Chapter1
pH and Buffer
pH is a numeric scale used to specify the acidity or alkalinity of
an aqueous solution. It is the negative of thelogarithm to base 10 of the
activity of the hydrogen ion. Solutions with a pH less than 7 are acidic
and solutions with a pH greater than 7 are alkaline or basic. Pure water is
neutral, being neither an acid nor a base. Contrary to popular belief, the
pH value can be less than 0 or greater than 14 for very strong acids and
bases respectively.
pH measurements are important in medicine, biology, chemistry,
agriculture, forestry, food science, environmental science,
oceanography, civil engineering, chemical engineering, nutrition, water
treatment & water purification, and many other applications.
The pH scale is traceable to a set of standard solutions whose pH is
established by international agreement.[2] Primary pH standard values
are determined using a concentration cell with transference, by
measuring the potential difference between a hydrogen electrode and a
standard electrode such as the silver chloride electrode. The pH of
aqueous solutions can be measured with a glass electrode and a pH
meter, orindicator.
pH is the negative of the logarithm to base 10 of the activity of the
(solvated) hydronium ion, more often (albeit somewhat inaccurately)
expressed as the measure of the hydronium ion concentration
The rest of this article uses the technically correct word "base" and
its inflections in place of "alkaline", which specifically refers to a base
dissolved in water and its inflections.
pH
pH is defined as the decimal logarithm of the reciprocal of the hydrogen
ion activity, aH+, in a solution.
This definition was adopted because ion-selective electrodes, which are
used to measure pH, respond to activity. Ideally, electrode potential, E,
follows the Nernst equation, which, for the hydrogen ion can be written
as
where E is a measured potential, E0 is the standard electrode
potential, R is the gas constant, T is the temperature in kelvin, F is
the Faraday constant. For H+ number of electrons transferred is one. It
follows that electrode potential is proportional to pH when pH is defined
in terms of activity. Precise measurement of pH is presented in
International Standard ISO 31-8 as follows:[8] A galvanic cell is set up to
measure the electromotive force (e.m.f.) between a reference electrode
and an electrode sensitive to the hydrogen ion activity when they are
both immersed in the same aqueous solution. The reference electrode
may be a silver chloride electrode or a calomel electrode. The hydrogenion selective electrode is a standard hydrogen electrode.
Reference electrode | concentrated solution of KCl || test solution | H2 |
Pt
Firstly, the cell is filled with a solution of known hydrogen ion activity
and the emf, ES, is measured. Then the emf, EX, of the same cell
containing the solution of unknown pH is measured.
The difference between the two measured emf values is proportional to
pH. This method of calibration avoids the need to know the standard
electrode potential. The proportionality constant, 1/z is ideally equal
to
the "Nernstian slope".
To apply this process in practice, a glass electrode is used rather than the
cumbersome hydrogen electrode. A combined glass electrode has an inbuilt reference electrode. It is calibrated against buffer solutions of
known hydrogen ion activity. IUPAC has proposed the use of a set of
buffer solutions of known H+ activity.[2] Two or more buffer solutions
are used in order to accommodate the fact that the "slope" may differ
slightly from ideal. To implement this approach to calibration, the
electrode is first immersed in a standard solution and the reading on
a pH meter is adjusted to be equal to the standard buffer's value. The
reading from a second standard buffer solution is then adjusted, using
the "slope" control, to be equal to the pH for that solution. Further
details, are given in the IUPAC recommendations.[2] When more than
two buffer solutions are used the electrode is calibrated by fitting
observed pH values to a straight line with respect to standard buffer
values. Commercial standard buffer solutions usually come with
information on the value at 25 °C and a correction factor to be applied
for other temperatures.
The pH scale is logarithmic and therefore pH is a dimensionless
quantity.
pH indicators
Indicators may be used to measure pH, by making use of the fact that
their color changes with pH. Visual comparison of the color of a test
solution with a standard color chart provides a means to measure pH
accurate to the nearest whole number. More precise measurements are
possible if the color is measured spectrophotometrically, using
a colorimeter of spectrophotometer.Universal indicator consists of a
mixture of indicators such that there is a continuous color change from
about pH 2 to pH 10. Universal indicator paper is made from absorbent
paper that has been impregnated with universal indicator.
pOH
pOH is sometimes used as a measure of the concentration of hydroxide
ions, OH−, or alkalinity. pOH values are derived from pH measurements.
The concentration of hydroxide ions in water is related to the
concentration of hydrogen ions by
where KW is the self-ionisation constant of water. Taking logarithms
So, at room temperature pOH ≈ 14 − pH. However this relationship is
not strictly valid in other circumstances, such as in measurements of soil
alkalinity.
Extremes of pH
Measurement of pH below about 2.5 (ca. 0.003 mol dm−3 acid) and
above about 10.5 (ca. 0.0003 mol dm−3 alkaline) requires special
procedures because, when using the glass electrode, the Nernst
law breaks down under those conditions. Various factors contribute to
this. It cannot be assumed that liquid junction potentials are independent
of pH.[11] Also, extreme pH implies that the solution is concentrated, so
electrode potentials are affected by ionic strength variation. At high pH
the glass electrode may be affected by "alkaline error", because the
electrode becomes sensitive to the concentration of cations such as
Na+ and K+ in the solution. Specially constructed electrodes are
available which partly overcome these problems.
A Universal indicator is a pH indicator composed of a solution of
several compounds that exhibits several smooth colour changes over
a pHvalue range from 1-14 to indicate the acidity or alkalinity of
solutions. Although there are several commercially available universal
pH indicators, most are a variation of a formula patented by Yamada in
1933.[1] Details of this patent can be found inChemical
Abstracts. Experiments with Yamada's Universal Indicator are also
described in the Journal of Chemical Education.
A universal indicator is typically composed of water, propan-1ol, phenolphthalein sodium
salt,
sodium
hydroxide, methyl
red, bromothymol blue monosodium salt, and thymol blue monosodium
salt.
A pH Meter is a device used for potentiometrically measuring the
pH, which is either the concentration or the activity of hydrogen ions, of
an aqueous solution. It usually has a glass electrode plus a calomel
reference electrode, or a combination electrode. pH meters are usually
used to measure the pH of liquids, though special probes are sometimes
used to measure the pH of semi-solid substances.
Buffer
A buffer is a solution that can resist pH change upon the addition
of an acidic or basic components. It is able to neutralize small amounts
of added acid or base, thus maintaining the pH of the solution relatively
stable. This is important for processes and/or reactions which require
specific and stable pH ranges. Buffer solutions have a working pH range
and capacity which dictate how much acid/base can be neutralized
before pH changes, and the amount by which it will change.
To effectively maintain a pH range, a buffer must consist of a weak
conjugate acid-base pair, meaning either a. a weak acid and its conjugate
base, or b. a weak base and its conjugate acid. The use of one or the
other will simply depend upon the desired pH when preparing the buffer.
For example, the following could function as buffers when together in
solution:



Acetic acid (weak organic acid w/ formula CH3COOH) and a salt
containing its conjugate base, the acetate anion (CH3COO-), such as
sodium acetate (CH3COONa)
Pyridine (weak base w/ formula C5H5N) and a salt containing its
conjugate acid, the pyridinium cation (C5H5NH+), such as Pyridinium
Chloride.
Ammonia (weak base w/ formula NH3) and a salt containing its
conjugate acid, the ammonium cation, such as Ammonium Hydroxide
(NH4OH)
How does a buffer work?
A buffer is able to resist pH change because the two components
(conjugate acid and conjugate base) are both present in appreciable
amounts at equilibrium and are able to neutralize small amounts of other
acids and bases (in the form of H3O+ and OH-) when the are added to the
solution.
To clarify this effect, we can consider the simple example of a
Hydrofluoric Acid (HF) and Sodium Fluoride (NaF) buffer.
Hydrofluoric acid is a weak acid due to the strong attraction between the
relatively small F- ion and solvated protons (H3O+), which does not
allow it to dissociate completely in water. Therefore, if we obtain HF in
an aqueous solution, we establish the following equilibrium with only
slight dissociation (Ka(HF) = 6.6x10-4, strongly favors reactants):

HF(aq)+H2O(l)⇌F−(aq)+H3O+(aq)
We can then add and dissolve sodium fluoride into the solution and
mix the two until we reach the desired volume and pH at which we
want to buffer. When Sodium Fluoride dissolves in water, the
reaction goes to completion, thus we obtain:

NaF(aq)+H2O(l)→Na+(aq)+F−(aq)
Since Na+ is the conjugate of a strong base, it will have no effect on
the pH or reactivity of the buffer. The addition of NaF to the solution
will, however, increase the concentration of F- in the buffer solution,
and, consequently, by Le Châtelier’s Principle, lead to slightly less
dissociation of the HF in the previous equilibrium, as well. The
presence of significant amounts of both the conjugate acid, HF, and
the conjugate base, F-, allows the solution to function as a buffer. This
buffering action can be seen in the titration curve of a buffer solution.
As we can see, over the working range of the buffer. pH changes very
little with the addition of acid or base. Once the buffering capacity is
exceeded the rate of pH change quickly jumps. This occurs because
the conjugate acid or base has been depleted through neutralization.
This principle implies that a larger amount of conjugate acid or base
will have a greater buffering capacity.
If acid were added:
F−(aq)+H3O+(aq)⇌HF(aq)+H2O(l)
In this reaction, the conjugate base, F-, will neutralize the added acid,
H3O+, and this reaction goes to completion, because the reaction of F with H3O+ has an equilibrium constant much greater than one. (In fact,
the equilibrium constant the reaction as written is just the inverse of the
Ka for HF: 1/Ka(HF) = 1/(6.6x10-4) = 1.5x10+3.) So long as there is
more F- than H3O+, almost all of the H3O+ will be consumed and the
equilibrium will shift to the right, slightly increasing the concentration of
HF and slightly decreasing the concentration of F-, but resulting in
hardly any change in the amount of H3O+ present once equilibrium is reestablished.
If base were added:
HF(aq)+OH−(aq)⇌F−(aq)+H2O(l)
In this reaction, the conjugate acid, HF, will neutralize added amounts of
base, OH-, and the equilibrium will again shift to the right, slightly
increasing the concentration of F- in the solution and decreasing the
amount of HF slightly. Again, since most of the OH- is neutralized,
little pH change will occur.
These two reactions can continue to alternate back and forth with
little pH change.
Chapter 2
UV-VISIBLE ABSORPTION SPECTROPHOTOMETRY
What Wavelength Goes With a Color?
Our eyes are sensitive to light which lies in a very small region of
the electromagnetic spectrum labeled "visible light". This "visible light"
corresponds to a wavelength range of 400 - 700 nanometers (nm) and a
color range of violet through red. The human eye is not capable of
"seeing" radiation with wavelengths outside the visible spectrum. The
visible colors from shortest to longest wavelength are: violet, blue,
green, yellow, orange, and red. Ultraviolet radiation has a shorter
wavelength than the visible violet light. Infrared radiation has a longer
wavelength than visible red light. The white light is a mixture of the
colors of the visible spectrum. Black is a total absence of light. Earth's
most important energy source is the Sun. Sunlight consists of the entire
electromagnetic spectrum.
Colors We Can't See
There are many wavelengths in the electromagnetic spectrum the
human eye cannot detect.
Energy with wavelengths too short for humans to see
Energy with wavelengths too short to see is "bluer than blue".
Light with such short wavelengths is called "Ultraviolet" light.
How do we know this light exists? One way is that this kind of light
causes sunburns. Our skin is sensitive to this kind of light. If we stay out
in this light without sunblock protection, our skin absorbs this energy.
After the energy is absorbed, it can make our skin change color ("tan")
or it can break down the cells and cause other damage.
Energy with wavelengths too long for humans to see
Energy whose wavelength is too long to see is "redder than red".
Light with such long wavelengths is called "Infrared" light. The term
"Infra-" means "lower than".
How do we know this kind of light
exists? One way is that we can feel energy with these wavelengths such
as when we sit in front of a campfire or when we get close to a stove
burner. Scientists like Samuel Pierpont Langley passed light through a
prism and discovered that the infrared light the scientists could not see
beyond red could make other things hot.
Very long wavelengths of
infrared light radiate heat to outer space. This radiation is important to
the Earth's energy budget. If this energy did not escape to space, the
solar energy that the Earth absorbs would continue to heat the Earth.
The Beer-Lambert Law
The Beer-Lambert law relates the attenuation of light to the
properties of the material through which the light is traveling. This page
takes a brief look at the Beer-Lambert Law and explains the use of the
terms absorbance and molar absorptivity relating to UV-visible
absorption spectrometry.
The Absorbance of a Solution
For each wavelength of light passing through the spectrometer, the
intensity of the light passing through the reference cell is measured. This
is usually referred to as Io - that's I for Intensity.
Figure 1: Light absorbed by sample in a cuvetter
The intensity of the light passing through the sample cell is also
measured for that wavelength - given the symbol, I. If I is less than Io,
then the sample has absorbed some of the light (neglecting reflection of
light off the cuvet surface). A simple bit of math is then done in the
computer to convert this into something called the absorbance of the
sample - given the symbol, A.
Deriving the Beer-Lambert Law
Assumption one relates the absorbance to concentration and can be
expressed as
A∝c
The absorbance (A) is defined via the incident intensity Io and
transmitted intensity I by
A = -log
𝑰
𝑰𝟎
Assumption two can be expressed as
A∝l
Combining Equations 1 and 3:
A∝cl
This proportionality can be converted into an equality by including a
proportionality constant.
A=kcl
This formula is the common form of the Beer-Lambert Law, although it
can be also written in terms of intensities:
A = -log
𝑰
𝑰𝟎
= kcl
Assumption two can be expressed as
The constant k is called molar absorptivity or molar extinction
coefficient and is a measure of the probability of the electronic
transition. On most of the diagrams you will come across, the
absorbance ranges from 0 to 1, but it can go higher than that.
An absorbance of 0 at some wavelength means that no light of that
particular wavelength has been absorbed. The intensities of the sample
and reference beam are both the same, so the ratio Io/I is 1. Log10 of 1 is
zero.
Spectrophotometry
Absorbance Spectrum
The extent to which a sample absorbs light depends strongly upon
the wavelength of light. For this reason, spectrophotometry is performed
using monochromatic light. Monochromatic light is light in which all
photons have the same wavelength.
In analyzing a new sample, a
chemist first determines the sample's absorbance spectrum. The
absorbance spectrum shows how the absorbance of light depends upon
the wavelength of the light. The spectrum itself is a plot of absorbance
vs wavelength and is characterized by the wavelength (λmax) at which the
absorbance is the greatest.The value of λmax is important for several
reasons. This wavelength is characteristic of each compound and
provides information on the electronic structure of the analyte. In order
to obtain the highest sensitivity and to minimize deviations from Beer's
Law (see subsequent pages on this topic), analytical measurements are
made using light with a wavelength of λmax.
Chapter 3
Amino acid and protein (part l)
Protein
Most proteins consist of linear polymers built from series of up to
20 different L-α-amino acids. All proteinogenic amino acids possess
common structural features, including α-carbon to which an amino
group, a carboxyl group, and a variable side chain are bonded. The side
chains of the standard amino acids, detailed in the list of standard amino
acids, have a great variety of chemical structures and properties; it is the
combined effect of all of the amino acid side chains in a protein that
ultimately determines its three-dimensional structure and its chemical
reactivity. The amino acids in a polypeptide chain are linked by peptide
bonds. Once linked in the protein chain.
Structure
The building blocks of proteins are amino acids, which are small
organic molecules that consist of an alpha (central) carbon atom linked
to an amino group, a carboxyl group, a
hydrogen atom, and a variable component
called a side chain (see below). Within a
protein, multiple amino acids are linked
together by peptide bonds, thereby forming
a
long chain. Peptide bonds are formed by a
biochemical reaction that extracts a water
molecule as it joins the amino group of one
amino acid to the carboxyl group of a
neighboring amino acid. The linear sequence of amino acids within a
protein is considered the primary structure of the protein. The primary
structure of a protein — its amino acid sequence — drives the folding
and intermolecular bonding of the linear amino acid chain, which
ultimately determines the protein's unique three-dimensional shape.
Hydrogen bonding between amino groups and carboxyl groups in
neighboring regions of the protein chain sometimes causes certain
patterns of folding to occur. Known as alpha helices and beta sheets,
these stable folding patterns make up the secondary structure of a
protein. Most proteins contain multiple helices and sheets, in addition to
other less common patterns (Figure 2). The ensemble of formations and
folds in a single linear chain of amino acids — sometimes called a
polypeptide — constitutes the tertiary structure of a protein. Finally,
the quaternary structure of a protein refers to those macromolecules
with multiple polypeptide chains or subunits.
Biuret test
The biuret test is a chemical test used for detecting the presence of
peptide bonds. In the presence of peptides, a copper(II) ion forms violetcolored coordination complexes in an alkaline solution. The biuret
reaction can be used to assess the concentration of proteins because
peptide bonds occur with the same frequency per amino acid in the
peptide. The intensity of the color, and hence the absorption at 540 nm,
is directly proportional to the protein concentration, according to the
Beer-Lambert law. Despite its name, the reagent does not in fact contain
biuret ((H2N-CO-)2NH). The test is so named because it also gives a
positive reaction to the peptide-like bonds in the biuret molecule.
Amino acid
Amino acids are organic compounds
combine to form proteins. Amino acids and
proteins are the building blocks of life.
proteins are digested or broken down, amino
are left. The human body uses amino acids
that
When
acids
to
make proteins to help the body:
Amino acids are classified into three groups:
1. Essential amino acids
2. Nonessential amino acids
3. Conditional amino acids
Essential amino acids
Essential amino acids cannot be made by the body. As a result,
they must come from food. The nine essential amino acids are: histidine,
isoleucine, leucine, lysine, methionine, phenylalanine, threonine,
tryptophan, and valine.
Nonessential amino acids
"Nonessential" means that our bodies produce an amino acid, even
if we don't get it from the food we eat. They include: alanine,
asparagine, aspartic acid, and glutamic acid.
Conditional amino acids
Conditional amino acids are usually not essential, except in times
of illness and stress. They include: arginine, cysteine, glutamine,
tyrosine, glycine, ornithine, proline,and serine.
20 Amino Acids
There are twenty amino acids required for human life to exist.
Adults need nine essential amino acids that they cannot synthesize and
must get from food. The other eleven can be produced within our bodies.
In addition to the twenty amino acids we show you, there are others
found in nature (and some very small amounts in us). These twenty are
the biggies for our species and defined as the standard amino acids.
Function of protein
Protein is termed the building block of the body. It is called this
because protein is vital in the maintenance of body tissue, including
development and repair. Hair, skin, eyes, muscles and organs are all
made from protein. This is why children need more protein per pound of
body weight than adults; they are growing and developing new protein
tissue.
Energy
Protein is a major source of energy. If you consume more protein
than you need for body tissue maintenance and other necessary
functions, your body will use it for energy. If it is not needed due to
sufficient intake of other energy sources such as carbohydrates, the
protein will be used to create fat and becomes part of fat cells.
Hormones
Protein is involved in the creation of some hormones. These
substances help control body functions that involve the interaction of
several organs. Insulin, a small protein, is an example of a hormone that
regulates blood sugar. It involves the interaction of organs such as the
pancreas and the liver. Secretin, is another example of a protein
hormone. This substance assists in the digestive process by stimulating
the pancreas and the intestine to create necessary digestive juices.
Enzymes
The creation of DNA could not happen without the action of
protein enzymes. Enzymes are proteins that increase the rate of chemical
reactions in the body. In fact, most of the necessary chemical reactions
in the body would not efficiently proceed without enzymes. For
example, one type of enzyme functions as an aid in digesting large
protein, carbohydrate and fat molecules into smaller molecules, while
another assists the creation of DNA.
Transportation and Storage of Molecules
Protein is a major element in transportation of certain molecules.
For example, hemoglobin is a protein that transports oxygen throughout
the body. Protein is also sometimes used to store certain molecules.
Ferritin is an example of a protein that combines with iron for storage in
the liver.
Antibodies
Antibodies formed by protein help prevent many illnesses and
infections. Protein forms antibodies that help prevent infection, illness
and disease. These proteins identify and assist in destroying antigens
such as bacteria and viruses. They often work in conjunction with the
other immune system cells. For example, these antibodies identify and
then surround antigens in order to keep them contained until they can be
destroyed by white blood cells.
Denaturation
Denaturation of proteins involves the disruption and possible
destruction of both the secondary and tertiary structures. Since
denaturation reactions are not strong enough to break the peptide bonds,
the primary structure (sequence of amino acids) remains the same after a
denaturation process.
Heat
Heat can be used to disrupt hydrogen bonds and non-polar
hydrophobic interactions. This occurs because heat increases the kinetic
energy and causes the molecules to vibrate so rapidly and violently that
the bonds are disrupted. The proteins in eggs denature and coagulate
during cooking. Other foods are cooked to denature the proteins to make
it easier for enzymes to digest them. Medical supplies and instruments
are sterilized by heating to denature proteins in bacteria and thus destroy
the bacteria.
Alcohol Disrupts Hydrogen Bonding:
Hydrogen bonding occurs between amide groups in the secondary
protein structure. Hydrogen bonding between "side chains" occurs in
tertiary protein structure in a variety of amino acid combinations. All of
these are disrupted by the addition of another alcohol.
Acids and Bases Disrupt Salt Bridges:
Salt bridges result from the neutralization of an acid and amine on
side chains. Review reaction. The final interaction is ionic between the
positive ammonium group and the negative acid group. Any
combination of the various acidic or amine amino acid side chains will
have this effect.
Heavy Metal Salts:
Heavy metal salts act to denature proteins in much the same
manner as acids and bases. Heavy metal salts usually contain Hg+2,
Pb+2, Ag+1 Tl+1, Cd+2 and other metals with high atomic weights. Since
salts are ionic they disrupt salt bridges in proteins. The reaction of a
heavy metal salt with a protein usually leads to an insoluble metal
protein salt.
Chapter 4
Amino acid and protein (part ll)
Qualitative analysis of protein
Amino acids are building blocks of all proteins, and are linked
in series by peptide bond (-CONH-) to form the primary structure of a
protein. Amino acids possess an amine group, a carboxylic acid group
and a varying side chain that differs between different amino acids.
There are 20 naturally occurring amino acids, which vary from
one another with respect to their side chains. Their melting points are
extremely high (usually exceeding 200°C), and at their pI, they exist as
zwitterions, rather than as unionized molecules.
Amino acids respond to all typical chemical reactions
associated with compounds that contain carboxylic acid and amino
groups, usually under conditions where the zwitter ions form is present
in only small quantities. All amino acids (except glycine) exhibit optical
activity due to the presence of an asymmetric α – Carbon atom. Amino
acids with an L – configuration are present in all naturally occurring
proteins, whereas those with D – forms are found in antibiotics and in
bacterial cell walls.
Fig1. Structure of amino acid
Ninhydrin test
In the pH range of 4-8, all α- amino acids react with ninhydrin
(triketohydrindene hydrate), a powerful oxidizing agent to give a purple
colored product (diketohydrin) termed Rhuemann’s purple. All primary
amines and ammonia react similarly but without the liberation of carbon
dioxide. The imino acids proline and hydroxyproline also react with
ninhydrin, but they give a yellow colored complex instead of a purple
one. Besides amino acids, other complex structures such as peptides,
peptones and proteins also react positively when subjected to the
ninhydrin reaction.
Xanthoproteic acid test
Aromatic amino acids, such as Phenyl alanine, tyrosine and
tryptophan, respond to this test. In the presence of concentrated nitric
acid, the aromatic phenyl ring is nitrated to give yellow colored nitroderivatives. At alkaline pH, the color changes to orange due to the
ionization of the phenolic group.
Pauly's diazo Test
This test is specific for the detection of Tryptophan or
Histidine. The reagent used for this test contains sulphanilic acid
dissolved in hydrochloric acid. Sulphanilic acid upon diazotization in the
presence of sodium nitrite and hydrochloric acid results in the formation
a diazonium salt. The diazonium salt formed couples with either tyrosine
or histidine in alkaline medium to give a red coloured chromogen (azo
dye).
Millon's test
Phenolic amino acids such as Tyrosine and its derivatives
respond to this test. Compounds with a hydroxybenzene radical react
with Millon’s reagent to form a red colored complex. Millon’s reagent is
a solution of mercuric sulphate in sulphuric acid.
Histidine test
This test was discovered by Knoop. This reaction involves
bromination of histidine in acid solution, followed by neutralization of
the acid with excess of ammonia. Heating of alkaline solution develops
a blue or violet coloration.
Hopkins cole test
This test is specific test for detecting tryptophan. The indole
moiety of tryptophan reacts with glyoxilic acid in the presence of
concentrated sulphuric acid to give a purple colored product. Glyoxilic
acid is prepared from glacial acetic acid by being exposed to sunlight.
Sakaguchi test
Under alkaline condition, α- naphthol (1-hydroxy naphthalene)
reacts with a mono-substituted guanidine compound like arginine, which
upon treatment with hypobromite or hypochlorite, produces a
characteristic red color.
Lead sulphide test
Sulphur containing amino acids, such as cysteine and cystine.
upon boiling with sodium hydroxide (hot alkali), yield sodium sulphide.
This reaction is due to partial conversion of the organic sulphur to
inorganic sulphide, which can detected by precipitating it to lead
sulphide, using lead acetate solution.
Folin's McCarthy Sullivan Test
Imino acids such as Proline and hydroxyproline condense with
isatin reagent under alkaline condition to yield blue colored adduct.
Addition to sodium nitroprusside[Na2Fe(CN)5NO] to an alkaline
solution of methionine followed by the acidification of the reaction
yields a red colour. This reaction also forms the basis for the quantitative
determination of methionine.
Isatin test
Imino acids such as Proline and hydroxyproline condense with
isatin reagent under alkaline condition to yield blue colored adduct.
Quantitative analysis of protein
The Biuret Method
In this method, the reagent, a dilute copper sulfate solution at alkaline
pH, reacts with peptides or proteins. This complex undergoes a blue to
violet color transformation that requires five minutes to complete. The
protein is quantitated at 540 nm. This method requires relatively large
quantities of protein (1 - 20 mg protein / mL) for detection. Additionally,
it is sensitive to a variety of nitrogen-containing substances that could be
in the protein solution, thereby increasing the likelihood of erroneous
results.
The Lowry Test
The Lowry test is the most sensitive quantitative colorimetric assay for
protein detection. This test requires only 0.005 to 0.3 mg protein per mL
for detection. It is a modification of the biuret method. The intense bluegreen color formed in the Lowry test comes from the reaction of the
phosphomolybdotungstate in the Lowry reagent with the W and Y
residues in the protein. This assay method, however, can give low values
if the protein under examination does not have a significant number of
W and Y residues.
The Bradford (Bio-Rad) Method
This method employs a dye that binds to the protein to form a blue
complex. It can detect from 0.2 to 1.4 mg of protein per mL. The
Bradford method uses the negatively charged dye Coomasie Brilliant
Blue G-250, which binds to positive chains of the protein, to give the
blue complex. The red form of this dye predominates in solution before
a protein is added. The color changes into the blue form upon
complexing to the protein – the more concentrated the protein, the more
intense the blue color. This method is more rapid and less susceptible to
interfering substances than either of the above methods. The color
develops within 2 - 5 minutes and is stable up to 24 hours. It is for these
reasons that it is the most popular method of protein quantitation.
Consequently, it is the method we will use in today’s experiment.
The Protein
The protein we will analyze is bovine serum albumin (BSA). Albumin is
a serum protein that transports fatty acids and is important in
maintaining plasma pH. In protein quantitation assays, BSA serves as a
reference protein that is used to construct protein standard curves. Other
proteins can be used depending on the physical/chemical properties of
your protein of interest.
Chapter 5
What are carbohydrates?
A carbohydrate is a biological molecule consisting of carbon (C),
hydrogen (H) and oxygen (O) atoms, usually with a hydrogen:oxygen
atom ratio of 2:1 (as in water); in other words, with the empirical
formula 𝐶𝑚 (𝐻2 𝑂)𝑛 (where m could be different from n). Some
exceptions exist; for example, deoxyribose, a sugar component of DNA,
has the empirical formula 𝐶5 𝐻10 𝑂4 . Carbohydrates are technically
hydrates of carbon; structurally it is more accurate to view them as
polyhydroxy aldehydes and ketones.
Chemical classification of carbohydrates
On the basis of the number of forming units, three major classes of
carbohydrates can be defined: monosaccharides, oligosaccharides and
polysaccharides.
Monosaccharides
Monosaccharides or simply sugars are formed by only one polyhydroxy
aldehydeidic or ketonic unit. (Glucose, fructose, galactose, mannose)
The most abundant monosaccharide is D-glucose, also called dextrose.
Oligosaccharides
Oligosaccharides are formed by short chains of monosaccharidic units
(from 2 to 20) linked one to the next by chemical bounds, called
glycosidic bounds.
The most abundant oligosaccharides are disaccharides, formed by two
monosaccharides, and especially in the human diet the most important
are sucrose (common table sugar), lactose and maltose. Within cells
many oligosaccharides formed by three or more units do not find
themselves as free molecules but linked to other ones, lipids or proteins,
to form glycoconjugates.(Sucrose ,maltose, lactose)
Polysaccharides
Polysaccharides are polymers consisting of 20 to 107 monosaccharidic
units; they differ each other for the monosaccharides recurring in the
structure, for the length and the degree of branching of chains or for the
type of links between units.
Whereas in the plant kingdom several types of polysaccharides are
present, in vertebrates there are only a small number.
For example of polysaccharides; Starch, Cellulose, Glycogen
Seliwanoff’s test
Seliwanoff’s test is a chemical test which distinguishes between aldose
and ketose sugars. Ketoses are distinguished from aldoses via their
ketone/aldehyde functionality. If the sugar contains a ketone group, it is
a ketose and if it contains an aldehyde group, it is an aldose. This test is
based on the fact that, when heated, ketoses are more rapidly dehydrated
than aldoses. It is named after Theodor Seliwanoff, the chemist that first
devised the test.
Molisch's test
Molisch's test (named after Austrian botanist Hans Molisch) is a
sensitive chemical test for the presence of carbohydrates, based on the
dehydration of the carbohydrate by sulfuric acid or hydrochloric acid to
produce an aldehyde, which condenses with two molecules of phenol
(usually α-naphthol, though other phenols (e.g. resorcinol, thymol) also
give colored products), resulting in a red- or purple-colored compound.
Chapter 6
Lipid
Lipids are an important component of living cells. Together with
carbohydrates and proteins, this differs from the carbohydrates and
proteins in being insoluble in water and soluble in certain organic
solvents. lipids are the main constituents of plant and animal cells.
Cholesterol and triglycerides are lipids. Lipids are easily stored in
the body. They serve as a source of fuel and are an important constituent
of the structure of cells.
Lipids include fatty acids, neutral fats, waxes and steroids (like
cortisone). Compound lipids (lipids complexed with another type of
chemical compound) comprise the lipoproteins, glycolipids and
phospholipids. The classification of lipids:
Figure 1 Classification of lipids
1. A simple lipid is a fatty acid ester of different alcohols and carries
no other substance. These lipids belong to a heterogeneous class of
predominantly nonpolar compounds, mostly insoluble in water, but
soluble in nonpolar organic solvents such as chloroform and
benzene.
Simple lipids: esters of fatty acids with various alcohols.
1.1 Glycerides, more correctly known as acylglycerols, are esters
formed from glycerol and fatty acids.
Glycerol has three hydroxyl functional groups, which can be
esterified with one, two, or three fatty acids to form monoglycerides,
diglycerides, and triglycerides.
Vegetable oils and animal fats contain mostly triglycerides, but are
broken down by natural enzymes (lipases) into mono and diglycerides
and free fatty acids.
Figure 2 Triglyceride
1.2 A wax is a simple lipid which is an ester of a long-chain
alcohol and a fatty acid. The alcohol may contain from 12-32 carbon
atoms. Waxes are found in nature as coatings on leaves and stems. The
wax prevents the plant from losing excessive amounts of water. Carnuba
wax is found on the leaves of Brazilian palm trees and is used in floor
and automobile waxes. Lanolin coats lambs, wool. Beeswax is secreted
by bees to make cells for honey and eggs. Spermaceti wax is found in
the head cavities and blubber of the sperm whale. Many of the waxes
mentioned are used in ointments, hand creams, and cosmetics.
2. Compound Lipids or Heterolipids
Heterolipids are esters of fatty acids with alcohol and possess additional
groups also.
2.1 Phospholipids or Phosphatids are compound containing fatty
acids and glycerol in addition to a phosphoric acid, nitrogen bases and
other substituents. They usually possess one hydrophilic head and tow
non-polar tails. They are called polar lipids and are amphipathic in
nautre.
Phospholipids can be phosphoglycerides, phosphoinositides and
phosphosphingosides.
Figure 3 Phospholipid
2.2 Sphingolipids, or glycosylceramides, are a class of lipids
containing a backbone of sphingoid bases, a set of aliphatic amino
alcohols that includes sphingosine. A sphingolipid with an R group
consisting of a hydrogen atom only is a ceramide. Other common R
groups include phosphocholine, yielding a sphingomyelin, and various
sugar monomers or dimers, yielding cerebrosides and globosides,
respectively. Cerebrosides and globosides are collectively known as
glycosphingolipids.
2.3 A lipoprotein is a biochemical assembly that contains both
proteins and lipids, bound to the proteins, which allow fats to move
through the water inside and outside cells. The proteins serve to
emulsify the lipid molecules. Many enzymes, transporters, structural
proteins, antigens, adhesins, and toxins are lipoproteins.
3. Derived Lipids
Derived lipids are the substances derived from simple and
compound lipids by hydrolysis. These includes fatty acids, alcohols,
monoglycerides and diglycerides, steroids, terpenes, carotenoids.
The most common derived lipids are steroids, terpenes and carotenoids.
Steroids do not contain fatty acids, they are nonsaponifiable, and
are not hydrolyzed on heating. They are widely distributed in animals,
where they are associated with physiological processes. Example:
Estranes, androstranes, etc.
Figure 4 Steroid
Saturated fatty Acids and Unsaturated Fatty Acids
A saturated fat is a fat that consists of triglycerides containing
only fatty acids that are saturated. Saturated fatty acids have no double
bonds between the individual carbon atoms of the fatty acid chain. That
is, the chain of carbon atoms is fully "saturated" with hydrogen atoms.
There are many kinds of naturally occurring saturated fatty acids, which
differ mainly in number of carbon atoms, from 3 carbons (propionic
acid) to 36 (hexatriacontanoic acid).
Various fats contain different proportions of saturated and
unsaturated fat. Examples of foods containing a high proportion of
saturated fat include animal fat products such as cream, cheese, butter,
ghee, suet, tallow, lard, and fatty meats.
Unsaturated Fatty Acids have one or more double bonds between
carbon atoms. (Pairs of carbon atoms connected by double bonds can be
saturated by adding hydrogen atoms to them, converting the double
bonds to single bonds. Therefore, the double bonds are called
unsaturated.)The two carbon atoms in the chain that are bound next to
either side of the double bond can occur in a cis or trans configuration.