This is a PPT for HPLC which I made for presenting my assigned topic for Practice School during 7 sem of my graduation . Hope it is useful for you guys :)
High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase).
Instrumentation of HPLC, principle by kk sahuKAUSHAL SAHU
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.
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.
High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase).
Instrumentation of HPLC, principle by kk sahuKAUSHAL SAHU
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.
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.
a type of an analyzer used in mass spectrometer. separates the ions based on mass to charge ratios. useful for the detection of ions present in the sample
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.
This Powerpoint presentation helps us to know the basic working principles, instrumentation an advantage of super critical fluid chromatography.
Contact Details:
Anbu Dinesh Jayakumar
M.Pharmacy ( Pharmaceutical Chemistry)
Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore
Mobile : 8838404664 / 8608890121( Whatsapp)
Email: anbudinesh007@gmail.com
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR HIGH PRESSURE LIQUID CHROMAT...Dr. Ravi Sankar
GASSCHROMATOGRAPHY[GC], ADVANCED STUDY OF THE FOLLOWING AND THEIR APPLICATIONS, INTRODUCTION, THEORY, COLUMN OPERATION,INSTRUMENTATION AND DETECTION,APPLICATIONS AND ADVANTAGES OF GC,PRINCIPLE OF SEPARATION IN GC, HOW GC MECHINE WORKS? COLUMN, DETECTORS.
BY P.RAVISANKAR, VIGNAN PHARMACY COLLEGE, VADLAMUDI, GUNTUR, A.P, INDIA.
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.
In this slide contains principle, instrumentation, methodology, and application of gel chromatography.
Presented by: SATHEES CHANDRA (Department of pharmaceutical analysis).
RIPER, anantapur
a type of an analyzer used in mass spectrometer. separates the ions based on mass to charge ratios. useful for the detection of ions present in the sample
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.
This Powerpoint presentation helps us to know the basic working principles, instrumentation an advantage of super critical fluid chromatography.
Contact Details:
Anbu Dinesh Jayakumar
M.Pharmacy ( Pharmaceutical Chemistry)
Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore
Mobile : 8838404664 / 8608890121( Whatsapp)
Email: anbudinesh007@gmail.com
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR HIGH PRESSURE LIQUID CHROMAT...Dr. Ravi Sankar
GASSCHROMATOGRAPHY[GC], ADVANCED STUDY OF THE FOLLOWING AND THEIR APPLICATIONS, INTRODUCTION, THEORY, COLUMN OPERATION,INSTRUMENTATION AND DETECTION,APPLICATIONS AND ADVANTAGES OF GC,PRINCIPLE OF SEPARATION IN GC, HOW GC MECHINE WORKS? COLUMN, DETECTORS.
BY P.RAVISANKAR, VIGNAN PHARMACY COLLEGE, VADLAMUDI, GUNTUR, A.P, INDIA.
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.
In this slide contains principle, instrumentation, methodology, and application of gel chromatography.
Presented by: SATHEES CHANDRA (Department of pharmaceutical analysis).
RIPER, anantapur
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.
Chromatography is a laboratory technique for the separation of a mixture. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase.
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.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
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.
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.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
1. 1
Supervised by :
Dr. Noel Rahul Shaw
Assistant Professor
Department of Quality Assurance
Presented by :
Lakshay Tayal
B.Pharm VII Sem
B.Pharm VII Sem (2020-2021)
HPLC
Affiliated to
Jai Narain Vyas University, jodhpur
3. Introduction
• Chromatography is a laboratory technique used for separation of mixture.
• HPLC is a type of Chromatography which shows high Performance
• HPLC is a process of separation of mixture containing two or more
components under high pressure by passing sample through a column
containing stationary solid bed by means of pressurized flow of liquid mobile
phase.
• The pressure used is 1000- 5000 psi.
• The Principle of separation of component of mixture depends upon their
relative affinity towards St. phase and m.phase or depends upon adsorption/
partition coefficient or depend upon charge or molecular size of mixture. 3
4. Types of HPLC
According to Phases:
• Liquid-solid chromatography or adsorption Chromatography
• Liquid-liquid chromatography or Partition Chromatography
• Ion exchange chromatography or Separation base on charge
• Size exclusion chromatography or Separation base on molecular size
of particle.
4
5. Types of HPLC
On the basis of modes:
1. Normal phase HPLC
• Stationary phase: High polar rigid silica, or silica based
compositions.( Hydrophilic, Polar)
• Mobile phase: Relatively non polar solvent, hexane, heptane etc.
2. Reverse phase HPLC ( Often used )
• Stationary phase: Bonded hydrocarbons (C18, C8 etc.)
• Mobile phase: Polar solvents or mixtures such as methanol-water or
acetonitrile -water.
• The most polar component is eluted first.
• It is useful for polar sample analysis (organic compounds)
5
9. Parts of HPLC
• Degasser: It is used to remove the air from solvent.
• Reservoir: These are glass or stainless steel containers capable of holding mobile
phase.
Pump: The pump provide a steady high pressure to the sample solution/mobile
phase flowing inside the column. Eg . Reciprocating pump/ constant flow pump
. Displacement pump/ syringe pump
Ideal pump property:
- Ability to generate high pressure
-Accurate control of flow
- Corrosion resistant
9
10. Injector: It is used to inject the sample into the continuously flowing mobile phase stream that carries the
sample into the HPLC column.
Column: They are constructed of stainless steel for highest pressure resistance.
• - Length (10-30 cm)
• - Internal diameter (4-10 mm)
• - Particles size (3-10 µm);
Detector: It is used to detect the separated compound bands as they elute from the HPLC column.
Generally UV detector is used in it.
• Characteristics of an ideal detector:
- Adequate sensitivity
- Good stability and reproducibility
- Gives linear response to analysts
- Short response time
Waste collector: The mobile phase/ sample solution exits from the detector and is collected in the waste
chamber where it is either collected or thrown, as desired.
Display unit: A device that records the electrical response of a detector on a computer screen in the form of
a chromatogram.
10
11. Applications
• Widely applicable to numerous fields of study; both academic and
industrial work.
• Separate, identify and quantify the active compounds.
• Qualitative and quantitative determination of sample.
• Separation of non-volatiles:
Amino acids, proteins, carbohydrates, pharmaceuticals, pesticides,
pigments, antibiotics, steroids, vitamins, and various other organic and
inorganic substances.
11
12. Applications
Food and Flavor Analysis:
• Ensuring the quality of drinking water.
• Sugar analysis in fruit juices
• Analysis of compounds in vegetables.
• Trace analysis of agricultural crops
Application in Clinical Tests :
1.Urine Analysis
2.Antibiotic analysis in blood
3.Identification of Steroids in blood and urine
12
13. Applications
In drug manufacturing stage:
• Identification tests of the drug product or its ingredients
• Assay/ Content Uniformity test
• Dissolution test
• Drug Impurity testing
• Drug Stability
• Cleaning Validation/testing of the manufacturing equipments such as blender, tablet
press, etc.
• In process quality control testing
•Data manipulation can be prevented 13
14. Advantages and Disadvantages
• Speed (analysis can be accomplished in 20 min. or less)
• Greater sensitivity (various detectors can be employed)
• Improved resolution (wide variety of stationary phases)
• Reusable columns (expensive columns but can be used for many analysis)
• Used for sepration of sample which tend to decompose at higher Temp
• Disadvantages:
1. It is Costly
2. It is complex
3. Low sensitivity for some compounds
14