in this slides contains principle and types of detectors used in Gas Chromatography.
Presented by: J.Vinay Krishna. (Department of industrial pharmacy),
RIPER, anantapur.
In this slide contains principle, instrumentation, methodology, and application of gel chromatography.
Presented by: SATHEES CHANDRA (Department of pharmaceutical analysis).
RIPER, anantapur
in this slides contains principle and types of detectors used in Gas Chromatography.
Presented by: J.Vinay Krishna. (Department of industrial pharmacy),
RIPER, anantapur.
In this slide contains principle, instrumentation, methodology, and application of gel chromatography.
Presented by: SATHEES CHANDRA (Department of pharmaceutical analysis).
RIPER, anantapur
Types of crystals & Application of x raykajal pradhan
some basic information:-
A crystal lattice is a 3-D arrangement of unit cells.
Unit cell is the smallest unit of a crystal, By stacking identical unit cells, the entire lattice can be constructed
A crystal’s unit cell dimensions are defined by six numbers, the lengths of the 3 axes, a, b, and c, and the three interaxial angles, α, β and γ.
If a unit cell has the same type of atom at the corners of the unit cell but not also in the middle of the faces nor in the centre of the cell, it is called primitive and given by symbol P
7 types of crystal system details
14 bravis lattice
APPLICATION X-RAY CRYSTALLOGRAPHY
1. Structure of crystals
2. Polymer characterisation
3. State of anneal in metals
4. Particle size determination
a) Spot counting method
b) Broadening of diffraction lines
c) Low-angle scattering
5.Applications of diffraction methods to complexes
a) Determination of cis- trans isomerism
b) Determination of linkage isomerism
6.Miscellaneous applications
In this slide contains types of HPLC Columns, Plate theory and Van Deemter Equation.
Presented by : Malarvannan.M (Department of pharmaceutical analysis).
RIPER,anantpur.
Hyphenated technique is a combination or coupling of two analytical techniques with the help of proper interface.
In this presentation Hyphenated techniques-LC-MS/MS, GC-MS/MS, HPTLC-MS has been discussed
Download and play it my friends it contain VIDEO
The technique of ion exchange chromatography is based upon the interaction between charged solute molecules and oppositely charged moieties covalently linked to chromatographic matrix.
The reasons for its widespread success is its applicability, high resolving power, high capacity and simplicity of the technique.
Separation in ion exchange chromatography depends upon the reversible adsorption of charged solute molecules to immobilized ion exchange groups of opposite charge. Most experiments are performed by following : Video For Understanding Play It
Types of crystals & Application of x raykajal pradhan
some basic information:-
A crystal lattice is a 3-D arrangement of unit cells.
Unit cell is the smallest unit of a crystal, By stacking identical unit cells, the entire lattice can be constructed
A crystal’s unit cell dimensions are defined by six numbers, the lengths of the 3 axes, a, b, and c, and the three interaxial angles, α, β and γ.
If a unit cell has the same type of atom at the corners of the unit cell but not also in the middle of the faces nor in the centre of the cell, it is called primitive and given by symbol P
7 types of crystal system details
14 bravis lattice
APPLICATION X-RAY CRYSTALLOGRAPHY
1. Structure of crystals
2. Polymer characterisation
3. State of anneal in metals
4. Particle size determination
a) Spot counting method
b) Broadening of diffraction lines
c) Low-angle scattering
5.Applications of diffraction methods to complexes
a) Determination of cis- trans isomerism
b) Determination of linkage isomerism
6.Miscellaneous applications
In this slide contains types of HPLC Columns, Plate theory and Van Deemter Equation.
Presented by : Malarvannan.M (Department of pharmaceutical analysis).
RIPER,anantpur.
Hyphenated technique is a combination or coupling of two analytical techniques with the help of proper interface.
In this presentation Hyphenated techniques-LC-MS/MS, GC-MS/MS, HPTLC-MS has been discussed
Download and play it my friends it contain VIDEO
The technique of ion exchange chromatography is based upon the interaction between charged solute molecules and oppositely charged moieties covalently linked to chromatographic matrix.
The reasons for its widespread success is its applicability, high resolving power, high capacity and simplicity of the technique.
Separation in ion exchange chromatography depends upon the reversible adsorption of charged solute molecules to immobilized ion exchange groups of opposite charge. Most experiments are performed by following : Video For Understanding Play It
Introduction to High Performance Liquid Chromatography (HPLC)Saurabh Arora
This presentation provides a brief introduction to HPLC and its parts. The technique has found immense scope of applications in both academic and industrial laboratories requiring identification and quantification of mixtures of organic compounds.It is essential for scientists working in any field to understand and know how to use a HPLC.
Detectors are the brain of any chromatograhic system. It help us to record the chromatogram based on certain characteristics of the analyte and help us in identifying that compound both qualitatively and quantitatively.
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
This document discusses various components of HPLC instrumentation including mobile phase reservoirs, pumps, sample introduction systems, columns, and detectors. It describes the basic components of an HPLC system including solvent bottles, pumps, autosamplers, columns, and detectors. It discusses different types of pumps including reciprocating pumps and syringe pumps. It also covers topics like column dimensions, fittings, packing materials, and sample introduction methods like manual injection and autosamplers.
High performance liquid chromatography (HPLC), also known as high pressure liquid chromatography, is essentially a form of column chromatography in which the stationary phase consists of small particle packings (3-50 µm) contained in a column with a small bore (2-5 mm), one end of which is attached to a source of pressurised liquid eluant (mobile phase)
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.
- 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
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.
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
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
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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.
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
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
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.
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.
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.
3. High Pressure Liquid Chromatography
or
High Performance Liquid Chromatography
What is it?
Separation technique based on solid stationary phase +
liquid mobile phase
How can achieve separation?
By partition
adsorption
ion exchange
5 October 2012 3
4. CLASSIFICATION OF HPLC:-
1. NORMAL PHASE: Stationary phase: Polar
Mobile phase: Non polar
Eg. Assay of Pilocarpine, Tacopherol, Piperazine
2. REVERSE PHASE: Stationary phase: Non polar
Mobile phase: Polar
Eg. Assay of Nifedipine, Sulphamethoxazole
Partition chromatography is used for hydrocarbon soluble
compound having molecular weight of lesser than
1000gm/mole.
5 October 2012 4
5. 3. ION-EXCHANGE CHROMATOGRAPHY (IEX)
Based on the different affinities of the ions for the
oppositely charged ions in the resin or adsorbed
counterions in the hydrophobic stationary phase.
Consider the exchange of two ions A and B between the
solution and exchange resin E :
A·E + B B·E + A
5 October 2012 5
6. 4. SIZE-EXCLUSION CHROMATOGRAPHY (SEC)
SEC is the method for dynamic separation of molecules
according to their size.
The separation is based on the exclusion of the molecules
from the porous space of packing material due to their
steric hindrance.
Hydrodynamic radius of the molecule is the main factor
determining its retention.
In general, the higher the hydrodynamic radius, the
shorter the retention.
5 October 2012 6
7. HPLC INTRUMENTATION CONSIST OF
Degasser
Solvent Reservoir( HPLC solvent reservoir systems)
Pumps
Pre Guard Column
Sample injection system
Columns
Detector
Recorder and integrators
5 October 2012 7
9. DEGASSER
Degassing of mobile phase is required because bubble has
property to expand or compress.
Degasser is needed to remove dissolved air
1) By Subjecting the mobile phase under vacuum.
2) By Purging with fine spray of an inert gas at lower
solubility such as Argon and Helium.
3) By heating and ultrasonic stirring.
5 October 2012 9
11. HPLC SOLVENT RESERVOIR SYSTEMS
These are the glass bottles use to store the mobile phase.
The mobile phase is pumped under pressure from one or several
reservoirs and flows through the column at a constant rate.
Desirable feature in the solvent delivery system is the capability
for generating a solvent gradient.
Filtration is needed to eliminate suspended particles and organic
impurities.
5 October 2012 11
12. PUMPS
Pass mobile phase through column at high pressure
and at controlled flow rate.
Performance of pump directly affects the Rt,
reproducibility, detector sensitivity.
5 October 2012 12
13. IDEAL CHARCETRISTIC OF A PUMP
Non corrosive and compatible with solvent.
Provide High pressure to push mobile phase
Provide constant flow rate to mobile phase.
Easy to change for one mobile phase to another.
Should have reproducible flow rate and independent of column
back pressure.
Should not leak & should be easy to dismantle and repair.
High pressure generated by pump 5 October 2012 should not lead to explosio1n3.
14. TYPE OF PUMP USED IN HPLC
1) Reciprocating pump
2) Displacement pump
3) Pneumatic pump
5 October 2012 14
16. WORKING
Contains reciprocating piston that moves back and forth in
hydraulic chamber.
By the movement of piston solvent flow into the column under
high pressure.
When piston moves backward inlet valve open while exit valve
closes. This result in mobile phase being drawn into the main
chamber (cylinder).
The reduction in volume in main chamber due to forward motion
of piston result in mobile phase moving out of the exit valve
u5n Odcteobre rh 2i0g12h pressure. 16
17. DISADVENTAGE
Pulsed flow which must be damped as they produce a base line
noise on the chromatogram
ADVANTAGES
Generate high output pressure (upto10000 poise).
Ready adaptability to gradient elusion.
Provide constant flow rate.
Pressure generated is so high that any back pressure generated in
the column due to higher viscosity of stationary phase can be
easily overcome.
5 October 2012 17
19. WORKING
Works on the principle of positive solvent pressure.
Consist of screw or plunger which revolves continuously driven
by motor.
Rotatory motion provides continuous movement of the mobile
phase which is propelled by the revolving screw at greater speed
and pushes solvent through small needle like outlet.
Consist of large syringe like chamber of capacity 250 – 500 ml.
Double syringe pumps have also been developed in which one
piston is delivering the solvent to the column while other one is
refilled from the reservoir.
5 October 2012 19
20. ADVANTAGES
Flow is pulse free.
Provide high pressure upto 200 – 475 atm.
Independent of column back pressure and viscosity of solvent.
Simple operation.
DISADVENTAGE
Limited solvent capacity
Gradient elution is not easy.
5 October 2012 20
22. WWOORRKKIINNGG
The driving air is applied, piston moves, inlet closes & outlet
open pushing mobile phase to the column.
Pressure on solvent is proportional to the ratio of piston usually
50: 1.
A lower pressure gas source of 1- 10 atm can be used to generate
high liquid pressure .( 1 – 400 atm )
About 70 ml of the mobile phase is pumped from every stroke.
ADVENTAGES: Pulse free flow & Generates high pressure.
DISADVANTAGES: 1) Limited volume capacity (70 ml )
2) Pressure output and flow rate depends on the viscosity and
column back pressure.
3) Gradient elusion is not possible. 22
5 October 2012
23. SAMPLE INJECTION SYSTEM
Septum injectors
Stop flow Septumless injection.
Rheodyne injector / loop valve type.
5 October 2012 23
24. SEPTUM INJECTION PORT.
Syringe is used to inject the sample through an inert septum
directly into the mobile phase.
Drawback: - leaching effect of the mobile phase in contact with
septum, which may give rise to ghost peaks.
STOP FLOW SEPTUMLESS INJECTION.
Flow of mobile phase through the column is stopped while
Syringe is used to inject the sample.
Drawback: formation of ghost peak.
5 October 2012 24
25. RHEODYNE INJECTOR / LOOP VALVE TYPE.
Sample is introduced in the column without causing interruption
to mobile phase flow.
Volume of sample ranges between 2 μl to over 100 μl.
Operation of sample loop.
Sampling mode
Injection mode.
Sample is loaded at atmospheric pressure
into an external loop in the micro volume
sampling valve, & subsequently injected
into mobile phase by 5 October 2012 suitable rotation of the valve. 25
26. COLUMN
Made up of stainless steel or heavy glass to withstand the
pressure.
The columns are usually long (10 – 30 cm) narrow tubes.
Contains stationary phase at particle diameters of 25 μm or less.
The interior of column should be smooth and uniform.
Column end fitting are designed to have a zero void volume.
5 October 2012 26
27. CLASSIFACTION OF CLOUMN
column
Main column Guard column
Analytical column Preparative column
Standard column
Narrow bore
Short fast column
Micro preparative
Preparative column
Macro preparative
27
A) BASED ON APPLICATION
5 October 2012
28. B) BASES OF COMPONENTS
Bonded phase column
Column where liquid is inpermagneted on solid inert
support
5 October 2012 28
29. ANALYTICAL COLUMN
STANDARD COLUMN
• Internal diameter 4 – 5 mm and length 10 – 30 cm.
• Size of stationary phase is 3 – 5 μm in diameter.
• Used for the estimation of drugs, metabolites, pharmaceutical
preparation and body fluids like plasma.
NARROW BORE COLUMN
Internal diameter is 2 – 4 mm.
Require high pressure to propel mobile phase.
Used for the high resolution analytical work of compounds with
very high Rt.
5 October 2012 29
30. SHORT FAST COLUMN
Length of column is 3 – 6 cm.
Used for the substances which have good affinity towards the
stationery phase.
Analysis time is also less (1- 4 min for gradient elusion & 15 –
120 sec for isocratic elusion).
PREPARATIVE COLUMN
Used for analytical separation i.e. to isolate or purify sample in
the range of 10-100 mg form complex mixture.
Length – 25- 100 cm
Internal diameter – 6 mm or more.
5 October 2012 30
31. TYPES OF PREPARATIVE COLUMN
Micro preparative or semi preparative column
Modified version of analytical column
Uses same packaging and meant for purifying sample less
then 100 mg.
Preparative column
Inner diameter – 25 mm .
Stationary phase diameter – 15- 100 μm
Macro Preparative Column
Column length – 20 – 30 cm
Inner diameter – 600 mm
5 October 2012 31
32. GUARD COLUMN
They are placed anterior to the separating column.
Serve as a protective factor that prolongs the life and
usefulness of the column.
They are dependable column designed to filter or remove
Particles that clog the separation column.
Compounds and ions that could ultimately cause baseline
drift, decrease resolution , decrease sensitivity and create
false peaks.
5 October 2012 32
33. BONDED PHASE COLUMN
Here the molecules, comprising the stationary phase i.e. the
surface of the silica particles, are covalently bonded to a silica
based support particles.
The most popular bonded phase, siloxanes, are formed by
heating the silica particles in dilute acid for the day so as to
generate the reactive Silonal group.
- OH OH OH
ו ו ו
- Si – O – Si - O - Si -
5 ו O c t o b e r 2 ו 0 1 33 ו ו 2
34. Silonal group is the treated with organochlorosilane.
These bonded phases are stable between the pH range 2 – 9 and
upto temperature of 80º C.
Bonded phase is made with a linear C 18 hydrocarbon, also
know as ODS (octadecyl silane) bonded phase. Used in
pharmaceutical analysis or separation of less polar components.
An alkyl nitrile column or cyano column which has 12 carbon
atoms with the last atom appearing as a nitrile group (CN),
moderately polar column.
Amino alkyl bonded phase column which is normally C 8, last C
atom bearing NH2 group, Polar column. Use full in separation of
CHO, peptides, amino acids.
5 October 2012 34
35. Advantages
Can withstand high pressure exerted by mobile phase.
Life of column is more.
No bleeding effect
Disadvantages
Very expensive
Manually can not be fabricated
5 October 2012 35
36. COLUMN WHERE LIQUID IS INPERMAGNETED
ON SOLID INERT SUPPORT.
These are not use widely now days.
Stationary phase dose not have the strength to stay in the
column on account of the physical forces exerted by the
mobile phase at very high pressure.
Amount of loading on inner support is minimum
Stationary phase starts bleeding out of the column and can
cause resistance to mass transfer.
5 October 2012 36
37. METHOD OF PACKING
Depends on the mechanical strength & Particle size of the
stationary phase.
Particle size greater then 20 μm – dry packing
Particle size lesser then 20 μm – slurry packing / wet packing.
WET / SLURRY PACKING
Particle size with diameter less then 20 μm can only be placed
wet as a suspension.
Suspension should be stable, it should not sediment, and
agglomentation should be avoided.
5 October 2012 37
38. DRY PACKING
Particle size greater then 20 μm filled into vertical clamped
column in small quantity.
Deposition is done by tapping or vibrating the column.
Column is unclamped and the tapped on the firm surface to
obtain dense and reproducible packing.
5 October 2012 38
40. DETECTORS
Based on the application, the detectors can be classified into
Bulk property detectors
Solute property detectors.
5 October 2012 40
41. BULK PROPERTY DETECTORS
Compare an over all change in physical property of mobile phase
with or without an eluting solute.
These types of detectors tend to be relatively low sensitive and
require temperature control.
e.g. Refractive index detector.
SOLUTE PROPERTY DETECTORS
They respond to a physical property of the solute that is not
exhibited by the pure mobile phase.
These detectors are more sensitive, detect the sample in
nanograms quantity.
e.g. Uv visible detector , Electrochemical detector, Fluorescence
d5e Otcetocbetro 2r0.12 41
42. ULTRAVIOLET VISIBLE DETECTOR
They measure the ability of a sample to absorb light. This can be
accomplished at one or several wavelengths.
A light source deliver a monochromatic parallel light beam
which passes through a cell swept by the column effluent, and
falls on photocell.
Selective in nature, detect only those solutes that absorb Uv/
visible radiation
E.g. alkenes, aromatic compounds and compound having
multiple bonds between C and O, N or S.
5 October 2012 42
43. BASICALLY THREE TYPES OF ABSORBANCE DETECTORS
ARE AVAILABLE
Fixed Wavelength Detector
Variable Wavelength Detector
Diode Array Detector
FIXED WAVELENGTH DETECTOR
5 October 2012 43
44. Detectors which do not allow changing the wavelength of the
radiation called fixed-wavelength detectors.
In this, most of the light may be emitted at a one wavelength,
with most single wavelength UV lamps.
Low-pressure mercury lamp emits very intense light at 254 nm.
By filtering out all other emitted wavelengths, utilize only 254
nm line to provide stable, highly sensitive detectors capable of
measuring subnanogram quantities of any components which
contains aromatic ring
The 254 nm was chosen since the most intense line of mercury
lamp is 254 nm, and most of UV absorbing compounds have
some absorbance at 254 nm.
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46. Variable wavelength detector employs a lamp that emits light
over a wide range of wavelengths and by using a
monochromator, light of a particular wavelength can be
selected for detection purposes.
Depending on the sophistication of the detector, wavelength
change is done manually or programmed on a time basis into
the memory of the system.
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47. DIODE ARRAY DETECTOR
It is also a multiwavelength UV detector, but functions on an
entirely different principle.
The UV photo diode-array detector.
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48. FLUORESCENCE DETECTORS
Very sensitive, but very selective.
By definition, it will detect only those materials that will
fluoresce or, by appropriate derivatization can be made to
fluoresce.
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49. Fluorescence occurs when compounds having specific functional
groups are excited by shorter wavelength energy and emit higher
wavelength radiation.
Fluorescence is often collected at right angle to excitation beam.
With all sample cells, scattered radiation from the excitation
source is selectively removed with cut off or band pass filters
placed before photomultiplier tube.
Most important detectors for use in trace analysis both in
environmental and forensic analysis.
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50. REFRACTIVE INDEX DETECTOR OR
DIFFERENTIAL REFRACTOMETER
The detection principle involves measuring of the change in
refractive index of the column effluent passing through the flow-cell.
It responds to any solute whose refractive index is significantly
different from that of the mobile phase.
Principle: it is based on two principles.
Deflection ( deflection type refractometer)
Reflection (reflection type refractometer)
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51. DEFLECTION TYPE REFRACTOMETER.
Measure the deflection of a beam of a monochromatic light by double
prism.
Eluent passes through one half of prism & pure mobile phase to other
half known as reference compartment.
Reference and sample compartment are separated by diagonal glass
divider.
Auto zero is used to set, out put signal to zero when mobile phase is in
both the compartments.
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52. Tungsten lamp provides beam of light collimated through lens
and passes through Eluent and reference compartment.
Reflected by the mirror through the same compartment again
The beam of light is focused on a beam splitter before passing
into the photo detector.
Refractive index of the mobile phase is changed due to the
presence of solute, the beam from the sample compartment is
deflected which produces the change signal that is proportional
to the concentration of solute.
Advantages
Wide range of linearity.
Covers entire refractive index range.
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53. REFLECTION TYPE REFRACTOMETER
Measure change in % of reflected light at glass liquid interface as
the reflective index of liquid changes.
Based on the Fresnel's law of reflection which states
“The amount of liquid reflected at a glass- liquid interface varies
with the angle of incidence and the refractive index of the liquid”
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54. working:
Two collimated beams from the projector (light source & lens)
illuminate the reference and sample cell.
Cells are formed of Teflon gasket, which is clamped between the
cell prism and a stainless steel reflecting back plate.
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55. As the light of beam is transmitted through the cell interfaces, it
passes through the liquid film and imposes on the surface of the
reflecting back plate.
Diffused, reflected light appears as two spots and passes through
the lens and detected by photo detector.
The ratio of the reflected light to transmitted light is function of
refractive index of the two liquid, the illumination of the cell
back plate is direct measure of the refractive index of the liquid
in each chamber
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56. ELECTROCHEMICAL DETECTOR OR
AMPEROMETRIC DETECTOR
It is based on the measurements of the current resulting from an
oxidation/reduction reaction of the analyte at a suitable electrode.
The level of the current is directly proportional to the analyte
concentration
Also called coulometric detector.
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57. RECORDER AND INTEGRATORS
Recorders are used to record the response obtained from the
detector after amplification. They record the baseline and all the
peaks obtained, with respect to time. Retention time for all the
peaks can be calculated.
Integrators are improved versions of recorder with data
processing capabilities. They can record the individual peaks
with retention time, height and width of peak, peak area, etc.
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58. DERIVATIZATION
The most commonly used detector in HPLC is 254 nm UV
detector, many methods have been developed to introduce or
enhance chromophores that will absorb light at this wavelength.
Also, reactions have been developed to produced a fluorophore
for the purposes of fluorimetric detection.
While it is common to derivatize analytes in order to improve
chromatographic properties, the emphasis in this section will be
on derivatization for the benefit of detectability.
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59. Derivatization may be either pre-column or post-column.
PRE-COLUMN: Derivatization-Injection-Separation-Detection.
Ex. Treatment of ketosteroids with 2, 4, DNP,
Benzoylation of hydroxysteroids,
Esterification of fatty acids.
POST-COLUMN: Injection-Separation-Derivatization-Detection.
Ex. Reaction of amino acid with ninhydrin,
Reaction of fatty acids with o-nitrophenol,
Thermal or acid/phenol treatment of carbohydrates.
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60. IDEAL CHARACTERISTICS
The ideal derivatization reaction is rapid, goes to completion,
produces a stable product.
Product has suitable chromatographic & spectral properties.
The unreacted derivatizing reagent should not interfere with the
chromatographic separation.
The derivatization reactions are characteristics of
functional group, their description will be classified according to
functional group.
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61. 1. CARBOXYLIC ACIDS
Ex.
PDBI (O-p-nitrobenzyl-N,N’-diisopropylisourea) &
1-(p-Nitro)benzyl-3-p-tolytriazine also reacts with carboxylic
acids to produce esters.
4-Bromomethyl-7-methoxycoumarin (BMC) reacts with
carboxylic acids to form a fluorigenic product.
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62. 2. ALCOHOL
Activated carboxylic acid derivatives such as acyl chlorides are
the most common reagents.
This reaction gives a product that has a molar absorptivity at 254
nm too low to be analytically useful.
P-nitrobenzoyl chloride, 3,5-dinitrobenzoyl chloride & anisyl
chloride form esters that have much higher molar absorptivity.
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63. 3. AMINES
The same acylating reagents used for alcohol can also be used
for amines.
R-NH2 + R’COCl R’CONHR + HCl
This reaction has been used for the analysis of tobramycin
in serum.
10 & 20 amines react with 7-chloro-4-nitrobenzyl-2-oxa-1,3-
diazole(NBD chloride) to produce a fluorescent derivative by
displacement of 7-chloro group.
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64. 4. ALDEHYDES & KETONES
Nucleophilic addition to a carbon-heteroatom double bond are
most frequently employed for derivatization of carbonyl
compounds.
A prototype reaction is the condensation of a ketone with
2,4-dinitrophenylhydrazine(2,4-DNPH) to form the hydrazone.
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65. REFERENCES
B.K.Sharma, Instrumental method of chemical
analysis, GOLE Publishing House, Page no 292-304.
Ashutosh kar, Pharmaceutical drug analysis -2nd
edition, page no 453 456,459,466
Dr.A.V.kasture, Pharmaceutical analysis vol-2, page
no 52, 53.
Elena katz, Roy Eksteen, Peter Schoenmarkers, Neil
Miller, Handbook of HPLC, volume 78, Special
Indian Edition, page no. 536-550.
Munson, Pharmaceutical Analysis, Page no. 76-80
http//hplc.chem.shu.edu/new/hplcbook/detector
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