moleular Biology

8/14/2019 moleular Biology

1/48

Training Report

On

Techniques in Plant Tissue Culture,

Molecular Biology & Chemical Analysis

Submitted byDipesh Kumar Trivedi

Under the supervision ofMrs. Varsha Parasharmi

Scientist,

AndDr. (Mrs.) Shubhada R Thengane

Senior Scientist,Plant Tissue Culture Division ,National Chemical Laboratory,

Pune.

1


2/48

ACKNOWLEDGEMENT

At the outset, I express my heartfelt gratitude to my training supervisiorMrs.Varsha A

Parasharami, National Chemical Laboratory for her untiring guidance at every step of thisstudy. Her everlasting cheerfulness and unbound enthusiasm rekindles a fresh interest at work

everyday.

My heartest gratitude is to Dr. Shubhada R Thengane, for her valuable suggestions, timely

remarks and research ideas. Moreover, her values and services for mankind is a constant source

of inspiration for research.

I extend my sincere gratitude toDr. B.M.Khan, Head of the Department, andDr. S. Sivaram,

Director who have permit to work in NCL.

I owe a high debt of gratitude toMrs. Swapna R. Sathe, SRF,Ms. Varsha R Dawande, Project

Assistant andMrs. Nitasha Singh, SRF for their kind support and technical insights in the lab

throughout the study.

I also extend my sincere gratitude to Software engineerMr.Vinod Jani (CDAC) andMr.Param

Priya Singh (Persistent) for their valuable help, encouragement and support.

I am also thankful to my lab matesMr. Blesson S.George, Mr.N. Luke Johnson and Ms. Anita

Patilfor their timely help in the lab.

My love to my family specially my NanjiSmt.iLaxmi Deviand NanajiShriBabulal Dave who

are God and ideal persons for me, my motherSmt. Sheela Trivedi, my father Kamlesh k.

Trivedi, and my Mamaji Pravin Dave and Bharat Dave and my all family members &

relatives for their constant moral support,insipiration and unfailing love.

My sincere gratitude goes to all my friends Amit, Santosh, Ankit, Vishal, Rakesh, Mangilal

and Pravin singh.

- Dipesh Kumar Trivedi

2


3/48

Contents: -

A.Plant Tissue Culture(1)Introduction.

(2)Aims and Objectives.

(3)Materials and Methods.

(4)Results and Conclusion.

B.Molecular Biology(1)Introduction.




C.Chemical Analysis

(1) Introduction.




3


4/48

PLANT TISSUE CULTURE

4


5/48

PLANT TISSUE CULTURE

1. INTRODUCTION:-

Plant tissue culture is a practice used to propagate plants under sterile conditions, often to

produce clones of a plant called clonal propagation or micropropagation. Different techniques

in plant tissue culture may offer certain advantages over traditional methods of propagation,

including:

The production of exact copies of plants that produce particularly good flowers,

fruits, or have other desirable traits.

To quickly produce mature plants.

The production of multiples of plants in the absence of seeds or necessary pollinators

to produce seeds.

The regeneration of whole plants from plant cells that have been genetically

modified.

The production of plants in sterile containers that allows them to be moved with

greatly reduced chances of transmitting diseases, pests, and pathogens.

The production of plants from seeds that otherwise have very low chances of

germinating and growing, i.e.;orchids and nepenthes.

To clean particular plant of viral and other infections and to quickly multiply these

plants as 'cleaned stock' for horticulture and agriculture.Plant tissue culture relies on the fact that many plant cells have the ability to regenerate a

whole plant (totipotency). Single cells, plant cells without cell walls (protoplasts), pieces of

leaves, or (less commonly) roots can often be used to generate a new plant on culture media

given the required nutrients and plant hormones.

Plant tissue culture is a novel and innovative technique to grow high quality, disease free

plants quickly and in a large quantity by culturing and maintaining plant cells or organs like

leaves, stem, root, branch shoot tip, petals, anther and pollen in sterile, nutritionally and

environmentally supportive conditions in vitro. This technique is especially useful when the

planting material is scarce or on the edge of extinction as thousands of plants can be

produced from a single selected plant. Plants prepared using micropropagation technique are

true to type resembling the mother plant in all traits, disease free, contains uniform growth

and can be obtained throughout the year.

5


6/48

Moreover, harvesting period for crops prepared with tissue culture plantations is earliest than

normal. So, cultivation using tissue culture especially micropropagation improves farmers

income and make economy stronger. The crop growth is also uniform with almost zero crop

loss due to the diseases, which consequently leads to more yield, and thereby more profit.

Micropropagation is the art of plant multiplication in vitro. The process includes manysteps--stock plant care, explant selection and sterilization, media manipulation to obtain

proliferation, rooting, acclimation, and growing on of liners. The use of biotechnological

methods to grow large numbers of plants from very small pieces of plants, often from single

cells using tissue culture methods allows to preserve and multiply rare species.

During training we took Garcinia species for micropropagation. Three species which have

been used for micropropagatio are (1) G.indica,(2) G.spicata,(3) G.talbotii.

Garcinia indica orkokum is a fruit tree, of culinary, pharmaceutical, and industrial uses. The

tree is also ornamental, with a dense canopy of green leaves and red-tinged tender emerging

leaves. It is indigenous to the Western Ghats region of India, along the western coast. Recently,

industries have started extracting hydroxycitric acid (HCA) from the rind of the fruit, which

have anticholestrol and antiobesity activity.

6


7/48

Fig 1 G.indica leaves, mature and immature fruits and dissecting fruits showing

seeds.

Garcinia spicata is a large, dense tree with beautiful glossy foliage and bright orange fruits. It

is relatively slow growing, and makes a nice ornamental. The fruits have an unpleasant smell,

and are probably not edible. It may have medicinal or other uses, but there is very little

information available on this species.

Fig 2 fruits and leaves ofG.spicata

G. talbotii is found to be distributed in evergreen forests of Western Ghats of India. Its a

medium sized, large, dense tree of 6-15m tall with beautiful glossy foliage. Fruits are berries

about 5cm in diameter, abounding in yellow latex. They have an unpleasant smell, and are

probably not edible. It may have medicinal or other uses, but there is very little information

available on this species.

Fig 3 Immature fruits ofG.talbotii

7
http://en.wikipedia.org/wiki/File:Garcinia_indica_-_fruits,_seeds,_pulp_and_rinds.jpg


8/48

(2) AIMS AND OBJECTIVES: -

Aims

Induction of sprouting of sterile mature shoots underin vitro condition for micropropagationstudies in all the 3 Garcina species collected during summer training period. And inoculation

of leaf and seed material of freshly collected G.indica for callus and germination initiation.

Objectives

The plant materials were collected from western ghat forest areas (Mulshi, Ratnagiri &

Satara district). The collection time May June was coincided with the fruiting in all the

three above species. To get sterile explants was challenging task. So, main objectives were as

follow-

Surface sterilization of shoot buds ofGarciniasps. and inoculation on WPM media

Surface sterilization of seeds of Garcinia indica and inoculation on already

standardized WPM media.

Surface sterilization of leaves ofG.indica and inoculation on WPM media.

(3) MATERIALS AND METHODS

1. Plant Material : - Explants are collected from Garcinia species. as G.indica, G.spicata,

G.talbotii from different locations of Maharashtra specially Mulshi, Dapoli region,

Pratapgarh.

2. Equipments:

The major equipments used include:

pH meter:pH is the negative logarithm of hydrogen ion concentration. The measurement of

pH in digital pH meter (Digital Instruments Corp Ltd.) is based on ion exchange in between

hydrated layers formed on glass surface. Change in ion exchange results in EMF or voltage

difference causing current flow. The current intensity gives the value of pH.

8


9/48

Electronic Balance: A manual top loading balance (Contech pvt Ltd.) used for quick weighing

and for analytical purposes. This is a single pan balance capacity 100-200gm, Sensitivity

0.1mg operating on 230 V 50 H2 AC mains. Precision of 0.005g, weighing range 0-1, 200g,

and digital read out was used for making stock solutions of growth regulators and for other

fine weighing.Autoclave: The autoclave (Nat Steel Equipment Private Limited, Bombay) was used for

sterilization of media, glassware, water, dissecting instruments etc and for decontamination of

contaminated cultures in culture vessels. It is based on application of steam under pressure.

Autoclaving was carried out at 121C temperature under 15 lb/in2 pressures. Except culture

media, all other media were autoclaved for one hour. The culture media were autoclaved for

15 min.

Laminar airflow ultra clean unit: All aseptic manipulations were carried out on this unit. In

laminar (Klenzoids/ Microfilt, India), with the help of air pump air is passed through HEPA

filters of pore size, 0.22 micron. Due to positive pressure, the entry of any contaminant is

restricted from the open side of the bench. The instrument is fitted with UV tubes in addition

to the fluorescent tubes.

Apart from these, instruments like Magnetic stirrer (Remi, India), Steamer (Ultradent, India),

Temperature controlled oven (Pathak Electricals, India), membrane filter sterilizing unit

(Laxbro, Pune) were used.

3.Tools for Inoculation-

Test tubes

50ml, 100ml, 250ml, 500 ml. Flasks.

Petri plates.

Measuring cylinders.

Scalpels, forceps.

Blade holder, Cotton plug, cline wrap, Spirit lamp etc.

In order to maintain sterile condition, all glassware & equipment were autoclaved at 121C at

15 lb for 60 mins.

9


10/48

4. Chemicals for Sterilization:-

Liquid detergent (Tween 20, Teepol): for cleaning dirt particles, and act as

surfactants.

10% Savlon (v/v): used as disinfectant.

1% Bavistin (w/v): used as a fungicide.

70% Ethanol (v/v): for surface sterilization (carries out dehydration of microbes).

Mercuric Chloride (HgCl2)(w/v) : used as sterilizing agent (against bacteria and

fungi)

Table 1 :Chemicals and Sources from where they were purchased

CHEMICAL

SOURCE

Macro - and Micro nutrients (AR grade)

Vitamins and amino acids.

Plant Growth Regulators

Amino acids.

Sucrose (ExelAR grade)

Tween 20

Mercuric chloride

Bavistin (Antifungal agent)

Savlon (Antiseptic Liquid)

Qualigens, India.

Sigma Chemical Co., USA.



Qualigens, India

Merck limited,India.

Qualigens,India.

BASF, India.

Johnson & Johnson Limited.

10


11/48

5. STOCK PREPARATION:-The concentrations of the macro and microelement salts,

organic constituents and amino acids of the basal media used are given below in Table

Table 2: Composition of basal media called Woody Plant Medium (Lloyds and

McKown, 1981)

i) Macro elements (Stocks were prepared in 20X).

CaCl2. 2H2O 72.50 mg/l

Ca(NO3)2.4H2O 386.80 mg/l

KH2PO4 170.00 mg/l

MgSO4.7H2O 180.54 mg/l

NH4NO3 400.00 mg/l

ii) K2SO4 (prepared in 50X)

K2SO4 990.00 mg/l

iii) Microelements (Stocks were prepared in 50X)

CuSO4.5H2O 0.25 mg/l

H3BO3 6.20 mg/l

MnSO4.7H2O 22.30 mg/l

Na2MoO4.2H2O 0.25 mg/l

ZnSO4.7H2O 8.60 mg/liv) Vitamins (Stocks were prepared in 50X)

Glycine 2.00 mg/l

Myo-inositol 100.00 mg/l

Nicotine 0.50 mg/l

Pyridoxine 0.50 mg/l

Thiamine 1.00 mg/l

v) Chelate (Stocks were prepared in 50X)

FeSO4.7H2O 27.80 mg/l

Na2EDTA.2H2O 37.3 mg/l

11


12/48

Add FeSO4 in boiling EDTA.All the stock solutions and media were prepared in glass

distilled water. Stock solutions of different elements were prepared as 20X or 50X

depending on the final concentration of the element in the medium.

Table 3: Stock Solutions

For the preparation of individual stock weigh the ingredients of stock and dissolve in distill

water.Na2FeEDTA stock (total volume 500ml) was prepared by dissolving Na2EDTA (100X) in

200 ml hot water and to this, 200 ml solution of FeSO4 (100X) was added drop-wise. The

resultant solution was mixed thoroughly and the final volume was made to 500 ml.

6. MEDIA PREPARATION:-

Culture media used for the in vitro cultivation of plant cells are composed of three basic

components:

(1) Essential elements, or mineral ions, supplied as a complex mixture of salts;

(2) An organic supplement supplying vitamins and/or amino acids; and

(3) A source of fixed carbon; usually supplied as the sugar sucrose.

Steps involved in media preparation (example of 2 BAP media)

200 ml of macro stock (20X) + 80 ml of remaining stocks (50X) of WPM (for 4 lit. basal

medium) were measured and mixed in 1000 ml of DW.

2% sucrose (80gm.) was added and volume was made to 2 lit with DW

2mg/l BAP (80ml) added in it, and volume was made to 3500ml. with DW.

Stock Concentration Storage

Major salts 20 X Refrigeration

Minor salts 50 X Refrigeration

Na2FeEDTA* 50 X Refrigeration

Vitamins 50 X - 20 C

12


13/48

75mg/l. Bavistin (300mg) added in it, and final volume was made to 4 lit. with DW.

pH was adjusted to 5.8 to 6.0

0.7% Agar-agar was added in each medium

All media were autoclaved for 20 min at 15 lb pressure at 120C

Add 400mg/l Cefotaxim (4ml) in autoclaved media in LAF bench under sterile conditions.

The media was then dispersed in test tubes (20 ml per tube) and the tubes were plugged andlabeled properly.

The following media were prepared during training work.-

Initiations Medium for Mature bud induction for G.indica:

A. 2 BAP WPM media + 2% sucrose + 75mg/l Bavistin + 400mg/l Cefotaxim +0.7% Agar.

B. Media for inoculation of seeds of G.indica

(i) DW + 2% Sucrose + 0.7% Agar.

(ii) WPM (Macro element half) + 2% sucrose + 0.7% Agar.

(iii) Full WPM 0.2 BAP + 2% Sucrose + 0.7% Agar.

C. Media for shoot inoculation ofG.spicata and G.talbotii.

Full WPM 1 BAP + 2% Sucrose + 0.7% Agar

D.. Media for shifting shoot buds ofG.indica.

Full WPM + 1 BAP + 2% Sucrose + 0.7% Agar.

7.STERILIZTION OF EXPLANT-

13


14/48

Surface sterilization of explants is done to remove the entire microbial load from them prior

to inoculation. The following experiments of surface sterilization were done during my

training work.-

Experiment-1: Sterilization treatment of shoots ofG.spicata and G.talbotii.

Wash under running tap water for 1 hr.

Detergent wash (Tween-20) for 1 minute

Treatment with 10% Savlon for 5 minutes

Treatment with 1.5% Bavistin for 1 hour

(Further procedure is carried out under LAF bench for sterile conditions.)

Treatment with 70% Ethanol for 30 seconds under

Treatment with 0.1% HgCl2 for 7-8 minutes

Wash with Distill Water three times and then inoculated.

Experiment-2: Sterilization treatment ofG.indica fruits.

1 hour wash in running tap water

Detergent wash (Tween-20) for 2 minutes

Treatment with 10% Savlon for 5 minutes

Treatment with 1% Bavistin for 1 hour

(Fruits to be cut inside the Laminar Air flow unit to get the seeds)

14


15/48

( Single fruit contains 5-6 seeds)

Treatment with 70% Ethanol for 30 seconds

Treatment with 0.05% HgCl2 for 7-10 minutes

Seeds ready to be inoculated.

Experiment-3: Sterilization treatment of leaves ofG.indica.

Leaves were cleaned by wet cotton to remove dust particles.

Soak the leaves in autoclaved distilled water for 1 hour

Detergent wash- (Tween 20) for 10 minutes

Wash with autoclaved single distill water

(Laminar air flow unit)

0.05 % HgCl2 treatment for 10 minutes

Wash with autoclaved DW with 3 times.

Leaves ready for inoculation.

8. INOCULATION OF EXPLANTS:-

15


16/48

Inoculation can be defined as Transfer of explant on suitable media under aseptic condition

(LAF bench). Two things have to be kept in mind prior to inoculation:-

To expose laminar airflow cabinet to UV light for at least 20 mins.

To properly sterilize all the equipments with absolute alcohol.

Inoculation of explants:-

1. Hands were rinsed with 70% alcohol.

2. Forceps and scalpel were heated & then cooled by dipping them in spirit, contained in

coupling jar. This procedure was repeated for 3 times.

3. Few sterile papers were taken out from sterile paper bag.

4. Forceps was taken out from coupling jar and then flamed to vaporize the alcohol.

5. Explants were transferred on sterile filter paper & allowed to dry for few seconds to remove

water/moisture from the surface of explants.6. The edges of the explants were cut with the help of scalpel and made to their appropriate size

suitable for inoculation.

7. After flaming the mouth of the culture tubes, explants were inoculated into the medium.

8. After inoculation, all the test tubes were properly flamed, plugged and labeled with the name

of the media and date of inoculation.

9. Then incubated the inoculated tubes in culture room.

Fig 4 Inoculation of Explant.

4. RESULTS AND CONCLUSION:-

16


17/48

After successfully inoculation, the test tubes were incubated under controlled environmental

conditions in culture room where 16 hrs light and 8 hrs dark periods and 252c aremaintained.

Seed inoculation and germination ofG.indica (Table; 4)Date of inoculation-25.05.09, after 20 days observation-

Total No. of Seeds

inoculated

No. of Contaminated seeds No. of sterile

seeds

% sterility

of seeds

14 seeds 6 seeds 8 seeds 57.14%

Observation of sterile shoots ofGarcinia species (Table;5).-

Date of inoculation- 23.05.09, After 30 days observation-

Total No. of

tubes

inoculated

Total No. of

contaminated

Tubes

Total No. of

sterile tubes

No. of tubes

for petiole

removal

% sterility of

inoculated

shoots

103 39 64 43 62.13%

According to above Tables data and graphical representation (A) it represents that % of

seeds sterility is above than 50%. Germination is started after 22 days. Initially inoculated

tubes kept in dark but when germination started these tubes were transferred to light and then

incubated for further propagation.

% sterility of shoot buds is good we obtained 62.13% sterile buds. These tubes were kept in

culture room for further propagation. In some inoculated tubes Petioles are fallen down these

petioles were removed and tubes were incubated again. First graph (A) shows representationof sterile seeds and second (B) shows representation of sterile shoots.

(A) (B)

0

20

40

60

80

100

120

1 2 3

Inoculated

Tubes

Sterile

tubes

Contamina

ted Tubes

17

0

2

4

6

8

10

12

14

1 2 3

Inoculated

Seeds

Sterile

Seeds

Contamina

ted Seeds


18/48

MOLECULAR

BIOLOGY

MOLECULAR BIOLOGY

18


19/48

(1) INTRODUCTION: -

Molecular biology is the study of biology at a molecular level. Deoxyribonucleic acid

(DNA) is a nucleic acid that contains the genetic instructions used in the development and

functioning of all known living organisms and some viruses. The main role of DNA molecules

is the long-term storage of information. DNA is often compared to a set of blueprints or a

recipe, or a code, since it contains the instructions needed to construct other components of

cells, such as proteins and RNA molecules. The DNA segments that carry this genetic

information are called genes, but other DNA sequences have structural purposes, or are

involved in regulating the use of this genetic information. Chemically, DNA consists of two

long polymers of simple units called nucleotides, with backbones made of sugars and

phosphate groups joined by ester bonds. These two strands run in opposite directions to each

other and are therefore anti-parallel. Attached to each sugar is one of four types of molecules

called bases. It is the sequence of these four bases along the backbone that encodes

information. This information is read using the genetic code, which specifies the sequence of

the amino acids within proteins. The code is read by copying stretches of DNA into the related

nucleic acid RNA, in a process called transcription.

19


20/48

Genetic diversity

It refers to any variation in the nucleotides, genes, chromosomes, or whole genomes of

organisms (thegenome is the entire complement of DNA within the cells or organelles of the

organism). Genetic diversity at its most elementary level is represented by differences in the

sequences of nucleotides (adenine, cytosine, guanine, and thymine) that form the DNA

(deoxyribonucleic acid) within the cells of the organism. Knowledge about germplasm

diversity and genetic relationships among breeding materials could be an invaluable aid in

crop improvement strategies. A number of methods are currently available for analysis of

genetic diversity in germplasm accessions, breeding lines, and populations. These methods

have relied on pedigreedata, morphological data, agronomic performance data, biochemical

data, and more recently molecular (DNA-based) data.

Morphological characters have commonly been used to measure genetic diversity. Molecular

methods provide an additional tool to measure genetic relatedness and evolution. With the

advent of molecular markers, a new generation of markers has been introduced over the last

two decades, which has revolutionized the entire scenario of biological sciences. DNA-based

molecular markers have acted as versatile tools and have found their own position in various

fields like taxonomy, physiology, embryology, genetic engineering, etc. They are no longer

looked upon as simple DNA fingerprinting markers in variability studies or as mere forensic

tools. Ever since their development, they are constantly being modified to enhance their

utility and to bring about automation in the process of genome analysis. The discovery of

PCR (polymerase chain reaction) was a landmark in this effort and proved to be an unique

process that brought about a new class of DNA profiling markers. This facilitated the

development of marker-based gene tags, map-based cloning of agronomically important

genes, variability studies, phylogenetic analysis, synteny mapping, marker-assisted selection

of desirable genotypes, etc. Thus giving new dimensions to concerted efforts of breeding and

marker-aided selection that can reduce the time span of developing new and better varieties

and will make the dream of super varieties come true. These DNA markers offer several

advantages over traditional phenotypic markers, as they provide data that can be analyzed

objectively. Plants have always been looked upon as a key

20
http://cnx.org/content/m12158/latest/#geneticdiversity#geneticdiversityhttp://cnx.org/content/m12158/latest/#geneticdiversity#geneticdiversity


21/48

source of energy for survival and evolution of the animal kingdom, thus forming a base for

every ecological pyramid.Over the last few decades plant genomics has been studied

extensively bringing about a revolution in this area. Molecular markers, useful for plant

genome analysis, have now become an important tool in this revolution.During the early

period of research, classical strategies including comparative anatomy, physiology and

embryology were employed in genetic analysis to determine inter- and intra-speciesvariability. In the past decade, however, molecular markers have very rapidly complemented

the classical strategies. Molecular markers include biochemical constituents (e.g. secondary

metabolites in plants) and macromolecules, viz. proteins and deoxyribonucleic acids (DNA).

Analysis of secondary metabolites is, however, restricted to those plants that produce a

suitable range of metabolites which can be easily analyzed and which can distinguish between

varieties. These metabolites which are being used as markers should be ideally neutral to

environmental effects or management practices. Hence, amongst the molecular markers used,

DNA markers are more suitable and ubiquitous to most of the living organisms. DNA is

unique to each individual like fingerprints. Thus, DNA can be mapped to reveal the genetic

make up of an organism. The technique of DNA fingerprinting was discovered by geneticist

Alec J. Jefferys in 1984. He was carrying out studies on the gene for myoglobin.

DNA fingerprinting in plants is used for protection of the ecosystem, identifying marker

traits, identification of gene diversity and variation and mutations. There are various methods

for plant DNA fingerprinting like Restriction fragment length polymorphisms (RFLPs),

Randomly Amplified Polymorphic DNAs (RAPDs), Amplified fragment length

polymorphism (AFLP) and Inter Simple Sequence Repeats (ISSRs).

ISSR Marker: -

"Microsatellites" or ISSR are defined as loci (or regions within DNA sequences) where short

sequences of DNA (nucleotides; adenine - A, thiamine - T, guanine - G, cytosine - C) are

repeated in tandem arrays. This means that the sequences are repeated one right after the

other. The lengths of sequences used most often are di-, tri-, or tetra-nucleotides. These

markers often present high levels of inter- and intra-specific polymorphism, particularly

when tandem repeats number ten or greater.Sequences amplified by ISSR-PCR can be used

for DNA fingerprinting.. We used University of British Columbia(UBC) ISSR primers.

21


22/48


Aims- Screening and annealing temperature standardization of ISSRs markers with DNA

templates ofPinus roxburghii from different locations for genetic diversity analysis.

Objectives

1. DNA isolation fromPinus roxburghii from different locations.

2. Quantification of isolated DNA by gel electrophoresis and UV-VIS spectrophotometer.

3. Screening of ISSRs for Amplification and Annealing temperature standardization of

different ISSR primers by PCR (Robocycler).

(3) MATERIAL AND METHODS: -

1. Sample material- DNA is isolated from the needles ofPinus roxburghii from different

location like Palampur (H.P.), Panchgani GureGhar(Maharashtra), and Rajmundari (A.P.).

2. Reagents-

A. Chemicals for DNA extraction and gel electrophoresis.

Extraction buffer :

2% CTAB (cetyltrimethylammonium bromide)

1.4M NaCl

100mM Tris HCl (pH-8.0).

20 mM EDTA (pH-8.0).

100mM PVP(polyvinylpyrolidone)

Dissolve CTAB by heating to 60C

Autoclave and store at 37C

Chloroform : isoamyl alcohol (24:1 v/v)

Liquid Nitrogen

5 M NaCl

TE buffer : Take 10mM Tris-HCl & 1mM EDTA, adjust pH to 8.0 & autoclave

22


23/48

RNase (10 mg/ml)

TBE Buffer (Tris HCl, Boric acid, EDTA)- For tank buffer during Gel

electrophoresis.

70% Ethanol- for DNA washing.

Agarose- for preparing gel.

B. Chemicals for PCR (Polymerase chain reaction)-

Taq buffer- (1x)

0.2 mM dNTPs (dATP, dCTP, dTTP, dGTP).

2.5 mM MgCl2.

0.6 ul Taq polymerase.

0.25 uM Primer.

C. Some other chemicals-

DNA Ladder (stock- 100ul DNA ladder + 600ul SMQ + 300 ul Bromophenol blue).

Bromophenol blue loading dye (6x buffer + 0.25%bromophenol blue + 0.25%

xylene cynoll ff + 15% ficoll type 400- all dissolve in water).

EtBr- ethidium bromide.

3. Equipments: -

(A) Water bath- It is electronic water heating equipment in which we maintain high temp.

for DNA extraction (65 c).

(B) Centrifuge It is used to centrifuge for separating DNA from other biomolecules at high

speed. (10000rpm for 10 minutes.).

(C) Micropipette It is used for taking desired volume.

(D) Gel electrophoresis system - Agarose gel electrophoresis is a method used in

biochemistry and molecular biology to separate DNA, or RNA molecules by size. This is

achieved by moving negatively charged nucleic acid molecules through an agarose matrix

with an electric field (electrophoresis). Shorter molecules move faster and migrate farther

than longer ones.

23


24/48

Fig 5 Gel Electrophoresis system

(E) UV-VIS Spectrophotometer: - It is used to quantify the ds DNA concentration

in solution. We used 260/280 nm program for quantify DNA purity also.

(F) PCR (Robocycler Gradient 96) - The polymerase chain reaction (PCR) is a technique

to amplify a single or few copies of a piece of DNA across several orders of magnitude,

generating millions or more copies of a particular DNA sequence. The method relies on

thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA

melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing

sequences complementary to the target region along with a DNA polymerase (after which the

method is named) are key components to enable selective and repeated amplification.

24


25/48

Fig 6 (A)PCR

steps,(B) Robocycler Gradient 96

Instrument

(F) Gel doc machine - Gel

documentation system is an easy-to-

use, high-performance gel

documentation system. It uses a CCD

camera to capture images in real time,

with a motorized zoom lens for

convenient zoom, focus, and iris

adjustments. Using BioRad's software,

images acquired with the Gel Doc can

be optimized, annotated, analyzed and

printed to a video printer or to your

local printer.

(4) DNA Extraction - . Protocol used for DNA Extraction (Doyle J, Doyle J 1990

method)

1. Weighed 1 gm of Pinus roxburghii young needles and crushed the needles in Liquid

nitrogen to make it fine powder and transferred to centrifuge tubes.

2. Add 20 ml of DNA Extraction buffer to the tubes.

2 % - C-TAB,

1.4 M - NaCl,

100mM - Tris HCl (pH 8.0),

25


26/48

20mM - EDTA (pH 8.0)).

Incubate the tubes for 1 hr at 65C.

3. After incubation add equal volume of Chloroform: Isoamylalcohol (24:1) and mix properly.

4. Centrifuge the tubes at 10,000 rpm at 16C for 10 min.

5. Collect the supernatant and repeat the extraction with equal volume of Chloroform:

Isoamylalcohol (24:1) and centrifuge the tubes at 10,000 rpm at 16C for 10 min.6. Collect the supernatant and to that add equal volume of Ice-cold Isopropanol and mix

gently.

7. Keep the tubes at room temperature for at least 1 hr for precipitation.

8. Recover the precipitate by centrifugation at 10,000 rpm for 10 min at 4C.

9. Dissolve the pellet in TE buffer (1mM Tris HCL pH 8.0 and 0.1 mM EDTA pH 8.0 ).

10. Add Rnase A incubates for 1hr at 37C.

11. Extraction with equal volume of Chloroform: Isoamylalcohol (24:1).

12. Centrifuge the eppendorf tubes at 10,000 rpm at 4C for 10 min..

13. Recover the upper phase and to that add 1/10th volume of 1M NaCl and then add Ice-cold

Isopropanol and mix gently.

14. Keep the tubes for precipitation and centrifuge at 10,000 rpm for 10min. at 4C.

15. Wash the DNA pellet with 70% alcohol.

16. Dry the pellet and dissolve it in minimum quantity of TE buffer.

17. Store the DNA preparation at 4C.

18. Genomic DNA extracted was checked on 0.8 % agarose gel.

19. DNA was quantified using a spectrophotometer.

(5) DNA Quantification: -

(i) By Agarose gel electrophoresis- Gel Electrophoresis is used to quantify the DNA. The

agarose gel sorts the DNA with the aid of an electrical current. Following is the protocol for

Gel Electrophoresis.

1. Make Agarose gel.

A. Put a 0.8% Agarose in to flask, Add TBE buffer solution. Then melt agarose in oven.

B Place comb in mold. This forms wells in the gel to insert the DNA

samples. add 1.5 ul/100 ml Etbr in it. And pour in it.

C. After solidify, remove comb carefully and keep it in electrophoresis box.

26


27/48


28/48

concentration. For preparation of 10 um working stock take 10 ul of major stock and add 90 ul

of SMQ.

2.Preparation of PCR DNA stock: - For the preparation of 5ng/ul of PCR DNA stock we

calculate from the formula N1V1=N2V2 where N1=dsDNA concentration (from

spectrophotometer), V1=? N2= PCR DNAFinal conc., V2= Final volume of stock.

3. Stock preparation of dNTPs: - For the preparation of dNTPs stock we add

1000ul SMQ in each dNTP vial (Major stock), for working stock we take 25ul

From each vial.

(7) Screening and annealing temperature standardization of ISSR primers by

PCR Gradient 96: -

To perform several parallel reactions, prepare a master mix containing water, buffer, dNTPs,

primers and Taq DNA polymerase in a single tube, which can then be aliquoted into

individual tubes. MgCl2 and template DNA solutions are then added. This method of setting

reactions minimizes the possibility of pipetting errors and saves time by reducing the number

of reagent transfers.

Reaction mixture for per reaction as follow- (MASTER MIX) Table : 6

PCR components Volume for/ reaction

SMQ 2.4ul.

Taq Buffer 3.5ul.

Mgcl2 3.5ul.

dNTPs 5.0ul.

Taq polymerase 0.6ul.

Primer 8.0ul.

DNA template 2.0ul.

The thermocycler was programmed for:

Initial denaturation of 5 mins at 95C

42 cycles of- denaturation at 95C for 1 min

- annealing at 47C to 58C for 2 min.

- extension at 72C for 1.30 min.

A final synthesis step at 72C for 10 mins was given

28


29/48

Fig 8 Polymerase Chain Reaction

The reaction product was analyzed by electrophoresis on 1.5% Agarose gel using 1X TBE

buffer.

(4)RESULTS AND CONCLUSIONS: -

Isolated DNA quantified by 0.8% Agarose Gel electrophoresis.

Fig 9 Gel diagrame showing DNA quantity (Palampur sample PA1-PA20)

According to above gel diagram we found that last five sample lane are highly dense and

showing smear it means either not completely dissolved or degraded.

Lane 1& 2 - good band and quantity.

Lane 3 - moderate quantity,

Lane 4 No DNA quantity.

Lane 5 very thick band and also show smear.

29


30/48

Lane 6 to 13 moderate DNA quantity.

Lane 16 to 22 highly dense DNA quantity.

The dsDNA concentration quantity are measured by UV-VIS Spectrophotometer and also

the purity of DNA.Table-7- dsDNA concentration and purity of DNA of Palampur DNA sample.

S.No. Tree

No.

260nm 280nm Ratio dsDNA

concentration ug/ml.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

PA-1

PA-2

PA-3

PA-4

PA-5

PA-6

PA-7

PA-8

PA-9

PA-10

PA-11

PA-12

PA-13

PA-14

PA-15

PA-16

PA-17

PA-18

PA-19

PA-20

0.0059

0.0398

0.0260

0.0252

0.0112

0.0121

0.0162

0.0149

0.0136

0.0349

0.0051

0.0068

0.0031

0.0250

0.0477

0.0699

0.0703

0.0832

0.1893

0.2452

0.0032

0.0211

0.0148

0.0149

0.0069

0.0080

0.0090

0.0103

0.0075

0.0200

0.0023

0.0032

0.0015

0.0136

0.0278

0.0411

0.0378

0.0459

0.1034

0.1353

1.8428

1.8858

1.7498

1.6875

1.6158

1.5000

1.8105

1.4486

1.8061

1.7477

2.2555

2.0898

1.9808

1.8401

1.7158

1.7015

1.8585

1.8143

1.8314

1.8126

58.600

398.10

259.50

251.60

112.30

120.60

162.40

149.50

136.00

349.20

51.200

67.500

30.700

249.70

477.00

699.30

702.70

832.00

1893.10

2451.60

Table-8- dsDNA concentration and purity of DNA of Panchgani (Gure Gharh-GG-

Research Station) DNA samples.

30


31/48

S.No. Tree

No.

260nm 280nm Ratio dsDNA

concentration ug/ml.

1.

2.

3.

4.5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

GG-1

GG-2

GG-3

GG-4GG-5

GG-6

GG-7

GG-8

GG-9

GG-10

GG-11

GG-12

GG-13

GG-14

GG-15

GG-16

GG-17

GG-18

GG-19

GG-20

0.0027

0.0126

0.0052

0.00460.0244

0.0231

0.0056

0.0098

0.0064

0.0047

0.0174

0.0137

0.0152

0.0050

0.0313

0.0874

0.1116

0.1333

0.1143

0.0942

0.0010

0.0096

0.0028

0.00260.0181

0.0195

0.0039

0.0081

0.0050

0.0037

0.0121

0.0083

0.0104

0.022

0.0195

0.0516

0.0640

0.0810

0.0690

0.0563

2.5481

1.3211

1.8804

1.79381.3444

1.1874

1.4326

1.2111

1.2826

1.2869

1.4372

1.6522

1.4545

2.2851

1.6047

1.6964

1.7445

1.6469

1.6579

1.6734

26.500

126.30

51.900

46.100244.00

231.30

56.300

98.100

64.000

47.100

173.90

137.30

152.00

50.500

313.40

874.50

1115.60

1333.3

1143.3

942.30

ISSR-PCR Amplification result: - We screened 31 ISSR Primers, out of which 18 primers

shows amplification on temperature gradients (By temp. gradients- we also noted annealing

temperature of that particular ISSR primer).

Table : 9- ISSR Markers and their amplification status, Annealing temperature and

bands scored

S.No. ISSR

Primer

Sequences No.

of

Amplification

status

Annealing

Temp.

No. of

Amplified

Bands

31


32/48

Bases scored

1. HB12 CAC CAC CAC GC 11 Good 45c 6-7

2. HB15 GTG GTG GTG GC 11 Good 45-47 c 7

3. 17901 GTGTGTGTGTGTCA 14 Good 45-47 c 7-8

4. 17899A CACACACACACAAG 14 Good 45-47 c 5-8

5. 17899B CACACACACACAGG 14 Good 45-47 c 7-8

6. UBC 809 AGA GAG AGA GAG

AGA GG

17 Good 51c 6-7 Bands

7. UBC 813 CTC TCT CTC TCT

CTC TT

17 Moderate 50-52c 1-2 Bands


CTC TA

17 Poor -- Smear


CTC TG

17 Moderate 52c 2 Bands

10. UBC 816 CAC ACA CAC ACA

CAC AT

17 Good 50c 4 Bands


CAC AG


12. UBC 819 GTG TGT GTG TGT

GTG TA

17 Poor 52c Smear


GTG TC

17 Poor -- No Bands


GTG TT

17 Poor -- No Bands

15. UBC 822 TCT CTC TCT CTC

TCT CA

17 Poor -- No Bands


TCT CC

17 Poor -- No Bands


TCT CG

17 Poor -- No Bands

18. UBC 825 ACA CAC ACA CAC

ACA CT

17 Poor 56c 1 Band


ACA CC

17 Poor -- No Bands


ACA CG

17 Good 48-50c 3-4 Bands

21. UBC 828 TGT GTG TGT GTG

TGT GA

17 Poor -- No Bands


TGT GC



TGT GG

17 Moderate 50c 1Thickband


AGA GYC

18 Moderate 48-50c 3 Bands


AGA GYA


26. UBC 842 GAG AGA GAG AGA

GAG AYG

18 Good 53c 3 Bands

27. UBC 843 CTC TCT CTC TCT 18 Good 51-53c 5 Bands

32


33/48

CTC TRA


CTC TRG

18 Poor -- 1 Bands


CAC ARC

18 Poor -- Smear


CAC ARG

18 Moderate 52c 2 Bands


GTG TYC

18 Poor -- No Bands

Fig. 10 It shows good amplification with primer HB12 (Lane 2,3&4) and HB15

(Lane6,7&8)

33


34/48

Fig 11- Primer UBC 809 shows amplifications with template GG-15.

34


35/48

CHEMICAL ANALYSIS

CHEMICAL ANALYSIS

(1) INTRODUCTION: -

All plants produce chemical compounds as part of their normal metabolic activities. These

include primary metabolites, such as sugars and fats, found in all plants, and secondary

metabolites found in a smaller range of plants, some useful ones found only in a particular

genus or species. Pigments harvest light, protect the organism from radiation and display

colors to attract pollinators. Many common weeds have medicinal properties.

The functions of secondary metabolites are varied. For example, some secondary metabolitesare toxins used to deter predation, and others are pheromones used to attract insects for

pollination. Phytoalexins protect against bacterial and fungal attacks. Allelochemicals inhibit

rival plants that are competing for soil and light.

Plants upregulate and downregulate their biochemical paths in response to the local mix of

herbivores, pollinators and microorganisms. The chemical profile of a single plant may vary

35


36/48

over time as it reacts to changing conditions. It is the secondary metabolites and pigments

that can have therapeutic actions in humans and which can be refined to produce drugs.

Plants synthesize a bewildering variety of phytochemicals but most are derivatives of a few

biochemical motifs.

Alkaloids contain a ring with nitrogen. Many alkaloids have dramatic effects on the

central nervous system. Caffeine is an alkaloid that provides a mild lift but the

alkaloids in datura cause severe intoxication and even death.

Phenolics contain phenol rings. The anthocyanins that give grapes their purple color,

the isoflavones, the phytoestrogens from soy and the tannins that give tea its

astringency are phenolics.

Terpenoids are built up from terpene building blocks. Each terpene consists of two

paired isoprenes. The names monoterpenes, sesquiterpenes, diterpenes and triterpenes

are based on the number of isoprene units. The fragrance of rose and lavenderis due

to monoterpenes. The carotenoids produce the reds, yellows and oranges ofpumpkin,

corn andtomatoes.

Glycosides consist of a glucose moiety attached to an aglycone. The aglycone is a

molecule that is bioactive in its free form but inert until the glycoside bond is broken

by water or enzymes

During training our main goal is to do chemical analysis of Garcinia sps. In which the

leaves, twigs and fruits ofG.indica contain () hydroxy citric acid, which performs anti-

cholesterol and anti-obesity activity.

Fig 12 - Structure of () Hydroxy citric acid.

Besides this there is no report about the medicinal importance and chemical composition of

G.spicata and G.talbotii. Two prenylated xanthones, (1)1,4,5,6-tetrahydroxy-7,8-di(3-

methylbut-2-enyl)xanthone and (2)1,2,6-trihydroxy-5-methoxy-7-(3-methylbut-2-

36
http://en.wikipedia.org/wiki/Soyhttp://en.wikipedia.org/wiki/Tanninshttp://en.wikipedia.org/wiki/Tanninshttp://en.wikipedia.org/wiki/Tanninshttp://en.wikipedia.org/wiki/Terpenehttp://en.wikipedia.org/wiki/Isoprenehttp://en.wikipedia.org/wiki/Monoterpenehttp://en.wikipedia.org/wiki/Sesquiterpenehttp://en.wikipedia.org/wiki/Diterpenehttp://en.wikipedia.org/wiki/Triterpenehttp://en.wikipedia.org/wiki/Rosehttp://en.wikipedia.org/wiki/Lavenderhttp://en.wikipedia.org/wiki/Carotenoidhttp://en.wikipedia.org/wiki/Pumpkinhttp://en.wikipedia.org/wiki/Maizehttp://en.wikipedia.org/wiki/Tomatoeshttp://en.wikipedia.org/wiki/Tomatoeshttp://en.wikipedia.org/wiki/Glycosideshttp://en.wikipedia.org/wiki/Glucosehttp://en.wikipedia.org/wiki/Aglyconehttp://en.wikipedia.org/wiki/Soyhttp://en.wikipedia.org/wiki/Tanninshttp://en.wikipedia.org/wiki/Terpenehttp://en.wikipedia.org/wiki/Isoprenehttp://en.wikipedia.org/wiki/Monoterpenehttp://en.wikipedia.org/wiki/Sesquiterpenehttp://en.wikipedia.org/wiki/Diterpenehttp://en.wikipedia.org/wiki/Triterpenehttp://en.wikipedia.org/wiki/Rosehttp://en.wikipedia.org/wiki/Lavenderhttp://en.wikipedia.org/wiki/Carotenoidhttp://en.wikipedia.org/wiki/Pumpkinhttp://en.wikipedia.org/wiki/Maizehttp://en.wikipedia.org/wiki/Tomatoeshttp://en.wikipedia.org/wiki/Glycosideshttp://en.wikipedia.org/wiki/Glucosehttp://en.wikipedia.org/wiki/Aglycone


37/48

enyl)xanthone , were isolated from the wood ofGarcinia xanthochymus along with a known

xanthone.By using various Biochemical techniques like Column chromatography, Thin layer

chromatography, Acid base titration, HPLC, and Gas chromatography,NMR and mass

spectroscopy we isolate these important chemicals and also quantify that chemicals in plants.


Aim Chemical analysis of leaves, twigs of Garcinia sps. Like G.indica, G.spicata,

G.talbotii and G.xanthochymus and isolation and determination of () Hydroxy citric acid

from the rinds of fruits ofG.indica.

Objectives In chemical analysis so many objectives as follow-

With the help of Column chromatography and thin layer chromatography separate out

individual compounds.

Isolation of () hydroxy citric acid from the rinds of fruits ofG.indica

Determination of () HCA in extract by High Performance Liquid Chromatography.

(3) MATERIALS AND METHODS: -

1. Sample material The powder of leaves and twigs ofG.indica, G.spicata and G.talbotii.

2. Chromatographic requirements The following things are required for column

chromatography and Thin layer chromatography

(i) Glass Column (ii) TLC plate (iii) Test tubes (iv) Funnel (v) Chromatogram

chamber (vi) Glass plates (vii) Cotton (viii) Capillary tubes etc.

3. Chemicals and equipments: -

For Column Chromatography and TLC Following reagents are required for Column

chromatography and TLC-

37


38/48

Acetone

Methanol

Ethanol

Chloroform

Petroleum ether

Combination of different ratio of Methanol and Chloroform.

For isolation and determination of () hydroxy citic acid Following reagents are

required-

Activated charcoal

Ethanol

10 mM Sulfuric acid

Following equipments are required-

Autoclave for extraction of HCA from fruits

Rota vapour for concentrate the extract.

Centrifuge for removing heavy particle and protein material from concentrated

extract with ethanol.

HPLC for determination of () HCA in extract.

4. Preparation of sample materials

Take Twigs and grind them and make fine powder in mixer or grinder

Weigh this powder and add silica five times of plant sample weight

Add some volume of Methanol for complete coating of plant sample with silica

Dry it and fill it in glass bottle or use as sample in Column Chromatography.

5. Elute out of different fraction by Column Chromatography

1. Take glass column and wash it with Methanol or Acetone. Fit it at stand.

2. Fit small cotton in its end and add silica and then sample material.

38


39/48

3. Pour 100ml. of each combination of different ratio of Methanol and Chloroform for

elution (as shown in below Table).

4. Collect different fraction in separate test tube (approx.-20ml.).

Fig 13 Elution of different fraction and graph showing time taken by each fraction

elution.

Table: 10 Different ratios of Methanol and Chloroform for elution

S.

No.

Reagents

Volume of

each

reagents(tot

al vl.-100ml)

Ratio

1. Methanol- Chloroform 0ml.100ml.

0:10

2. Methanol- Chloroform 10ml.

90ml.

1:9


80ml.

2:8


70ml.

3:7

39


40/48


60ml.

4:6


50ml.

5:5


40ml.

6:4

8. Methanol- Chloroform 70ml.30ml.

7:3


20ml.

8:2


10ml.

9:1


0ml.

10:0

.

6. Separation of different compounds by Thin layer Chromatography: -

Thin layer chromatography (TLC) is among the most useful tools for following the progress of

organic chemical reactions and for assaying the purity of organic compounds. Thin layerchromatography (TLC) is a chromatography technique used to separate mixtures. Thin layer

chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated

with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose. This

layer of adsorbent is known as the stationary phase.

After the sample has been applied on the plate, a solvent or solvent mixture (known as the

mobile phase) is drawn up the plate via capillary action. Because different analytes ascend

the TLC plate at different rates, separation is achieved.

Experiment: -

Take the TLC plates are usually commercially available, with standard particle size ranges to

improve reproducibility.

The plate is activatedby heating in an oven for ten minutes at 110 C.

40


41/48

A small spot of solution containing the sample is applied to a plate, about one centimeter

from the base

After all spoting the plate is then dipped in to a suitable Mobile phase, and placed in a sealed

container.

The solvent moves up the plate by capillary action and meets the sample mixture, which is

dissolved and is carried up the plate by the solvent. Different compounds in the sample

mixture travel at different rates due to the differences in their attraction to the stationary

phase, and because of differences in solubility in the solvent.

After movement take out TLC plate and dry it and then spraying is done for different

colour bands production.

Fig 14 Thin Layer Chromatography and movement of sample on stationary phase by

Mobile phase.

41
http://en.wikipedia.org/wiki/Solventhttp://en.wikipedia.org/wiki/Capillary_actionhttp://en.wikipedia.org/wiki/Chemical_compoundhttp://en.wikipedia.org/wiki/Solventhttp://en.wikipedia.org/wiki/Capillary_actionhttp://en.wikipedia.org/wiki/Chemical_compound


42/48

For the separation of different fraction of twigs of G.indica on TLC plate (by column

chromatography) we used different Mobile phase as follow -

Table: 11- Different mobile phases

S.No. Mobile phase reagents and their ratios

1. 1.5% Acetone-Chloroform

2. 20% Acetone-Pet ether 3. 30% Methanol- Chloroform

4. 15% Methanol- Chloroform

5. 5% Acetone- 10% methanol in Chloroform

6. 1% Methanol-Chloroform



9. 7% Methanol- Chloroform


Spraying reagents: - 5%H2SO4in Methanol is used as a spraying agent.

TLC plates are also dipped in a solution as follow for band appearance.

2% Acetic acid + 3.2% Anisaldehyde + 3.2% H2SO4

in 100 ml. Ethanol solution

(7)Isolation of () Hydroxy citric acid from the rinds ofG.indica fruits : -

25gm.ofG.indica fruit rinds were autoclaved at 15lbs/inch2 pressure with 50ml. of water for

20 min. and filtered.

Autoclaving and filtration was repeated 2-3 times for complete extraction of the () HCA.

Then the dark brown extract was decolorized using activated charcoal and filtered.

The decolorized extract was then concentrated in vacuum on rota-vapour.

Then concentrated extract was treated with 50ml. of Ethanol to remove pectinaceous

materials and centrifuged at10000rpm for 10 min.

42
http://en.wikipedia.org/wiki/Hydrogenhttp://en.wikipedia.org/wiki/Hydrogenhttp://en.wikipedia.org/wiki/Sulfatehttp://en.wikipedia.org/wiki/Sulfatehttp://en.wikipedia.org/wiki/Hydrogenhttp://en.wikipedia.org/wiki/Sulfatehttp://en.wikipedia.org/wiki/Sulfatehttp://en.wikipedia.org/wiki/Hydrogenhttp://en.wikipedia.org/wiki/Sulfatehttp://en.wikipedia.org/wiki/Hydrogenhttp://en.wikipedia.org/wiki/Sulfate


43/48

Take supernatant and again concentrate it under reduced pressure and then stored at 4C.

Then this sample can applied for HPLC for determination of quantity and quality.

(8) Working Mechanism of High Performance Liquid Chromatography (HPLC): -

High performance liquid chromatography is a powerful tool in analysis.

Introduction

High performance liquid chromatography is basically a highly improved form of column

chromatography. Instead of a solvent being allowed to drip through a column under gravity,

it is forced through under high pressures of up to 400 atmospheres. That makes it much

faster.

The column and the solvent

Confusingly, there are two variants in use in HPLC depending on the relative polarity of the

solvent and the stationary phase. There are of two types of HPLC.

1. Normal phase HPLC- This is essentially just the same as we already have read about in

thin layer chromatography or column chromatography. Although it is described as "normal",

it isn't the most commonly used form of HPLC.

The column is filled with tiny silica particles, and the solvent is non-polar - hexane, for

example. A typical column has an internal diameter of 4.6 mm (and may be less than that),

and a length of 150 to 250 mm.

43


44/48

2. Reversed phase HPLC - In this case, the column size is the same, but the silica is

modified to make it non-polar by attaching long hydrocarbon chains to its surface - typically

with either 8 or 18 carbon atoms in them. A polar solvent is used - for example, a mixture of

water and an alcohol such as methanol.

In this case, there will be a strong attraction between the polar solvent and polar molecules in

the mixture being passed through the column. There won't be as much attraction between the

hydrocarbon chains attached to the silica (the stationary phase) and the polar molecules in

the solution. Polar molecules in the mixture will therefore spend most of their time moving

with the solvent.

In it the polar molecules that will travel through the column more quickly.

Reversed phase HPLC is the most commonly used form of HPLC.

A flow scheme for HPLC

Injection of the sample

Injection of the sample is entirely automated, and you wouldn't be expected to know how this

is done at this introductory level. Because of the pressures involved, it is notthe same as in

gas chromatography (if you have already studied that).

44


45/48

Retention time

The time taken for a particular compound to travel through the column to the detector is

known as its retention time. This time is measured from the time at which the sample is

injected to the point at which the display shows a maximum peak height for that compound.

The detector

There are several ways of detecting when a substance has passed through the column. A

common method which is easy to explain uses ultra-violet absorption. Many organic

compounds absorb UV light of various wavelengths. If you have a beam of UV light shining

through the stream of liquid coming out of the column, and a UV detector on the opposite

side of the stream, you can get a direct reading of how much of the light is absorbed.

The amount of light absorbed will depend on the amount of a particular compound that is

passing through the beam at the time.

45


46/48

You might wonder why the solvents used don't absorb UV light. They do! But different

compounds absorb most strongly in different parts of the UV spectrum.

The output will be recorded as a series of peaks - each one representing a compound in the

mixture passing through the detector and absorbing UV light.

(4) RESULTS AND CONCLUSIONS: -

By Column Chromatography different fractions are collected on the basis of different colors

and up to 20ml. The flow rate of elution is-

20ml. elution in 6 minutes so, flow rate is-

20/6 = 6.66

Then these fractions are used as samples for Thin Layer Chromatography and did separation

of these samples. We used different mobile phase for clear and appropriate separation.

46


47/48

Fig 15- First lane leaf sample elution and second lane twig sample elution separated

in 1% Methanol-Chloroform mobile phase.

We used different Mobile phase, in which we found that 1% Methanol-Chloroform is good

mobile phase. In this Mobile phase the compound was separated easily and well defined (in

figure A). As we shown that in figure (B) there are not good separation.

(B)

Fig 16 Leaves and twigs elution in 20% Acetone-Petroleum Ether.

47


48/48

This Report is due to my God Jai Shree Hanuman

Documents

moleular Biology