High performance liquid chromatography (HPLC) is a separation technique used to separate mixtures. It uses columns packed with small particle sizes under high pressure, allowing better separation than traditional liquid chromatography. HPLC involves pumping a mobile phase through a column containing a stationary phase, separating components as they flow through at different rates based on interactions with the phases. Components are then detected and quantified as they exit the column. HPLC provides rapid, sensitive, and precise separation of mixtures and is widely used in fields like pharmaceuticals, chemistry, and environmental analysis.
HPLC is a High Performance liquid Chromatography.
High Pressure Liquid Chromatography.
High Priced Liquid Chromatography.
It is column chromatography.
It is Liquid Chromatography.
It is modified from of gas chromatography, it is applicable for both Volatile as well as Non volatile compound.
It can mainly divided by two types 1. Normal phase HPLC 2. Reversed Phase HPLC.
It is having a high resolution and separation capacity.
HPLC Principle,Instrumentation and ApplicationAlakesh Pradhan
HPLC Chromatography and its principle
Liquid chromatography
High Performance Liquid Chromatography ( HPLC )
The components of the high performance liquid chromatograph (HPLC).
The separation process.
The chromatogram
ION EXCHANGE CHROMATOGRAPHY
ByM.Vharshini
B.Sc. Bio Medical Science
Sri Ramachandra University
ION EXCHANGE CHROMATOGRAPHY
Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger.
It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
Cations or Anions can be separated using this method.
PRINCIPLE
It is based on the reversible electrostatic interaction of ions with the separation matrix (i.e.)
The separation occurs by reversible exchange of ions between the ions present in the solution and those present in the ion exchange resin.
CLASSIFICATION OF RESINS
According to the chemical nature they classified as-
1. Strong cation exchange resin
2. Weak cation exchange resin
3. Strong anion exchange resin
4. Weak anion exchange resin
According to the Source they can -
Natural resins : Cation - Zeolytes, Clay
Anion - Dolomite
Synthetic resins: Inorganic & Organic resins
◘Organic resins are polymeric resin matrix.
The resin composed of –
Polystyrene (sites for exchangeable functional groups)
Divinyl benzene(Cross linking agent)-offers stability.
Ion exchange resin should have following requirements
»It must be chemically stable.
»It should be insoluble in common solvents.
» It should have a sufficient degree of cross linking.
»The swollen resin must be denser than water.
»It must contain sufficient no. of ion exchange groups.
Physical properties of ion exchange resins
Cross linking:
It affects swelling & strength & solubility
Swelling:
When resin swells, polymer chain spreads apart
Polar solvents → swelling
Non-polar solvents → contraction
Swelling also affected electrolyte concentration.
Particle size and porosity
Increase in surface area & decrease in particle size will increase the rate of ion exchange.
Regeneration
Cation exchange resin are regenerated by treatment with acid, then washing with water.
Anion exchange resin are regenerated by treatment with NaOH, then washing with water until neutral.
EXPERIMENTAL SETUP OF ION EXCHANGE CHROMATOGRAPHY
Metrohm 850 Ion chromatography system
Instrumentation of ion exchange chromatography
PRACTICAL REQUIREMENTS
1.Column
» glass, stainless steel or polymers
2.Packing the column
» Wet packing method:
A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles.
3.Application of the sample
After packing, sample is added to the top of the stationary phase, use syringe or pipette.
This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent.
4.Mobile phase
Acids, alkalis, buffers…
6.Stationary phase
The ionic
HPLC is a High Performance liquid Chromatography.
High Pressure Liquid Chromatography.
High Priced Liquid Chromatography.
It is column chromatography.
It is Liquid Chromatography.
It is modified from of gas chromatography, it is applicable for both Volatile as well as Non volatile compound.
It can mainly divided by two types 1. Normal phase HPLC 2. Reversed Phase HPLC.
It is having a high resolution and separation capacity.
HPLC Principle,Instrumentation and ApplicationAlakesh Pradhan
HPLC Chromatography and its principle
Liquid chromatography
High Performance Liquid Chromatography ( HPLC )
The components of the high performance liquid chromatograph (HPLC).
The separation process.
The chromatogram
ION EXCHANGE CHROMATOGRAPHY
ByM.Vharshini
B.Sc. Bio Medical Science
Sri Ramachandra University
ION EXCHANGE CHROMATOGRAPHY
Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger.
It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
Cations or Anions can be separated using this method.
PRINCIPLE
It is based on the reversible electrostatic interaction of ions with the separation matrix (i.e.)
The separation occurs by reversible exchange of ions between the ions present in the solution and those present in the ion exchange resin.
CLASSIFICATION OF RESINS
According to the chemical nature they classified as-
1. Strong cation exchange resin
2. Weak cation exchange resin
3. Strong anion exchange resin
4. Weak anion exchange resin
According to the Source they can -
Natural resins : Cation - Zeolytes, Clay
Anion - Dolomite
Synthetic resins: Inorganic & Organic resins
◘Organic resins are polymeric resin matrix.
The resin composed of –
Polystyrene (sites for exchangeable functional groups)
Divinyl benzene(Cross linking agent)-offers stability.
Ion exchange resin should have following requirements
»It must be chemically stable.
»It should be insoluble in common solvents.
» It should have a sufficient degree of cross linking.
»The swollen resin must be denser than water.
»It must contain sufficient no. of ion exchange groups.
Physical properties of ion exchange resins
Cross linking:
It affects swelling & strength & solubility
Swelling:
When resin swells, polymer chain spreads apart
Polar solvents → swelling
Non-polar solvents → contraction
Swelling also affected electrolyte concentration.
Particle size and porosity
Increase in surface area & decrease in particle size will increase the rate of ion exchange.
Regeneration
Cation exchange resin are regenerated by treatment with acid, then washing with water.
Anion exchange resin are regenerated by treatment with NaOH, then washing with water until neutral.
EXPERIMENTAL SETUP OF ION EXCHANGE CHROMATOGRAPHY
Metrohm 850 Ion chromatography system
Instrumentation of ion exchange chromatography
PRACTICAL REQUIREMENTS
1.Column
» glass, stainless steel or polymers
2.Packing the column
» Wet packing method:
A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles.
3.Application of the sample
After packing, sample is added to the top of the stationary phase, use syringe or pipette.
This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent.
4.Mobile phase
Acids, alkalis, buffers…
6.Stationary phase
The ionic
Chromatography is a laboratory technique for the separation of a mixture. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase.
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
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
- Video recording of this lecture in English language: https://youtu.be/lK81BzxMqdo
- Video recording of this lecture in Arabic language: https://youtu.be/Ve4P0COk9OI
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
- Link to NephroTube website: www.NephroTube.com
- Link to NephroTube social media accounts: https://nephrotube.blogspot.com/p/join-nephrotube-on-social-media.html
The prostate is an exocrine gland of the male mammalian reproductive system
It is a walnut-sized gland that forms part of the male reproductive system and is located in front of the rectum and just below the urinary bladder
Function is to store and secrete a clear, slightly alkaline fluid that constitutes 10-30% of the volume of the seminal fluid that along with the spermatozoa, constitutes semen
A healthy human prostate measures (4cm-vertical, by 3cm-horizontal, 2cm ant-post ).
It surrounds the urethra just below the urinary bladder. It has anterior, median, posterior and two lateral lobes
It’s work is regulated by androgens which are responsible for male sex characteristics
Generalised disease of the prostate due to hormonal derangement which leads to non malignant enlargement of the gland (increase in the number of epithelial cells and stromal tissue)to cause compression of the urethra leading to symptoms (LUTS
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
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.
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
Factory Supply Best Quality Pmk Oil CAS 28578–16–7 PMK Powder in Stockrebeccabio
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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.
Title: Sense of Smell
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 primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
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
2. • A form of column chromatography to
separate, identify, and quantify the
compounds.
• Developed in 1970s.
• The most widely used analytical
separation technique.
3. Chromatography
• Chromatography is a technique which
separates components in a mixture due to
the differing time taken for each
component to travel through a
stationary phase when carried through it
by a mobile phase.
4. Basically, all chromatographic systems
consists of two phases.
•Mobile phase - liquid or gaseous and
flows over or through the stationary phase
•Stationary phase - solid, liquid or a
solid/liquid mixture which is immobilized
5. Some chromatography terms
Analyte
•Substance that is to be separated during
chromatography
Immobilized phase
•Stationary phase which is immobilized on
the support particles or on the inner wall of
the column tubing
6. Mobile phase
•Phase which moves in a definite direction.
(liquid/gas/fluid).
•Consists of the sample being separated/
analyzed and the solvent that moves the
sample through the column.
Effluent
•Mobile phase leaving the column.
Some chromatography terms
7. Different types of chromatography methods
• Paper chromatography
• Liquid chromatography
• Gas chromatography
• High performance liquid chromatography
8. High performance liquid chromatography
• HPLC is an extension of conventional
liquid chromatography.
• Powerful tool in analytical techniques
• Columns are tightly packed, and the
eluent is forced through the column under
high pressure(up to 5,000 psi) by a pump.
9. • Allows to use a very smaller particle size
for the column packing material which
gives a much greater surface area for
interactions between the stationary phase
and the molecules flowing through it.
• Allows a much better separation of the
components of the mixture.
10. HPLC Technique
• Utilizes liquid mobile phase to separate
the mixture
• Analytes are first dissolved in a solvent
then through the column under high
pressure of up to 400 atm
• Mixture is resolved into its components in
the column
11. • The total separation time is often 5 or 10
minutes rather than hours or even days
required for some separations by gravity
flow with the larger systems.
12. Components of HPLC
• Pump
• Injector
• Column
• Detector
• Recorder or data system
15. Pump
• A pump forces the mobile phase through
the column at a much greater velocity than
gravity-flow columns.
• The pump can be pneumatic, syringe-
type, reciprocating, or hydraulic amplifier.
16. Pump (cont.)
• Pneumatic pumps are used for
preoperative purposes.
• The most widely used pump today is the
multihead pump with two or more
reciprocating pistons.
17. • Pumps are designed in order to maintain a
stable flow rate, avoiding pulsations even
when the composition of the mobile phase
varies
• flow range – 0.01-10 ml/min
19. Injectors
• Inject the liquid sample within range of
0.1- 100 ml of volume under high pressure
• Produce minimum band broadening
• Produce possible flow disturbances
• Volume must be small (0.1-500 uL)
22. Columns
• Smooth-bore stainless steel or heavy-
walled glass tubing.
• Hundreds of packed columns differing in
size and packing are available from
manufacture.
23. Columns
• E.g. Column packing vary in size (3 to 20
um) with the smaller particles used mostly
for analytical separations and the larger
ones for preparative separation.
• The most common material used for
column packing is silica gel.
26. Detector
• HPLC detectors monitor the elute as it
leaves the column
• Produce an electronic signal proportional
to the concentration of each separated
component
27. Detector
• Crucial in trace analysis
• High sensitivity
• Fast response
• Simplifies quantitation
• Insensitive to changes in type of solvent,
flow rate and temp.
28. The most widely used detection methods
• Spectrophotometers
• Fluorometers
• Electrochemical detectors
• Mass spectrometer
• Refractive index detector
29. Detectors used in HPLC
Type Principle Detection
limit
Comments
Spectro-
photometer
Measure
absorbance of
light
<1 ng Analyte must
absorb UV or
visible light
Fluorometers Measures
fluorescence
pg to ng Analyte must
fluoresce
Electro-
chemical
detectors
Electrochemically
measures
oxidized/ reduce
analyte
pg to ng Useful for
catecholamine
s
30. Detectors used in HPLC
Type Principle Detection
limit
Comments
Mass
spectrometer
Detects ions after
separation by
mass-to-charge
ration
fg to ng Analyte must
be converted
to ionized
form
Refractometer Measure change
in refractive index
1 µg Detection of
most
compounds
but relatively
low sensitivity
31. Depending on the relative polarity of the
solvent and stationary phase, there are two
variants in use in HPLC
1.Normal phase HPLC
• Utilize polar adsorbent surface and non-
polar eluent
• Polar substance in the mixture sticks to polar
adsorbent than non-polar
• Non-polar ones will pass more quickly
through the column
32. 2. Reversed phase HPLC
• Utilize non-polar adsorbent surface and
polar eluent
• Attraction between non-polar compound in
the mixture and non-polar adsorbent
33. 2. Reversed phase HPLC (cont.)
• Polar molecules will travel through the
column more quickly because there is
strong attraction between polar solvent
and polar molecules when pass through
the column
• Reversed phase HPLC is the most
commonly used form of HPLC
34. Solvents used in mobile phase
• hexane, heptane, cyclohexane, carbon
tetrachloride, benzene, toluene, diethyl
ether, chloroform etc.
Adsorbents used in stationary phase
• silica gel, alumina, celite, cellulose powder,
ion-exchange, cellulose, starch
35. Retention time
• The time taken for a particular compound
to travel through the column to the
detector
• From the time at which the sample is
injected to the point at which the display
shows a maximum peak height for that
compound.
38. Adsorption Chromatography
• Competition for adsorption sites occurs
between the molecules of the mixture to
be separated and the molecules of the
mobile phase
• Mobile phase can be either a single
solvent or two or more solvents depend on
the analytes to be desorbed
• Speed of migration of the component
along the column depend on adsorptive
affinity
39. Ion- exchange Chromatography
• Molecules can be separated by their ionic
charges in a process known as Ion-
exchange Chromatography.
• Ion-exchange resins are used as the
column packing materials.
• This method is used for separation of ionic
species, such as amino acids.
40.
41. • Known as gel permeation chromatography
or gel filtration chromatography.
• Packing material with very small pore is
used.
• Precisely controlled pore size materials in
the column
• Large molecules, such as polymers are
physically prevented from passing through
the column
Size Exclusion Chromatography
42. Applications
HPLC is used for
• Chemistry and biochemistry research
analyzing complex mixtures,
• Purifying chemical compounds
• Quality control to ensure the purity of raw
materials
• Analyzing air and water pollutants,
43. Applications (cont.)
• Monitoring materials that may jeopardize
occupational safety or health
• Monitoring pesticide levels in the
environment.
• To survey food and drug products,
• To identify confiscated narcotics
• To determine the amount of such chemical
compounds found in new drugs in
pharmaceutics
44. HPLC as compared with the classical
technique
• Small diameter, reusable stainless steel
columns
• Column packing with very small particles
• Control flow of mobile phase
• Precise sample introduction
45. HPLC as compared with the classical
technique (cont.)
• Good pumping system
• Special continuous flow detectors- can
handle small flow rates and detect very
small amounts
• Rapid analysis
• High resolution
46. Disadvantages of HPLC
• Cost
• Complexity
• Low sensitivity for some compounds
• Irreversibly adsorbed compounds not
detected
• Co-elution difficult to detect
47. Summary
• The modern day technique is greatly
enhanced in terms of selectivity,
resolution, through miniaturization and the
use of very elaborate stationary phases.
• Therefore HPLC is widely used for
separation of molecules in biological,
pharmaceutical, food, environmental and
industrial process
Different compounds have different retention times.
For a particular compound, the retention time will vary depending on:
Pressure used -(affects flow rate of the solvent)
Nature of stationary phase (material, particle size)
Exact composition of the solvent
Temperature of the column