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
HPLC is a High Performance liquid Chromatography.
High Pressure Liquid Chromatography.
High Priced Liquid Chromatography.
It is column chromatography.
It is Liquid Chromatography.
It is modified from of gas chromatography, it is applicable for both Volatile as well as Non volatile compound.
It can mainly divided by two types 1. Normal phase HPLC 2. Reversed Phase HPLC.
It is having a high resolution and separation capacity.
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
HPLC is a High Performance liquid Chromatography.
High Pressure Liquid Chromatography.
High Priced Liquid Chromatography.
It is column chromatography.
It is Liquid Chromatography.
It is modified from of gas chromatography, it is applicable for both Volatile as well as Non volatile compound.
It can mainly divided by two types 1. Normal phase HPLC 2. Reversed Phase HPLC.
It is having a high resolution and separation capacity.
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
This presentation gives you thorough knowledge about the IR Spectroscopy. This include basic principle, type of vibrations, factors influencing vibrational frequency, instrumentation and applications of IR Spectroscopy. This is the most widely used technique for identifying unknown functional group depending on the vibrational frequency.
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.
HPTLC- Principle, Instrumentation and Software (Abhishek Gupta)Abhishek Gupta
HPTLC is the improved method of TLC which utilizes the conventional technique of TLC in more optimized way
It is also known as planar chromatography or Flat-bed chromatography.
principle, application and instrumentation of UV- visible Spectrophotometer Ayetenew Abita Desa
This Presentation powerpoint includes the principle, application, and instrumentation of UV- Visible Spectrophotometer. It covers beer-lambert low and its quantitative applications. It also includes the qualitative applications in different fields of study. Presented at Addis Ababa University, School of medicine, department of medical biochemistry.
This presentation gives you thorough knowledge about the IR Spectroscopy. This include basic principle, type of vibrations, factors influencing vibrational frequency, instrumentation and applications of IR Spectroscopy. This is the most widely used technique for identifying unknown functional group depending on the vibrational frequency.
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.
HPTLC- Principle, Instrumentation and Software (Abhishek Gupta)Abhishek Gupta
HPTLC is the improved method of TLC which utilizes the conventional technique of TLC in more optimized way
It is also known as planar chromatography or Flat-bed chromatography.
principle, application and instrumentation of UV- visible Spectrophotometer Ayetenew Abita Desa
This Presentation powerpoint includes the principle, application, and instrumentation of UV- Visible Spectrophotometer. It covers beer-lambert low and its quantitative applications. It also includes the qualitative applications in different fields of study. Presented at Addis Ababa University, School of medicine, department of medical biochemistry.
High-performance liquid chromatography (HPLC), is a technique in analytical chemistry used to separate, identify, and quantify individual components from a mixture.
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.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
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 .
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.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
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.
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.
Lateral Ventricles.pdf very easy good diagrams comprehensive
HPLC Principle,Instrumentation and Application
1. High Performance LiquidHigh Performance Liquid
ChromatographyChromatography
ALAKESH PRADHANALAKESH PRADHAN
COCHIN UNIVERSITY OFCOCHIN UNIVERSITY OF
SCIENCE AND TECHNOLOGYSCIENCE AND TECHNOLOGY
School of IndustrialSchool of Industrial
FisheriesFisheries
M.Sc IInd Sem.M.Sc IInd Sem.
2. Overview:
Chromatography and its principleChromatography and its principle
Liquid chromatographyLiquid chromatography
High Performance Liquid Chromatography ( HPLC )High Performance Liquid Chromatography ( HPLC )
The components of the high performance liquid chromatograph (HPLC).The components of the high performance liquid chromatograph (HPLC).
The separation process.The separation process.
The chromatogram.The chromatogram.
4. BackgroundBackground
Chromatography and its Principle
Chromatography is a separation technique which is used toChromatography is a separation technique which is used to
separate a mixture of compounds into its individual componentsseparate a mixture of compounds into its individual components
based on certain physical and chemical properties.based on certain physical and chemical properties.
5. Some important terms:Some important terms:
Mobile phase: The solvent system which carries the mixture to beMobile phase: The solvent system which carries the mixture to be
separated.separated.
Stationary phase: Immobile surface which is particulate in nature. This is theStationary phase: Immobile surface which is particulate in nature. This is the
region over which the compound gets separated.region over which the compound gets separated.
6. Principle:
The process involves the interaction of the compounds in the analyte (whichThe process involves the interaction of the compounds in the analyte (which
travels along with a mobile phase) across an immobile surface (stationarytravels along with a mobile phase) across an immobile surface (stationary
phase).phase).
The compounds bind at specific regions of stationary phase based onThe compounds bind at specific regions of stationary phase based on
certain physical and chemical properties. These bound molecules are thencertain physical and chemical properties. These bound molecules are then
eluted with a suitable buffer and the same are collected with time.eluted with a suitable buffer and the same are collected with time.
These are –These are –
PolarityPolarity
ChargeCharge
Molecular weightMolecular weight
Present of functional groupPresent of functional group
7. IntroductionIntroduction
HPLC is a form of liquid chromatography used toHPLC is a form of liquid chromatography used to
separate compounds that are dissolved in solution.separate compounds that are dissolved in solution.
HPLC instruments consist of a reservoir of mobileHPLC instruments consist of a reservoir of mobile
phase, a pump, an injector, a separation column, and aphase, a pump, an injector, a separation column, and a
detector.detector.
Compounds are separated by injecting a sample mixtureCompounds are separated by injecting a sample mixture
onto the column.onto the column.
The different component in the mixture pass through theThe different component in the mixture pass through the
column at differentiates due to differences in theircolumn at differentiates due to differences in their
partition behavior between the mobile phase and thepartition behavior between the mobile phase and the
stationary phase.stationary phase.
The mobile phase must be degassed to eliminate theThe mobile phase must be degassed to eliminate the
formation of air bubbles.formation of air bubbles.
8. Continued…
What is Liquid Chromatography?
Liquid chromatography is a separation technique that involves:Liquid chromatography is a separation technique that involves:
•• the placement (injection) of a small volume of liquid samplethe placement (injection) of a small volume of liquid sample
•• into a tube packed with porous particles (stationary phase)into a tube packed with porous particles (stationary phase)
•• where individual components of the sample are transported along thewhere individual components of the sample are transported along the
packed tube (column) by a liquid moved by gravity.packed tube (column) by a liquid moved by gravity.
The components of the sample are separated from one another by theThe components of the sample are separated from one another by the
column packing that involves various chemical and/or physical interactionscolumn packing that involves various chemical and/or physical interactions
between their molecules and the packing particles.between their molecules and the packing particles.
The separated components are collected at the exit of this column andThe separated components are collected at the exit of this column and
identified by an external measurement technique , such as aidentified by an external measurement technique , such as a
spectrophotometer that measures the intensity of the color , or by anotherspectrophotometer that measures the intensity of the color , or by another
device that can measure their amount.device that can measure their amount.
Note:Note: The modern form of liquid chromatography is now referred to asThe modern form of liquid chromatography is now referred to as
““flash chromatographyflash chromatography
10. Terminologies for HPLC
HPLC : High Performance Liquid Chromatography : High Pressure LCHPLC : High Performance Liquid Chromatography : High Pressure LC
Now, before we go in depth of principle, lets have a basic look at fewNow, before we go in depth of principle, lets have a basic look at few
terms as follows:terms as follows:
Resolving Power: The extent of separation of the compounds presentResolving Power: The extent of separation of the compounds present
in the mixture across the column.in the mixture across the column.
Theoretical plates : An imaginary division of the column intoTheoretical plates : An imaginary division of the column into
equilength plates.equilength plates.
11. Principles of HPLC
Principle:Principle:
The table shows relation between variousThe table shows relation between various
parameters of HPLC.parameters of HPLC.
Trendline:Trendline:
Stationary phase have small particulate size andStationary phase have small particulate size and
high surface areas.high surface areas.
Columns: 20 cm or lessColumns: 20 cm or less
Mobile phase pumped at high pressures ofMobile phase pumped at high pressures of
200Bar, 3000 psi.200Bar, 3000 psi.
Flow rates: 1-3 cmFlow rates: 1-3 cm33 per minper min
Column length No. of theoretical plates
per unit area
Resolving power Column length
Particle size Surface area
12. What is HPLC?
HPLC is a separation technique that involves:HPLC is a separation technique that involves:
••the injection of a small volume of liquid samplethe injection of a small volume of liquid sample
••into a tube packed with tiny particles (3 to 5 micron ( μm ) in diameterinto a tube packed with tiny particles (3 to 5 micron ( μm ) in diameter
called thecalled the stationary phase)stationary phase)
••where individual components of the sample are moved down thewhere individual components of the sample are moved down the
packed tube (packed tube (column) with a liquid (mobile phase) forced throughcolumn) with a liquid (mobile phase) forced through
the column by high pressure delivered by a pump.the column by high pressure delivered by a pump.
TheseThese components are separated from one another by the columncomponents are separated from one another by the column
packing that involves various chemical and/or physical interactionspacking that involves various chemical and/or physical interactions
between their molecules and the packing particles.between their molecules and the packing particles.
These separated components are detected at the exit of this tube (These separated components are detected at the exit of this tube (column)column)
by a flow-through device (detector) that measures their amount. Anby a flow-through device (detector) that measures their amount. An
output from this detector is called a “liquid chromatogram”.output from this detector is called a “liquid chromatogram”.
In principle, LC and HPLC work the same way except the speed ,In principle, LC and HPLC work the same way except the speed ,
efficiency, sensitivity and ease of operation of HPLC is vastlyefficiency, sensitivity and ease of operation of HPLC is vastly
superior.superior.
14. Varian 9010 Solvent Delivery
System
Rheodyne
Injector
%A %B %C Flow Rate Pressure
{H2O} {MeOH} (mL/min) (atmos.)
Ready
Ternary Pump
A
C
B
from solvent
reservoir
Column
to
detector
to column
through
pulse
dampener
to injector
through pump
load
inject
16. COMPOSITION OF A LIQUID CHROMATOGRAPH SYSTEMCOMPOSITION OF A LIQUID CHROMATOGRAPH SYSTEM
SolventSolvent
Solvent Delivery System (Pump)Solvent Delivery System (Pump)
InjectorInjector
SampleSample
ColumnColumn
DetectorsDetectors
Waste CollectorWaste Collector
Recorder (Data Collection)Recorder (Data Collection)
17. Instrumentation of HPLC
( Describing the 5 major components and their
functions….)
1
2
3
4
5
Solvent
reservoirs
and degassing
Not shown
here
1 – Pump
2 – Injector
3 – Column
4 – Detector
5 – Computer
18. 1. Pump:
•The role of the pump is to force a liquid (called the mobile phase)
through the liquid chromatograph at a specific flow rate, expressed
in milliliters per min (mL /min).
•Normal flow rates in HPLC are in the 1-to 2-mL/min range.
•Typical pumps can reach pressures in the range of 6000-
9000
psi (400-to 600-bar).
•During the chromatographic experiment, a pump can deliver a
constant mobile phase composition (isocratic) or an increasing
mobile phase composition (gradient).
19. Pump Module–typesPump Module–types::
Isocratic pump - Delivers constant mobile phase composition;Isocratic pump - Delivers constant mobile phase composition;
••solvent must be pre-mixed;solvent must be pre-mixed;
••lowest cost pumplowest cost pump
Gradient pump - Delivers variable mobile phase composition;Gradient pump - Delivers variable mobile phase composition;
••can be used to mix and deliver an isocratic mobile phase or acan be used to mix and deliver an isocratic mobile phase or a
gradient mobile phasegradient mobile phase
––Binary gradient pumpBinary gradient pump –delivers two solvents–delivers two solvents
––Quaternary gradient pumpQuaternary gradient pump –four solvents–four solvents
20.
21. 2. Injector:
•The injector serves to introduce the liquid sample into the flow stream of
the mobile phase.
•Typical sample volumes are 5-to 20-microliters (μL).
•The injector must also be able to withstand the high pressures of
the liquid system.
•An auto sampler is the automatic version for when the user has many
samples to analyze or when manual injection is not practical .
22. Sample Injection
……how is a sample actually put into an LC system
Manual InjectorManual Injector::
1.User manually loads sample into the injector using a syringe1.User manually loads sample into the injector using a syringe
2.and then turns the handle to inject sample into the flowing mobile2.and then turns the handle to inject sample into the flowing mobile
phase… which transports the sample into the beginning (head) of thephase… which transports the sample into the beginning (head) of the
column, which is at high pressurecolumn, which is at high pressure
Auto samplerAuto sampler::
1.User loads vials filled with sample solution into the auto sampler tray1.User loads vials filled with sample solution into the auto sampler tray
(100 samples)(100 samples)
2.and the auto sampler automatically2.and the auto sampler automatically
a. measures the appropriate sample volume,a. measures the appropriate sample volume,
b. injects the sample,b. injects the sample,
c. then flushes the injector to be ready for the next sample,c. then flushes the injector to be ready for the next sample,
etc., until all sample vials are processed ……etc., until all sample vials are processed ……
………….for unattended automatic operation.for unattended automatic operation
25. 3.3. Column:Column:
•• Considered the “heart of the chromatograph” the column’s stationaryConsidered the “heart of the chromatograph” the column’s stationary
phase separates the sample components of interest using various physicalphase separates the sample components of interest using various physical
and chemical parameters.and chemical parameters.
••The small particles inside the column are what cause the highThe small particles inside the column are what cause the high
back pressure at normal flow rates.back pressure at normal flow rates.
••The pump must push hard to move the mobile phase through theThe pump must push hard to move the mobile phase through the
column and this resistance causes a high pressure within thecolumn and this resistance causes a high pressure within the
chromatograph.chromatograph.
26. Several Column Types
( can be classified as)
Normal phaseNormal phase
Reverse phaseReverse phase
Size exclusionSize exclusion
Ion exchangeIon exchange
27. Normal phase
In this column type, the retention is governed byIn this column type, the retention is governed by
the interaction of the polar parts of the stationarythe interaction of the polar parts of the stationary
phase and solute.phase and solute.
For retention to occur in normal phase, theFor retention to occur in normal phase, the
packing must be more polar than the mobilepacking must be more polar than the mobile
phase with respect to the samplephase with respect to the sample
28. 28
HO Si
O
O
STATIONARY PHASES
(NORMAL POLARITY)
Silica or alumina possess polar sites that
interact with polar molecules.
Most polar…….Least polar
Components elute in increasing
order of polarity.
Components elute in increasing
order of polarity.
Polar Group
silica
29. Reverse phase
In this column the packing material is relatively nonpolar and the solvent isIn this column the packing material is relatively nonpolar and the solvent is
polar with respect to the sample. Retention is the result of the interaction ofpolar with respect to the sample. Retention is the result of the interaction of
the nonpolar components of the solutes and the nonpolar stationary phase.the nonpolar components of the solutes and the nonpolar stationary phase.
Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bondedTypical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded
hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-
organic mixtures such as methanol-water or acetonitrile-water.organic mixtures such as methanol-water or acetonitrile-water.
Common Reverse Phase SolventsCommon Reverse Phase Solvents ––
Methanol
Acetonitrile
Tetrahydrofuran
Water
CH3OH
CH3CN
H2O
30. 30
STATIONARY PHASES
(REVERSE POLARITY)
If the polar sites on silica or alumina are capped with non-polar
groups, they interact strongly with non-polar molecules.
Most non-polar…….Least non-polar
Components elute in decreasing
order of polarity.
Components elute in decreasing
order of polarity.
C18 phase
silica
Si
Me
Me
O Si
O
O
31. Size exclusion
In size exclusion the HPLC column is consisted ofIn size exclusion the HPLC column is consisted of
substances which have controlled pore sizes andsubstances which have controlled pore sizes and
is able to be filtered in an ordinarily phaseis able to be filtered in an ordinarily phase
according to its molecular size.according to its molecular size.
Small molecules penetrate into the pores withinSmall molecules penetrate into the pores within
the packing while larger molecules only partiallythe packing while larger molecules only partially
penetrate the pores. The large molecules elutepenetrate the pores. The large molecules elute
before the smaller moleculesbefore the smaller molecules..
32. 32
STATIONARY PHASES
(SIZE EXCLUSION)
Size exclusion gels separate on the basis of molecular size.
Individual gel beads have pores of set size, that restrict
entry to molecules of a minium size.
Larger molecules…….Smaller molecules
Large molecules elute fast (restricted path),
while small molecules elute slowly (long path length)
Large molecules elute fast (restricted path),
while small molecules elute slowly (long path length)
33. Ion exchange
In this column type the sample componentsIn this column type the sample components
are separated based upon attractive ionicare separated based upon attractive ionic
forces between molecules carrying chargedforces between molecules carrying charged
groups of opposite charge to those chargesgroups of opposite charge to those charges
on the stationary phase.on the stationary phase.
Separations are made between a polar mobileSeparations are made between a polar mobile
liquid, usually water containing salts or smallliquid, usually water containing salts or small
amounts of alcohols, and a stationary phaseamounts of alcohols, and a stationary phase
containing either acidic or basic fixed sites.containing either acidic or basic fixed sites.
34. 34
STATIONARY PHASES
(CATION EXCHANGE)
Silica is substituted with anionic residues that interact
strongly with cationic species (+ve charged)
Most +ve…….Least +ve
+ve charged species adhere to the support
and are later eluted with acid (H+
)
+ve charged species adhere to the support
and are later eluted with acid (H+
)
Cations exchange Na+
silica
S
O
O
ONa
35. 35
STATIONARY PHASES
(ANION EXCHANGE)
Silica is substituted with cationic residues that interact
strongly with anionic species (-ve charged)
Most -ve…….Least -ve
-ve charged species adhere to the support
and are later eluted with acid (H+
)
-ve charged species adhere to the support
and are later eluted with acid (H+
)
Anions exchange Cl-
silica
Me N
Me
Me
CH2
Cl
36. HPLC Columns
Within the Column is where separation occurs.Within the Column is where separation occurs.
Key Point –Proper choice of column is critical for success in HPLCKey Point –Proper choice of column is critical for success in HPLC
Materials of construction for the tubingMaterials of construction for the tubing
Stainless steel (the most popular; gives high pressure capabilities)Stainless steel (the most popular; gives high pressure capabilities)
Glass (mostly for biomolecules)Glass (mostly for biomolecules)
PEEK polymer (biocompatible and chemically inert to most solventsPEEK polymer (biocompatible and chemically inert to most solvents
Packing material:Packing material:
TheThe packing material is prepared from SILICA particle, ALUMINA particlepacking material is prepared from SILICA particle, ALUMINA particle
and ion exchange RESIN.and ion exchange RESIN.
Porous plug of stainless steel or Teflon are used in the end of the columnsPorous plug of stainless steel or Teflon are used in the end of the columns
to retain the packing material.to retain the packing material.
According to the mode of HPLC , they are available in different size ,According to the mode of HPLC , they are available in different size ,
diameters, pore size or they can have special materials attached ( such asdiameters, pore size or they can have special materials attached ( such as
antigen or antibody ) for immuno affinity chromatography.antigen or antibody ) for immuno affinity chromatography.
37. 3737
Modes of High Performance LiquidModes of High Performance Liquid
ChromatographyChromatography
Types of Compounds Mode Stationary
Phase
Mobile Phase
Neutrals
Weak Acids
Weak Bases
Reversed
Phase
C18, C8, C4
cyano, amino
Water/Organic
Modifiers
Ionics, Bases, Acids Ion
Pair
C-18, C-8 Water/Organic
Ion-Pair Reagent
Compounds not
soluble in water
Normal
Phase
Silica, Amino,
Cyano, Diol
Organics
Ionics Inorganic Ions Ion
Exchange
Anion or Cation
Exchange
Resin
Aqueous/Buffer
Counter Ion
High Molecular Weight
Compounds
Polymers
Size
Exclusion
Polystyrene
Silica
Gel Filtration-
Aqueous
Gel Permeation-
Organic
38. Types of columns in HPLCTypes of columns in HPLC::
Guard ColumnGuard Column
Fast ColumnFast Column
Preparative(i.d. > 4.6 mm; lengths 50 –250 mm)Preparative(i.d. > 4.6 mm; lengths 50 –250 mm)
Capillary(i.d. 0.1 -1.0 mm; various lengths)Capillary(i.d. 0.1 -1.0 mm; various lengths)
Nano(i.d. < 0.1 mm, or sometimes stated as < 100 μm)Nano(i.d. < 0.1 mm, or sometimes stated as < 100 μm)
Analytical[internal diameter (i.d.) 1.0 -4.6-mm; lengths 15 –250 mm]Analytical[internal diameter (i.d.) 1.0 -4.6-mm; lengths 15 –250 mm]
39. Guard Column
These are placed anterior to the separating column. This serves asThese are placed anterior to the separating column. This serves as
protective factor.protective factor.
They are dependable columns designed to filter or remove :They are dependable columns designed to filter or remove :
Particles that clog the separation columnParticles that clog the separation column
Compounds and ions that could ultimately cause “ Baseline drift ”,Compounds and ions that could ultimately cause “ Baseline drift ”,
decreased resolution, decreased sensitivity and create false peaks.decreased resolution, decreased sensitivity and create false peaks.
These columns must be changed on a regular basis in order to optimizeThese columns must be changed on a regular basis in order to optimize
their protective function.their protective function.
40. Fast Column
One of the primary reasons for using these column is to obtain improvedOne of the primary reasons for using these column is to obtain improved
sample output ( amount of compound per unit time).sample output ( amount of compound per unit time).
Fast column are designed to decrease the time of chromatographic analysisFast column are designed to decrease the time of chromatographic analysis
Here internal diameter is same but length is short and packed with smallerHere internal diameter is same but length is short and packed with smaller
particles , that are 3particles , that are 3 μm diameter.μm diameter.
Advantages-Advantages-
Increased sensitivityIncreased sensitivity
Decreased analysis timeDecreased analysis time
Decreased mobile phase usageDecreased mobile phase usage
Increase reproducibilityIncrease reproducibility
41. Capillary Column
It is also known as micro columnsIt is also known as micro columns
It has a diameter much less than a millimeter and there 3 types:It has a diameter much less than a millimeter and there 3 types:
Open tubularOpen tubular
Partially packedPartially packed
Tightly packedTightly packed
They allow the user to work with nanoliter sample volume , decreasedThey allow the user to work with nanoliter sample volume , decreased
flow rate and decreased solvent usage volume , led to cost effectivenessflow rate and decreased solvent usage volume , led to cost effectiveness
42. Preparatory Column
Used when objective is to prepare bulk ( milligrams) of sample forUsed when objective is to prepare bulk ( milligrams) of sample for
laboratory preparatory application.laboratory preparatory application.
It has usually a large column diameter , which is designed to facilitateIt has usually a large column diameter , which is designed to facilitate
large volume injections into the HPLC systemlarge volume injections into the HPLC system
43. 4.4. DetectorDetector::
•• The detector can see (detect) the individual molecules that comeThe detector can see (detect) the individual molecules that come
out (elute) from the column.out (elute) from the column.
••A detector serves to measure the amount of those moleculesA detector serves to measure the amount of those molecules
so that the chemist can quantitatively analyze the sampleso that the chemist can quantitatively analyze the sample
components.components.
••The detector provides an output to a recorder or computerThe detector provides an output to a recorder or computer
that results in the liquid chromatogram(i.e., the graph of thethat results in the liquid chromatogram(i.e., the graph of the
detector response).detector response).
46. 46
UV-Vis Detectors
b
c
Detector Flow Cell
I0 I
Log I0 = A = abc
I
Principles: The fraction of light transmitted through the detector cell is
related to the solute concentration according to Beer’s Law.
Characteristics: Specific, Concentration Sensitive, good stability,
gradient capability.
Special: UV-Vis Spectral capability (Diode Array Technology ).
47. 47
Fluorescence Detection
Emission
Monochromator
signal &
spectra mode
PMT detector
Reference Diode
8 µl Flow Cell, auto-recognition
Trigger pack
Exitation
Monochromator,
signal &
spectra mode
Mirror
Lens
(condensor EX)
Lens (condensor EM)
Slit EM Slit PMT
Slit EX
Diffuser
Xenon
flash Lamp,
15 W
48. 48
Electrochemical Detectors
• Gold for carbohydrates.
• Platinum for chlorite, sulfate, hydrazine, etc.
• Carbon for phenols, amines.
• Silver for chloride, bromide, cyanide.
50. 5.5. ComputerComputer::
•• Frequently called the data system,Frequently called the data system,
The computer not only controls all the modules of theThe computer not only controls all the modules of the
HPLC instrument but it takes the signal from theHPLC instrument but it takes the signal from the
detector and uses it to:detector and uses it to:
1. determine the time of elution (retention time) of the1. determine the time of elution (retention time) of the
sample components (qualitative analysis) andsample components (qualitative analysis) and
2. the amount of sample ( quantitative analysis) .2. the amount of sample ( quantitative analysis) .
52. HPLC is optimum for the separation of chemical and biologicalHPLC is optimum for the separation of chemical and biological
compounds that are non-volatile .compounds that are non-volatile .
NOTE: If a compound is volatile (i.e. a gas, fragrance, hydrocarbon inNOTE: If a compound is volatile (i.e. a gas, fragrance, hydrocarbon in
gasoline, etc.), gas chromatography is a better separation technique .gasoline, etc.), gas chromatography is a better separation technique .
Typical non-volatile compounds are:Typical non-volatile compounds are:
Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol)Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol)
Salts like sodium chloride and potassium phosphateSalts like sodium chloride and potassium phosphate
Proteins like egg white or blood proteinProteins like egg white or blood protein
Organic chemicals like polymers (e.g. polystyrene, polyethylene)Organic chemicals like polymers (e.g. polystyrene, polyethylene)
Heavy hydrocarbons like asphalt or motor oilHeavy hydrocarbons like asphalt or motor oil
Many natural products such as ginseng, herbal medicines, plant extractsMany natural products such as ginseng, herbal medicines, plant extracts
Thermally unstable compounds such as trinitrotoluene (TNT), enzymesThermally unstable compounds such as trinitrotoluene (TNT), enzymes
What is HPLC used for ?
Separation and analysis of non-volatile or
thermally unstable compounds
54. 54
How can We Analyze the Sample?
For example:
Carbohydrates
1. fructose
2. Glucose
3. Saccharose
4. Palatinose
5. Trehalulose
6. isomaltose
1
2
3
4
5
mAU
time
6
55. 55
Separations
Separation in based upon differential
migration between the stationary and
mobile phases.
Stationary Phase - the phase
which remains fixed in the
column, e.g. C18, Silica
Mobile Phase - carries the sample
through the stationary phase as it
moves through the column.
Injector
Detector
Column
Solvents
Mixer
Pumps
High Performance Liquid Chromatograph
Waste
75. HPLC uses
This technique is used for -This technique is used for -
chemistry and biochemistry research analyzingchemistry and biochemistry research analyzing
complex mixturescomplex mixtures
purifying chemical compoundspurifying chemical compounds
developing processes for synthesizing chemicaldeveloping processes for synthesizing chemical
compoundscompounds
isolating natural products, or predicting physicalisolating natural products, or predicting physical
properties.properties.
It is also used in quality control to ensure the purity ofIt is also used in quality control to ensure the purity of
raw materials, to control and improve process yields, toraw materials, to control and improve process yields, to
quantify assays of final products, or to evaluate productquantify assays of final products, or to evaluate product
stability and monitor degradation.stability and monitor degradation.
76. In addition,
it is used for analyzing air and water pollutants, for
monitoring materials that may jeopardize occupational
safety or health, and for monitoring pesticide levels in
the environment.
78. References
HPLC instrumentation – Agilent TechnologiesHPLC instrumentation – Agilent Technologies
Introduction to HPLC – Agilent TechnologiesIntroduction to HPLC – Agilent Technologies
Principles and Technique of Biochemistry and Moleculer Biology –Principles and Technique of Biochemistry and Moleculer Biology –
Wilson.Keith and Walker. JohnWilson.Keith and Walker. John
HPLC THEORY INTRODUCTION AND INSTRUMENTATION HARDWAREHPLC THEORY INTRODUCTION AND INSTRUMENTATION HARDWARE
6th October 2008. L1 - Dr Cristina Legido-Quigley, Lecturer in Pharmaceutical6th October 2008. L1 - Dr Cristina Legido-Quigley, Lecturer in Pharmaceutical
Chemistry (Separation Science) at KCLChemistry (Separation Science) at KCL
WEB REFERENCESWEB REFERENCES
http://192.215.107.101/ebn/942/tech/techfocus/1071main.htmlhttp://192.215.107.101/ebn/942/tech/techfocus/1071main.html
Skoog, Holler, and Neiman.Skoog, Holler, and Neiman. Principles of Instrumental AnalysisPrinciples of Instrumental Analysis. 5th ed.. 5th ed.
Orlando: Harcourt Brace & Co., 1998.Orlando: Harcourt Brace & Co., 1998.
http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htmhttp://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htm
http://www.chemistry.nmsu.edu/Instrumentation/Lqd_Chroma.htmlhttp://www.chemistry.nmsu.edu/Instrumentation/Lqd_Chroma.html
http://weather.nmsu.edu/Teaching_Material/SOIL698/Student_Material/HPLChttp://weather.nmsu.edu/Teaching_Material/SOIL698/Student_Material/HPLC
HP1090/HPLCINJ.HTMHP1090/HPLCINJ.HTM
http://test-http://test-
equipment.globalspec.com/LearnMore/Labware_Scientific_Instruments/Analytiequipment.globalspec.com/LearnMore/Labware_Scientific_Instruments/Analyti
cal_Instruments/Chromatographs/HPLC_Columnscal_Instruments/Chromatographs/HPLC_Columns