Académique Documents
Professionnel Documents
Culture Documents
Faculty of Science
Department of Biochemistry
Girls Section
General Biochemistry
Lab
BIOC 201
1 Page
Table of Contents
Lab #
Experiment name
Laboratory Wares
12
3
4
Page #
21
26
Proteins
Qualitative Tests for Proteins
34
42
CARBOHYDRATES
QUALITATIVE TESTS FOR CARBOHYDRATES
39
48
58
Lipids
70
72
11
2 Page
13
Paper chromatography
75
14
80
3 Page
Laboratory Wares
Flasks
Glass Beakers
borosilicate glass
measuring, storing
sides for
measurement. These
graduated cylinders,
measuring pipets or
burets.
Dropping Pipet
laboratory.
Burets
Used to transfer
liquids in qualitative
a accurately
feature durable,
measurement
permanent
markings; fine,
sharp lines and
large, easy-to-read
numbers. Our
Burets meet ASTM
specifications.
4 Page
Pipets
Pipets are used to
measure and transfer
small volumes (10
mL or less) of liquids.
Pipets are long
graduated tubes that
allow one to
accurately measure
and transfer small
volumes.
Test Tubes
Pippet Bulbs
The orange pipette
bulb can be used
with the 10ml pipet.
The thumbwheel
pipetter is designed
for use with the 2ml
pipettes but will
also work with
small sizes. Neither
work with corrosive
liquids.
Graduated
Cylinders
Glass graduated
from borosilicate
for accurate
measurements of
heat resistance.
small volumes of
Optional marking
cloud if exposed to
notations.
materials such as
concentrated NaOH,
or any hydrocarbon
5 Page
Volumetric flask
Centrifuges
A centrifuge is a
A volumetric flask
is a type of laboratory
flask (piece of
piece of equipment,
generally driven by a
motor, which puts an
object in rotation around
laboratory glassware)
used to contain or
force perpendicular to
measure a very
precise and accurate
amount of a liquid.
Spectrophotometer
Hot Plates
A spectrophotometer
is an instrument
an efficient source
of infrared heat.
amount of radiant
by molecules.
6 Page
pH Meter
Scale
A pH meter a device
an instrument or
used for
potentiometric pH
measurements, which
measures essentially
the electro-chemical
potential between a
known liquid inside
the glass electrode
(membrane) and an
unknown liquid
outside.
Microscope
Vortex
A microscope is an
vortex is an
instrument for
instrument for
mixing substances
or chemicals in a
test tube.
unaided eye.
Pipets
There are several different types and sizes of pipets, which are used for slightly different
purposes. Be sure that you know how to identify the different types of pipets and that you
can determine the total volume and the gradations on each.
7 Page
Types of Pipets
1- Volumetric or transfer pipettes are designed to deliver a single volume precisely (the
volume will be indicated near the top of the pipet (i.e., 2 mL).
2- Mohr or measuring pipets are graduated but stop at a baseline before the pipet begins
to narrow.
3- Serological pipets are graduated to deliver (there is no base mark).
The appropriate amount of fluid is drawn into the pipet (with the meniscus precisely on
the correct mark) and the entire amount is transferred.
4- Mechanical
Most mechanical pipets can be set to draw and dispense different volumes. They are
usually set by turn the knurled nob near the top. The volume is read in the window.
Mechanical pipets are operated by depressing the plunger. On the downward stroke of the
plunger there are two stops. The first offers firm resistance, and the second is a hard stop.
To take up a volume in the pipet, place a tip on the end of the pipet. Depress the plunger
to the first stop and insert into the sample to be transferred. Draw the liquid into the pipet
by slowly releasing the plunger. To dispense the liquid from the pipet, place the tip of the
pipet into the opening of the well and slowly depress the plunger all the way to the
second stop. When the liquid has been dispensed withdraw the pipet tip from the well
before releasing the plunger.
:Reference
www.biology.lsu.edu/.../1208_pipet.php
8 Page
9 Page
Title Page
This should be the first (cover) page of the report. When writing the title page of a lab
report, the following should be included:
1. The title of the experiment.
2. The students name in full.
3. The instructor or person for whom the lab report is being compiled.
4. The date on which the experiment was performed or the date the lab report was
written.
Introduction Page
10 Page
Under this heading should be an overview of what the experiment was about. A sound
definition of what was learned about the process being carried out during the experiment
should be included.
Materials and Methods
This section should contain a description, in the students own words, of the experimental
procedure that was followed in the performance of the experiment. The materials and
methods section should be complete enough so that another student with the same
background, but unfamiliar with the experiment, could perform the same experiment
without additional instructions. Procedures and equipment used should be written in a
sentence form. Do not list!
Results
The result section should contain raw data. Raw data consist of actual measured values
recorded during the experiment. Use tables to present this information. All tables should
have descriptive titles, and they should show the units of data entries clearly. The data
section should also contain any graphs that are required. This is an effective method for
communicating experimental results. The following steps should be taken into
consideration while plotting a graph:
1. Do not use tiny dots, use symbols like X or O.
2. Do not draw a series of straight line segments between experimental data points
plotted on
a graph. The purpose of many of the experiments is to verify theoretical relationships
between variables.
3. All graphs should have descriptive titles. These titles should tell what the graph is
intended
to show. Each axis of a graph should be labeled with the variable and unit it
represents.
Always use graph paper and always label graph coordinate lines so that it is easy to
see how
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12 Page
Units of Concentrations:
I- Mass unit / volume unit:
- Example: mg/ml, mg/l, g/l, g/dl, mg%, etc.
- To convert from:
g>>>>>mg >>>>X 1000
mg>>>>g >>>> / 1000
g >>>> g >>>> X 1000000
13 Page
Problem:
Convert 0.2 mg/ml to
- g/l
- g/dl
- mg%
- mg/ l
- g/ l
moles of solute
liter of solution
- Rather than writing out moles per liter, these units are abbreviated as M or M.
M = wt X 1000
MW X Vml
- To convert from mg% to mmol/l
mmol/l = mg X 10
14 Page
MWsubstance
wt
Eq.wt X
1000
Vml
Problem:
1- How many grams of glucose are needed to make 100 ml of a 0.6 mol/l solution? (MW
glucose
= 180).
2- How can you prepare 0.1 M NaOH solution?
3- Describe the preparation of 5 L of 0.1 M Na2CO3 (MW = 105.99) from the primary
standard solid.
Never pour or transfer chemicals over the balance. Spilled chemicals can damage the
balances, which are very expensive to repair or replace. Never weigh warm or hot
objects; if you can feel any heat, the weighing will not be accurate. Always use a container
such as a vial, beaker, flask, or watch glass to weigh a solid or liquid chemical on the
balance to protect the balance pan.
15 Page
2.
Make sure your hands are clean and dry before you handle containers or objects that
are to be weighed. The outside of these containers or objects must also be clean and dry.
Clean up any spills on the balance pan or lab bench around the balance immediately.
3.
First open or remove the draft lid or cover (if there is one) and check to make sure
that the balance pan is clean. If the pan is dirty, have your TA show you how to clean it
and gently place it back on the balance.
4.
Close or put the draft lid back on the balance and zero the balance by pressing the
"T" or "on/tare" button (re-zero bar on Mettler balances). Wait 5-10 seconds for the
weight display to stabilize. (If the object to be weighed is so large that the draft lid can't
be used, do this step without the draft lid in place.)
5.
Open or remove the draft lid and place the object to be weighed on the balance pan.
Then close or place the draft lid back on the balance. (As long as it does not touch the
object to be weighed, leave the lid off if it does touch the object.) After 5-10 seconds the
weight display will stabilize and you can record the mass to 0.001 g.
6.
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(v/v)
volume
of
solute
100
volume of solution
Example:
Rubbing alcohol is generally 70% by volume isopropyl alcohol. That means that 100 ml
of solution contains 70 ml of isopropyl alcohol. That also means that a liter (or 1000 ml)
of this solution has 700 ml of isopropyl alcohol plus enough water to bring it up a total
volume of 1 liter, or 1000 ml.
17 Page
weight of solute
100
weight of solution
Question:
What is the weight percent of glucose in a solution made by dissolving 4.6 g of glucose in
145.2 g of water?
Analysis:
To get weight percent we need the weight of the solute and the total weight of the
solution.
Determine total weight of solution: 4.6 g+ 145.2 g = 149.8 g
Calculate percent:
Weight % glucose =
4.6 g glucose
149.8 g solution
weight
of
solute
100
volume of solution
Dilution:
M conc X V conc
M dil X V dil
Problem:
How can we prepare 100 ml of 0.04M K2Cr2O7 from 0.2M K2Cr2O7?
Serial Dilution:
- This technique involves the removal of a small amount of an original solution to another
container which is then brought up to the original volume using the required buffer or water.
In the example below, if you have 1 mL of your original solution and you remove 10 L and
place it in a tube containing 990 L of water or media you have made a 1:100 dilution.
Dilution factor =
Final volume
Initial volume
Standard Curve:
- A standard curve is a quantitative research tool, a method of plotting assay data that is
used to determine the concentration of a substance.
- The assay is first performed with various known concentrations of a substance similar
to that being measured. For example a standard curve for protein concentration is
often created using known concentrations of bovine serum albumin.
- The assay procedure may measure absorbance, optical density, luminescence,
fluorescence, radioactivity, or something else.
- This data is used to make the standard curve, plotting concentration on the X axis,
and assay measurement on the Y axis. The same assay is then performed with samples
of unknown concentration.
- To analyze the data, one locates the measurement on the Y-axis that corresponds to the
assay measurement of the unknown substance and follows a line to intersect the
standard curve.
20 Page
Concentration
545nm
mg/ml
0.1
0.5
0.2
1.0
0.3
1.5
0.4
2.5
0.5
5.0
0.6
10.0
0.7
25.0
21 Page
22 Page
pH
1-3
2.4- 3.4
5.5- 7.5
6.3- 6.6
6.5- 7.5
7
7.35- 7.45
8- 9
11.7
23 Page
24 Page
Definition of buffer:
Is a solution that resists change in pH when limited amounts of an acid
or a base are added to it; buffers consist of weak acids +
corresponding salts Or weak base + their salt.
Applications
Their resistance to changes in pH makes buffer solutions very useful
for chemical manufacturing and essential for many biochemical
processes. Buffer solutions are necessary to keep the right pH for
enzymes in many organisms to work. Many enzymes work only under
very precise conditions. Industrially, buffer solutions are used in
fermentation processes and in setting the correct conditions for dyes
used in coloring fabrics.
The acid-base balance or pH of the body fluids is maintained by a
closely regulated mechanism. This involves the body buffers, the
respiratory system and the kidney.
Selection of the buffer:
The selection of a particular buffer for a given application is based on
two consideration:
1- the desired pH
2- chemical compatibility of the buffer components with the sample
25 Page
Volume of
1M
Volume of 1M
Na2HPO4 NaH2PO4 (mL)
(mL)
7.9
92.1
12.0
88.0
17.8
82.2
25.5
74.5
35.2
64.8
46.3
53.7
57.7
42.3
68.4
31.6
77.4
22.6
84.5
15.5
89.6
10.4
93.2
6.8
References:
MN Chatterjea, Text book of biochemistry. Jaypee.2004
Bettelheim, FA, Introduction to general, organic and
biochemistry.2007
26 Page
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Measurement of Extinction:
- The earliest colorimeters relied on the human eye to match the color of a solution with
that of one of a series of colored discs. The results obtained were too subjective and not
particularly accurate.
The Colorimeter:
- Colorimeter is generally any tool that characterizes colour samples to provide an
objective measure of colour characteristics. In chemistry, the colorimeter is an apparatus
that allows the absorbance of a solution at a particular frequency (colour) of visual light to
be determined. Colorimeters hence make it possible to determine the concentration of a
known solute, since it is proportional to the absorbance.
- Different chemical substances absorb varying frequencies of the visible spectrum.
Colorimeters rely on the principle that the absorbance of a substance is proportional to its
concentration i.e., a more concentrated solution gives a higher absorbance reading.
- Filter in the colorimeter is used to select the color of light which the solute absorbs the
most, in order to maximize the accuracy of the experiment. Note that the colour of the
absorbed light is the 'opposite' of the colour of the specimen, so a blue filter would be
appropriate for an orange substance. Sensors measure the amount of light which has
passed through the solution, compared to the amount entering, and a display reads the
amount absorbed.
- A quantitative reading for the concentration of a substance can be found by making up a
series of solutions of known concentration of the chemical under study, and plotting a
29 Page
graph of absorbance against concentration. By reading off the absorbance of the specimen
substance on the graph, a value for its concentration is found.
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Light source
slit
condenser
cuvette
filter
photocell
galvanometer
Lens
The Spectrophotometer:
- Is a sophisticated type of colorimeter where monochromatic light is provided by prism.
The band with of the light passed by a filter is quite board, so that it may be difficult to
distinguish between two components of closely related absorption with a colorimeter. A
spectrophotometer is then needed.
31 Page
- All types require a Blank: which is a solution that contains the entire reagents except the
substance to be measured. It is used to adjust the device to zero.
Here is a summary of the steps of operation of a Spec 20 & spectrophotometer:
1- Power >>>>>>>>Turn on power.
2-Warmup>>>>>>Allow about 5 minutes when first turned on.
3-Wavelength>>>>>Select appropriate wavelength.
4- Zero>>>>>With sample holder empty and closed, adjust meter needle to 0%T (or
infinite A) using zero control knob.
5- Blank >>>>Fill tube half full with water. Place in sample holder and close cover. Adjust
meter needle to 100%T (or 0 A) using light control knob.
6- Standard>>>>Measure absorbance (or %T) of known solution. Fill tube half full with
sample of known concentration. Place in sample holder and close cover. Read absorbance
value (or %T) from meter. Repeat this step if making a calibration curve or verifying
proportionality (Beer's Law).
7- Sample>>>>>Measure absorbance (or %T) of solution with unknown concentration as
in previous step.
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Types of spectrophotometer:
1- Visible spectrophotometer.
2- Ultraviolet (UV) spectrophotometer.
References:
www.wikipedia.org
33 Page
COLORIMETRIC DETERMINATION OF
VITAMIN B-12
- Vitamin B12 is water soluble vitamin.
- RDA= 2.4 g /day for adult.
- It is found only in animal sources.
Functions:
1- Aids folic acid in synthesis of heme.
2- It prevents anemia.
3- Required for protein digestion and absorption.
Method:
- Prepare series of tubes according to the following table
Tube
Standard
B
-
1
1ml
2
2ml
3
3ml
4
4ml
5
5ml
UNK
-
vitamin
Unk
H2O
concentration
5ml
4ml
2ml
2ml
5ml
- Vortex.
- Read the absorbance at 555nm.
35 Page
Cunk = Aunk
A std
X C std
PROTEINS
Introduction:
- Protein (from the Greek protas meaning "of primary importance") is a complex, highmolecular-weight organic compound that consists of amino acids joined by peptide bonds.
- Proteins are natural polymer molecules consisting of amino acid units. The number of
amino acids in proteins may range from two to several thousand.
- Proteins are probably the most important class of biochemical molecules, although of
course lipids and carbohydrates are also essential for life. Proteins are the basis for the
major structural components of animal and human tissue.
- Proteins are essential to the structure and function of all living cells and viruses. Many
proteins are enzymes or subunits of enzymes, catalyzing chemical reactions. Other proteins
play structural or mechanical roles, such as those that form the struts and joints of the
cytoskeleton, serving as biological scaffolds for the mechanical integrity and tissue
signaling functions.
- Proteins can be hydrolyzed by acids, bases or specific enzymes.
1- Primary structure: the amino acid sequence. In order to function properly, peptides and
proteins must have the correct sequence of amino acids.
3- Tertiary structure: is the entire three-dimensional shape of the protein. This shape is
determined by the sequence of amino acids. The overall shape of a single protein molecule
primarily formed by hydrophobic interactions, but hydrogen bonds, ionic interactions, and
disulfide bonds are usually involved too.
4- Quaternary structure: the shape or structure that results from the union of more than
one protein molecule, usually called protein subunits in this context, which function as part
of the larger assembly or protein complex.
38 Page
Denaturation of Proteins:
- Denaturation is the disruption of secondary, tertiary and quaternary structure of proteins
leading to loss of their biological activity.
- Proteins denature when they lose their three-dimensional structure - their chemical
conformation and thus their characteristic folded structure. Proteins may be denatured at
the secondary, tertiary and quaternary structural levels, but not at the primary structural
level.
- Denaturation may be caused by:
1- Physical factors such as heating.
2- Chemical factors such as strong acid or base.
39 Page
40 Page
1- Biuret Test:
- It is the general test for all proteins.
- Biuret reagent is dilute CuSO4 in strong alkaline medium.
- Alkaline CuSO4 reacts with all compounds containing 2 or more peptide bonds to give a
blue-violet color.
Method:
1 ml of biuret reagent + 1 ml of protein mix well>>>> blue-violet color.
41 Page
References:
www.chemtopics.com
www.wikipedia.org
42 Page
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There are a number of qualitative tests to detect the presence of amino acids and these are
largely dependent on the nature of R-group.
Experiment-1:
Ninhydrin Reaction:
-A color reaction given by amino acids and peptides on heating with the chemical ninhydrin. The
technique is widely used for the detection and quantitation (measurement) of amino acids and
peptides.
-Ninhydrin is a powerful oxidizing agent which reacts with all amino acids between pH 4-8 to
produce a purple colored-compound.
-The reaction is also given by primary amines and ammonia but without the liberation of Co2
-The amino acids proline and hydroxyproline also reacts but produce a yellow color.
Method:
1 ml AA + 1 ml NH---- heat in boiling WB for 5min-----Purple color.
Alpha-amino acid + 2 ninhydrin ---> CO2 + aldehyde + final complex (purple) + 3H2O
In summary, ninhydrin, which is originally yellow, reacts with amino acid and turns deep
purple. It is this purple color that is detected in this method.
Experiment-2:
Xanthoproteic Reaction:
44 Page
- This reaction involves the nitration of benzene nucleus in alkaline medium. As a result AAs that
contain aromatic nucleus undergo this reaction.
- Aromatic AAs form yellow nitro derivative on heating with conc. nitric acid, the salts of these
derivatives are orange.
Phenylalanine
Tryptophan
Tyrosine
Method:
1 ml AA + 1 ml conc. HNO3----- heat the mixture in WB for 30s--cool--add drop-wise 40%
NaOH to render the solution alkaline--- Yellow to orange color.
45 Page
Experiment-3:
Millon Reaction:
- This reaction is used to detect the presence of phenol (hydroxybenzene) which reacts with
Millon's reagent to form red complexes.
- The only phenolic AA is tyrosine.
Tyrosine
Method:
1 ml AA + 5 drops of Millon reagent ----- heat the mixture in BWB for 10min--cool too
room temp--add 5 drops of NaNO2---Brick red color.
Experiment-4:
Hopkin-cole Reaction:
- This reaction is used to detect the presence of indol group
46 Page
- The indol group of tryptophan reacts with glyoxalic acid in the presence of conc. H 2SO4 to
give purple color.
- The reagent is glyoxalic acid in conc. H2SO4 (Glacial acetic acid which has been exposed to
light contains glyoxalic acid).
Tryptophan
Method:
1 ml AA + 1 ml Hopkin-cole reagent -----mix well--Carefully pour conc. H2SO4 down the
side of the tube so as to form two layers --Purple ring at the interface.
Experiment-5:
Sulfur Test:
- This reaction is specific to detect the presence of sulfur.
- The sulfur of cystein and cystine is converted to inorganic sulfide with conc. NaOH. Lead
acetate is added and a ppt of black lead sulfide indicates a +ve reaction.
Cystein
47 Page
Method:
2 ml AA + 1 ml 40% NaOH + 1-3 drops of lead acetate solution----- heat the mixture in WB
for 3min -----cool--observe any change ----- Black ppt.
Experiment-6:
Sakaguchi Reaction:
- This reaction is used to detect the presence of guanidine group.
- The only AA that contains guanidine group is arginine which reacts with -naphthol and an
oxidizing agent such as bromide water to give a red color.
Arginine
Method:
2 ml AA + 1 ml 2M NaOH + 1 ml ethanolic 0.02% -naphthol ----- mix wellcool in
ice-----add 1 ml of alkaline hypochlorite solution---- Red color
References:
www.wikipedia.org
www.chemtopics.com
48 Page
- You are supplied with samples of different amino acids, identify them.
-Present your results in a good and full lab report.
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CARBOHYDRATES
General Information:
Carbohydrates are the most abundant class of organic compounds found in living organisms.
They originate as products of photosynthesis, an endothermic reductive condensation of
carbon dioxide requiring light energy and the pigment chlorophyll.
+ n H2O + energy
CnH2nOn + n O2
As noted here, the formulas of many carbohydrates can be written as carbon hydrates,
Cn(H2O)n, hence their name. The carbohydrates are a major source of metabolic energy, both
for plants and for animals that depend on plants for food. Aside from the sugars and starches
that meet this vital nutritional role, carbohydrates also serve as a structural material
(cellulose), a component of the energy transport compound ATP, recognition sites on cell
surfaces, and one of three essential components of DNA and RNA.
Carbohydrates are called saccharides or, if they are relatively small, sugars.
A- Simple Sugars
1- Contain the elements carbon, hydrogen, and oxygen.
- The name carbohydrate literally means water compounds of carbon.
- The general formula for simple sugars is Cn(H2O)n.
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HC
HO
H
CH2OH
glyceraldehyde
O
HOH2C
CH2OH
dihydroxyacetone
A - Methods of Classification:
- Several methods are used to classify carbohydrates.
51 Page
HC
OH
OH
CH2OH
erythrose
B- Ketose - a monosaccharide with a ketone group.
CH 2OH
C O
HO C H
H
C OH
C OH
CH 2OH
esotcurf
- Usually combine the carbonyl classification and the number classification together.
H
C
H
C
H
OH
HO
O
OH
glyceraldehyde
aldotriose
HO
OH
OH
OH
OH
CH2OH
CH2OH
CH2OH
glucose
aldohexose
CH2OH
fructose
ketohexose
2- If the OH group is found on the left side of the chain of carbons, the sugar is designated
as an L sugar.
H O
C
H O
C
H C OH
HO C H
CH2OH
CH2OH
D-glyceraldehyde
L-glyceraldehyde
D-aldotriose
L-aldotriose
CHO
H C OH
CH 2OH
edyhedlarecylg-D
CHO
CHO
H C OH
HO C H
H C OH
H C OH
CH 2OH
CH 2OH
esorhtyre-D
esoerht-D
CHO
HO C H
H C OH
H C OH
H C OH
HO C H
H C OH
HO C H
H C OH
CH 2OH
CHO
HO C H
HO C H
CHO
H C OH
HO C H
H C OH
CH 2OH
CH 2OH
esolyx-D
esoxyl-D
CHO
CHO
HO C H
H C OH
H C OH
H C OH
HO C H
H C OH
HO C H
HO C H
HO C H
HO C H
CHO
CHO
CHO
CHO
H C OH
HO C H
H C OH
H C OH
HO C H
HO C H
H C OH
H C OH
H C OH
H C OH
H C OH
H C OH
H C OH
H C OH
H C OH
H C OH
H C OH
H C OH
CH 2OH
CH 2OH
CH 2OH
CH 2OH
CH 2OH
CH 2OH
esonnam-D
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H C OH
HO C H
H C OH
CH 2OH
esobir-D
esonibara-D
CHO
CHO
CHO
esoculg-D
CH 2OH
esortla-D
CH 2OH
esolla-D
esollat-D esotcalag-D
esodi-D
esolug-D
CH2OH
C
CH2OH
dihydroxyacetone
CH2OH
H
HO
OH
OH
CH2OH
D-erythrulose
CH2OH
C
CH2OH
C
OH
OH
CH2OH
D-ribulose
CH2OH
D-xylulose
CH2OH
CH2OH
CH2OH
CH2OH
HO
OH
HO
OH
HO
HO
OH
OH
OH
OH
OH
OH
CH2OH
D-tagatose
CH2OH
D-sorbose
CH2OH
CH2OH
D-fructose
D-psicose
Cyclic Structures:
- Five membered sugar rings are known as furanose rings.
- Six membered sugar rings are known as pyranose rings.
H O
C
H C OH
HO CH 2
H H
H C OH
H C OH
54 Page
H H
+
OH
OH OH
CH 2OH
esobir-D
HO CH 2 OH
O
O H
esonarufobir-D
H
OH OH
esonarufobir-D-
H O
C
H C OH
HO C H
H C OH
H C OH
CH 2OH
esoculg-D esonarypoculg-D-
HO CH 2
H OH
OH H
HO CH 2
H OOH
OH H
+
HO OH
H OH
HO
H
H OH
esonarypoculg-D-
Carbohydrate Anomers:
- Formation of either of the cyclic form has created a new stereocenter.
- These stereoisomeric ring forms of carbohydrates are called Anomers.
- Anomers are carbohydrates that differ by the stereo-configuration of the carbon involved in
ring formation.
- The greek letters and are used to describe the configuration about the ring forming
carbon.
- The anomer always has the OH group oriented in a downward fashion on the anomeric
carbon of a D-sugar.
- The anomer always has the OH group oriented in an upward fashion on the anomeric
carbon of a D-sugar.
Important Carbohydrates:
Monosaccharides
- composed of three to seven carbon atoms.
1- Glucose - most abundant hexose in our diet.
- The building block of complex carbohydrates.
- Component of the disaccharides: sucrose, maltose and lactose.
- Found in the polysaccharides: starch, cellulose and glycogen.
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CHO
H
CH 2OH
C OH
HO C H
H
C OH
C OH
H
OH
HHO,H
OH
H
CH 2OH
OH
2- Galactose
- Found in the disaccharide, lactose.
- Found in the cellular membranes of the brain and nervous system.
- Galactose is the C-4 epimer of glucose.
CHO
H C OH
HO C H
HO
CH 2OH
HO
C H
H C OH
CH 2OH
H
OH
O
HHO,H
H
H
OH
3- Fructose
- Sweetest of the carbohydrates.
- Component of the disaccharide sucrose.
- Fructose is a keto sugar.
Disaccharides
- composed of two monosaccharide units.
1- Maltose - malt sugar.
Used in cereals, candies and the brewing of beverages.
Composed of two D-glucose sugars joined by an -1,4 linkage.
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CH 2OH
H
CH 2OH
O H
H
OH
H
O
OH
H
OH
O OH
H
OH
OH
CH 2OH
OH
H
OH
H
O
OH
OH
O OH
H
OH
H
OH
CH 2OH
O
O H
H
OH
OH
OH
H
OH
CH 2OH
Polysaccharides
- composed of many (more than 10) monosaccharide units.
1- Cellulose:
Major structural material of plant cells.
Consists of many glucose units joined by -1,4 linkages.
2- Starch:
Storage form of glucose found in rice wheat, potatoes, grains and cereals.
Consists of many glucose units joined by -1,4 linkages.
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3- Glycogen:
Animal starch. Storage form of glucose found in the liver and muscle of animals.
Contains many highly branched glucose units.
Joined by -1,4 linkages and branched by -1,6 linkages.
4- Dextrin:
- Mixture of branched and un-branched soluble polysaccharides produced by partial
hydrolysis of starch by acids or amylases.
Reducing sugars:
Any sugar that contains either:
1-A free aldehyde group.
2- An -hydroxy ketone group.
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Experiment-1:
Molisch Test:
- It is the general test for all carbohydrates.
- All carbohydrates. Monosaccharides give a rapid positive test. Disaccharides and
polysaccharides react slower.
- The Molisch reagent dehydrates pentoses to form furfural (top reaction) and dehydrates
hexoses to form 5-hydroxymethyl furfural (bottom reaction). The furfurals further react
with
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Method:
1ml test solution + 2 drops of -naphthol >> mix well >> add conc. H2SO4 down the side of
the tube to form the ring at the interface of the two layers.
-ve
+ve
Experiment-2:
Fehling's Test:
- This test is used to differentiate between reducing and non reducing sugars.
- A reducing sugar reacts with Fehling's reagent in alkaline medium to form an orange to red
precipitate. Fehling's reagent is commonly used for reducing sugars but is known to be not
specific for aldehydes.
- Positive result is detected by reduction of the deep blue solution of cupric (II) to a red
precipitate of insoluble cuprous oxide (Cu2O).
- The sucrose does not react with Fehling's reagent. Sucrose is a disaccharide of glucose and
fructose. Most disaccharides are reducing sugars (e.g. lactose and maltose)
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- Sucrose is non-reducing sugar because the anomeric carbon of glucose is involved in the
glucose- fructose bond and hence is not free to form the aldehyde in solution.
Experiment-3:
Benedict's Test:
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- This test is used also to differentiate between reducing and non reducing sugars.
- It works on the same principle but Benedict is more stable than Fehling's reagent.
- Benedict's reagent contains blue copper(II) sulfate (CuSO4) 5H2O which is reduced to red
copper(I) oxide by aldehydes, thus oxidizing the aldehydes to carboxylic acids.
- The copper oxide is insoluble in water and so precipitates. The colour of the final solution
ranges from green to brick red depending on how many of the copper (II) ions are present.
Method:
1ml test solution + 1ml Benedict's reagent > heat the mixture in BWB (5min)>>Reddish
brown ppt
Experiment-4:
Barfoid Test:
- It is a test used to differentiate between monosaccharides and disaccharides. This reaction
will detect reducing monosaccharides in the presence of disaccharides. This reagent uses
copper ions to detect reducing sugars in an acidic solution. Barfoed's reagent is copper
acetate in dilute acetic acid (pH 4.6)
- Reducing monosaccharides are oxidized by the copper ions in a weak acidic medium to
form a carboxylic acid and a reddish ppt of Cu2O (cuprous oxide).
- Reducing disaccharides (lactose but not sucrose) undergo the same reaction but at slower
rate.
Method:
- 1 ml of the solution to be tested + 2 ml of freshly prepared Barfoed's reagent. Place test
tubes into a boiling water bath and heat for 2 minutes. Remove the tubes from the bath and
allow to cool.
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Experiment-5:
Seliwanoff Test:
- The test reagent dehydrates ketohexoses to form 5-hydroxymethylfurfural. 5hydroxymethylfurfural further reacts with resorcinol present in the test reagent to produce a
red product within two minutes (reaction not shown). Aldohexoses react to form the same
product, but do so more slowly.
Method:
1/2 ml of a sample + 2ml of Seliwanoff's reagent (a solution of resorcinol and HCl) is added.
The solution is then heated in a boiling water bath for two minutes.
- A positive test is indicated by the formation of a red product.
- In case of sucrose, avoid over-boiling because sucrose may be hydrolyzed to its component
(glucose and fructose) and gives false positive result.
-ve
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+ve
Experiment-5:
Hydrolysis test:
- Sucrose is the only non-reducing sugar so it does not reduce the alkaline Cu solutions (Fehling
and Benedict). Sucrose must first be hydrolyzed to its component and then test for.
Method:
6 ml of 1% sucrose in a test tube + 2 drops of concentrated hydrochloric acid (HCl). Heat the tube
in a boiling water bath for 15 minutes.
- Then test for Fehling, Benedict and all the previous tests.
Experiment-6:
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Experiment-6:
NHNH 2
O
N
H+
N
RCR
RCR
H
C
OH
HO
CNNH
NHNH 2
CNNH
HO
OH
OH
OH
OH
CH 2OH
glucose
CH 2OH
osazone
- Because both carbons 1 and 2 are involved in the reaction C-2 epimers produce the same
osazone.
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H
C
CNNH
NHNH 2
HO
OH
OH
OH
OH
OH
HO
HO
HO
OH
HO
OH
CH 2OH
CH 2OH
CH 2OH
mannose
osazone
glucose
HO
CNNH
NHNH 2
Ketoses with configurations identical to aldoses below C-2 give the same osazones e.g.
glucose and fructose.
H
C
OH
HO
CNNH
NHNH 2
CNNH
CH 2OH
NHNH 2
HO
HO
OH
OH
OH
OH
OH
OH
CH 2OH
CH 2OH
osazone
glucose
CH 2OH
fructose
Characteristics of osazones:
1- Have a characteristic shape.
2- Have characteristic melting points.
3- Specific time and whether the osazone is formed from hot solutions or only on cooling.
- Glucose + Phenyl hydrazine>>>>>>>>>>> Glucosazone (broomed shape)
- Fructose + Phenyl hydrazine>>>>>>>>>>> Fructosazone (broomed shape)
- Maltose + Phenyl hydrazine>>>>>>>>>>> Maltosazone (spherical shape)
- Lactose + Phenyl hydrazine>>>>>>>>>>> Lactosazone
- Sucrose + Phenyl hydrazine>>>>>>>>>>> -ve (WHY?)
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Method:
1/2 g of phenyl hydrazine + 1 spoon sodium acetate + 2ml of glucose solution >>>>>BWB (45 min)
until yellow crystals appear >>> cold and examine a sample of crystals under microscope. Compare
the crystal shapes with the supplied photographs.
References:
www.wikipedia.org
www.chemtopics.com
- You are supplied with samples of different sugars carry on all the previous experiments.
- Record your methods, observations and inferences in the three-column format
- Draw the form of crystals.
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Iodine test
Iodine test
+ve
-ve
Starch
Non-reducing
-ve
+ve
Reducing
Reducing
Disaccharide
monosaccharides
(sucrose)
and disaccharides
Dextrin
*Hydrolysis of sucrose
(5ml of sugar + o.5 ml conc. Hcl
heat in boiling bath for 5 min.
Then perform Fehling, Benedict,
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+ve
Reducing disaccharides
monosaccharides
(Lactose)
Seliwanoff`s test
(1ml sugar+1ml Seliwanoff reagent>>>boiling 2 min.)
+ve
Keto-sugar
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-ve
Aldo-sugar
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LIPIDS
-The lipids are a large and diverse group of naturally occurring organic compounds that
are related by their solubility in non-polar organic solvents (e.g. ether, chloroform, acetone
& benzene) and general insolubility in water.
- Lipids are defined by their physical behavior rather than by their chemical structures. As
a result there is great structural variety among the lipid class.
- Fats and oils are made from two kinds of molecules: glycerol (a type of alcohol with a
hydroxyl group on each of its three carbons) and three fatty acids joined by dehydration
synthesis. Since there are three fatty acids attached, these are known as triglycerides.
- The main distinction between fats and oils is whether theyre solid or liquid at room
temperature, and this is based on differences in the structures of the fatty acids they
contain.
- The main functions of lipids include:
1- Source of energy.
2- Some fat soluble vitamins have regulatory or coenzyme functions.
3- Structural functions (cell membrane).
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are generally referred to by their common names, which in most cases reflect their sources.
Natural fatty acids may be saturated or unsaturated.
- The terms saturated, mono-unsaturated, and poly-unsaturated refer to the number of
hydrogen atoms attached to the hydrocarbon tails of the fatty acids as compared to the
number of double bonds between carbon atoms in the tail.
- Fats, which are mostly from animal sources, have all single bonds between the carbons in
their fatty acid tails, thus all the carbons are also bonded to the maximum number of
hydrogen atoms possible. Since the fatty acids in these triglycerides contain the maximum
possible amount of hydrogen atoms, these would be called saturated fats. The hydrocarbon
chains in these fatty acids are, thus, fairly straight and can pack closely together, making
these fats solid at room temperature.
- Oils, mostly from plant sources, have some double bonds between some of the carbons in
the hydrocarbon tail, causing bends or kinks in the shape of the molecules. Because some
of the carbons share double bonds, theyre not bonded to as many hydrogen atoms as they
could if they werent double bonded to each other. Therefore these oils are called
unsaturated fats. Because of the kinks in the hydrocarbon tails, unsaturated fats cant pack
as closely together, making them liquid at room temperature.
Method:
1- Weigh out 2g of the test compound (fresh and rancid).
2- Suspend the melted fat in about 10ml of fat solvent.
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References:
www.wikipedia.org
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- You are provided with the sample of fresh and rancid oil.
- Calculate the acid value for fresh and rancid oil.
Calculations:
1M KOH 1L contains 56 g/ L of fat
0.1M KOH 1L contains 5.6 mg/ ml of fat
So:
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PAPER CHROMATOGRAPHY
Types of Chromatography:
1- Adsorption chromatography.
2- Partition chromatography e.g. paper chromatography
3- Gel-filtration chromatography.
Uses of chromatography:
- Government laboratories used to check
Mixtures can be solids, liquids or gases but their components must be able to dissolve in
stationary phase
mobile phase
solvent or a gas
- Test mixture is applied onto the chromatography paper and a solvent is then allowed to
pass over the paper. As the solvent does so, the components of the mixture travel along with
it.
- The stationary phase retards the passage of the components of the sample. When
components pass through the system at different rates they become separated in time.
The solvent used depends on the substance to be separated
The components will travel at different rates over paper depending on:
Generally, the more soluble the component is in the solvent and the less it adsorb onto the
chromatography paper, the faster it would move with the solvent on the paper and hence
the spot appears further up the paper
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Stationary Phase:
- In paper chromatography, cellulose in the form of paper sheets makes an identical
support medium. >>>> WHY?
- Because it has the ability to adsorb water molecules between cellulose fibers and forms a
stationary hydrophilic phase.
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- Paper: Watman No. 1 of high quality is the paper most frequently used for analytical
purposes.
Mobile Phase:
- In paper chromatography, mobile phase is a mixture of solvents.
- The choice of solvent depends on the mixture investigated:
1- If the compounds move close to solvent (A) front >>>>> these compounds are highly
soluble in solvent A
2- If the compounds are crowded around the origin >>>>> these compounds are not
sufficiently soluble in solvent B.
Therefore, a suitable solvent for separation would be an appropriate mixture of both
solvent A & B. As a result R f values of the components of the mixture are spread across the
length of the paper.
Retention Factor(R f ):
- The retention is measured as the retention factor Rf, the run length of the compound
divided by the run length of the solvent front:
laboratories due to variations of the solvent, the stationary phase, temperature, and the
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setup. It is therefore important to compare the retention of the test compound to that of one
or more standard compounds under absolutely identical conditions.
Detection of Spots:
- After development, the spots corresponding to different compounds may be located by
their color
- However, most compounds are colorless and are visualized by:
1- Spraying the paper with specific reagents.
2- Dipping the paper in a solution of the reagent in a volatile solvent.
3- Fluorescent substances can be visualized by ultraviolet (UV) light.
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- Separation and identification of amino acids are operations that must be performed
frequently by biochemists. The 20 amino acids present in proteins have similar structures.
However, each amino acid is unique in polarity and ionic characteristics. In this
experiment, we will use paper chromatography to separate and identify the components of
an unknown amino acid mixture.
- The solvent mixture contains several components, one of which is usually water and
another of which is a more non-polar solvent. As the solvent mixture moves up the paper
by capillary action, the water in the mixture binds to the hydrophilic paper (cellulose) and
creates a liquid stationary phase of many small water droplets. The non-polar solvent
continues to move up the paper forming a liquid mobile phase. Since amino acids have
different R-groups, they also have different degrees of solubility in water vs. the non-polar
solvent. An amino acid with a polar R-group will be more soluble in water than in the nonpolar solvent, so it will dissolve more in the stationary water phase and will move up the
paper only slightly. An amino acid with a hydrophobic R-group will be more soluble in the
mobile non-polar solvent than in water, so it will continue to move up the paper. Different
amino acids will move different distances up the paper depending upon their relative
solubilities in the two solvents, allowing for separation of amino acid mixtures.
- The movement of amino acids can be defined by a quantity known as Rf value, which
measures the movement of an amino acid compared to the movement of the solvent. At the
start of the chromatography, the amino acid is spotted at what is called the origin. The
chromatography is then performed, and the procedure is stopped before the solvent runs
all the way up the paper. The level to which the solvent has risen is called the solvent front.
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The Rf value of an amino acid is the ratio of the distance traveled by the amino acid from
the origin to the distance traveled by the solvent from the origin.
- Since Rf value for an amino acid is constant for a given chromatography system, an
unknown amino acid can be identified by comparing its Rf value to those of known amino
acids.
Application:
Materials:
1- Filter paper: Watman No.1.
2- Solvent system: Butanol: glacial acetic acid: water.
3- Ninhydrine reagent.
4- Standard amino acids and mixture of unknown.
Procedure:
1- Draw a light pencil line 1-2cm from the bottom of the paper.
2- Place a single drop of compound at intervals 2cm.
3- Dry with hair dryer.
4- Dip the paper in the jar with one of the edges of the paper to which the sample of the
spot is adjacent into the solvent.
5- Allow to run.
6- Remove the paper.
7- Determine the solvent front.
8- Dry.
9- Spray the paper with ninhydrin.
10- Dry the paper.
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References:
www.wikipedia.org
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- Calculate the R f and then identify the unknown amino acids in the mixture.
- Present your results in a good and full lab report.
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