COLUMN CHROMATOGRAPHY - SEPERATION OF THE MIXTURE OF COMPONENTS IJN TO INDIVIDUAL COMPONENTS BY USING STATIONARY PHASE AND MOBILE PHASE UPON THE USING OF COLUMN
Chromatography : A seperation techniqueSHIVANEE VYAS
Chromatography is a method of seperating mixture of components into individual components through equlibrium distribution between two phases.
Each chromatographic method essentially consists of 2 phases a staionary phase and a mobile phase.
Stationary phase : solid or liquid
Mobile phase : liquid or gas
A chromatogrpahic technique widely employed for identification of certain organic compounds. Applied in laboratories of colleges as a teaching tool for easy understanding the thechnique of chromatography.
• Chromatography is a method of separation in which the components to be separated are distributed between two phases, one of these is called a stationary phase and the other is a mobile phase which moves on stationary phase in a definite direction
Paper chromatography is an analytical method used to separate colored chemicals or substances. It is primarily used as a teaching tool, having been replaced by other chromatography methods, such as thin-layer chromatography.
COLUMN CHROMATOGRAPHY - SEPERATION OF THE MIXTURE OF COMPONENTS IJN TO INDIVIDUAL COMPONENTS BY USING STATIONARY PHASE AND MOBILE PHASE UPON THE USING OF COLUMN
Chromatography : A seperation techniqueSHIVANEE VYAS
Chromatography is a method of seperating mixture of components into individual components through equlibrium distribution between two phases.
Each chromatographic method essentially consists of 2 phases a staionary phase and a mobile phase.
Stationary phase : solid or liquid
Mobile phase : liquid or gas
A chromatogrpahic technique widely employed for identification of certain organic compounds. Applied in laboratories of colleges as a teaching tool for easy understanding the thechnique of chromatography.
• Chromatography is a method of separation in which the components to be separated are distributed between two phases, one of these is called a stationary phase and the other is a mobile phase which moves on stationary phase in a definite direction
Paper chromatography is an analytical method used to separate colored chemicals or substances. It is primarily used as a teaching tool, having been replaced by other chromatography methods, such as thin-layer chromatography.
intoduction to lumiscence
introduction and principle of chemilumiscence
different types of lumiscence
detail of the electrochemilumiscence, working, principle, instrumentation, measurin.
application in medical field
difference between chemilumiscence and elecrochemiluminescence
This presentation include information about electron microscope & types of electron microscope i.e. SEM (Scanning electron microscope) & TEM (Transmission electron microscope).
An electron microscope is a microscope that uses a beam of scattered electrons as a source of illumination. It is used to get information about structure, topology, morphology & composition of materials. It has many advantages. Basically there are 4 types of electron microscope but here we will discuss only 2 types.
Transmission electron microscopy is a microscopy technique in which a beam of electrons is transmitted through an ultra-thin specimen, interacting with the specimen as it passes through it. Its resolution & magnification is about 10,000,000x. There are 5 types of transmission electron microscope i.e. BFTEM (Bright field transmision electron microscope), DFTEM (Dark field transmission electron microscope), HRTEM (High resolution transmission electron microscope), EFTEM (Energy filtered transmission electron microscope), ED (Electron diffraction). there are 4 techniques of TEM i.e. negative staining, shadow casting, Freeze fracture replication, freeze etching. It has many applications e.g, for the study of Cancer research, virology, chemical industry, electronic structure etc.
A scanning electron microscope is a type of electron microscope that produces images of a sample by scanning it with a focused beam of electrons. Types of signals produce by SEM include secondary electrons, back scattered electrons, X-rays, light rays. There are many advantages of SEM e.g, Btter resolution, fast imaging easy to operate, work with low voltage etc.
electrophoresis-
principle
types
details on paper electrophoresis
cellulose acetate electrophoresis
zone electrophoresis
SDS-PAGE
iso-electric focussing gel electrophoresis
two-dimensional gel electrophoresis
pulsed gel electrophoresis
isotachophoresis
capillary electrophoresis
microchip electrophoresis
Thin layer chromatography technique - easier, cheaper.
Handling is easy. Used as an identification test also purity test. It comprises of stationary and mobile phase. There are various types of chromatography technique. TLC consists of three steps - spotting, development, and visualization. The Rf value is used to quantify the movement of the materials along the plate. Rf is equal to the
distance traveled by the substance divided by the distance traveled by the solvent. Its value is
always between zero and one. A TLC analysis might be summarized something like, "Using a silica
gel plate and ethyl acetate as the development solvent, unknown mixture X showed three spots
having Rf's of 0.12, 0.25, and 0.87". CThere are three components in TLC:
(1) the TLC plate (stationary phase), the development solvent (mobile phase), and the sample to be
analyzed (solute). In our experiment the TLC plate consists of a thin plastic sheet covered with a
thin layer of silica gel.
INTRODUCTION TO PAPER CHROMATOGRAPHY
Cellulose filter paper is often used as the statioary phase in the paper chromatography. Since it is hydrophillic, it is usually covered with thin film of water. The procedure is often regarded as liquid-liquid cromatography
Other liquids can be encorporated in place of water, thus provides different type of stationary phase. Eg. Paper treated with silicone or paraffin oil permits reverse phase-paper chromatography, in which mobile phase is a polar solvent.
There are some commercially available papers that contain an adsorbent or an ion-exchange resin, thus permmits adsorption and ion-exhange paper chromatography.
PRINCIPLE OF CHROMATOGRAPHY
This is type of partition chromatography in which the substance are distributed between two liquids that is one is the stationary liquid (usually water) which is held in the fibres of paper and called the stationary phase; the other is the moving liquid or developing solvent and called the mobile phase. The components of mixture to be separated migrates at different rates as its solubility between two phases and appear as spot at different points on the paper.
In this technique, a drop of the test solution is applied as a small spot on a filter paper and the spot is dried. The paper is kept in close chamber and the edge of filter is dipped into a solvent called as developing solvent. As soon as filter paper gets liquids via capillary action and reaches to the spot of the test solution then various substances are moved by solvent with various speeds. When solvent move up to suitable height (15-18) the paper is dried and various spot are visualised by suitable reagent called visualising reagent.
MIGRATION PARAMETERS
1) RF VALUE(RETENSION FACTOR) :- It is ratio of the solute’s distance travelled to solvent’s distance travelled.
It is constant for a given substance, provided the conditions of chromatographic system are kept constant with respect to tempreture, type of paper, duration and direction of development, nature and the shape and the size of the wick used (i.e., radial chromatography), the amount of liquid in the reservoir, humidity etc.
The Rf of of a substance depends upon a number of factors which are:
The solvent employed
The medium used for separation i.e., the quality of paper chromatography
The nature of mixture
The tempreture
Size of vessel in which operation has been carried out
It is possible to compare the Rf Values of different substances keeping above factor constant
types of paper chromatogtaphy
ascending, descending, ascending-descending, radial , two diamentional chromatography
describe about planar chromatography-classification,separation,procedure,application.
it is the mixing of both paper chromatography and thin layer chromatography.
Describe about planar chomatography- classificatn,separation ,procedure nd application.planar chromatography is the different technique is the mixing of both paper chromatography and thin layer chromatography.
Chromatography- Principles and application of chromatographySanchit Dhankhar
Laboratory technique for the Separation of mixtures
Chroma -"color" and graphein - "to write”.
Colour bands - separation of individual compounds
Measured or analysed.
Analytical
Determine Chemical composition of a sample
Preparative
Used to purify sufficient quantities of a substance
Chromatograph - equipment that enables a sophisticated
separation
EX. Gas chromatography or Liquid chromatography
Eluent - Fluid entering column/ solvent that carries the analyte.
Eluate - Mobile phase leaving the column.
Stationary phase - Immobilized phase
Immobilized on the support particles or on the inner wall of the column tubing.
Examples : Silica layer - Thin Layer Chromatography
Detailed chapter on Medical Lipid chemistry under different heading. The content is designed keeping the course in the view - MBBS, BDS, BPT, Nursing, BSc, MSc etc
Vitamin C and Vit B1 to B6 by Dr Anurag YadavDr Anurag Yadav
Details related to the Vitamin C and Vitamin B1 to B6. The biochemistry of these water soluble vitamins are explained under all the necessary heading.
Useful for students of MBBS, BDS, BPT, Nursing, BSc, MSc etc
Biochemistry of Calcium metabolism covering the source, factors effecting absorption, normal level of calcium, regulation of the calcium, hypercalcemia, hypocalcemia, disorders related to calcium and bone markers.
Useful for students of MBBS, BDS, BSc, MSc, MLT, Physiotherapy (BPT), Nursing etc.
Total Quality Management (TQM) by Dr Anurag YadavDr Anurag Yadav
Laboratory Total Quality Management, Concept of Laboratory errors, the quality control material, quality assurance program, factors affecting the quality of report, Steps in quality management, PDCA cycle, accuracy, precision, EQAS, IQAS, Proficiency testing.
the details are related to medical laboratory and help MBBS, MD, BSc MLT, MSc MLT, etc
The brief classification, types, physical properties, chemical properties, mucopolysaccherides type, disorders related to GAG.
the Topic covered with the interest of MBBS, BDS, BPT, Nursing, Bsc and MSc Biochemistry and MLT students
Plasma proteins, the components of plasma proteins, the protein fractions and condition causing the alteration in the each protein fraction. Clinical implications of the each fraction, the electrophorotic pattern of plasma protein. Acute phase proteins which include the positive and negative phase proteins.
Prix Galien International 2024 Forum ProgramLevi Shapiro
June 20, 2024, Prix Galien International and Jerusalem Ethics Forum in ROME. Detailed agenda including panels:
- ADVANCES IN CARDIOLOGY: A NEW PARADIGM IS COMING
- WOMEN’S HEALTH: FERTILITY PRESERVATION
- WHAT’S NEW IN THE TREATMENT OF INFECTIOUS,
ONCOLOGICAL AND INFLAMMATORY SKIN DISEASES?
- ARTIFICIAL INTELLIGENCE AND ETHICS
- GENE THERAPY
- BEYOND BORDERS: GLOBAL INITIATIVES FOR DEMOCRATIZING LIFE SCIENCE TECHNOLOGIES AND PROMOTING ACCESS TO HEALTHCARE
- ETHICAL CHALLENGES IN LIFE SCIENCES
- Prix Galien International Awards Ceremony
These lecture slides, by Dr Sidra Arshad, offer a quick overview of physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar leads (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
ARTIFICIAL INTELLIGENCE IN HEALTHCARE.pdfAnujkumaranit
Artificial intelligence (AI) refers to the simulation of human intelligence processes by machines, especially computer systems. It encompasses tasks such as learning, reasoning, problem-solving, perception, and language understanding. AI technologies are revolutionizing various fields, from healthcare to finance, by enabling machines to perform tasks that typically require human intelligence.
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
Pulmonary Thromboembolism - etilogy, types, medical- Surgical and nursing man...VarunMahajani
Disruption of blood supply to lung alveoli due to blockage of one or more pulmonary blood vessels is called as Pulmonary thromboembolism. In this presentation we will discuss its causes, types and its management in depth.
Factory Supply Best Quality Pmk Oil CAS 28578–16–7 PMK Powder in Stockrebeccabio
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MANAGEMENT OF ATRIOVENTRICULAR CONDUCTION BLOCK.pdfJim Jacob Roy
Cardiac conduction defects can occur due to various causes.
Atrioventricular conduction blocks ( AV blocks ) are classified into 3 types.
This document describes the acute management of AV block.
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
Ethanol (CH3CH2OH), or beverage alcohol, is a two-carbon alcohol
that is rapidly distributed in the body and brain. Ethanol alters many
neurochemical systems and has rewarding and addictive properties. It
is the oldest recreational drug and likely contributes to more morbidity,
mortality, and public health costs than all illicit drugs combined. The
5th edition of the Diagnostic and Statistical Manual of Mental Disorders
(DSM-5) integrates alcohol abuse and alcohol dependence into a single
disorder called alcohol use disorder (AUD), with mild, moderate,
and severe subclassifications (American Psychiatric Association, 2013).
In the DSM-5, all types of substance abuse and dependence have been
combined into a single substance use disorder (SUD) on a continuum
from mild to severe. A diagnosis of AUD requires that at least two of
the 11 DSM-5 behaviors be present within a 12-month period (mild
AUD: 2–3 criteria; moderate AUD: 4–5 criteria; severe AUD: 6–11 criteria).
The four main behavioral effects of AUD are impaired control over
drinking, negative social consequences, risky use, and altered physiological
effects (tolerance, withdrawal). This chapter presents an overview
of the prevalence and harmful consequences of AUD in the U.S.,
the systemic nature of the disease, neurocircuitry and stages of AUD,
comorbidities, fetal alcohol spectrum disorders, genetic risk factors, and
pharmacotherapies for AUD.
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
2. The word derived from Greek:
Initial described by Mikhail Tswett in 1903.
• colorChroma
• To writegraphein
2
DrAnuragYadav
3. CHROMATOGRAPHY
“Chromatography is a technique for separating
mixtures into their components in order to analyze,
identify, purify, and/or quantify the mixture or
components.”
Separate
• Analyze
• Identify
• Purify
• Quantify
ComponentsMixture 3
DrAnuragYadav
4. TERMINOLOGIES:
Chromatograph - equipment that enables a sophisticated
separation
EX. Gas chromatography or Liquid chromatography
Eluent - Fluid entering column/ solvent that carries the analyte.
Eluate - Mobile phase leaving the column.
Stationary phase - Immobilized phase
Immobilized on the support particles or on the inner wall of
the column tubing.
Examples : Silica layer - Thin Layer Chromatography
4
DrAnuragYadav
5. TERMINOLOGIES:
Mobile phase
Moves in a definite direction. Liquid (LC), Gas (GC).
The mobile phase moves through the chromatography column (the
stationary phase) where the sample interacts with the stationary phase
and is separated.
Retention time : Time takes for a particular analyte to pass through the
system (from the column inlet to the detector) under set conditions.
Sample (Anylate) :Substance analyzed in chromatography.
Solvent : Any substance capable of solubilizing another
substance.
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6. Chromatogram
Visual output of the chromatograph.
Separation - Different peaks or patterns on the chromatogram
correspond to different components of the separated mixture.
6
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7. CLASSIFICATION:
1. Based on supporting medium
2. Based on mobile & stationary phase
3. Based on mechanism of separation
7
DrAnuragYadav
8. 1. BASED ON SUPPORTING MEDIUM
Supporting
medium
planar column
8
DrAnuragYadav
9. 2. BASED ON MOBILE & STATIONARY PHASE:
MOBILE AND STATIONARY
PHASE
GC
GLC GSC
LC
LLC LSC
9
DrAnuragYadav
11. 3. BASED ON MECHANISM OF SEPARATION:
MECHANISM
ION
EXCHANGE
PARTITION
ADSORPTION
AFFINITY
SIZE
EXCLUSION
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DrAnuragYadav
12. PLANAR CHROMATOGRAPHY:
Based on principle of the partition
Chromatography.
“The differential distribution of solute between two
immiscible liquids on plane.”
12
DrAnuragYadav
13. partition chromatography a process of separation of solutes utili
zing the partition of the solutes between two liquid phases, namely
the original solvent and the film of solvent.
When substance mixed
with immiscible solvent,it
will distribute such that,
At equilibirum the ratio of
its conc In two phase is
constant
13
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14. This ratio is termed as partition coefficient & is
characteristic of a particular substance for a given
pair of solvent.
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15. TYPES OF PARTITION CHROMATOGRAPHY
Normal phase
Reverse phase: Ion suppression & Ion pair
chromatography
Normal phase LC, stationary phase is polar & mobile
phase is non-polar. water is the stationary phase; hexane,
benzene, chloroform or butanol form the mobile phase.
Reverse phase LC, stationary phase is non-polar (eg.
octadecyl silane packing in a column) and mobile phase
is polar (solvents like methanol, acetonitrile used in
column mode of chromatography). 15
DrAnuragYadav
16. ION-SUPPRESSION CHROMATOGRAPHY
Ionic character of weakly acidic/basic→suppressed(by
modification of mobile phase PH →solutes become less
polar →interact with nonpolar stationary phase →reverse
phase chromatography
16
DrAnuragYadav
17. ION-PAIR CHROMATOGRAPHY
Counter ion of analyte → added to mobile phase →ionic
pair with analyte →neutralysed analytes are separated by
reverse phase chromatography
Uses : separation of therapeutic drugs & metabolites.
17
DrAnuragYadav
18. PAPER CHROMATOGRAPHY :
Paper chromatography is a variant of partition
chromatography procedure in which the cellulose
support is in the form of a sheet or paper
Cellulose contain a large amount of bound water even
when extensively dried
Partitioning occurs between the bound water and the
developing solvent
In paper chromatography the mixture to be separated is
spotted onto the paper and dried
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DrAnuragYadav
20. Paper Chromatography has different types or
modes:
Ascending chromatography: As
the name indicates, the
chromatogram ascends. Here the
development of paper occurs due
the solvent movement or travel in
upward direction on the paper.
Descending chromatography: Here the development of paper occurs
due to solvent travel downwards on the paper.
Ascending- descending mode: Here solvent first travels
upwards and then down wards on the paper. 20
DrAnuragYadav
21. Radial mode: Here the solvent travels from center(mid point) towards
periphery of Circular chromatography paper.
Two dimensional chromatography: Here
the chromatogram development occurs in
two directions at right angles.
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23. INSTRUMENTATION
Chromatography jar:
It is made of glass and has a lid on it. Jar maintains
proper environment that is required for separation.
Capillary tube:
It is used to apply sample mixture.
Stationary phase:
liquid impregnated paper
23
DrAnuragYadav
24. INSTRUMENTATION
Mobile phase:
Mobile phase may be a single liquid or a mixture of
liquids.
Commonly used mobile phases are;
Methanol
Ethanol
Ethyl acetate
Diethyl ether
Acetone
Chloroform 24
DrAnuragYadav
26. 1. PREPARING THE PAPER STRIPS
• Cut the filter paper into 5
x4 measurement.
• Draw a line 0.5 cm above
the bottom edge of the
strip with the pencil.
• Label each strip with its
corresponding solution.
•
• Place a spot from each
pen on your starting line.
26
DrAnuragYadav
27. 2. DEVELOPING THE CHROMATOGRAMS
• Place the strips in the beakers.
• Make sure the solution does not come
above your start line.
• Keep the beakers covered.
• Let strips develop until the ascending
solution front is about 2 cm from the top
of the strip.
• Remove the strips and let them dry.
27
DrAnuragYadav
28. More polar!
Less polar!
solvent front
origin mixture
solvent front
component B
component A
origin
solvent front
component B
component A
origin
Increasing Development Time
28
DrAnuragYadav
29. VISUALIZATION OF CHROMATOGRAPHY
If the sample is separated into colored components, then
the location is dried in ordinary light. But in case of
colorless components following are used;
Uv lamp
Iodine crystals
Spraying agents: Ninhydrin for aminoacids and
proteins , sulfuric acid for phospholipids ,
diphenylamine for sugars 29
DrAnuragYadav
30. DOCUMENTATION
Storage of chromatogram.
Calculating Rf values
Calculate Rf value & interpret.
Defined : “as the ratio of the distance travelled by
the substance & the distance travelled by solvent
front”
30
DrAnuragYadav
31. RF VALUE IMPORTANCE:
Ratio of distance travelled by the solute to the
distance travelled by the solvent
Rf value is constant for a particular solvent system
at a given temperature
Spots of the unknown substance can be identified
by comparing those of the pure standards
31
DrAnuragYadav
32. APPLICATIONS
It is used for separation and identification of;
Amino acids
Carbohydrates
Tannins
Glycosides
Alkaloids etc.
32
DrAnuragYadav
33. THIN-LAYER CHROMATOGRAPHY (TLC)
“ The technique which involves flowing of mobile phase over a
thin layer of adsorbent, applied on solid support, where
separation of components occur by differential migration
which occurs when solvent flows along fine powder spread on
glass plates, is called thin –layer chromatography.”
Silica / Alumina layered over a glass plate ----- uniform thin
layer(0.2mm)
Procedure same as that for paper
chromatography.
Better resolution
HPTLC: particle size-4.5μm. 33
DrAnuragYadav
35. Chromatography jar:
It is made of glass and has a lid on
it.
Jar maintains proper environment
that is required for separation.
Capillary tube:
It is used to apply sample mixture
on TLC plate.
TLC plate:
Borosilicate glass plates are
preferred. Most commonly used
sizes are;
20 X 20cm
20 X 10cm
20 X 5cm
Mobile phase:
Mobile phase may be a single
liquid or a mixture of liquids.
Commonly used mobile phases
are;
Methanol
Ethanol
Ethyl acetate
Diethyl ether
Acetone
Chloroform
35
DrAnuragYadav
37. APPLICATION:
It is used for separation and identification of;
Amino acids
Peptides and proteins
Alkaloids
Carbohydrates
Fats and fatty acids
Antibiotics
Narcotic analgesics
Glycosides 37
DrAnuragYadav
38. ADVANTAGES OF TLC
Simple
Rapid
Ability to process large number of samples in minimal
time
Low cost in terms of reagent & equipment.
38
DrAnuragYadav
39. APPLICATIONS OF CHROMATOGRAPHY.
• In clinical diagnosis : detection & estimation of amino
acids, metabolites, sugars, mucopolysaccharides in urine
& blood.
• Useful for screening and diagnosis of inborn metabolic
disorders : Aminoacidurias, hemoglobinopathies,
mucopolysaccharidoses, etc.
39
DrAnuragYadav
40. APPLICATIONS OF CHROMATOGRAPHY
In clinical diagnosis
Paper chromatography,TLC –qualitative
HPLC, GC –For quantitation
In clinical diagnosis
Assay of Hormones, drugs, vitamins,
metabolites ---HPLC and GC
40
DrAnuragYadav
41. APPLICATIONS OF CHROMATOGRAPHY
Chromatography in protein research –
1) Purification –adsorption, ion exchange ,affinity, gel
filtration chromatography.
2) Sequencing – ion-exchange chromatography.
3) Mol.wt. determination – gel filtration chromatography.
41
DrAnuragYadav
42. TROUBLESHOOTING
It seems to be easy procedure, but error do
occur like;
- Compound runs as streak rather than spot
(sample was overloaded)
- Sample run as smear/upward crescent :
compound possess strongly acidic or basic
group- add few drops of ammonium hydroxide
or acetic acid to the solvent.
- Sample runs downward crescent- adsorbent
was disturbed during spotting.
streak
More polar
Cross placed
Less sample
Normal
42
DrAnuragYadav
43. - Plate solvent front runs crookedly:
either the adsorbent has flaked off the sides
or the side of plate are touching sides of
container.
- Many random spots
- No spots are seen on plate
- Blur blue spots on plate : use of ink pen
instead of pencil to mark the origin.
streak
More polar
Cross placed
Less sample
Normal
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DrAnuragYadav
46. ADSORBENTS:
“ An adsorbent is a substance, usually porous in nature and with a high surface area that can adsorb substances onto its surface by intermolecular forces.”
AN IDEAL ADSORBENT:
The Ideal adsorbent must fulfill the following requirements:
Insoluble in mobile phase
Inert to solutes (adsorptive)
Colorless especially when work with colored mixtures
Suitable particle size enough to give good separation and reasonable flow rate
COMMON ADSORBENTS:
Hydrated silica gel
Silica gel G
Silica gel S
Silica gel GF 254
Silica gel H
Silica gel N
Silica gel HF 254
Silica gel PF 254
Modified silica gel
Alumina
Kieselghur (Diatomaceous earth)
Cellulose MN 300
Cellulose microcrystalline
46
DrAnuragYadav
47. THIN LAYER CHROMATOGRAPHY (TLC)
In TLC, any substance that can be finely divided and
formed into a uniform layer can be used.
Both organic and inorganic substances can be used
to form a uniform layer for TLC.
Organic substances include: cellulose, polyamide,
polyethylene
Inorganic: silica gel, aluminum oxide and magnesium
silicate
47
DrAnuragYadav
48. THIN-LAYER CHROMATOGRAPHY: A
TWO-COMPONENT MIXTURE
More polar!
Less polar!
solvent front
origin mixture
solvent front
component B
component A
origin
solvent front
component B
component A
origin
Increasing Development Time
48
DrAnuragYadav
49. APPLICATIONS
1. Separation of carbohydrates:
Mobile phase:
acetonitrile : water (85:15)
Detection:
sulfuric acid : methanol (1:3)
heat for 10 min at 110 C to see
brown spots
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DrAnuragYadav
50. Separation of Total Lipid into
different Classes
Mobile Phase: hexane: diethyl ether: formic acid (80:20:2)
Cholesteryl esters
TAG
Free fatty acids
Cholesterol
1,3-DAG
1,2-DAG
Monoacyl glycerols
Phospholipids
50
DrAnuragYadav
51. Separation of Triacylglycerols
Mobile Phase: Pet ether: diethyl ether: acetic acid (90:9:1)
Tristearin
2-oleodistearin
1-stereodiolein
Triolein
Trolinolein
With HUFA
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DrAnuragYadav
Editor's Notes
All of the above (including the procedure page) might sound like TLC is quite an easy procedure. But
what about the first time you run a TLC, and see spots everywhere and blurred, streaked spots? As
with any technique, with practice you get better. One thing you have to be careful Examples of
common problems encountered in TLC:The compound runs as a streak rather than a spot
The sample was overloaded. Run the TLC again after diluting your sample. Or, your sample might
just contain many components, creating many spots which run together and appear as a streak.
Perhaps, the experiment did not go as well as expected.
The sample runs as a smear or a upward crescent.
Compounds which possess strongly acidic or basic groups (amines or carboxylic acids) sometimes
show up on a TLC plate with this behavior. Add a few drops of ammonium hydroxide (amines) or
acetic acid (carboxylic acids) to the eluting solvent to obtain clearer plates.
The sample runs as a downward crescent.
Likely, the adsorbent was disturbed during the spotting, causing the crescent shape.
The plate solvent front runs crookedly.
Either the adsorbent has flaked off the sides of the plate or the sides of the plate are touching the
sides of the container (or the paper used to saturate the container) as the plate develops.
Crookedly run plates make it harder to measure Rf values accurately.
Many, random spots are seen on the plate.
Make sure that you do not accidentally drop any organic compound on the plate. If get a TLC
plate and leave it laying on your workbench as you do the experiment, you might drop or splash
an organic compound on the plate.
No spots are seen on the plate.
You might not have spotted enough compound, perhaps because the solution of the compound is
too dilute. Try concentrating the solution, or, spot it several times in one place, allowing the
solvent to dry between applications. Some compounds do not show up under UV light; try
another method of visualizing the plate. Or, perhaps you do not have any compound because
your experiment did not go as well as planned.
If the solvent level in the developing jar is deeper than the origin (spotting line) of the TLC plate,
the solvent will dissolve the compounds into the solvent reservoir instead of allowing them to
move up the plate by capillary action. Thus, you will not see spots after the plate is developed.
You see a blur of blue spots on the plate as it develops.
Perhaps, you used an ink pen instead of a pencil to mark the origin?