1) HPLC is a form of liquid chromatography that uses high pressure to push a mobile phase through a column containing a stationary phase to separate complex mixtures.
2) The main components of an HPLC system are the pump, injector, analytical column, and detector. The pump delivers the mobile phase at high pressure. The injector introduces the sample into the column. Separation occurs in the analytical column. The detector then detects the separated components.
3) The main advantages of HPLC are its ability to accurately analyze complicated samples with speed, precision and sensitivity. It can separate both polar and non-polar compounds.
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)
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)
Basavarajeeyam is a Sreshta Sangraha grantha (Compiled book ), written by Neelkanta kotturu Basavaraja Virachita. It contains 25 Prakaranas, First 24 Chapters related to Rogas& 25th to Rasadravyas.
Best Ayurvedic medicine for Gas and IndigestionSwastikAyurveda
Here is the updated list of Top Best Ayurvedic medicine for Gas and Indigestion and those are Gas-O-Go Syp for Dyspepsia | Lavizyme Syrup for Acidity | Yumzyme Hepatoprotective Capsules etc
New Drug Discovery and Development .....NEHA GUPTA
The "New Drug Discovery and Development" process involves the identification, design, testing, and manufacturing of novel pharmaceutical compounds with the aim of introducing new and improved treatments for various medical conditions. This comprehensive endeavor encompasses various stages, including target identification, preclinical studies, clinical trials, regulatory approval, and post-market surveillance. It involves multidisciplinary collaboration among scientists, researchers, clinicians, regulatory experts, and pharmaceutical companies to bring innovative therapies to market and address unmet medical needs.
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
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.
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.
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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!
CDSCO and Phamacovigilance {Regulatory body in India}NEHA GUPTA
The Central Drugs Standard Control Organization (CDSCO) is India's national regulatory body for pharmaceuticals and medical devices. Operating under the Directorate General of Health Services, Ministry of Health & Family Welfare, Government of India, the CDSCO is responsible for approving new drugs, conducting clinical trials, setting standards for drugs, controlling the quality of imported drugs, and coordinating the activities of State Drug Control Organizations by providing expert advice.
Pharmacovigilance, on the other hand, is the science and activities related to the detection, assessment, understanding, and prevention of adverse effects or any other drug-related problems. The primary aim of pharmacovigilance is to ensure the safety and efficacy of medicines, thereby protecting public health.
In India, pharmacovigilance activities are monitored by the Pharmacovigilance Programme of India (PvPI), which works closely with CDSCO to collect, analyze, and act upon data regarding adverse drug reactions (ADRs). Together, they play a critical role in ensuring that the benefits of drugs outweigh their risks, maintaining high standards of patient safety, and promoting the rational use of medicines.
The Gram stain is a fundamental technique in microbiology used to classify bacteria based on their cell wall structure. It provides a quick and simple method to distinguish between Gram-positive and Gram-negative bacteria, which have different susceptibilities to antibiotics
- Video recording of this lecture in English language: https://youtu.be/lK81BzxMqdo
- Video recording of this lecture in Arabic language: https://youtu.be/Ve4P0COk9OI
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- Video recording of this lecture in English language: https://youtu.be/kqbnxVAZs-0
- Video recording of this lecture in Arabic language: https://youtu.be/SINlygW1Mpc
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Basavarajeeyam is an important text for ayurvedic physician belonging to andhra pradehs. It is a popular compendium in various parts of our country as well as in andhra pradesh. The content of the text was presented in sanskrit and telugu language (Bilingual). One of the most famous book in ayurvedic pharmaceutics and therapeutics. This book contains 25 chapters called as prakaranas. Many rasaoushadis were explained, pioneer of dhatu druti, nadi pareeksha, mutra pareeksha etc. Belongs to the period of 15-16 century. New diseases like upadamsha, phiranga rogas are explained.
2. Pharmaceutical Analysis
•High Performance Liquid
Chromatography
•High Pressure Liquid Chromatography
•HPLC says
•If you are able to dissolve it
•We are able to resolve it
4. Importance
• Chromatography has application in every branch of
the physical and biological sciences
– 12 Nobel prizes were awarded between 1937 and 1972
alone for work in which chromatography played a vital role
4
5. Chromatography Definition
• Chromatography is defined as the physical
method of separation, in which the mixture
of analytes is separated using two phases,
one is stationary phase and other a mobile
phase which percolates through the
stationary phase. The separation occurs
because of difference in affinity between
analytes and stationary phase.
5
6. Introduction
High-performance liquid chromatography
(HPLC) is a form of liquid chromatography to
separate compounds that are dissolved in
solution
• HPLC is characterized by the use of
high pressure to push a mobile phase solution
through a column of stationary phase allowing
separation of complex mixtures with high
performance /resolution
6
7. • The different components in the mixture pass
through the column at different rates due to five
different reasons:
• 1: Polarity of solutes 2: ionic nature 3:Mol.Wt
4: partitioning ability 5: Ability to form affinity
complexes
• This requires sophisticated instrument.
• Provides specific, sensitive and precise method for
analysis of different complicated samples.
7
9. Advantages of HPLC
Sensitive method for analysis of different
complicated samples
There is ease of sample preparation and introduction
There is speed of analysis
Accuracy and precision
Operating at low temperature so can be used for
separation of thermolabile, high polarity substances
9
10. Basic Components
• Pump to deliver mobile phase at high
pressure
• Injection system to introduce sample
• Column for separating sample components
• Detector to detect the separated components
• Data System to operate, control instrument
and process the signal given by the detector
10
12. HP/LC
LC- Liquid Chromatography (Liquid- mobile phase)
•LSC •LLC
•Adsorption
Chromatography
•Partition
Chromatography
•on nature of stationary phase
•Normal
Phase
•Reversed
Phase
•on polarity of stationary phase &
mobile phase
13.
14. Chromatography Stationary Phases
•relatively polar surface
O O O
| | |
OSiOSiOSiOH
| | |
O O O
| | |
OSiOSiOSiOH
| | |
O O O
•bulk (SiO2)x
•surface
• relatively nonpolar surface
•Silica Gel
O O O
| | |
OSiOSiOSiOR
| | |
O O O
| | |
OSiOSiOSiOR
| | |
O O O
•bulk (SiO2)x
•surface
•Derivatized Silica Gel
•Where R = C18H37
•hydrocarbon chain
•(octadecylsilyl deriv.
•silica or “C18”)
•“normal phase” •“reversed phase”
15. Normal vs. Reversed Phase Chromatography
Normal Phase Reversed Phase
Stationary phase Polar (silica gel) Non-polar (C18)
Mobile phase
Non-polar
(organic solvents)
Polar
(aqueous/organic)
Sample movement Non-polar fastest Polar fastest
Separation based on
Different polarities
(functionality)
Different
hydrocarbon content
16. 16
HPLC
• HPLC as compared with the classical technique is
characterized by:
Small diameter (2-5 mm), reusable stainless steel columns;
Column packings with 3, 5 and 10 µ particles.
High inlet pressures and controlled flow of the mobile phase;
Precise sample introduction
Special continuous flow detectors capable of handling small
flow rates and detecting very small amounts.
Automated standardized instruments;
Rapid analysis; and
High resolution.
17. 17
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
• Very small particles of narrow distribution range.
• Uniform pore size and distribution.
• High pressure column slurry packing technique.
•
• Accurate low volume sample injectors.
• Sensitive low volume detector.
•
• Good pumping systems.
•
18. Instrumentation in HPLC
Mobile phase reservoir
Pump and Gradient Controller
Mixing Chamber
Sample Conditioning Column
Sample injector unit
Precolumn
Analytical column
Detectors
18
21. Classification of HPLC on the internal
diameter of column
• Semi Micro (0.3-1mm ID)
• micro (1-3mm ID)
• Conventional (4-8mm ID)
• Semi preparative (10-20mm ID)
• Preparative (20-50mm ID)
• Process (less than 50mm ID)
21
22. Solvent/ mobile phase reservoirs
• Glass or stainless-steel containers capable of
holding up to 1 liter mobile phase (pure
organic solvents or aqueous solutions of salts
and buffers)
• Inert to a variety of aqueous and non aqueous
mobile phases.
• Stainless steel should be avoided for use with
solvents containing halide ions.
23. • In many cases, aqueous solvents & some organic
solvents are degassed prior to use
• Degassing is done to prevent formation of gas
bubbles in the pump or detector ( Mobile phases are
degassed by stirring of the mobile phase under
vacuum, sonication or sparging with helium gas)
• The mobile phase are filtered to remove particulate
matter that may clog the system
Degassing & filtration of mobile phase
24. Tubing
• Should be inert,
• have ability to withstand pressure
• able to carry sufficient volume
25. 25
PUMPS
– FUNCTION
– Deliver constant , reproducible flow of eluent to the column under
high pressure.
– What is a good HPLC pump?
– Capable of delivering a range of flow rates suitable for different modes of
HPLC.
– Reproducibility of flow rates.
– Ability to operate under high pressure - 6000 psi
– Stable flow without pulsations to minimise detector noise.
– Compatibility of components with the wide range of eluents
– Easy access for regular maintenance.
26. Solvent delivery system (Pump)
• Must be constructed from material that are
inert to all mobile phases
• Materials commonly used are glass, stainless
steel, Teflon, and sapphire.
• The solvent flow rate produced by pump
should be pulseless or should be dampened in
order to remove pulses (pulses may cause
spurious results with some detectors)
27. •HPLC
Pump
•Constant flow rate (Mechanical) Pump •Constant Pressure Pump
(Pneumatic)
•Manner in which they
operate
•syringe
pump
(screw
driven)
•Reciprocating
pump
•Single
piston
reciprocatin
g pump
•Double
piston
reciprocatin
g pump
•Reciprocati
ng
diaphragm
pump
•Direct pressure
pump
•Amplifier
pump
28. 28
Constant Pressure Pumps
• Advantages of constant pressure pumps
Simple
Free from pulsation's resulting in smooth baselines.
Inexpensive, easy to operate, and easy to maintain.
• Disadvantages
Flow rate must be monitored carefully and constantly,
Factors affecting flow rate -
Solvent viscosity due to a temperature or composition change.
• Changes in flow rate can affect-
• Qualitative - dependent on retention time.
• Quantitative analysis - detectors are concentration dependent, affects the peak
area to be taken for calculation.
29. Pumps
• Pneumatic pump : which produce a constant pressure
• Gas displacement type: which use direct pressure from
a highly compressed gas to force solvent out of a tube
• Pneumatic amplifier type: in which compressed gas at a
lower pressure impinges on the large end of the piston
to force the smaller end to deliver the liquid.
• The pneumatic pumps have the advantage of pulseless
operation.
30. 30
Constant Flow Pumps
• Advantages -
Ability to repeat elution volume and peak area, regardless of viscosity
changes.
• Two types
- Reciprocating piston -
1. Can maintain a liquid flow for indefinitely long time.
2. Causes flow and pressure pulsation.
Used - Most of the HPLC applications.
- Positive displacement (syringe) pumps -
1. Has to be refilled after it displaces the whole syringe volume.
2. Does not have any flow and pressure pulsation.
Used - Micro-HPLC applications where a constant flow at the
micro-liter per minute flow rate range is essential.
31. 31
Reciprocating Piston Pumps
.
•Disadvantage -
•Pressure pulsations.
•Pulse dampers are used to reduce pulsation.
The piston expels liquid through a one-way valve (check valve).
The pumping rate is usually adjusted by controlling the distance the piston retracts.
32. 32
Reciprocating dual piston pump
• Dual Piston Pumps - provides a constant and almost pulse free flow.
• Both pump chambers are driven by the same motor through a common
eccentric cam; this common drive allows one piston to pump while the
other is refilling.
• As a result, the two flow-profiles overlap each other significantly
reducing the pulsation downstream of the pump.
33. 33
]
Advantages of dual piston pumps
• A computer-designed camshaft is used to achieve maximum overlap of
pump strokes, eliminating pulsation or ripple.
• Use of staggered inlet/outlet lines to allow complete flushing when
liquids are changed or if air is drawn through the pump.
• Small-volume check valves are used to allow the pumps to function
reliably at flow rates as low as 0.001 mL/min.
• Fewer moving parts, with maintenance-requiring components pump
seals, check valves) readily accessible from the front of the instrument.
• A wide flow rate range (0.01 to 10 ml/min) is provided without gear
change.
34. 34
MODES OF ELUTION
1. Isocratic elution.
– Solvent composition remains constant.
2. Gradient elution.
– Steady changes in the mobile phase composition.
– Used when solutes of different retention times need to be analysed.
– Reduce retention times of the later eluting peaks by increasing the solvent
strength of the eluent.
– Stepwise switching from one eluent to another after a certain interval of time.
– Continuos gradient of solvent strength.
35. • If gradient analysis is necessary for separation, most
common way of forming gradient is to include
second reservoir and pump and a gradient controller.
• Gradient controller is an electronic device combines
the operation of two pumps, to provide mobile phase
mixture of desired conc. E.g. if 50:50 mixture of
solvent in two reservoirs is desired at an flow rate of
1mL/min., controller adjust the rate of delivery of
each pump to 0.5 mL/min. The solvents then are
combined in the mixing chamber.
35
36. • Solvent Conditioning Column:
• HPLC column materials are prepared from Silica
gel, which dissolves in solvents having pH values
are below 2 or above 7.
• This results in shrinkage of column gives void
spaces in which separated solute remixed or
diluted results low resolution.
• To minimize this and protect expensive silica
based packing materials,
36
37. • A small column (5 to 10 cm) packed with HPLC
grade silica gel is inserted into liquid stream
after the pump but before the injector.
• The material in this column is dissolved and
saturating the mobile phase and preserving the
analytical column.
• So the conditioning column need not be used
always.
37
38. 38
INJECTORS
Injection device should deliver
1. Sample within the range of 0.1 to 100 ml of volume.
2. High reproducible volumes under high pressure (up to the 4000 psi).
3. Produce minimum band broadening.
4. Minimize possible flow disturbances.
• Rheodyne injector
• Valves are commonly used.
• Samples introduced reproducibly into pressurized columns without significant
interruption of flow, even at elevated temperatures
39. Injection device
• The solute mixture is introduced into the
chromatograph by means of suitable injection device.
•Septum injectors : are available, in which the sample
solution is injected through a self sealing rubber or Teflon disk
using a micro liter syringe.
•-This may be done while the mobile phase is flowing or while
it has been stopped temporarily.
•-Inexpensive and easy to use
•-Problem : lack of reproducibility
•Type of injector
•syringe •Stopped flow
•Injection device
•Solvent flowing,
•(Loop injectors)
40.
41. Rotary-valve and loop injector
• This consist of a stainless-steel and Teflon
block that has been drilled to provide two
alternative paths for solvent flow each
selectable by a rotating valve.
43. 43
INJECTORS
•Advantages of valve injection - rapid, reproducible, and operator independent
delivery of a wide range of sample volumes from 60 nl up to several milliliters at
pressures up to 7000 psi .
•Disadvantage - sample loop must be changed to obtain various sample volumes.
44.
45. Precolumn:
It contains a packing chemically identical to that in
analytical column.
Mainly used to remove the impurities from the
solvent and thus prevents contamination of the
analytical column.
It is also called as guard column.
it is having large particle size.
It is having short length of 2 to 10 cm, so does not
affect separation.
45
46. Analytical column:
The success or failure of analysis depends upon choice of
column.
Actual separation is carried out here.
Stainless –steel tube
size – length -25 to 100 cm
Internal diameter – 2 to 4.6 mm
Column is filled with small particles 5 – 10 micron. The
solid support can be silica gel, alumina.
The separation is result of different components adhering to
or diffusion into the packing particles when the mobile
phase is forced through column.
46
47. Analytical column:
• Both C8 and C18 columns are considered as examples of
reversed phase liquid chromatography (RP).
• The stationary phase here is seen as a thin film of non-polar
liquid phase that has been designed to be chemically similar
to an inert material (Silica gel particles).
• The non-polar layer is chemically linked to the silica
particles surface by reaction with the polar silanol groups on
the stationary phase surface and so rendering them less
polar or non-polar.
47
48. • The difference between the two columns will be in the
length of the carbon-chain attached to the silica surface.
• Acoordingly C8 hplc columns have packing material
composed of silica particles attached to C8 carbon units
• C18 will, of course, have packing materials coated with C18
hydrophobic units.
• Categorically both are reversed phase but C18 columns will
definitely be more "hydrophobic rather than the C8
columns.
48
49. 49
COLUMN
Must operate in high pressure
Usually constructed of metals
Typical dimensions
10-30 cm long
1-3 cm ID
Contains packing material which
holds the stationary phase
Many types exist
Typical packing materials are 5-
10 µm in diameter
Guard column used to extend life of
main column
50. 50
DETECTORS
• Function of a detector -
– High Precision, High sensitivity & High stability.
• Basic detector should -
1) be capable of detecting 1 part or less of solute in 106 parts of eluent.
2) cause no re-mixing of solute bands.
3) have a wide linear dynamic range to ensure good quantitative analysis.
4) have low noise level and drift.
5) have fast response time to record rapidly eluting peaks.
6) be insensitive to flow rate and temperature changes.
7) be insensitive to eluent composition changes to allow gradient elution.
8) be reliable and reproducible.
9) be easy to operate and maintain.
10) be non-destructive.
51. 51
DETECTORS
• What is Noise ?
• Any disturbance of the detector baseline which is not related to eluted solute
is termed as ‘Noise’.
• Short term noise is the short variation of the baseline from a straight line
caused by
– electric signal fluctuations, lamp instability,temperature fluctuations and
other factors.
– Noise usually has much higher frequency than actual chromatographic
peak.
Drift is continuos increase or decrease of the detector signal as a
result of temperature or mobile phase composition changes.
52. Detectors:
Features of good detectors are
1. should respond to all components in
the mixture
2. should not respond to mobile phase
3. should be unaffected by the changes
in temp. and flow rate.
4. should have high sensitivity, low
noise 52
53. •Major Types of HPLC Detectors
•Solute Property
•UV-Vis
•Electrochemical
•Fluorescence
•Fixed l
•Variable l
•Photodiode Array
•Amperometry
•Pulse Amperometry
•Voltammetry
•Coulometry
•Bulk Property
•Refractive Index
•Conductivity
•Suppressed
•Non-suppressed
•Deflectance Type
•Reflectance Type
54. 54
OPTICAL DETECTORS
• Ultraviolet (UV) - Most popular and widely used.
– The solutes that contain a chromophore at the monitoring wavelength, absorb the
incident light as they pass through the flow cell.
– Amount of light absorbed produces a signal proportional to the concentration of solute.
–
» Absorbance (A) = ECL
55. 55
FIXED WAVELENGTH DETECTOR
• HPLC detectors which does not allow to change the wavelength of the
radiation called fixed-wavelength detectors.
• Cost effective.
• Low-pressure mercury vapor lamp emit very intense light at 253.7 nm.
By filtering out all other emitted wavelengths, the 254 nm line is utilised
to provide stable, highly sensitive detector.
• 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.
58. Characteristics:
•These detectors are nondestructive.
•These are insensitive to changes in solvent flow rate
and temperature.
•Drawback:
•Nonuniformity of response for different compounds.
58
59. •Photodiode Array Detectors (PDA) or DAD
•--Even much more rapid scanning of the absorption spectra of the eluted peak is
•possible using a photodiode array detector
•--The optical arrangement of the photodiode array detection is shown below:
•--Optical arrangement is referred to as
•“reverse optics”. This is because the
•dispersion device (holographic gratings) is
•placed after the flow cell (opposite to UV-Vis)
• Working of DAD
a) Light from a continuum source (e.g., D2
• Lamp) passes through a lens system which
• focusses polychromatic light onto the
• flow cell (containing the sample)
•b) The transmitted light then falls on a
•holographic gratings where it is dispersed
•into a photodiode array (PDA).
•c)PDA is a several hundreds of photodiodes
•arranged in a linear fashion. A typical
•photodiode array has 512 diodes to cover a
•range of wavelength (190-800 nm), each photodiode has a bandwidth of 2 nm.
•d) A range of wavelengths of light falls on a photodiode array and each diode picks
•up a different wavelength of light.
60. 60
PHOTO DIODE ARRAY DETECTOR-PDA
• Special feature is the ability to perform spectroscopic scanning.
•Precise absorbance readings at a variety of wavelengths while the peak is passing
though the flow cell.
•Allows for the best wavelength(s) to be selected for actual analysis.
•Allows for the determination of peak purity when the peak shape in itself does not
reveal that it actually corresponds to two (or even more) components.
61. 61
REFRACTIVE INDEX DETECTOR
• Principle- Measuring of the change in refractive index of the column
effluent passing through the flow-cell.
• The deflection of a light beam is changed when the composition in the
sample flow-cell changes in relation to the reference side.
• When no sample is present in the cell, the light passing through both
sides is focused on the photo-detector.
• As sample elutes through one side, the changing angle of refraction
moves the beam.
62. 62
R I DETECTOR
Advantages :
– (1) universal response
– (2) low sensitivity to dirt and air bubbles in the cells
– (3) the ability to cover the entire refractive index range from 1.000
to 1.750 RI with a single, easily balanced cell.
Disadvantages:
– Low Sensitivity
– Complex mixtures, may cover a wide range of refractive index
values and some may closely match that of the mobile phase,
becoming invisible to the detector.
– Changes in the eluent composition require the re-balancing of the
detector.
– Cannot be used in the analyses requiring the gradient elution.
– Disability to easily remove and clean or replace the cell when
filming or clogging occurs.
63. 63
FLUORESCENCE DETECTORS
• Fluorescence detectors are very specific and selective
• Most sensitive detector.
• Fluorescence intensity depends on both the excitation and emission
wavelength, allowing selectively detect some components while suppressing
the emission of others.
64. • Fluorescence detector:
• These are very sensitive and selective
• Certain compounds emit light when excited by
UV light.
• In this detector the photodetector is placed at
right angles to the excitation light in order to
detect only fluorescence light.
• Wavelength selection is for the excitation and
emission can be accomplished by either filters or
monochromators.
64
65. • The sensitivity of this detector is increased by
increasing power of excitation source.
• This is widely used in pharmaceutical analysis
for analysis of biological fluids and this is 100
times more sensitive than UV detection
• Drawback:
• Its Relatively narrow linear dynamic range.
65
67. 67
– Based on the measurements of the current resulting from
oxidation/reduction reaction of the analyte at a suitable electrode.
– Used for analyzing phenols and organic acids.
68. 68
•The conductivity of the column effluent is continuously
measured and the appearance of the analyte in the cell is
indicated by a change in conductivity.
•Used most successfully in ion-exchange chromatography of
anions and cations.
69.
70. Principle
• A liquid mobile phase is pumped under pressure
through a stainless steel column containing particles of
stationary phase with a different diameter of 3-10 μm.
• The analyte is loaded onto the head of the column via
a loop valve and separation of a mixture occurs
according to the relative lengths of time spent by its
components in the stationary phase.
• It should be noted that all components in a mixture
spend more or less the same time in mobile phase in
order to exit the column.
• Monitoring of the column effluent can be carried out
with a variety of detectors.
71. Applications
• The combination of HPLC with monitoring by UV/Visible
detection provides an accurate, precise and robust method
for quantitative analysis of pharmaceutical products. It is
the industry standard method for this purpose.
• Monitoring of the stability of pure drug substances and of
drugs in formulations, with quantitation of any degradation
products.
• Measurement of drugs and their metabolites in biological
fluids.
• Determination of partition coefficients and pKa values of
drugs and drug protein binding.
74. Strengths
• Easily controlled and precise sample introduction ensures
quantitative precision.
• HPLC is the chromatographic technique which has seen
the most intensive development in recent years, leading to
improved columns, detectors and software control.
• The variety of columns and detectors means that the
selectivity of the method can be readily adjusted.
• Compared to GC there is less risk of sample degradation
because heating is not required in the chromatographic
process.
• Readily automated.
75. Limitations
• There is still a requirement for a reliable and
inexpensive detectors which can monitor
compounds that lack a chromophore.
• Drugs have to be extracted from their formulations
prior to analysis.
• Large amounts of organic solvent waste are
generated, which are expensive to dispose of.