Department of Chemistry /College of Sciences/ University of Baghdad
Subject: Analytical Chemistry 4
Second stage
2nd semester
Dr. Ashraf Saad Rsaheed
2017-2018
Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis.
The Russian botanist Mikhail Tswett coined the term chromatography in 1906.
The first analytical use of chromatography was described by James and Martin in 1952, for the use of gas chromatography for the analysis of fatty acid mixtures.
A wide range of chromatographic procedures makes use of differences in size, binding affinities, charge, and other properties to separate materials.
It is a powerful separation tool that is used in all branches of science and is often the only means of separating components from complex mixtures.
hromatography is based on the principle where molecules in mixture applied onto the surface or into the solid, and fluid stationary phase (stable phase) is separating from each other while moving with the aid of a mobile phase.
The factors effective on this separation process include molecular characteristics related to adsorption (liquid-solid), partition (liquid-solid), and affinity or differences among their molecular weights.
Because of these differences, some components of the mixture stay longer in the stationary phase, and they move slowly in the chromatography system, while others pass rapidly into the mobile phase, and leave the system faster.
Three components thus form the basis of the chromatography technique.
1. Stationary phase: This phase is always composed of a “solid” phase or “a layer of a liquid adsorbed on the surface solid support”.
2. Mobile phase: This phase is always composed of “liquid” or a “gaseous component.”
3. Separated molecules
Types of Chromatography
Substances can be separated on the basis of a variety of methods and the presence of characteristics such as size and shape, total charge, hydrophobic groups present on the surface, and binding capacity with the stationary phase.
This leads to different types of chromatography techniques, each with their own instrumentation and working principle.
For instance, four separation techniques based on molecular characteristics and interaction type use mechanisms of ion exchange, surface adsorption, partition, and size exclusion.
Other chromatography techniques are based on the stationary bed, including column, thin layer, and paper chromatography.
Applications of Chromatography
Pharmaceutical sector
To identify and analyze samples for the presence of trace elements or chemicals.
Separation of compounds based on their molecular weight and element composition.
Detects the unknown compounds and purity of mixture.
In drug development.
Chemical industry
In testing water samples and also checks air quality.
HPLC and GC are very much used for detecting various contaminants such as polychlorinated biphenyl (PCBs) in pesticides and oils.
In various life sciences applications.
In forensic pathology and crime scene testing like analyzing blood and hair samples.
After reading this ppt we knowing about column chromatography techniques. In this chromatography process, the molecule mixture is separated depending on its differentials partitioning between a stationary phase and a mobile phase.
Department of Chemistry /College of Sciences/ University of Baghdad
Subject: Analytical Chemistry 4
Second stage
2nd semester
Dr. Ashraf Saad Rsaheed
2017-2018
Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis.
The Russian botanist Mikhail Tswett coined the term chromatography in 1906.
The first analytical use of chromatography was described by James and Martin in 1952, for the use of gas chromatography for the analysis of fatty acid mixtures.
A wide range of chromatographic procedures makes use of differences in size, binding affinities, charge, and other properties to separate materials.
It is a powerful separation tool that is used in all branches of science and is often the only means of separating components from complex mixtures.
hromatography is based on the principle where molecules in mixture applied onto the surface or into the solid, and fluid stationary phase (stable phase) is separating from each other while moving with the aid of a mobile phase.
The factors effective on this separation process include molecular characteristics related to adsorption (liquid-solid), partition (liquid-solid), and affinity or differences among their molecular weights.
Because of these differences, some components of the mixture stay longer in the stationary phase, and they move slowly in the chromatography system, while others pass rapidly into the mobile phase, and leave the system faster.
Three components thus form the basis of the chromatography technique.
1. Stationary phase: This phase is always composed of a “solid” phase or “a layer of a liquid adsorbed on the surface solid support”.
2. Mobile phase: This phase is always composed of “liquid” or a “gaseous component.”
3. Separated molecules
Types of Chromatography
Substances can be separated on the basis of a variety of methods and the presence of characteristics such as size and shape, total charge, hydrophobic groups present on the surface, and binding capacity with the stationary phase.
This leads to different types of chromatography techniques, each with their own instrumentation and working principle.
For instance, four separation techniques based on molecular characteristics and interaction type use mechanisms of ion exchange, surface adsorption, partition, and size exclusion.
Other chromatography techniques are based on the stationary bed, including column, thin layer, and paper chromatography.
Applications of Chromatography
Pharmaceutical sector
To identify and analyze samples for the presence of trace elements or chemicals.
Separation of compounds based on their molecular weight and element composition.
Detects the unknown compounds and purity of mixture.
In drug development.
Chemical industry
In testing water samples and also checks air quality.
HPLC and GC are very much used for detecting various contaminants such as polychlorinated biphenyl (PCBs) in pesticides and oils.
In various life sciences applications.
In forensic pathology and crime scene testing like analyzing blood and hair samples.
After reading this ppt we knowing about column chromatography techniques. In this chromatography process, the molecule mixture is separated depending on its differentials partitioning between a stationary phase and a mobile phase.
Chromatography: Principle, types, application.
A complete description of Chromatography along with all the types including HPLC, GAS, COLUMN, ION EXCHANGE, AFFINITY, COLUMN, PAPER, THIN LAYER CHROMATOGRAPHY - Techniques, Steps, principles, application.
Imagine a race where the runners are molecules from a mysterious mixture. HPLC sets the stage for this separation marathon. The sample, dissolved in a solvent, acts as the starting line. A pressurized stream of solvent pushes the molecules through a packed column, like an obstacle course. Each molecule interacts uniquely with the obstacles and the solvent, causing them to separate along the way. Finally, a detector acts as the finish line, identifying and measuring each molecule as it emerges. This powerful technique allows scientists to unmask unknown competitors (compounds), determine the number of each type of runner (quantify components), and even check if anyone cheated (assess purity).
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
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.
Chromatography: Principle, types, application.
A complete description of Chromatography along with all the types including HPLC, GAS, COLUMN, ION EXCHANGE, AFFINITY, COLUMN, PAPER, THIN LAYER CHROMATOGRAPHY - Techniques, Steps, principles, application.
Imagine a race where the runners are molecules from a mysterious mixture. HPLC sets the stage for this separation marathon. The sample, dissolved in a solvent, acts as the starting line. A pressurized stream of solvent pushes the molecules through a packed column, like an obstacle course. Each molecule interacts uniquely with the obstacles and the solvent, causing them to separate along the way. Finally, a detector acts as the finish line, identifying and measuring each molecule as it emerges. This powerful technique allows scientists to unmask unknown competitors (compounds), determine the number of each type of runner (quantify components), and even check if anyone cheated (assess purity).
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
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.
NVBDCP.pptx Nation vector borne disease control programSapna Thakur
NVBDCP was launched in 2003-2004 . Vector-Borne Disease: Disease that results from an infection transmitted to humans and other animals by blood-feeding arthropods, such as mosquitoes, ticks, and fleas. Examples of vector-borne diseases include Dengue fever, West Nile Virus, Lyme disease, and malaria.
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
Knee anatomy and clinical tests 2024.pdfvimalpl1234
This includes all relevant anatomy and clinical tests compiled from standard textbooks, Campbell,netter etc..It is comprehensive and best suited for orthopaedicians and orthopaedic residents.
Recomendações da OMS sobre cuidados maternos e neonatais para uma experiência pós-natal positiva.
Em consonância com os ODS – Objetivos do Desenvolvimento Sustentável e a Estratégia Global para a Saúde das Mulheres, Crianças e Adolescentes, e aplicando uma abordagem baseada nos direitos humanos, os esforços de cuidados pós-natais devem expandir-se para além da cobertura e da simples sobrevivência, de modo a incluir cuidados de qualidade.
Estas diretrizes visam melhorar a qualidade dos cuidados pós-natais essenciais e de rotina prestados às mulheres e aos recém-nascidos, com o objetivo final de melhorar a saúde e o bem-estar materno e neonatal.
Uma “experiência pós-natal positiva” é um resultado importante para todas as mulheres que dão à luz e para os seus recém-nascidos, estabelecendo as bases para a melhoria da saúde e do bem-estar a curto e longo prazo. Uma experiência pós-natal positiva é definida como aquela em que as mulheres, pessoas que gestam, os recém-nascidos, os casais, os pais, os cuidadores e as famílias recebem informação consistente, garantia e apoio de profissionais de saúde motivados; e onde um sistema de saúde flexível e com recursos reconheça as necessidades das mulheres e dos bebês e respeite o seu contexto cultural.
Estas diretrizes consolidadas apresentam algumas recomendações novas e já bem fundamentadas sobre cuidados pós-natais de rotina para mulheres e neonatos que recebem cuidados no pós-parto em unidades de saúde ou na comunidade, independentemente dos recursos disponíveis.
É fornecido um conjunto abrangente de recomendações para cuidados durante o período puerperal, com ênfase nos cuidados essenciais que todas as mulheres e recém-nascidos devem receber, e com a devida atenção à qualidade dos cuidados; isto é, a entrega e a experiência do cuidado recebido. Estas diretrizes atualizam e ampliam as recomendações da OMS de 2014 sobre cuidados pós-natais da mãe e do recém-nascido e complementam as atuais diretrizes da OMS sobre a gestão de complicações pós-natais.
O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
Prof. Marcus Renato de Carvalho
www.agostodourado.com
Ozempic: Preoperative Management of Patients on GLP-1 Receptor Agonists Saeid Safari
Preoperative Management of Patients on GLP-1 Receptor Agonists like Ozempic and Semiglutide
ASA GUIDELINE
NYSORA Guideline
2 Case Reports of Gastric Ultrasound
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.
Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
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.
MANAGEMENT OF ATRIOVENTRICULAR CONDUCTION BLOCK.pdf
Chromatography by Anita Yadav
1. Pokhara University
School of Health and Allied Sciences
Chromatography
Anita Yadav
First Semester, M Pharm(Clinical Pharmacy)
School of health and allied Sciences
Pokhara University, Dhungepatan, Lekhnath-12, Kaski, Nepal
2. Pokhara University
School of Health and Allied Sciences
Topics under discussion
• Introduction
• Background
• Discussion
August 8, 2017 2INS 591: Presentation
3. Pokhara University
School of Health and Allied Sciences
Introduction
• Chromatography is an analytical technique where in a
sample mixture is separated into different
components.
• This is both a qualitative and quantitative method.
• The sample gets separated under the influence of a
mobile phase (moving phase) over a stationary phase.
• These separated components are later identified and
also quantified.
August 8, 2017 3INS 591: Presentation
4. Pokhara University
School of Health and Allied Sciences
Origin of chromatography:-
• The Russian botanist Mikhail Tswett had
invented chromatography, a word he derived from
the Greek words for color (chroma) and writing
(graphe) in 1906.
August 8, 2017 INS 591: Presentation 4
5. Pokhara University
School of Health and Allied Sciences
• A. J. P. Martin (1910-2002) and R. L. M. Synge (1914-
1994) developed the first theoretical explanations.
• chromatography did not come into wide use until
1952, when Martin, working with A. T. James,
described a way of using a gas instead of a liquid as
the mobile phase, and a highly viscous liquid coated
on solid particles as the stationary phase
August 8, 2017 INS 591: Presentation 5
6. Pokhara University
School of Health and Allied Sciences
Principle of Chromatography:-
• All chromatographic methods require one static part
(the stationary phase) and one moving part (the
mobile phase).
• Interaction between the stationary and mobile phase
cause separation of compounds from the mixture.
• Rely on one of the following phenomena: adsorption;
partition; ion exchange; or molecular exclusion.
August 8, 2017 INS 591: Presentation 6
7. Pokhara University
School of Health and Allied Sciences
• The preferential separation is done due to
differential affinities of compounds towards
stationary and mobile phase. After separation of the
compounds, they are identified by
suitable detection methods.
• The differences in affinities arise due to
relative adsorption or partition coefficient in
between components towards the both phases.
August 8, 2017 INS 591: Presentation 7
8. Pokhara University
School of Health and Allied Sciences
Classifications of chromatography
(1)Based on the nature of mobile and stationary phase:
• Liquid chromatography: LLC, LSC
• Gas chromatography: GLC, GSC
August 8, 2017 INS 591: Presentation 8
9. Pokhara University
School of Health and Allied Sciences
August 8, 2017 INS 591: Presentation 9
• Based on the principle of separation:
• Adsorption chromatography: - It is based on the
different ability of component to adsorb on the
surface of SP.E.g. TLC, CC, GSC
• Partition chromatography: - It is based on the
different solubility of sample of component in the
stationary and mobile phase. E.g. GLC, LLC
• Ion exchange chromatography: - it is based on the
exchange of ionic sample with ionic group of
stationary phase and is governed by electrostatic
interaction.
• E.g. IC, IEC, TLC
10. Pokhara University
School of Health and Allied Sciences
• Gel chromatography: - Based on the size and shape
of their molecules as well as the size and shape of
the pores of the stationary phase.
• Affinity chromatography:- based on the molecular
recognition of only those components which are
complementary to stationary phase, are absorbed by
their affinity
August 8, 2017 INS 591: Presentation 10
11. Pokhara University
School of Health and Allied Sciences
• Thin-layer chromatography (TLC) is
a chromatography technique used to separate
non-volatile mixtures.
• It is performed on a sheet of glass, plastic, or
aluminium foil, which is coated with a thin
layer of adsorbent material, usually silica
gel, aluminium oxide (alumina), or cellulose.
• This layer of adsorbent is known as
the stationary phase.
Thin Layer Chromatography
August 8, 2017 11INS 591: Presentation
12. Pokhara University
School of Health and Allied Sciences
STATIONARY PHASE
• Silica is commonly used as stationary phase
• The separation of sample mixture will be depent on the
polarity of sample.
• Some modified silica is also used in certain purposes.
13. Pokhara University
School of Health and Allied Sciences
Stationery phase Description Application
Silica gel G Silica gel with average
particle size 15µm
containing ca 13%
calcium sulfate binding
agent
Used in wide range
pharmacopoeial test
Silica gel G254 Silica gel G with
fluorescence added
Same application with
Silica gel G where
visualization is to be
carried out under UV
light.
Cellulose Cellulose powder of less
than 30µm particle size.
Identification of
tetracyclines
14. Pokhara University
School of Health and Allied Sciences
• The ability of mobile phase to move up is
depent on the polarity itself
• Volatile organic solvents is preferably used as
as mobile phase.
MOBILE PHASE
15. Pokhara University
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SOLVENT POLARITY INDEX
Heksana 0
Butanol 3.9
Chloroform 4.1
Methanol 5.1
Ethanol 5.1
Acetonitrile 5.8
Air 9.0
MOBILE PHASE
16. Pokhara University
School of Health and Allied Sciences
August 8, 2017 INS 591: Presentation 17
The compounds in the sample on the TLC plate (the analyte)
can do two things as the solvent moves up the plate:
If a compound is attracted to the
coating it sticks and does not move
up the plate:
If a compound is not attracted to
the coating it will not stick and it
does move up the plate:
17. Pokhara University
School of Health and Allied Sciences
August 8, 2017 INS 591: Presentation 18
• Capillary action pulls the solvent (the mobile phase)
slowly up the plate like water being soaked up by a
sponge.
• Actually, most compounds will be partially attracted
to both the coating and the solvent.
• Compounds more attracted to the coating move up
the plate slowly, while those more attracted to the
solvent travel more quickly and separation is
achieved.
18. Pokhara University
School of Health and Allied Sciences
• A major factor is polarity of the bonds.
• For example if, the coating is composed of aluminum
oxide (Al2O3).
• The aluminum - oxygen bonds are very polar. The
solvent is usually a nonpolar or very moderately polar
organic solvent.
• In general, the more polar bonds a compound has then
the more attracted it is to the very polar aluminum
oxide and the more slowly it moves up the plate.
August 8, 2017 INS 591: Presentation 19
What features cause some compounds to prefer the stationary phase to the
mobile phase?
19. Pokhara University
School of Health and Allied Sciences
Uses
• To determine the number of components in a mixture.
• To determine the identity of two substances.
• To monitor the progress of a reaction.
• To determine the effectiveness of a purification.
• To determine the appropriate conditions for a column
chromatographic separation.
• To monitor column chromatography.
August 8, 2017 INS 591: Presentation 20
20. Pokhara University
School of Health and Allied Sciences
• TLC plate
• ‘Developing container’
- chamber/ jar/ glass beaker
• Pencil
• Ruler
• Capillary pipe
• Solvents / mobile phase
- organic solvents
• UV lamp
Materials
22. Pokhara University
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• It includes three methods:
• Spotting
• Developing
• Visualizing
August 8, 2017 INS 591: Presentation 23
23. Pokhara University
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Spotting
• Spotting: A drop of a mixture of three compounds, A,
B, and C was spotted on the TLC plate with a capillary
tube at the point marked spotting line or origin.
• The plate consists of a piece of plastic coated with
silica gel, a fine grade of sand.
August 8, 2017 INS 591: Presentation 24
Fig.Spotting
24. Pokhara University
School of Health and Allied Sciences
Developing
• The spotted plate was placed in a beaker containing a
solvent.
• The solvent rose on the plate.
• The amount of solvent in the beaker was enough so
that the spotting line did not dip into the solvent.
• The solvent passed through the spotting line and
continued to move up the plate until the plate was
removed from the beaker.
August 8, 2017 INS 591: Presentation 25
25. Pokhara University
School of Health and Allied Sciences
August 8, 2017 INS 591: Presentation 26
• The dotted line labeled solvent front in Figure 1
shows how far the solvent moved (i.e., from below
the spotting line to the upper dotted line) when it was
removed.
• As soon as the plate was removed from the beaker,
the experimenter scratched a line with a pencil and
straight edge across the plate to indicate the solvent
front.
• The solvent is still visible on a plate immediately after
the plate is removed from the beaker.
26. Pokhara University
School of Health and Allied Sciences
• However, the solvent quickly evaporates.
• Therefore, the solvent front must be marked with a
pencil before the solvent dries.
• After the solvent dried, the pencil line (solvent front)
and origin were all that was visible.
August 8, 2017 INS 591: Presentation 27
Fig. Developing
27. Pokhara University
School of Health and Allied Sciences
Visualizing
• As the solvent moved up the plate, compounds A, B,
and C also moved up the plate but at different rates.
• Of the three compounds, A moved furthest up the
plate because it traveled faster.
• How fast a compound, referred to as a spot, moves
up a plate is a measure of its mobility in that
particular coating and solvent combination.
August 8, 2017 INS 591: Presentation 28
28. Pokhara University
School of Health and Allied Sciences
• To see the individual spots, the plate must be
visualized. The color samples are easy to be seen and
no need to use UV lamp to detect them.
August 8, 2017 INS 591: Presentation 29
Fig. visualizing
29. Pokhara University
School of Health and Allied Sciences
• For colorless spots,following procedure applied,
• a. Iodination
• b. Ninhydrin
• c.KMnO4
• d.Alkaline tetrazolium blue
August 8, 2017 INS 591: Presentation 30
30. Pokhara University
School of Health and Allied Sciences
• The coating contains a fluorescent indicator, which is
visible under ultraviolet (UV) light.
• After the plate was visualized, the experimenter had
the chromatogram shown in Figure 1. The final step
was to analyze the plate.
August 8, 2017 INS 591: Presentation 31
31. Pokhara University
School of Health and Allied Sciences
Analyzing
• After a fixed time, as measured by how far the
mobile phase has moved, the process is stopped, and
the dissolved substances have moved different
distances.
• The ratio of how far the substance moved to how far
the solvent moved is called the Rf.
August 8, 2017 INS 591: Presentation 32
32. Pokhara University
School of Health and Allied Sciences
Thin-Layer Chromatography:
Determination of Rf Values
solvent front
component B
component A
origin
dS
dB
dA
Rf of component A =
dA
dS
Rf of component B =
dB
dS
The Rf value is a decimal
fraction, generally only
reported to two decimal
places
More polar!
Less polar!
33. Pokhara University
School of Health and Allied Sciences
The use of Retention Faactor(Rf) as
separation parameter
• The distance taken through by the solvent to move
up will be assigned as solvent front
• The distance taken trrough by the sample to move
up will be assign as sample front
• Rf value is obtained by dividing the sample front
toward solvent front
• Rf = sample front
• solvent front
August 8, 2017 INS 591: Presentation 34
34. Pokhara University
School of Health and Allied Sciences
August 8, 2017 INS 591: Presentation 35
A
B
C
y = distance
solvent moved
x = distance
compound A
moved
xy
The Rf of compound A is x over y (x/y).
35. Pokhara University
School of Health and Allied Sciences
• In general, low polarity compounds have higher Rf
values than higher polarity compounds
• Typically an effective solvent is one that gives Rf in
the range of 0.3-0.7.
August 8, 2017 INS 591: Presentation 36
36. Pokhara University
School of Health and Allied Sciences
Experimental Techniques of Thin Layer
Chromatography:
• Pouring
• Dipping
• Spraying
• Spreading
August 8, 2017 INS 591: Presentation 37
37. Pokhara University
School of Health and Allied Sciences
Choice of solvents system in TLC:
• The choice of solvent or a mixture of solvents used in
TLC is solely guided by two important factors:
• (a) the nature of the constituent to be separated
i.e. whether it is polar or non-polar; and
• the nature of the process involved i.e. whether it is
a case of ‘ adsorption’ or ‘partition chromatography’.
August 8, 2017 INS 591: Presentation 38
39. Pokhara University
School of Health and Allied Sciences
a. The spot shape is too broad
- Diameter is supposed to be < 1-2mm
b. The movement of solvent
- should be straight up
- unproportionality in stationary phase surface will
inhibit the movement of solvent
c. streaking formation
- caused by too concentrated sample
Three Common Problems in TLC
40. Pokhara University
School of Health and Allied Sciences
Advantages of TLC
• Low cost and short analysis time
• Ease of sample preparation
• All spots can be visualized
• Sample cleanup is seldom necessary
• Adaptable to most pharmaceuticals
August 8, 2017 INS 591: Presentation 41
41. Pokhara University
School of Health and Allied Sciences
• Uses small quantities of solvents
• Requires minimal training
• Reliable and quick
• Minimal amount of equipment is needed
• Densitometers can be used to increase accuracy of
spot comcentration
August 8, 2017 INS 591: Presentation 42
42. Pokhara University
School of Health and Allied Sciences
Applications of TLC
• To identify the presence of undesirable specific
organic compounds present as impurities in a
number of pharmaceutical substances, namely:
morphin in apomorphin hydrochloride; hydrazine in
carbidopa; 3-aminopropanol in dexampanthenol;
etc.
• Related substances present in official drugs,
namely: related substances present in a wide
number of potent pharmaceutical substances e.g.,
aminophylline; baclofen; chloramphenicol;
carbamazepine,etc.August 8, 2017 INS 591: Presentation 43
43. Pokhara University
School of Health and Allied Sciences
• Foreign alkaloids present in alkaloidal drugs, for
example: atropine sulphate; codeine
• Foreign steroids present in steroidal drugs, for
example betamethasone valerate;
• Ninhydrin positive substances in official amino acids
e.g.glutamic acid; leucine.
August 8, 2017 INS 591: Presentation 44
44. Pokhara University
School of Health and Allied Sciences
References
• http//orgchem.colorado.edu/hndbooksupport/TLC/T
LCprocedure.html.
• kalasz Huba, Bathori Maria, LC.GC Int., 1-8(2001)
• Kar Ashutosh(2015) Pharmaceutical Drug Analysis(3
Ed.) New Age International Publishers, pp471-492.
August 8, 2017 INS 591: Presentation 45