HPLC is a technique used to separate components in a mixture using pumps to pass a pressurized liquid solvent containing the sample through a column filled with an adsorbent material. Each component interacts slightly differently with the material, causing different flow rates and separation. HPLC has applications in manufacturing like pharmaceutical production, legal purposes like drug testing, research like separating biological samples, and medical uses like detecting vitamin levels. It provides superior resolving power over traditional chromatography to distinguish between compounds.
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 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
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 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
In this slide contains types of HPLC Columns, Plate theory and Van Deemter Equation.
Presented by : Malarvannan.M (Department of pharmaceutical analysis).
RIPER,anantpur.
The Power Point Presentation includes The Size Exclusion Chromatography and Its Method. These Slides may be helpful for master of science students. The Syllabus for the slides was prepared by following as KSV, Gandhinagar. Paper Code is CH-AC-302, Unit-01
HPLC- introduction, principle, types, working, instrumentation and operations of HPLC has been included with appropriate gifs and images for better understanding. What are all the things need to be known by a science student about HPLC (basics and working) is clearly given in this presentation.
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.
The Power Point Presentation includes The Size Exclusion Chromatography and Its Method. These Slides may be helpful for master of science students. The Syllabus for the slides was prepared by following as KSV, Gandhinagar. Paper Code is CH-AC-302, Unit-01
HPLC- introduction, principle, types, working, instrumentation and operations of HPLC has been included with appropriate gifs and images for better understanding. What are all the things need to be known by a science student about HPLC (basics and working) is clearly given in this presentation.
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.
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.
Introduction to High Performance Liquid Chromatography (HPLC)Saurabh Arora
This presentation provides a brief introduction to HPLC and its parts. The technique has found immense scope of applications in both academic and industrial laboratories requiring identification and quantification of mixtures of organic compounds.It is essential for scientists working in any field to understand and know how to use a HPLC.
HPLC Principle,Instrumentation and ApplicationAlakesh Pradhan
HPLC Chromatography and its principle
Liquid chromatography
High Performance Liquid Chromatography ( HPLC )
The components of the high performance liquid chromatograph (HPLC).
The separation process.
The chromatogram
Chromatography is based on the principle where molecules in mixture applied onto the surface or into the solid, and fluid stationary phase (stable phase) is separating from each other while moving with the aid of a mobile phase.
The factors effective on this separation process include molecular characteristics related to adsorption (liquid-solid), partition (liquid-solid), and affinity or differences among their molecular weights
Because of these differences, some components of the mixture stay longer in the stationary phase, and they move slowly in the chromatography system, while others pass rapidly into mobile phase, and leave the system faster.
High Performance Liquid Chromatography HPLC is a process of separating components in a liquid mixture. A liquid sample is injected into a stream of solvent mobile phase flowing through a column packed with a separation medium stationary phase . Sample components separate from one another by a process of differential migration as they flow through the column.As bands emerge from the column, flow carries them to one or more detectors which deliver a voltage response as a function of time. This is called a chromatogram. For each peak, the time at which it emerges identifies the sample constituent with respect to a standard. The peak’s area represents the quantity .HPLC provides a highly specific, reasonably precise, and fairly rapid analytical method for a plethora of complicated samples.This is difficult in detecting compounds. Low sensitivity of some compounds towards the stationary phase in the columns is difficult. Mohd Ali | Panjak Chasta | Dr. Kausal Kishore Chandrul "High Performance Liquid Chromatography (HPLC)" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-5 | Issue-5 , August 2021, URL: https://www.ijtsrd.com/papers/ijtsrd45146.pdf Paper URL: https://www.ijtsrd.com/pharmacy/other/45146/high-performance-liquid-chromatography-hplc/mohd-ali
Definition of biopharmaceuticals and biosimilars, Steps involved in manufacturing biopharmaceuticals, Points of differences between Biosimilars and Chemical Generics, Related issues with biosimilars
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
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.
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.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
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.
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.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...
High performance liquid chromatography
1. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
(HPLC)
Figure 1: HPLC instrument
INTRODUCTION
High-performance liquid chromatography (HPLC), is a technique in analytical chemistry used to
separate, identify, and quantify each component in a mixture. It relies on pumps to pass a
pressurized liquid solvent containing the sample mixture through a column filled with a
solid adsorbent material. Each component in the sample interacts slightly differently with the
adsorbent material, causing different flow rates for the different components and leading to
the separation of the components as they flow out the column.
HPLC has been used for manufacturing (e.g. during the production process of pharmaceutical
and biological products), legal (e.g. detecting performance enhancement drugs in urine),
research (e.g. separating the components of a complex biological sample, or of similar synthetic
chemicals from each other), and medical (e.g. detecting vitamin D levels in blood serum)
purposes.
2. HPLC is distinguished from traditional ("low pressure") liquid chromatography because
operational pressures are significantly higher (50–350 bar), while ordinary liquid
chromatography typically relies on the force of gravity to pass the mobile phase through the
column. Due to the small sample amount separated in analytical HPLC, typical column
dimensions are 2.1–4.6 mm diameter, and 30–250 mm length. Also HPLC columns are made
with smaller sorbent particles (2–50 micrometer in average particle size). This gives HPLC
superior resolving power (the ability to distinguish between compounds) when separating
mixtures, which makes it a popular chromatographic technique.
The schematic of an HPLC instrument typically includes a sampler, pumps, and a detector. The
sampler brings the sample mixture into the mobile phase stream which carries it into the
column. The pumps deliver the desired flow and composition of the mobile phase through the
column. The detector generates a signal proportional to the amount of sample component
emerging from the column, hence allowing for quantitative analysis of the sample components.
A digital microprocessor and user software control the HPLC instrument and provide data
analysis. Some models of mechanical pumps in a HPLC instrument can mix multiple solvents
together in ratios changing in time, generating a composition gradient in the mobile phase.
Various detectors are in common use, such as UV/VIS, photodiode array (PDA) or based
on mass spectrometry. Most HPLC instruments also have a column oven that allows for
adjusting the temperature the separation is performed at.
Figure 2: Principle of HPLC
3. TYPES OF HPLC
Partition chromatography
Partition chromatography uses a retained solvent, on the surface or within the grains or fibers
of an "inert" solid supporting matrix as with paper chromatography; or takes advantage of
some coulombic and/or hydrogen donor interaction with the stationary phase. Analyte
molecules partition between a liquid stationary phase and the eluent. Just as in Hydrophilic
Interaction Chromatography(HILIC; a sub-technique within HPLC), this method separates
analytes based on differences in their polarity. HILIC most often uses a bonded polar stationary
phase and a mobile phase made primarily of acetonitrile with water as the strong component.
Partition HPLC has been used historically on unbonded silica or alumina supports. Each works
effectively for separating analytes by relative polar differences. HILIC bonded phases have the
advantage of separating acidic, basic and neutral solutes in a single chromatographic run.
The polar analytes diffuse into a stationary water layer associated with the polar stationary
phase and are thus retained. The stronger the interactions between the polar analyte and the
polar stationary phase (relative to the mobile phase) the longer the elution time. The
interaction strength depends on the functional groups part of the analyte molecular structure,
with more polarized groups (e.g. hydroxyl-) and groups capable of hydrogen bonding inducing
more retention. Coulombic (electrostatic) interactions can also increase retention. Use of more
polar solvents in the mobile phase will decrease the retention time of the analytes, whereas
more hydrophobic solvents tend to increase retention times.
Figure 3: Partition chromatography
4. NORMAL PHASE CHROMATOGRAPHY
Also known as normal-phase HPLC (NP-HPLC) this method separates analytes based on their
affinity for a polar stationary surface such as silica; hence it is based on analyte ability to engage
in polar interactions (such as hydrogen bonding or dipole-dipole type of interactions) with the
sorbent surface. NP-HPLC uses a non-polar, non-aqueous mobile phase (e.g. chloroform), and
works effectively for separating analytes readily soluble in non-polar solvents. The analyte
associates with and is retained by the polar stationary phase. Adsorption strengths increase
with increased analyte polarity. The interaction strength depends not only on the functional
groups present in the structure of the analyte molecule, but also on stearic factors. The effect
of steric hindrance on interaction strength allows this method to resolve (separate) structural
isomers.
Figure 4: Normal phase chromatography
DISPLACEMENT CHROMATOGRAPHY
The basic principle of displacement chromatography is: A molecule with a high affinity for the
chromatography matrix (the displacer) will compete effectively for binding sites, and thus
displace all molecules with lesser affinities. There are distinct differences between
displacement and elution chromatography. In elution mode, substances typically emerge from
a column in narrow, Gaussian peaks. Wide separation of peaks, preferably to baseline, is
desired in order to achieve maximum purification. The speed at which any component of a
mixture travels down the column in elution mode depends on many factors. But for two
substances to travel at different speeds, and thereby be resolved, there must be substantial
differences in some interaction between the biomolecules and the chromatography matrix.
Operating parameters are adjusted to maximize the effect of this difference. In many cases,
baseline separation of the peaks can be achieved only with gradient elution and low column
loadings. Thus, two drawbacks to elution mode chromatography, especially at the preparative
scale, are operational complexity, due to gradient solvent pumping, and low throughput, due to
low column loadings. Displacement chromatography has advantages over elution
5. chromatography in that components are resolved into consecutive zones of pure substances
rather than “peaks”. Because the process takes advantage of the nonlinearity of the isotherms,
a larger column feed can be separated on a given column with the purified components
recovered at significantly higher concentration.
Figure 5: Displacement chromatography
REVERSED PHASE CHROMATOGRAPHY
Reversed phase HPLC (RP-HPLC) has a non-polar stationary phase and an aqueous, moderately
polar mobile phase. One common stationary phase is silica which has been surface-modified
with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or C8H17. With such
stationary phases, retention time is longer for molecules which are less polar, while polar
molecules elute more readily (early in the analysis). An investigator can increase retention
times by adding more water to the mobile phase; thereby making the affinity of the
hydrophobic analyte for the hydrophobic stationary phase stronger relative to the now more
hydrophilic mobile phase. Similarly, an investigator can decrease retention time by adding more
organic solvent to the eluent. RP-HPLC is so commonly used that it is often incorrectly referred
to as "HPLC" without further specification. The pharmaceutical industry regularly employs RP-
HPLC to qualify drugs before their release.
RP-HPLC operates on the principle of hydrophobic interactions, which originates from the high
symmetry in the dipolar water structure and plays the most important role in all processes in
life science. RP-HPLC allows the measurement of these interactive forces. The binding of the
6. analyte to the stationary phase is proportional to the contact surface area around the non-polar
segment of the analyte molecule upon association with the ligand on the stationary phase.
This solvophobic effect is dominated by the force of water for "cavity-reduction" around the
analyte and the C18-chain versus the complex of both. The energy released in this process is
proportional to the surface tension of the eluent (water: 7.3×10−6 J/cm², methanol:
2.2×10−6 J/cm²) and to the hydrophobic surface of the analyte and the ligand respectively. The
retention can be decreased by adding a less polar solvent (methanol, acetonitrile) into the
mobile phase to reduce the surface tension of water. Gradient elution uses this effect by
automatically reducing the polarity and the surface tension of the aqueous mobile phase during
the course of the analysis.
Figure 6: Reverse phase chromatography
SIZE EXCLUSION CHROMATOGRAPHY
Size-exclusion chromatography (SEC), also known as gel permeation chromatography or gel
filtration chromatography separates particles on the basis of molecular size (actually by a
particle's Stokes radius). It is generally a low resolution chromatography and thus it is often
reserved for the final, "polishing" step of the purification. It is also useful for determining
the tertiary structure and quaternary structure of purified proteins. SEC is used primarily for the
analysis of large molecules such as proteins or polymers. SEC works by trapping these smaller
molecules in the pores of a particle. The larger molecules simply pass by the pores as they are
too large to enter the pores. Larger molecules therefore flow through the column quicker than
smaller molecules, that is, the smaller the molecule, the longer the retention time.
7. This technique is widely used for the molecular weight determination of polysaccharides. SEC is
the official technique (suggested by European pharmacopeia) for the molecular weight
comparison of different commercially available low-molecular weight heparins
Figure 7: Size exclusion chromatography
ION EXCHANGE CHROMATOGRAPHY
In ion-exchange chromatography (IC), retention is based on the attraction between solute ions and
charged sites bound to the stationary phase. Solute ions of the same charge as the charged sites on
the column are excluded from binding, while solute ions of the opposite charge of the charged sites
of the column are retained on the column. Solute ions that are retained on the column can be eluted
from the column by changing the solvent conditions (e.g. increasing the ion effect of the solvent
system by increasing the salt concentration of the solution, increasing the column temperature,
changing the pH of the solvent, etc.).
Types of ion exchangers include:
Polystyrene resins – These allow cross linkage which increases the stability of the chain. Higher
cross linkage reduces swerving, which increases the equilibration time and ultimately improves
selectivity.
Cellulose and dextran ion exchangers (gels) – These possess larger pore sizes and low charge
densities making them suitable for protein separation.
Controlled-pore glass or porous silica
In general, ion exchangers favor the binding of ions of higher charge and smaller radius.
8. An increase in counter ion (with respect to the functional groups in resins) concentration reduces the
retention time. A decrease in pH reduces the retention time in cation exchange while an increase in
pH reduces the retention time in anion exchange. By lowering the pH of the solvent in a cation
exchange column, for instance, more hydrogen ions are available to compete for positions on the
anionic stationary phase, thereby eluting weakly bound cations.
This form of chromatography is widely used in the following applications: water purification,
preconcentration of trace components, ligand-exchange chromatography, ion-exchange
chromatography of proteins, high-pHanion-exchange chromatography of carbohydrates and
oligosaccharides, and others.
Figure 8: ion exchange chromatography
BIOAFFINITY CHROMATOGRAPHY
This chromatographic process relies on the property of biologically active substances to form stable,
specific, and reversible complexes. The formation of these complexes involves the participation of
common molecular forces such as the Vander waalsinteraction, electrostatic interaction, dipole-
dipole interaction, hydrophobic interaction, and the hydrogen bond. An efficient, biospecific bond is
formed by a simultaneous and concerted action of several of these forces in the complementary
binding sites.
Figure 9: Bioaffinity chromatography
9. AQUEOUS NORMAL PHASE CHROMATOGRAPHY
Aqueous normal-phase chromatography (ANP) is a chromatographic technique which
encompasses the mobile phase region between reversed-phase chromatography (RP) and
organic normal phase chromatography (ONP). This technique is used to achieve unique
selectivity for hydrophilic compounds, showing normal phase elution using reversed-phase
solvents.
Figure 10: Aqueous normal phase chromatography
APPLICATIONS OF HPLC
MANUFACTURING
As briefly mentioned, HPLC has many applications in both laboratory and clinical science. It is a
common technique used in pharmaceutical development as it is a dependable way to obtain
and ensure product purity. While HPLC can produce extremely high quality (pure) products, it is
not always the primary method used in the production of bulk drug materials. According to the
European pharmacopoeia, HPLC is used in only 15.5% of syntheses. However, it plays a role in
44% of syntheses in the United States pharmacopoeia. This could possibly be due to differences
in monetary and time constraints, as HPLC on a large scale can be an expensive technique. An
increase in specificity, precision, and accuracy that occurs with HPLC unfortunately corresponds
to an increase in cost.
LEGAL
This technique is also used for detection of illicit drugs in urine. The most common method of
drug detection is an immunoassay. This method is much more convenient. However,
convenience comes at the cost of specificity and coverage of a wide-range of drugs. As HPLC is a
method of determining (and possibly increasing) purity, using HPLC alone in evaluating
concentrations of drugs is somewhat insufficient. With this, HPLC in this context is often
performed in conjunction with mass spectrometry. Using liquid chromatography instead of gas
chromatography in conjunction with MS circumvents the necessity for derivitizing with
10. acetylating or alkylation agents, which can be a burdensome extra step. This technique has
been used to detect a variety of agents like doping agents, drug metabolites, glucuronide
conjugates, amphetamines, opioids, cocaine, BZDs, ketamine, LSD, cannabis, and
pesticides. Performing HPLC in conjunction with Mass spectrometry reduces the absolute need
for standardizing HPLC experimental runs.
RESEARCH
Similar assays can be performed for research purposes, detecting concentrations of potential
clinical candidates like anti-fungal and asthma drugs. This technique is obviously useful in
observing multiple species in collected samples, as well, but requires the use of standard
solutions when information about species identity is sought out. It is used as a method to
confirm results of synthesis reactions, as purity is essential in this type of research. However,
mass spectrometry is still the more reliable way to identify species.
MEDICAL
Medical use of HPLC can include drug analysis, but falls more closely under the category of
nutrient analysis. While urine is the most common medium for analyzing drug concentrations,
blood serum is the sample collected for most medical analyses with HPLC. Other methods of
detection of molecules that are useful for clinical studies have been tested against HPLC,
namely immunoassays. In one example of this, competitive protein binding assays (CPBA) and
HPLC were compared for sensitivity in detection of vitamin D. Useful for diagnosing vitamin D
deficiencies in children, it was found that sensitivity and specificity of this CPBA reached only
40% and 60%, respectively, of the capacity of HPLC. While an expensive tool, the accuracy of
HPLC is nearly unparalleled.
BIBLIOGRAPHY
www.wikipedia.com
www.slideshare.com
www.wikihow.com
Images downloaded through Google image search.
ABU SUFIYAN CHHIPA BPHARM VI SEM