fragments of
suitable size. In a direct chemical method, four
53
samples of a given restriction fragment can each be
treated with a selective reagent, causing cleavage at
a given base. The resulting mixtures can be
analyzed by gel electrophoresis, which separates,
on the basis of size, the oligonucleotides produced
by this treatment. The base sequence of the
oligonucleotide can be "read" directly from the
sequencing gel.
54
55
56