INTRODUCTION
Instrumentation of HPLC
TYPES OF HPLC
PARAMETERS
APPLICATION
CONCLUSION
REFERENCE
High-performance liquid chromatography ( HPLC) is a specific form of column chromatography generally used in biochemistry and analysis to separate, identify, and quantify the active compounds.
HPLC mainly utilizes a column that holds packing material (stationary phase), a pump that moves the mobile phase(s) through the column, and a detector that shows the retention times of the molecules.
In this slide contains principle, instrumentation, methodology, and application of gel chromatography.
Presented by: SATHEES CHANDRA (Department of pharmaceutical analysis).
RIPER, anantapur
In this slide contains principle, instrumentation, methodology, and application of gel chromatography.
Presented by: SATHEES CHANDRA (Department of pharmaceutical analysis).
RIPER, anantapur
Gas chromatography and its instrumentationArgha Sen
Gas chromatography is an unique technology which helps us in separating volatile analytes. Its is an easy and reproduciple method for detecting residual solvents found in APIs.
fluid chromatography (SFC) can be used on an analytical
scale.
It is a combination of High performance liquid chromatography (HPLC)
and Gas chromatography (GC).
It can be used with non-volatile and thermally labile analytes.
It can be used with the universal flame ionization detector.
It is important to producing narrower peaks due to rapid diffusion.
It is important for the chiral separations and analysis of high-molecularweight
hydrocarbons.
Supercritical fluids are suitable as a substitute for organic solvents in a
range of industrial and laboratory processes.
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
In this slide contains types of HPLC Columns, Plate theory and Van Deemter Equation.
Presented by : Malarvannan.M (Department of pharmaceutical analysis).
RIPER,anantpur.
Gas chromatography and its instrumentationArgha Sen
Gas chromatography is an unique technology which helps us in separating volatile analytes. Its is an easy and reproduciple method for detecting residual solvents found in APIs.
fluid chromatography (SFC) can be used on an analytical
scale.
It is a combination of High performance liquid chromatography (HPLC)
and Gas chromatography (GC).
It can be used with non-volatile and thermally labile analytes.
It can be used with the universal flame ionization detector.
It is important to producing narrower peaks due to rapid diffusion.
It is important for the chiral separations and analysis of high-molecularweight
hydrocarbons.
Supercritical fluids are suitable as a substitute for organic solvents in a
range of industrial and laboratory processes.
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
In this slide contains types of HPLC Columns, Plate theory and Van Deemter Equation.
Presented by : Malarvannan.M (Department of pharmaceutical analysis).
RIPER,anantpur.
All you need to know about the HPLC Chromatography!
HPLC (High-performance liquid chromatography) also referred as high-pressure liquid chromatography, is a technique used in analytical labs to separate, identify, and quantify each component in a mixture. HPLC separates and purifies compounds according to their polarity. I have tried to simplify the whole process of HPLC Chromatography and explain in simple terms. Let’s look at the main components involved in HPLC in this video.
I discussed about Normal Phase Chromatography, Reverse Phase Chromatography and their comparison and differences. Watch this video for more details.
If you have any doubts: Please leave a comment in the comments section below.
A complete description about HPLC and its mechanism. it will help you to understand HPLC technique very easily.The individual dye or colour band are used for explanation.Actually the sample are colour less and separation can not see by eye.
HPLC
Chromatography
Mobile Phase & Stationary Phase
CLASSIFICATION OF CHROMATOGRAPHY
Characteristics of HPLC
Purpose
Superiority of HPLC
TYPES OF HPLC TECHNIQYES
Principle
PHASING SYSTEM & (normal vs reversed phase)
INSTRUMENTATION
Flow diagram of HPLC instrument
Advantages of HPLC
Introduction
History
Tumor suppressor gene- pRB
- RB gene
- Role of RB in regulation of cell cycle
- Tumor associated with RB gene mutation
Tumor suppressor gene- p53
- What is p53 gene?
- Function of p53 gene
- How it regulates cell cycle
- What happen if p53 gene inactivated
- Cancer associated with p53 mutation
- Conclusion
- References
Introduction
Definition
History
Two hit hypothesis
Functions
Mutation in tumor suppressor genes
What is mutation
Inherited mutation of TSGs
Acquired mutation of TSGs
What is Oncogenes?
TSGs and Oncogenes : Brakes and accelerators
Stop and go signal
Examples of TSGs:
RB-The retinoblastoma gene
P53 protein
TSGs &cell suicide
Conclusion
References
Introduction
Protein synthesis
Synthesis of secretory proteins on membrane-bound ribosomes
Processing of newly synthesized proteins in the ER
Synthesis of integral membrane protein on membrane bound ribosomes
Maintenance of membrane asymmetry
Conclusion
Reference
Introduction
Definition
Factors required for Translation
Formation of aminoacyl t-RNA
1)Activation of amino acid
2) Transfer of amino acid to t-RNA
Translation involves following steps:-
1)Initiation
2)Elongation
3)Termination
Conclusion
Reference
Introduction
Definition
History
central dogma
Major components
mRNA,tRNA,rRNA
Energy source
Amino acids
Protien factor
Enzymes
Inorganic ions
Step involves in translation:
Aminoacylation of tRNA
Initiation
Elongation
termination
Importance of translation
Conclusion
Reference
Introduction
Protein modifications
Folding
Chaperon mediated
Enzymatic
Cleavage
Addition of functional groups
Chemical groups
Hydrophobic groups
Proteolysis
Conclusion
Reference
INTRODUCTION
HISTORY
WHAT IS TRANSCRIPTION
PROKARYOTIC TRANSCRIPTION
STEPS OF TRANSCRIPTION
HOW TRANSCRIPTION OCCURS
PROCESS OF TRANSCRIPTION
Initiation
Elongation
Termination
CONCLUSION
REFRENCES
Enzyme Kinetics and thermodynamic analysisKAUSHAL SAHU
Introduction
Kinetics and thermodynamicSG
Thermodynamic in enzymatic reactions
balanced equations in chemical reactions
changes in free energy determine the direction & equilibrium state of chemical reactions
the rates of reactions
Factors effecting enzymatic activity
(i) Enzyme concentration.
(ii) Substrate concentration.
(iii)Temperature
(iv) pH.
(v) Activators.
(vi)Inhibitors
Michaelis-menten equation
CONCLUSIONS
REFERENECES
Recepter mediated endocytosis by kk ashuKAUSHAL SAHU
INTRODUCTION
DEFINITION OF RECEPTOR MEDIATED ENDOCYTOSIS
WHAT TYPE OF LIGANDS ENTER BY RME?
FORMATION OF CLATHRIN-COATED VESICLES
TRISKELIONS
ROLE OF DYNAMIN IN THE FORMATION OF CLATHRIN-COATED VESICLES
ROLE OF PHOSPHOLIPIDS IN THE FORMATION OF COATED VESICLES
ENDOCYTIC PATHWAY
LDLs AND CHOLESTROL METABOLISM
CONCLUSION
REFERENCES
The delivery of newly synthesized protein to their proper cellular destination, usually referred to as protein targeting or sorting.
The mode of protein transport depends chiefly on the location in the cell cytoplasm of the polysomes involved in protein synthesis.
There are two modes of protein sorting:-
1) Co - translational Transportation.
2) Post - translational Transportation.
Prokaryotic translation machinery by kk KAUSHAL SAHU
Introduction
Definition
Factors required for Translation
Formation of aminoacyl t-RNA
1)Activation of amino acid
2) Transfer of amino acid to t-RNA
Translation involves following steps:-
1)Initiation
2)Elongation
3)Termination
Conclusion
Reference
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
3. INTRODUCTION
• High-performance liquid chromatography ( HPLC) is a
specific form of column chromatography generally
used in biochemistry and analysis to separate, identify,
and quantify the active compounds.
• HPLC mainly utilizes a column that holds packing
material (stationary phase), a pump that moves the
mobile phase(s) through the column, and a detector
that shows the retention times of the molecules.
• The sample to be analyzed is introduced in small
volume to the stream of mobile phase and is retarded
by specific chemical or physical interactions with the
stationary phase.
7. Mobile Phase Reservoirs
• The type and composition of the mobile phase affects
the separation of the components.
• Different solvents are used for different types of
HPLC.
• For normal-phase HPLC, the solvent is usually
nonpolar, and in reverse-phase HPLC, the solvent is
normally a mixture of water and a polar organic
solvent.
8. Pumps
• High-pressure pumps are needed to push the
mobile phase through the packed stationary
phase.
• Pumps are typically known to be robust, but
adequate maintenance must be performed to
maintain that characteristic.
• Inability to build pressure, high pressures or
leakage could indicate that the pump is not
functioning correctly.
• Proper maintenance of the pump system will
minimize down time.
10. Sample Injector
• The injector can be a single injection or an
automated injection system.
• An injector for an HPLC system should provide
injection of the liquid sample within the range
of 0.1-100mL of volume with high
reproducibility and under high pressure (up to
4000 psi).
• For liquid chromatography, liquid samples can
be directly injected and solid samples need
only to be diluted in the appropriate solvent.
11.
12. Column(s)
• The column or stationary phase is the core of
any chromatographic system.
• Columns are commercially available in
different lengths and packing materials.
• The use of the correct combination of length
and packing material in correlation with the
appropriate mobile phase can assist in the
most effective separation of a sample
compound.
13. Detector
• The detector is used to sense the presence
of a compound passing through and to
provide an electronic signal to a data-
acquisition device.
• The main types of detectors used in HPLC
are refractive index (RI), ultraviolet (UV-Vis)
and fluorescence, but there are also diode
array, electrochemical and conductivity
detectors.
• Each detector has its assets, limitations and
sample types for which it is most effective.
14. Data Display Systems
• Since the detector signal is electronic, use of
modern data- acquisition techniques can aid
in the signal analysis.
• The data- acquisition system of most HPLC
systems is a computer.
• The computer integrates the response of the
detector to each component and places it
into a chromatograph that is easy to read
and interpret.
15. TYPES OF HPLC
• Normal phase chromatography: Also known
Normal phase HPLC (NP-HPLC), this method
separates analytes based on polarity. NP-HPLC
uses a polar stationary phase and a non-polar
mobile phase.
• Reversed phase chromatography: Reversed
phase HPLC (RP-HPLC or RPC) has a non-polar
stationary phase and an aqueous, moderately
polar mobile phase. RPC operates on the
principle of hydrophobic interactions, which
result from repulsive forces between a polar
eluent, the relatively non-polar analyte, and the
non-polar stationary phase.
16. • Size exclusion chromatography: Size exclusion
chromatography (SEC), also called as gel
permeation chromatography or gel filtration
chromatography mainly separates particles on
the basis of size. It is also useful for determining
the tertiary structure and quaternary structure
of proteins and amino acids.
• Ion exchange chromatography: In Ion-exchange
chromatography, retention is based on the
attraction between solute ions and charged
sites bound to the stationary phase. Ions of the
same charge are excluded.
17. • Bio-affinity chromatography: Separation
based on specific reversible interaction of
proteins with ligands. Ligands are covalently
attached to solid support on a bio-affinity
matrix, retains proteins with interaction to
the column-bound ligands.
18. PARAMETERS
• For the accurate analysis of a compound,
there are some parameters which are used
as a standard for a particular compound.
• Internal diameter
• Particle size
• Pore size
• Pump pressure
19. APPLICATION
• Chemical Separations:- It is based on the
fact that certain compounds have different
migration rates given a particular column
and mobile phase, the extent or degree of
separation is mostly determined by the
choice of stationary phase and mobile
phase.
20. Purification:- Purification is defined as the
process of separating or extracting the
target compound from a mixture of
compounds or contaminants.
Identification :- Generally assay of
compounds are carried using HPLC. The
parameters of this assay should be such that
a clean peak of the known sample is
observed from the chromatograph.
21. Other applications of HPLC
Pharmaceutical applications
Shelf-life determinations of pharmaceutical products .
Identification of active ingredients of dosage forms.
Pharmaceutical quality control
Environmental applications
Detection of phenolic compounds in Drinking Water
Bio-monitoring of pollutant
Forensics
Quantification of the drug in biological samples.
Identification of anabolic steroids in serum, urine, and hair
Determination of cocaine and metabolites in blood
22. Clinical
Quantification of ions in human urine Analysis
of antibiotics in blood plasma.
Estimation of bilirubin and bilivirdin in blood
plasma in case of hepatic disorders.
Food and Flavor
Ensuring the quality of soft drink and drinking
water.
Analysis of beer.
Sugar analysis in fruit juices.
Analysis of polycyclic compounds in vegetables.
23. CONCLUSION
• It can be concluded that HPLC is a versatile,
reproducible chromatographic technique for
the estimation of drug products.
• It has wide applications in different fields in
term of quantitative and qualitative
estimation of active molecules.