This document discusses chromatography and PCR techniques. It provides details on:
- The principles and types of chromatography including TLC, HPLC, and their components and procedures. HPLC allows for quantitative analysis and is commonly used for pharmaceutical quality control.
- PCR amplification which uses DNA polymerase to exponentially replicate DNA sequences. It requires template DNA, primers, nucleotides, and DNA polymerase. Repeated heating and cooling cycles allow for target DNA replication.
- Applications of chromatography and PCR include pharmaceutical analysis, forensic analysis, detection of genetic disorders, microbial detection, and molecular biology research techniques. Both provide powerful tools for separation, detection, and analysis of biological molecules.
The slides are informative of HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY & its thorough components further its advantages and applications. The comparison of HPLC and HPTLC is explained.
The slides are informative of HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY & its thorough components further its advantages and applications. The comparison of HPLC and HPTLC is explained.
Introduction points in High Performance Liquid Chromatography.
Principle
Theory of HPLC
Kinetic Theory (Van Deemter)
Factors affecting Column Performance
Capacity Factor
Selectivity Factor
Factors affecting Resolution
Mobile phase composition effect on separation in HPLC
Standard Addition Method
Internal Standard Method
Types of HPLC
-Prajakta
Introduction points in High Performance Liquid Chromatography.
Principle
Theory of HPLC
Kinetic Theory (Van Deemter)
Factors affecting Column Performance
Capacity Factor
Selectivity Factor
Factors affecting Resolution
Mobile phase composition effect on separation in HPLC
Standard Addition Method
Internal Standard Method
Types of HPLC
-Prajakta
A brief review on development and validation of hplc method.adhirajain
the slides in the ppt gives a brief review on product development and its validation in HPLC method. Contents are with advantages, disadvantages, application , classification and methods for development.
Various chromatographic techniques can be employed for the qualitative and quantitative analysis of the drug. It is observed that HPTLC is the most widely used Chromatography instrument as it consumes less solvent for developing in less time. Reproducibility of results is also easy.
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
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
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Ve...kevinkariuki227
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
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.
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
Dr. Vidisha Kumari, a leading epidemiologist in Bangalore, emphasizes the importance of getting vaccinated. "The flu vaccine is our best defense against the influenza virus. It not only protects individuals but also helps prevent the spread of the virus in our communities," he says.
This year, the flu season is expected to coincide with a potential increase in other respiratory illnesses. The Karnataka Health Department has launched an awareness campaign highlighting the significance of flu vaccinations. They have set up multiple vaccination centers across Bangalore, making it convenient for residents to receive their shots.
To encourage widespread vaccination, the government is also collaborating with local schools, workplaces, and community centers to facilitate vaccination drives. Special attention is being given to ensuring that the vaccine is accessible to all, including marginalized communities who may have limited access to healthcare.
Residents are reminded that the flu vaccine is safe and effective. Common side effects are mild and may include soreness at the injection site, mild fever, or muscle aches. These side effects are generally short-lived and far less severe than the flu itself.
Healthcare providers are also stressing the importance of continuing COVID-19 precautions. Wearing masks, practicing good hand hygiene, and maintaining social distancing are still crucial, especially in crowded places.
Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
Couples presenting to the infertility clinic- Do they really have infertility...Sujoy Dasgupta
Dr Sujoy Dasgupta presented the study on "Couples presenting to the infertility clinic- Do they really have infertility? – The unexplored stories of non-consummation" in the 13th Congress of the Asia Pacific Initiative on Reproduction (ASPIRE 2024) at Manila on 24 May, 2024.
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.
Anti ulcer drugs and their Advance pharmacology ||
Anti-ulcer drugs are medications used to prevent and treat ulcers in the stomach and upper part of the small intestine (duodenal ulcers). These ulcers are often caused by an imbalance between stomach acid and the mucosal lining, which protects the stomach lining.
||Scope: Overview of various classes of anti-ulcer drugs, their mechanisms of action, indications, side effects, and clinical considerations.
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
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
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4. Chromatography
It is defined as analytical method in which separation of
active constituent from complex mixture occurs, while
mixture is distributed in two phases.
This technique is used in :
SEPERATION IDENTIFICATION QUANTIFICATION
13. Mobile Phase
As it moves, so called as Mobile Phase
Liquid mobile phase is used in HPLC
Is mixture of solvent- polar/non-polar
Contained is glass of stainless steel, capable of holding 1L of solvent
Solvent selection, depends on Stationary phase
14. Degassing of Mobile Phase
De-gassing is done to prevent
formation of gas-bubbles in
the pump or detector prior to
use
De-gassing is done by vacuum
or sparing with helium gas
Then, mobile phase are filtered to remove any
particulate matter that may clog the system
15. Tubing System in HPLC
Ability to
withstand
pressure
Should be
Inert
Able to
carry
sufficient
volume
20. Made of stainless steel
Can withstand pressure up to 50 MP
Length : 5-25 cm
Internal diameter : 4.6 mm
Flow of solvent : 1-3 ml/minute
Contains adsorbent (Stationary phase ) of smaller particle size
21. Reason for smaller particle size
?
Small Particle Size
More surface/ volume ratio
More surface area for
Interaction with Solvent
More efficient Separation
23. Stationary Phase material
Silica or Alumina ( of 5-10 micron ) commonly used
The separation is the result of different components
adhering to or diffusion into the packing particles when
the mobile phase is forced through the column
Selection of mobile phase depends on stationary phase
material
27. Here Silica is made non-polar by adding long
Hydrocarbon chain of 8 to 18 Carbon atoms (C8 or C18)
C18 being more hydrophobic than C8, commonly used
Mobile phase used is polar (Methanol)
First elute will be Polar
Now a days, Reverse phase HPLC is commonly used
30. Detector System
Is coupled to column and detects substances
It sends signals to processing unit (Computer)
Ideal detector must be :
Sensitive
Non-
destructive
Insensitive to
temperature
Reliable
31. Most commonly used detector system is UV detector
system
Other detector system :
1- Electro chemical
2- Florescence
3- Chemiluminence
4- Optical rotation
5- Refractive index
34. Interpretation of
Chromatogram
It is the time required for individual compound to pass
from the column
Measured from time of sample administration to peak of
that particular compound
Different compounds have different unique R.T.
35. Continued…
Retention time depends on
1- Pressure applied by pump
2- Nature and size of stationary phase molecules
3- Composition of solvent
4- Temperature of Column
36.
37. Quantification of
Compounds
Concentration of compound
is measured using AUC
of that particular peak
More AUC More is the
concentration
Measured automatically by computer
Computers are connected to printers
38. Coupling of HPLC with Mass
spectrometer
When detector is showing the peak, some of what
substance is passing through the detector at that time
can be diverted to a mass spectrometer
There it will form Fragmentation pattern
Pattern can be compared against a computer database
of known pattern
Identity of substance can be found without having to
know their retention time
39.
40. Application of HPLC
Sugar analysis in fruit
juices
To detect
pharmaceuticals
in water
To detect
pesticides in
drinking water
To ensure quality
of soft drinks
Analysis of
alcohol
percentage in
beers
To analyze
polycyclic
compounds in
vegetables
To detect
Phenolic
compound in
drinking water
45. Principle of Action
Separation
depends on
relative affinity
of compounds
towards 2 phase
Compound with higher
affinity for Stationary
phase will travel slowly
Compound
under influence
of mobile phase
travel over
stationary phase
49. TLC Plates
1
• Readymade available in market, where aluminum base is used
2
• Can also prepare in lab by using glass slide as a base, where
Stationary phase is applied over it
3
• Readymade plate is preferred
4
• Any side is taken as base, and above 1 cm from base line by pencil is
made
5
• TLC plate, after preparing kept in TLC Chambers
51. TLC Chamber
Contains mobile phase as a solvent
Mobile phase used, should be particulate free and of
highest purity for proper development of Spots
Solvent used, should be chemically inert to sample and
stationary phase
A moistened filter paper is placed onto wall of chamber
to maintain humidity and Edge effect
52. Procedure
TLC plates are
prepared
Filter paper is
applied over the
wall of chamber
Mobile phase is
added and chamber
is saturated
Lid is closed, to
allow movement of
mobile phase over
stationary phase
Spots are
developed. After
washing, the
process is repeated
53. Mistake to avoid !!!
Side of plate with base line is placed facing mobile phase
Prevent immersion of TLC plate’s sample line into mobile
phase , to prevent its dissolution into mobile phase
55. Outcome
TLC plate are removed and allowed them to dry
Spots are visualized by various techniques
56. Polyphenol are
visualized by putting
it in UV light
chamber
For Basic substance,
dip in acid, allow to
dry and then heat it
Brown spots
Iodine vapor
treatment for C=C
compounds
Valinine stain for
alcohol and
aldehyde
Ninhydrin test for
amino acids
57. How to identify compound
?
Measured using Retention Factor (RF)
Range from 0 to 1
RF = Distance covered by individual compound / Distance
covered by solvent
Calculated RF is matched with Standards and substance is
identified
In Normal TLC, larger RF value will be of less polar substance
59. Types of TLC
Stationary phase is Polar Stationary phase Non-polar
1st elute – Non-polar 1st elute - Polar
60. Application of TLC
To check
purity of
sample
To identify
sample like
acid,
alcohol,
amines To identify
pesticides
in drinking
water
For rapid
determinati
on of
petroleum
products in
sample
To identify
drug in
biological
samples
61. DISADVANTAGES
• Time consuming
• Spots larger than 2mm
overlap
• Long streak sometimes
formed rather than spots
ADVANTAGES
• Simple process
• Cheaper
• Purity standards of given
sample can be assessed
62. HPLC VS TLC
FEATURES : HPLC TLC
INSTRUMENTATION Much Minimal
SAMPLE APPLICATION Automated, injection Manual, spotting
MOBILE PHASE MOVEMENT By High pressure Capillary action
FORM OF RESULTS Peak Spot
COST Costly Cheaper
ANALYSIS Qualitative and quantitative Qualitative only
COMMON METHOD Reverse phase HPLC Normal Phase TLC
SAMPLE PREPERATION NO YES
TIME 3-6 Minutes 3-4 Hours
64. PCR (Polymerase Chain
Reaction)
Used to amplify single copy of DNA to thousands
of copies
Amplify specific DNA fragments from minute
quantity, even that DNA is of poor quality
On of the those few scientific revolutionary
development
69. Properties of DNA Template
Sample
DNA
It contains DNA region we need to amplify
Concentra
tion
Required
30- 50 ng with 50-55 % of GC content,
Low concentration – False negative result,
High concentration - False positive result
Purification
Required
Before procedure, template is purified and
impurities are removed by either anion exchange
technique or phenol-chloroform technique
73. Is short sequence of
Nucleotide
20 Nucleotide
containing single DNA
strand
< 10 and > 20
Nucleotide are not
recommended
2 Primers are used in
each PCR reaction
They are designed
(given sequence) that
they bind to opposite
strand of template
74. DNTP is building block of DNA molecule
• Consist of Phosphate group, deoxyribose sugar, and
nitrogenous base – adenine, guanine (Purine), thymine
and cytosine (Pyrimidine)
Function of DNTP is to expand DNA strand in
presence of Taq polymerase
• It binds with complementary DNA strand by Hydrogen
bond
76. Denaturation
Heat is applied to
reaction (70-75 °C)
To separate DNA strands
Single stranded template
formation occur
77. Annealing
Now Taq
Polymerase
will extend
the primer ,
synthesizing
new strand
of DNA
Taq
Polymerase
Primer
binds to its
complimen
tary
sequence
on the
single
stranded
DNA
Binding
After
heating,
reaction is
cooled (55
to 60 °C to
allow
primer to
bind
Cooling
78.
79. Extension
ELONGATION
Synthesis of new DNA Strand occurs with extension of primer
TAQ POLYMERASE
Being enzyme of DNA replication, assembles nucleotide and so extends primer
RAISING OF TEMPERATURE
After Annealing, temperature is raised to 70 °C for action of Taq Polymerase
80.
81.
82. Cycle and Timing
Cycle repeats 25-35 times in one PCR reaction
One PCR reaction takes 3-4 hrs. to complete
Time duration, depends upon length of DNA
region to be copied
If reaction is efficient, target region can go from
one or a few copies to billions
83.
84. APPLICATION
Genetic engineering
Diagnosing genetic
disorder and so
preventing disease
by screening before
birth, e.g - CF
Genetic
fingerprinting –
from sample of
blood, semen, hair
In Archaeology to
identify human or
animals from
samples
Detection of
Infection in the
Environment
Detection of
infectious diseases,
e.g – Covid 19
Personalized
medicine
85. Types of PCR
Here Complimentary DNA (CDNA) is created by
transcription from RNA
Then formation of dsDNA done, then by PCR, Gene
expression is qualitatively studied using RTPCR
Test for qualitative detection of nucleic acid from SARS-
CoV-2
86. Aka Real time PCR
Allow quantification of target species, that is total
bacterial count in chemical sample
By adding dye like Sybr green, the florescence signal is
proportional to amount of DNA synthesized
87. Used for the identification of species that inhabit humans
, e.g- Malaria
Multiple primer pairs are used for concomitant
detection of different species
88. Is modification of PCR, that reduces non-specific binding
of primary product, limiting non specific products
89. References
• Murray, Robert K. Harper's Illustrated Biochemistry. New York: McGraw-Hill, 2003. Print.
• Harvey, Richard A., Ph. D. Lippincott's Illustrated Reviews: Biochemistry. Philadelphia :Wolters Kluwer
Health, 2011.
• Kumar, Vinay, et al. Robbins Basic Pathology. 10th ed., Elsevier - Health Sciences Division, 2017.
• Postgraduate Pharmacology 1st Edition 2020 by Sougata Sarkar.
• Hearn, Milton TW. "High–Performance Liquid Chromatography and Its Application to Protein
Chemistry." Advances in chromatography (2021): 1-82.
• Jalil, Abduladheem Turki, et al. "Polymerase chain reaction technique for molecular detection of HPV16
infections among women with cervical cancer in Dhi-Qar Province." Materials Today:
Proceedings (2021).