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King Abdulaziz UniversityFaculty of Science

Department of Biochemistry Girls Section

General Biochemistry Lab

BIOC 201

Organized by: Sharifa Al-Ghamdi& Huda Al-Shaibi

Table of Contents

Lab # Experiment name Page #

1 Laboratory Wares 3

2 TERMS USED IN BIOCHEMISTRY & CALCULATIONS 12

3 Definition of pH and buffer 21

4 COLORIMETRY & SPECTROPHOTOMETR 26

5 ProteinsQualitative Tests for Proteins

3439

8 Qualitative Tests for Amino Acids 42

9 CARBOHYDRATESQUALITATIVE TESTS FOR CARBOHYDRATES

48

58

11Lipids 70

Determination of acid value 72

13 Paper chromatography 75

14 SEPARATION OF AMINO ACIDS BY PAPER CHROMATOGRAPHY 80


Laboratory Wares

Flasks

flasks are commonly

used for simple

measuring, storing

and mixing of liquids.

They are of similar to

beakers but less than

graduated cylinders,

measuring pipets or

burets.

Glass Beakers

The beakers are

borosilicate glass

(heat resistant) and

graduated along the

sides for

measurement. These

are perfect for

heating liquids and

storing solids in the

laboratory.

Dropping Pipet

Used to transfer

liquids in qualitative

test. It does not allow

a accurately

measurement

Burets

Ground and finished

stopcocks for leak-

free operation. They

feature durable,

permanent

markings; fine,

sharp lines and

large, easy-to-read

numbers. Our

Burets meet ASTM

specifications.


http://www.startracksmedical.com/supplies/erlenmeyerflask.jpg

http://www.startracksmedical.com/supplies/glassbeakers.jpg

http://www.startracksmedical.com/supplies/buret.jpg

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.

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.

Test Tubes

A glass test tube is

the most common of

lab supplies. Made

from borosilicate

glass for strength &

heat resistance.

Optional marking

spot allows for pencil

notations.

Graduated

Cylinders

Glass graduated

cylinders are handy

for accurate

measurements of

small volumes of

liquid and will not

cloud if exposed to

materials such as

concentrated NaOH,

or any hydrocarbon


http://www.startracksmedical.com/supplies/pipetbulbs.jpg

http://www.startracksmedical.com/supplies/testtubes.jpg

http://www.startracksmedical.com/supplies/graduatedcylinders.jpg

Volumetric flask

A volumetric flask

is a type of laboratory

flask (piece of

laboratory glassware)

used to contain or

measure a very

precise and accurate

amount of a liquid.

Centrifuges

A centrifuge is a piece of equipment,

generally driven by a

motor, which puts an

object in rotation around

a fixed axis, applying

force perpendicular to

the axis. The centrifuge

works using the

sedimentation principle,

where the centripetal

acceleration is used to

separate substances of

greater and lesser

density.

Spectrophotometer

A spectrophotometer

is an instrument

designed to detect the

amount of radiant

light energy absorbed

by molecules.

Hot Plates

hot plate provides

an efficient source

of infrared heat.

Most have solid

state push button

auto ignition and

heat output is fully

adjustable.


http://www.startracksmedical.com/supplies/benchtopcentrifuge.jpg

http://www.startracksmedical.com/supplies/hotplate.jpg

pH Meter

A pH meter a device

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.

Scale

an instrument or

machine for weighing.

Microscope

A microscope is an

instrument for

viewing objects that

are too small to be

seen by the naked or

unaided eye.

Vortex

vortex is an

instrument for

mixing substances

or chemicals in a

test tube.

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.


http://www.startracksmedical.com/supplies/biologicalmicroscope.jpg

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


http://www.biology.lsu.edu/introbio/tutorial/webtutorial/1208_pipet.php

CALIBRATION OF VOLUMETRIC EQUIPMENT:

In any experiment, you must ascertain the limits and range of your

measuring instruments.

Buret

1. Calibrating the Buret. Use de-ionized water for all operations. Check your buret for

leakage and drainage time.

Pipet

1. Calibrating the Pipet. Just as in the case of the buret, check your pipet for proper

drainage time. Fill it to the mark with de-ionized water and observe the time for it

to empty while it is held vertically.

Read pipette volume at eyelevel with pipette held vertically.


How to Write Lab ReportsThe ability to report technical information in a clear and concise manner is one of the

most important practical skills that a technically trained person can develop. This is true

because, the result and conclusions drawn from experimental methods are of little value

unless they can be communicated to others.

Writing lab reports that describe experimental methods, results, discussions, and

conclusions that can be drawn from those results is an excellent way to gain the practice

and experience needed to become an effective technical writer. It is only by writing and

being corrected that one can learn to write. A beginner will find it helpful to follow a

certain format for his or her reports. This will help ensure that the report is complete and

well organized. Written lab reports should consist of the following parts:

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


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


many units each division represents.

Discussions and Conclusions

This is the interpretation-and-conclusion of your report. This section should include the

following:

1. How the conduct of the experiment met the objectives.

2. What took place during the process.

3. All questions should be answered within this section in a very logical and clear

manner.

The questions should be put into statement form.

4. The conclusions should be relevant to the experiment that was performed and should

be

based on facts learned as a result of the experiment.

5. You should also include any recommendations that you feel would improve the

experimental

procedure. If you have any further investigations that might be suggested by the data,

you should also include them here.

References:

http://water.me.vccs.edu/reports.htm


http://water.me.vccs.edu/reports.htm

TERMS USED IN BIOCHEMISTRY & CALCULATIONS

Tips to Success in Biochemistry Lab:

1. Write down a clear plan before you start working.

2. Keep very careful notes so you can recheck what you did if you get unexpected results.

3. Label all tubes and samples so they don't get mixed up.

4. Work slowly and carefully. Accurate, reproducible results in a biochemical test like this

requires care on your part. If you are sloppy or careless your results will suffer.

Terms used in biochemistry lab:

Liter-------volume unit.

1L = 1000ml = 106 μl (micro liter).

Gram--------mass unit (weight).

1g = 1000mg = 106 μg

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 >>>> μg >>>> X 1000000


mg>>>> μg >>>> X1000

L>>>>>ml >>> X 1000

L>>>> µl >>>> X 1000000

ml>>>> µl >>>> X 1000

dl >>> ml >>>>> X 100

Problem:Convert 0.2 mg/ml to

- g/l

- g/dl

- mg%

- mg/ µl

- g/ µl

II- Molarity (M):

- Another way of expressing concentration is called molarity. Molarity is the number of

moles of solute dissolved in one liter of solution. The units, therefore are moles per liter,

specifically it's moles of solute per liter of solution.

Molarity = 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


MWsubstance

III- Normality (N):

- When you need to compare solutions on the basis of concentration of specific ions or the

amount of charge that the ions have, a different measure of concentration can be very useful.

It is called normality.

- No. of equivalent weight per liter of solution.

Eq.wt = molecular weight (MW)

Valance

- N = wt X 1000

Eq.wt X 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.

Balance Rules and Instructions:The Figure below illustrates one type of top loading electronic balance. Refer to this

figure when following the steps and precautions for using the balance listed below:

1. 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.


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. Never unplug the balance.

Top loading electronic balance


IV- Percent Concentration (%):

1-Volume percent is usually used when the solution is made by mixing two liquids.

- The use of percentages is a common way of expressing the concentration of a solution.

- The percentages can be calculated using volumes as well as weights, or even both

together.

- One way of expressing concentrations is by volume percent. Another is by weight

percent. Still another is a hybrid called weight/volume percent.

Volume percent (v/v) = volume of solute X 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.

2- Weight percent: is expressing the concentration of a solution in weight percent (or

mass percent).


Weight percent (w/w) = weight of solute X 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 x 100 = 3.1% glucose

149.8 g solution

3- Weight- volume percent: Another variation on percentage concentration is

weight/volume percent or mass/volume percent. This variation measures the amount of

solute in grams but measures the amount of solution in milliliters. An example would be

a 5 %( w/v) NaCl solution. It contains 5 g of NaCl for every 100 mL of solution.

Weight-Volume percent (w/v) = weight of solute X 100

volume of solution

Dilution:

- Diluted solutions can be prepared from concentrated solutions

M conc X V conc = M dil X V dil


Moles taken from concentrated solution = Moles placed in diluted solution

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 µL and

place it in a tube containing 990 µL of water or media you have made a 1:100 dilution.

Here is an example of how to do a series of serial dilutions:

1 ml extract + 4 ml water = 1/5 dilution

1 ml 1/5 dilution + 4 ml water = 1/25 dilution

1 ml 1/25 dilution + 4 ml water = 1/125 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.

- The corresponding value on the X-axis is the concentration of substance in the

unknown sample.

- Here is an example of how construct a standard curve.


PROTEIN STANDARD CURVE... a plot of absorbance @ 545nm vs. protein concentration

Absorbance 545nm

Concentrationmg/ml

0.10.5

0.21.0

0.31.5

0.42.5

0.55.0

0.610.0

0.725.0


Definition of pH and buffer

pH:- is a measure of acidity and the alkalinity of a solution in terms of hydrogen ion H+ or (hydronium ion concentration) pH= -log[H3O+]

Thus, it is evident that the pH is inversely proportional to the acidity. Lower the pH, higher the acidity or hydrogen ion concentration while higher the pH, the acidity is lower.Just as pH is convenient way to represent the concentration of H3O+ pOH is convenient way to express the concentration of OH-.

pOH= -log[OH-]A solution is acidic if its pH is less than 7 A solution is basic if its pH is grater than 7 (base is any substance that accept H+) A solution is neutral if its pH is equal to 7pH value of some common materials

Material pHGastric juice 1-3

Vinegar 2.4- 3.4Urine 5.5- 7.5Milk 6.3- 6.6

Saliva 6.5- 7.5Pure water 7

Blood 7.35- 7.45Sea water 8- 9ammonia 11.7


There are tow ways to measure the pH:1- By using a pH paper which is a plane paper soaked with mixture of

pH indicator.

Some acid-base indicators.

2- By using pH meter this method is more accurate and more precise.


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.ApplicationsTheir 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 pH2- chemical compatibility of the buffer components with the sample

Example of the Preparation of phosphate buffer Phosphate buffer is composed of NaH2PO4 (acid) and Na2HPO4 (base)

Preparation of 0.1M sodium phosphate buffer at 25ºC

Desired pH

Volume of 1M

Na2HPO4 (mL)

Volume of 1M NaH2PO4 (mL)


5.8 7.9 92.1 6.0 12.0 88.0 6.2 17.8 82.26.4 25.5 74.5 6.6 35.2 64.8 6.8 46.3 53.77.0 57.7 42.3 7.2 68.4 31.67.4 77.4 22.67.6 84.5 15.57.8 89.6 10.48.0 93.2 6.8

References:MN Chatterjea, Text book of biochemistry. Jaypee.2004Bettelheim, FA, Introduction to general, organic and biochemistry.2007


RESULTS & LAB REPORT


COLORIMETRY & SPECTROPHOTOMETR

- Many biochemical experiments involve the measurements of compound or group of

compounds present in a complex mixture.

- The most widely used method for determining the concentration of biochemical

compounds is colorimetry, which makes use of the property that when white light passes

through a colored solution, some wavelength are absorbed more than others.

- Many compounds are not themselves colored but can be made to absorb light in visible

region by reaction with suitable reagents.

- These reactions are fairly specific and in most cases very sensitive, so that quantities of

material in the region of mM / L concentrations can be measured.

- The big advantage of is that complete isolation of compound is not necessary and the

constituents of a complex mixture such as blood can be determined after little treatment.

- The depth of the color is proportional to the concentration of the compound being

measured, while the amount of light is proportional to the intensity of the color and hence

the concentration.


http://en.wikipedia.org/wiki/Image:Spektrofotometri.jpg

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


http://en.wikipedia.org/wiki/Colour

http://en.wikipedia.org/wiki/Filter

http://en.wikipedia.org/wiki/Proportional

http://en.wikipedia.org/wiki/Visible_spectrum

http://en.wikipedia.org/wiki/Light

http://en.wikipedia.org/wiki/Frequency

http://en.wikipedia.org/wiki/Absorbance

http://en.wikipedia.org/wiki/Chemistry

http://en.wikipedia.org/wiki/Colour

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

graph of absorbance against concentration. By reading off the absorbance of the specimen

substance on the graph, a value for its concentration is found.

How colorimeter works?

1- White light from a tungsten lamp passes through a slit, then a condenser lens, to give a

parallel beam which falls on the solution under investigation contained in an absorption

cell or cuvette. The cell is made of glass with the sides facing the beam cut parallel to each

other.

2- Beyond the absorption cell is the filter, which is selected to allow maximum transmission

of the color absorbed. If a blue solution is under examination, then red is absorbed and a

red filter is selected.

NOTE: The color of the filter is complementary to the solution.

3- The light then falls on to a photocell which generates an electrical current in direct

proportion to the intensity of light falling on it.

4- This small electrical signal is increased by the amplifier which passes to a galvanometer

of digital readout to give absorbance reading directly.


-Among the simplest and most common colorimeters are the Spectronic 20 and Spectronic

21. They are commonly called the Spec 20 and Spec 21.

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.


- 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.


Types of spectrophotometer:1- Visible spectrophotometer.

2- Ultraviolet (UV) spectrophotometer.

References:

www.wikipedia.org


COLORIMETRIC DETERMINATION OF VITAMIN B-12

- Vitamin B12 is water soluble vitamin.

- RDA= 2.4 µg /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 B 1 2 3 4 5 UNK

Standard

vitamin

- 1ml 2ml 3ml 4ml 5ml -

Unk - - - - - - 5ml

H2O 5ml 4ml 2ml 2ml 1 -

concentration

- Vortex.

- Read the absorbance at 555nm.



- You are supplied with the sample of unk vitamin.

- Plot the standard curve to calculate the concentration of unk.

- Calculate the concentration by using the equation>>>> Cunk = Aunk X C std

A std


http://www.clipartguide.com/_pages/0060-0505-2613-1558.html

PROTEINSIntroduction:

- Protein (from the Greek protas meaning "of primary importance") is a complex, high-

molecular-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.

Primary Protein Structure:

- Proteins are biopolymers built from 20 different L-alpha-amino acids.

- The two ends of the amino acid chain are referred to as the carboxy terminus (C-

terminus) and the amino terminus (N-terminus) based on the nature of the free group on

each extremity.

- Biochemists refer to four distinct aspects of a protein's structure:


http://en.wikipedia.org/wiki/N-terminal_end

http://en.wikipedia.org/wiki/C-terminal_end

http://en.wikipedia.org/wiki/Amino_acid

http://en.wikipedia.org/wiki/Cytoskeleton

http://en.wikipedia.org/wiki/Protein_subunit

http://en.wikipedia.org/wiki/Enzyme

http://en.wikipedia.org/wiki/Virus

http://en.wikipedia.org/wiki/Cell_(biology)

http://en.wikipedia.org/wiki/Peptide_bond

http://en.wikipedia.org/wiki/Amino_acid

http://en.wikipedia.org/wiki/Organic_compound

http://en.wikipedia.org/wiki/Greek_language

1- Primary structure: the amino acid sequence. In order to function properly, peptides and

proteins must have the correct sequence of amino acids.

2- Secondary structure: is the specific geometric shape caused by intramolecular and

intermolecular hydrogen bonding of amide groups. Some combinations of amino acids will

tend to form:

Alpha Helix : In the alpha helix, the polypeptide chain is coiled tightly in the fashion of a

spring. The "backbone" of the peptide forms the inner part of the coil while the side chains

extend outward from the coil. The helix is stabilized by hydrogen bonds between the >N-H

of one amino acid and the >C=O on the 4th amino acid away from it.

Beta Pleated Sheet : In this structure, individual protein chains are aligned side-by-side

with every other protein chain aligned in an opposite direction. The protein chains are held

together by intermolecular hydrogen bonding, that is hydrogen bonding between amide

groups of two separate chains. This intermolecular hydrogen bonding in the beta-pleated

sheet is in contrast to the intramolecular hydrogen bonding in the alpha-helix.


http://en.wikipedia.org/wiki/Secondary_structure

http://en.wikipedia.org/wiki/Peptide_sequence

http://en.wikipedia.org/wiki/Primary_structure

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.


http://en.wikipedia.org/wiki/Protein_complex

http://en.wikipedia.org/wiki/Protein_subunit

http://en.wikipedia.org/wiki/Quaternary_structure

http://en.wikipedia.org/wiki/Disulfide_bond

http://en.wikipedia.org/wiki/Hydrogen_bond

http://en.wikipedia.org/wiki/Hydrophobe

http://en.wikipedia.org/wiki/Tertiary_structure

http://en.wikipedia.org/wiki/Image:ProteinStructure.jpg

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.

Denaturated proteins are characterized by:

1-Loss of function: Most biological proteins lose their biological function when denatured,

for example, enzymes lose their catalytic activity.

2- They become less soluble. As a result, they are easily precipitated.


http://en.wikipedia.org/wiki/Catalysis

http://en.wikipedia.org/wiki/Enzyme

http://en.wikipedia.org/wiki/Primary_structure

http://en.wikipedia.org/wiki/Quaternary_structure

http://en.wikipedia.org/wiki/Tertiary_structure

http://en.wikipedia.org/wiki/Secondary_structure

http://en.wikipedia.org/wiki/Protein_folding

http://en.wikipedia.org/wiki/Chemical_conformation

http://en.wikipedia.org/wiki/Chemical_conformation

http://en.wikipedia.org/wiki/Image:Hemoglobin.jpg

3- Reversibility and irreversibility: In many proteins (unlike egg whites), denaturation is

reversible (the proteins can regain their native state when the denaturing influence is

removed).


Qualitative Tests for Proteins

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.

2- Denaturation by heat and extreme pH:

- Extreme heating and pH (conc. acids) denature proteins leading to precipitation of

proteins.

Method:

3ml Protein >>>>>>BWB-10min >>>>>> ppt of protein.

3ml Protein >>>>>> drops conc.HCL >>>>>> ppt of protein.

3- Precipitation of proteins by heavy metals:



- Proteins are precipitated in alkaline medium with heavy metals due to the direct union of

cation (Cu++, Ag+, Ba++, Pb++) with anionic groups of proteins, which are formed in basic

medium.

- At alkaline pH 7 and above, proteins are usually negatively charged so the addition of

positively charged ions will neutralize this charge and the proteins come out of solution (i.e.

heavy metals combine with proteins forming insoluble metalloproteine).

Method:

Few drops of heavy metals + 2ml protein + few drops 10% NaOH>>>>ppt

4- Precipitation of proteins by acidic reagent: - Proteins are precipitated in acidic medium with some reagents such as TCA, picric acid

and tannic acid due to the direct union of the anionic group with the cationic groups of the

proteins, which are formed in acidic medium.

- These compounds carry large negative charges which neutralize the positively charged

protein to form insoluble salt complex with protein.

- The acidic reagents are therefore most effective at acidic medium where proteins are

positively charged.

Method:

Few drops of acidic reagent + 2ml protein >>>slowly add dilute NaOH and observe the

result as the pH increase.

3- Detection of Amino acids contents of Protein:Carry on all the experiments you have done in amino acids lab on proteins to detect the

amino acid content of each protein.

References:www.chemtopics.com

www.wikipedia.org



http://www.wikipedia.org/

http://www.chemtopics.com/

- Present your results in a good and full lab report.



Qualitative Tests for Amino Acids

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:- 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.


http://en.wikipedia.org/wiki/Image:L-Phenylalanine.png

http://en.wikipedia.org/wiki/Image:L-Tryptophan.png

http://en.wikipedia.org/wiki/Image:L-Tyrosine.png

Nitrated tyrosine (a) and tryptophan (b)

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

- The indol group of tryptophan reacts with glyoxalic acid in the presence of conc. H2SO4 to

give purple color.


http://en.wikipedia.org/wiki/Image:L-Tyrosine.png

- 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

Method:


http://en.wikipedia.org/wiki/Image:L-Tryptophan.png

http://en.wikipedia.org/wiki/Image:L-Cysteine.png

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 α-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% α-naphthol ----- mix wellcool in

ice-----add 1 ml of alkaline hypochlorite solution---- Red color

References:www.wikipedia.org

www.chemtopics.com





http://en.wikipedia.org/wiki/Image:L-Arginine.png

- You are supplied with samples of different amino acids, identify them.

-Present your results in a good and full lab report.



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.


- This class of compounds is better described as Polyhydroxy aldehydes and ketones.

- The simplest carbohydrates are glyceraldehyde and dihydroxyacetone.

A - Methods of Classification:

- Several methods are used to classify carbohydrates.

1-One method of classification is based on whether the carbohydrate can be broken down into smaller units.

Monosaccharides - cannot be broken down into smaller units by hydrolysis. Sometimes

called simple sugars.

Disaccharides - can be broken down (hydrolyzed) into two monosaccharide units.

Oligosaccharides - can be broken into three to six monosaccharide units.

Polysaccharides - composed of 7 or more mono-saccharide units.

2-Another method is based on the number of carbons found in a simple sugar.

- If it has three carbons it is called a triose.

- If it has four carbons it is called a tetrose.

- If it has five carbons it is called a pentose.

- If it has six carbons it is called a hexose.

3-Another method uses the kind of carbonyl group.

A- Aldose - a monosaccharide with an aldehyde group.


B- Ketose - a monosaccharide with a ketone group.

CCC

CH 2OH

CCH 2OH

OH

HH

HOOHOH

esotcurf

- Usually combine the carbonyl classification and the number classification together.

B- Stereoconfigurations of simple sugars.

- Carbohydrates contain many stereocenters.

1- If the OH group is found on the right side of the carbon chain, the sugar is designated as

a D sugar.


2- If the OH group is found on the left side of the chain of carbons, the sugar is designated

as an L sugar.

CCCC

CHOHH

HOH

OHOH

HOH

CH 2OH

CCCC

CHOH

HHOH

OHHOHOH

CH 2OH

CCCC

CHOOH

HOHOH

HOH

HH

CH 2OH

CCCC

CHOOH

OHOH

HOH

HHH

CH 2OH

CCCC

CHOH

HHOH

HOOH

HOH

CH 2OH

C OHHCH 2OH

CHO

CC

HOH

HOH

CH 2OH

CHO

CCCC

CHOH

OHOH

HOHO HHH

CH 2OH

CC

OHOH

HH

CH 2OH

CHO

CCC

HHOH

OHHOH

CH 2OH

CHO

CCC

HHOH

HOHOH

CH 2OH

CHOCCC

OHOH

HO HHH

CH 2OH

CHO

CCC

OHOH

H OHHH

CH 2OH

CHO

CCCC

CHOHO

HHOH

HHOHOH

CH 2OH

CCCC

CHOH

HOHOH

HOOH

HH

CH 2OH

edyhedlarecylg-D

esorhtyre-D esoerht-D

esonibara-D esobir-D esolyx-Desoxyl-D

esoculg-Desonnam-D esolla-Desortla-D esollat-D esotcalag-D esodi-D esolug-D


CC OHHCH2OH

OH

D-glyceraldehyde

D-aldotriose

CC HHOCH2OH

OH

L-glyceraldehyde

L-aldotriose

Cyclic Structures:

- Five membered sugar rings are known as furanose rings.

- Six membered sugar rings are known as pyranose rings.

esonarufobir-D- esonarufobir-D-

O

OHH

H

H

OH

CH 2HO

OHH

O

OHH

H

OH

CH 2HO

HH

OH

+

CCCC

CH 2OH

HH

OH

HOHOH

OH

esobir-D


esonarypoculg-D-esonarypoculg-D-

+O

H

OH

OHH

OHHCH 2HO

HO

HO

OH

H

OHH

OHHCH 2HO

HO

H

esoculg-D

CCCC

COH

OHOH

HOH

HHH

CH 2OH

OH

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 α and β are used to describe the configuration about the ring forming

carbon.

- The α anomer always has the OH group oriented in a downward fashion on the anomeric

carbon of a D-sugar.

- The β 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.


CCCC

CHOOH

OHOH

HOH

HHH

CH 2OH

O

H

H

H

OHOH

CH 2OH

HOH HO,H

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.

CCCC

CHOOH

HOH

HOH

HHOH

CH 2OH

HO,H

O

H

H

OH

CH 2OH

HOH

HO

H

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 α-1,4 linkage.


O OHH

H

HH

H

O

HOHOH H H

OH OHOH

OH

CH 2OH CH 2OH

H

2-Lactose - milk sugar.

- Found in milk and milk products.

- Composed of one galactose and one glucose unit joined by a β-1,4 linkage.

OH

H

H

OH H

OH

OH

CH 2OH

HO

CH 2OH

OH

HOHH

H

OH

H

O

3- Sucrose - table sugar.

- Product of sugar cane and sugar beets.

- Composed of one glucose and one fructose unit.

- Linkage is at both anomeric carbons.

O O

OH

H

OH

H

H

O

HH

H

OH H

HOHCH 2OHOH

CH 2OHCH 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 β-1,4 linkages.

2- Starch:

Storage form of glucose found in rice wheat, potatoes, grains and cereals.

Consists of many glucose units joined by α-1,4 linkages.


Maltose is the disaccharide starting material.

3- Glycogen:

Animal starch. Storage form of glucose found in the liver and muscle of animals.

Contains many highly branched glucose units.

Joined by α-1,4 linkages and branched by α-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 α-hydroxy ketone group.


3-A hemiacetal linkage

-The presence of any of these groups allows the carbohydrate to undergo easy oxidation.

- If the sugar gets oxidized it causes reduction.

- Thus the name “reducing sugar”.


QUALITATIVE TESTS FOR CARBOHYDRATES

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 -naphthol present in the test reagent to produce a purple product.

→ Condensation with α-naphthol >>>> Purple ring.


Method:

1ml test solution + 2 drops of α-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)


- 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.

Fehling's Reagent: Two solutions are required:

Fehling's "A" uses 7 g CuSO4.5H2O dissolved in distilled water containing 2 drops of dilute

sulfuric acid.

Fehling's "B" uses 35g of potassium tartrate and 12g of NaOH in 100 ml of distilled water.

Method:

1ml test solution + 1ml Fehling's reagent > heat the mixture in BWB (5min)>>Reddish

brown ppt

Experiment-3:Benedict's Test:

- 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.

- Formation of a green, red, or yellow precipitate is a positive test for reducing

monosaccharides. Do not heat the tubes longer than 3 minutes, as a positive test can be

obtained with disaccharides if they are heated long enough.


http://en.wikipedia.org/wiki/Copper

http://en.wikipedia.org/wiki/Precipitate

http://en.wikipedia.org/wiki/Oxide

http://en.wikipedia.org/wiki/Carboxylic_acid

http://en.wikipedia.org/wiki/Copper(I)_oxide

http://en.wikipedia.org/wiki/Water

http://en.wikipedia.org/wiki/Oxygen

http://en.wikipedia.org/wiki/Sulfur

http://en.wikipedia.org/wiki/Copper

http://en.wikipedia.org/wiki/Copper(II)_sulfate

Experiment-5: Seliwanoff Test:- The test reagent dehydrates ketohexoses to form 5-hydroxymethylfurfural. 5-

hydroxymethylfurfural 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 +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: Iodine test : Test for Polysaccharides

1- Starch:

- 1/2 mL of the fresh starch solution + 1 drop of the iodine solution.

- A dark blue color indicates a positive test for starch. If the yellow color of the iodine

reagent simply becomes diluted, no starch is present. Record the observation as positive

(blue) or negative (yellow).

2- Dextrin:

- 1/2 mL of the fresh dextrin solution + 1 drop of the iodine solution.

- A violet color indicates a positive test for dextrin. If the yellow color of the iodine reagent

simply becomes diluted, no dextrin is present. Record the observation as positive (violet) or

negative (yellow).

Experiment-6: The preparation of osazone :


- Phenyl hydrazine reacts with normal carbonyls to produce phenyl hydrazones.

1- Sugars undergo a variation of this reaction in which 3 molecules of phenylhydrazine

react with the sugar to produce a 1,2-diphenylhydrazone.

2- These 1,2-diphenylhydrazones are known as osazones.

- Because both carbons 1 and 2 are involved in the reaction C-2 epimers produce the same

osazone.


Ketoses with configurations identical to aldoses below C-2 give the same osazones e.g.

glucose and fructose.

Explain glucose and fructose form the same osazone?

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?)

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.orgwww.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.



SCHEME FOR UNKNOWN SUGARSolution of carbohydrate

Benedict’s test

(2ml sugar+2ml Benedicts reagent….boiling 5 min.)

-ve for non reducing sugars +ve for reducing sugars

Iodine test Iodine test

(1ml of iodine + 5 drops of sugar) (1ml of iodine + 5 drops of sugar)

+ve -ve -ve +ve

Starch Non-reducing Reducing

Reducing

Disaccharide monosaccharides

Dextrin

(sucrose) and disaccharides

*Hydrolysis of sucrose

(5ml of sugar + o.5 ml conc. Hcl

heat in boiling bath for 5 min.

Then perform Fehling, Benedict,

Seliwanoff and Barfoid tests)

Barfoed`s Test

(2ml Barfoed reagent+ 1ml sugar >>boiling 5min)

-ve +ve

Reducing disaccharides monosaccharides

(Lactose)


Seliwanoff`s test

(1ml sugar+1ml Seliwanoff reagent>>>boiling 2 min.)

+ve -ve

Keto-sugar Aldo-sugar

RESULTS & LAB REPORTOrganized by: Sharifa Al-Ghamdi& Huda Al-Shaibi

- You are supplied with samples of unknown samples, identify them.

- Record your methods, observations and inferences in the three-column format



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 they’re 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).

1. Fatty Acids (FA):

- The “tail” of a fatty acid is a long hydrocarbon chain, making it hydrophobic. The “head”

of the molecule is a carboxyl group which is hydrophilic. These long-chain carboxylic acids

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, they’re not bonded to as many hydrogen atoms as they

could if they weren’t double bonded to each other. Therefore these oils are called

unsaturated fats. Because of the kinks in the hydrocarbon tails, unsaturated fats can’t pack

as closely together, making them liquid at room temperature.

Essential Fatty Acids:

- Are those that the body can not synthesize them and therefore must be supplied in the

diet.

- Two of FAs are essential in humans>>>Linoleic acid & Linolenic acid.

DETERMINATION OF ACID VALUE Organized by: Sharifa Al-Ghamdi& Huda Al-Shaibi

Sensitivity of Fats to Oxidation (Rancidity):

- Saturated FAs are relatively resistant to oxidation outside the body.

- Unsaturated FAs are slowly but spontaneously oxidize in the presence of air.

Rancidity:

- Oxidative cleavage of the double bonds in unsaturated FA and peroxide formation which

results in unpleasant taste and smell.

- It produces aldehyde and carboxylic acids of shorter length.

Rancidity is caused by:

1- Atmospheric air.

2- Hydrolysis by microorganisms.

- The amount of free fatty acids present therefore gives an indication of the:

1- Age.

2- Quality of oil.

Acid Value:

- Is the number of mg KOH required to neutralize the free fatty acids present in 1g of fat.

Method:

1- Weigh out 2g of the test compound (fresh and rancid).

2- Suspend the melted fat in about 10ml of fat solvent.

3- Add 2 drops of ph.ph. (Indicator).



4- Mix well.

5- Titer with 0.1M KOH.

6- End point>>>> faint pink color persists 20-30s

7- Record the volume of KOH required.

References:www.wikipedia.org



- 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: 5.6 mg >>>>>>>>>>> 1ml

? mg >>>>>>>>>>>> titer No.

5.6 X titer No. = Y (No. of mg of KOH)

Y >>>>>>>>> 2g of fat

? >>>>>>>>> 1g of fat

Y X 1 / 2 = No of mg of KOH that required to neutralize 1g of fat (i.e. acid value).



PAPER CHROMATOGRAPHY

- Chromatography is a family of analytical chemistry techniques for the separation of

mixtures.

- It was the Russian botanist Mikhail Tsvet (Mikhail Semyonovich Tsvet) who invented the

first chromatography technique in 1901.

- The separation of molecules depends on differences of 1- size 2- shape 3- mass 4- charges

5- solubility and 6- adsorption.

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

for approved dyes in food

that vegetables contained tiny amounts of pesticides and herbicides

Advantages of using chromatography:

1. Require very minute amount for identification.

2. Can be used to identify substances that cannot be easily melted or distilled.


All types of chromatography involve interaction between:

1- The mixture to be separated.

2- The stationary phase.

3- The mobile phase.

Principle of Paper Chromatography:

- Method of separating and identifying both colored and colorless mixtures.

- Mixtures can be solids, liquids or gases but their components must be able to dissolve in

the same solvent to different extents.

- Generally involves 2 phases:

stationary phase solid support e.g. water on paper

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:

their solubility in the solvent

how well the dyes adsorb on the chromatography paper

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


Result of chromatography is known as the >>>> Chromatogram

Types of Paper Chromatography:

- There are three types of paper chromatography:

I- Ascending Paper Chromatography:

- Solvent running up the paper by capillary action.

II- Descending Paper Chromatography:

- Solvent running down the paper by both capillary action and gravity.

Advantage of the descending method over the ascending method:

- Good for long pieces of paper thus better separation.

- Aided by gravity thus faster.

III- Two-Dimensional Paper Chromatography:

- The mixture is separated in the first solvent which should be volatile then after drying the

paper is turned through 90 and separation is carried out in the second solvent. After

location, a map is obtained and compounds can be identified by comparing their position

with a map of known compounds developed under the same conditions.

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.


- 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:

- Unknown substances could be identified by the Rf values

Rf = dist. Moved by the substance dist. Moved by the solvent

- The Rf of a compound often differs considerably between experiments and

laboratories due to variations of the solvent, the stationary phase, temperature, and the


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.


SEPARATION OF AMINO ACIDS BY PAPER CHROMATOGRAPHY

- 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 non-

polar 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.


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.


After some time

References:

www.wikipedia.org




- 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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