UPLC is a chromatographic technique that can separate mixtures faster and with higher resolution than HPLC using columns packed with 1.7μm particles operating at pressures up to 15,000 psi. It provides increased speed, sensitivity, and resolution for applications in various fields including drug discovery, toxicology, and cancer research. Key components of a UPLC system include binary solvent manager pumps, sample manager, column heater, and detectors. UPLC's use of small particle sizes and high pressures allows for rapid analysis times, less solvent usage, and improved performance over traditional HPLC.
In this slide contains Factors Affecting Resolution In HPLC and its criteria's.
Presented by: M.Sudheeshna. (Department of pharmaceutical analysis).
RIPER,anantpur.
In this slide contains Factors Affecting Resolution In HPLC and its criteria's.
Presented by: M.Sudheeshna. (Department of pharmaceutical analysis).
RIPER,anantpur.
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.
This presentation covers an introduction to UPLC, its general chemistry, and laws behind it. It also discusses the instrumentation of UPLC, advantages, disadvantages, and application of UPLC.
In this slide contains types of HPLC Columns, Plate theory and Van Deemter Equation.
Presented by : Malarvannan.M (Department of pharmaceutical analysis).
RIPER,anantpur.
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.
This presentation covers an introduction to UPLC, its general chemistry, and laws behind it. It also discusses the instrumentation of UPLC, advantages, disadvantages, and application of UPLC.
In this slide contains types of HPLC Columns, Plate theory and Van Deemter Equation.
Presented by : Malarvannan.M (Department of pharmaceutical analysis).
RIPER,anantpur.
Chromatography is an analytical method in which compounds are physically separated and measured.
The main purpose of chromatography is to separate and quantify the target sample.
The Chromatography technique used to separate a mixture of compounds in pharmaceutical sciences , analytical analytical Chemistry with the purpose of identifying, quantifying and purifying the individual components of the mixture.
UPLC is an advance analytical technique where it takes advantage of innovation in various technologies such as instrumentation and particle size to achieve dramatic increases in resolution, speed and sensitivity of the liquid chromatography. It operates at higher pressure than that used in HPLC and uses fine particles (less than 2.5µm) & mobile phases at high linear velocities. It can be hyphenated with other techniques such as (MS), (IC), (NMR) and (IR) etc. This is used in all industries and has found application in various fields such as pharmaceutical, food, environmental, forensic, toxicology and pesticide.
Development and Validation of Reverse Phase Liquid Chromatography Method for ...IOSR Journals
A simple, precise and reversed phase liquid chromatographic method was developed and it is validated for estimation of losartan in bulk drug. Losartan is use for treatment of hypertension. The separation was achieved on Acquity BEH C18 1.7μ, (2.1 X 100) mm, analytical column with mobile phase consisted of buffer (adjust pH 3.0 of water with dilute formic acid) : Acetonitrile (50:50 v/v) at isocratic flow of 0.3ml/min with UV detection wavelength was at 230 nm. The method was successfully validated in accordance to ICH guidelines for accuracy, precision, specificity, linearity. The linear regression analysis data for calibration plots showed good linear relationship in the concentration range 25-75μg/mL for losartan. The % Recovery/Accuracy was within the range between 98% and 102%. The percentage RSD for precision method was found to be less than 2%. The method was successfully applied for routine analysis of losartan in bulk samples
Similar to ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY (20)
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.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
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.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
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.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
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 .
2. INTRODUCTION
UPLC means Ultra Performance Liquid Chromatography
UPLC is a chromatographic technique, in which it can separate a mixture of
compounds and is used in biochemistry and analytical chemistry to identify, quality,
purity in the individual components of a mixture utilizing a small packaging particle
and columns with high pack pressure.
4. Advancements in various areas
Nutrition
Drug Discovery
Toxicology
Natural product discovery
Cancer
5. Advantages
Operation cost is reduced
Less solvent consumption
Run time and increase sensitivity
Maintains resolution performance
Disadvantages
High price of instruments, spare parts and
columns
Number of stationary phases still limited
Lack of variety in commercial columns
6. Principle of uplc
The UPLC is based on the principle of use of stationary phase consisting of particles less than 2 μm (while
HPLC columns are typically filled with particles of 3 to 5 μm). The underlying principles of this evolution are
governed by the van Deemter equation, which is an empirical formula that describes the relationship between
linear velocity (flow rate) and plate height (HETP or column efficiency).
Where
A ,B,C are constants
V - linear velocity
A - Eddy mixing
B - Axial diffusion
H=A+B/v+CV
8. Various parts present in Uplc instrument are
Columns
Column heater
Sample manager
Binary solvent manager
Pumps
Optional Sample organizer
9. Binary solvent manager
The binary solvent manager uses two individual serial flow pumps to deliver a parallel binary
gradient.
The binary solvent manager is a high pressure pump that moves solvent through the system.
The binary solvent manager delivers solvent at flow rates of 1 ml/min at 103421 Kрa [ 1034 bar,
1500 psi] and up to 2ml/min at reduced pressures to 62053 Kpa [621 bar, 9000 psF] . The solvent
manager can pump two solvents immediately.
Sample manager:
The Acuity sample manager injects the sample it draws from Micro titer plates or vials in to the
chromatographic flow stream.
A locating mechanism uses a probe to access sample locations and draw sample from them.
The Sample manager can perform an injection in approximately 15 seconds. The sample manager
also controls the column heater. Column temperatures up to 65°С can be attained.
10. Column heater:
The column heater is of a modular design and its foot print is identical to that of the sample manager.
Thus it attaches to the top of the sample manager and serves as the instrument’s top cover.
Columns:
The UPLC columns are made up of small particles having size less than 2 μm. The role played by small
particle size in UPLC technique has been mentioned above. The particles are bonded in matrix as the
bonded stationary phase is required for providing both retention and selectivity.
11. Micro bore column Optional sample organizer
The optional sample organizer stores micro
miter or vial plates
It transfers them to and from the sample
manages, automating their processing and
increasing throughput
Optional sample organizer holds the sample
12. Pumps:
The UPLC pump is considered to be one of the most important components in a liquid chromatography
system which has to provide a continuous constant flow of the eluent through the UPLC injector,
column, and detector.
The two basic classifications are
Constant pressure pump
Constant flow pump
Standard UPLC pump requirements :
Sample injection volume is as less as 3-5 micro liters
Pumps operates at 10000 psi pressure
Particle size in stationary phase packing material is less than
2 micro meter
15. Applications of Uplc
Rapid analysis of products
Bio equivalence studies
Dissolution testing
Impurity profiling
Identification of metabolites
High through put qualitative analysis
Drug discovery
Analysis of dosage form
Study of metabolomics
Forced degradation studies
In finger print analysis
ADME screening
Determination of pesticides
Analysis of amino acids
In QA control
16. References
1. Jerkovich A.D., Mellors J.S., and Jorgenson J.W., LCGC 21(7), 660–611(2003).
2. Wu N Lippert J.A. and Lee M.L., J. Chromatogram. 911(1) (2001).
3. Unger K. K., Kumar D. , Grun M., Buchel G., Ludtke S., AdamTh., Scumacher K., and Renker S., J. Chromatogram., A
892(47)(2000).
4. Swartz M. E. and Murphy B., Lab Plus Int., 18(6) (2004).
5. Swartz M. E. and Murphy B., Pharm. Formulation Quality 6(5), p. 40 (2004)
6. Gerber F. , Krummen M., Potgeter H., Roth A. , Siffrin C., and Spoendlin C., J. Chromatogr., A1036, 127-133 (2004).
7. Tanaka N. , Kobayashi H., Nakanishi K., Minakuchi H., and Ishizuka N., Anal. Chem. 73, 420A–429A (2001).
8. Wu N. , Dempsey J. , Yehl P.M., Dovletoglu A., Ellison A., and Wyvratt J., Anal. Chim. Acta 523, 149–15 -6 (2004).
9. Preeti vinod gaikwad, Ultra performance liquid chromatography: A recent novel development in HPLC. Pharmacie globale
International journal of comprehensive pharmacy, 01, 2012, 1-3.
10. Swartz M., LCGC 23(1), 46–53 (2005).
